Influence of B cell-lineage targeted CAR-T cell therapy on humoral immunity and vaccine-induced antibody response

Domain: chemistry
Published: 2026-04-07
Editorial outcome: minor_revision

Abstract
Humoral immune-related adverse events, including hypogammaglobulinemia and B cell depletion, pose long-term infection risks after chimeric antigen receptor T cell therapy (CARTx) for hematologic malignancies. This prospective study evaluates the kinetics of pathogen-specific humoral immunity prior to and up to a year after CARTx targeting CD19 and CD20 (B cells) or BCMA (plasma cells) in 100 and 28 individuals, respectively. Antibodies are tested for 12 vaccine-preventable pathogens and using comprehensive high-throughput antibody profiling. A subset of 72 participants are evaluated for post-CARTx vaccine responses. Here, we show pathogen-specific humoral immunity does not significantly change after CD19-, CD20-, or BCMA-targeted CAR-T cell therapy (CARTx). However, seroprotective antibodies are absent for up to one-third of routine vaccine-preventable pathogens in CD19- and CD20-CARTx recipients and for nearly half of vaccine-preventable pathogens in BCMA-CARTx recipients by one-year post-CARTx. Pre-vaccination B cell count is the main predictor of vaccine response.

Introduction
The development of chimeric antigen receptor T cell therapy (CARTx) for B cell and plasma cell malignancies is a major milestone in cancer immunotherapy with high rates of complete remission1,2. Five products targeting CD19 are approved to treat children and/or adults with refractory or relapsed acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), and chronic lymphocytic leukemia (CLL)3,4, and two products targeting B cell maturation antigen (BCMA) are approved for adults with multiple myeloma (MM)5,6. These products are also being studied as potentially transformational therapies for autoimmune diseases7,8. Thus, the uses for CARTx are rapidly increasing.

Although cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are the earliest and most serious immune related adverse events (irAEs) after CARTx, severe manifestations affect a minority of patients, are typically transient, and can usually be effectively managed9. In contrast, on-target, off-tumor depletion of non-malignant B lineage cells affects the majority of B lineage targeted CARTx recipients and is a principal irAE of CARTx10,11. These humoral irAEs, along with immune effector cell-associated hematotoxicity (ICAHT), can persist for years and result in increased risk for infections; indeed, infections are the most common cause of non-relapse mortality after CARTx9–16.

The long-term effects of CARTx on pre-existing pathogen-specific immunity are poorly understood. CD19 expression is found on early and mature B cell lineages but declines as B cells differentiate into plasma cells17. Plasma cells can live for decades18 and are the primary long-lived source of antibodies to previously encountered pathogens17,19–23. Small studies have suggested that pre-existing pathogen-specific IgG may persist despite depletion of CD19+ B cells after CD19-CARTx or treatment with rituximab (anti-CD20) due to preservation of fully differentiated CD19- plasma cells24–28. In contrast, BCMA is expressed by long-lived plasma cells29,30, and depletion of non-malignant BCMA+ plasma cells after BCMA-CARTx may result in loss of pre-existing pathogen-specific IgG but preserve the naïve and memory B cell compartments necessary for vaccine response31. Additionally, pathogen-specific B or T cells may recover after CAR-T cell therapy, which may also impact antibody titers26,32,33. To date, the published literature on the effects of CARTx on pre-existing pathogen-specific immunity are limited and inconclusive, particularly for BCMA-CARTx.

In light of these uncertainties, patients often receive prophylactic immunoglobulin replacement therapy (IGRT) to address hypogammaglobulinemia, although this blood product is expensive, has frequent side effects, can reduce vaccine efficacy, and is of unclear benefit in this population34–36. Alternatively, vaccination after CARTx may be a durable and cost-effective tool to mitigate irAEs, as approximately one third of infections may be from vaccine-preventable infections37. Vaccine responses after CD19-CARTx are biologically plausible given observations that patients can respond to vaccines ≥6 months after rituximab, even in the absence of measurable peripheral blood B cells38–40. Similar observations have been made for CARTx recipients receiving SARS-CoV-2 and influenza vaccines26,33,41. Additionally, CD19-CARTx recipients can recover poly T clonal CD19+ B cells without relapse, typically ≥6 months after therapy42, although persistent B cell aplasia is common1. While responses to SARS-CoV-2 vaccination have been well studied after CARTx43–45, data on response to other routine vaccines in CARTx recipients are very limited41,46,47. Improved understanding of the need and strategies for vaccinating CARTx recipients is critical given pre-existing and emergent gaps in humoral immunity related to their treatment.

We hypothesized that CD19 or CD20-CARTx targeting B cells (hereafter referred to as CD19/20-CARTx) would not impact pre-existing pathogen-specific antibody levels, but these individuals would have poor vaccine responses. Conversely, we hypothesized that BCMA-CARTx targeting plasma cells would result in loss of pathogen-specific antibody levels but have less impact on vaccine responses. Here, we report the results of a prospective observational study evaluating the impact of CD19/20 and BCMA-targeted CARTx on multiple metrics of humoral and pathogen-specific immunity, as well as the ability of routine vaccines to improve pathogen-specific immunity in CARTx recipients.

Results
Participant characteristics
We prospectively enrolled 279 individuals who were planning to receive CARTx for a B cell or plasma cell malignancy from 2019-2023 at Fred Hutchinson Cancer Center (Fred Hutch) and Seattle Children’s Hospital (SCH). We collected blood pre-CARTx (pre-lymphodepleting chemotherapy) and at approximately six months and/or one year in 128 participants who remained in remission without additional anti-tumor therapy and who had not received IGRT in the preceding eight weeks (Fig.1; Supplementary Table 1). One hundred participants received CD19/CD20-CARTx and 28 received BCMA-CARTx. Demographic and clinical characteristics are detailed in Table 1. The median age was 62 years old (range, 18-83 years). The BCMA cohort was more heavily pre-treated with a median of 8 (range, 1-14) prior cancer therapies compared to a median of 4 (range, 1-9) in the CD19/20 cohort. Forty-four participants (34%) received a hematopoietic cell transplant (HCT) before CARTx with a higher proportion in the BCMA-CARTx cohort (75%) compared to the CD19/20-CARTx cohort (23%). CARTx indications in the CD19/20 cohort included B cell NHL, ALL, and CLL; the BCMA cohort was treated for MM.

To evaluate pre-CARTx pathogen-specific antibody titers in children, adolescents, and young adults (CAYA) treated with B cell-targeted CARTx for ALL who were underrepresented in the longitudinal cohort due to frequent use of IGRT and additional anti-tumor therapies, we included baseline samples from 22 participants treated at SCH who did not have subsequent samples meeting inclusion criteria (Supplementary Table 2). Thirteen individuals in this subgroup received CD19-CARTx, two received CD22-CARTx, and six received CD19/22 dual-targeting therapy.

CAR-T cell persistence
We measured CAR-T cell persistence at approximately six-months (n=30, CD19/20; n=9, BCMA) and one-year (n=23, CD19/20; n=8, BCMA) in a subset of participants for whom a PCR assay targeting their CAR-T cell product was available. For both CD19- and BCMA-CARTx recipients with ongoing cancer remission, most participants had detectable CAR-T cells through one year (Supplementary Fig.1a,b). Thus, our results are interpreted in the context of ongoing CAR-T cell mediated B cell and plasma cell depletion as further demonstrated below.

Kinetics of immunoglobulins, B cell subsets, and T cell subsets before and after CARTx
We evaluated changes in total IgA, IgM, and IgG subsets, as well as T and B cell subsets, stratified by receipt of CD19/CD20 or BCMA-CARTx (Fig.2a-i). Baseline immunoglobulin levels were below the lower limit of normal for most participants in both groups. Baseline IgA and IgM (Fig.2a,b) titers were significantly lower in participants receiving BCMA- compared to CD19/20-CARTx, whereas IgG levels were similar (Fig.2c). IgA levels significantly decreased at six months after CARTx in both groups, with modest recovery by one year that was no longer significantly different between CARTx target but remained below pre-CARTx levels (Supplementary Fig.2a-c). IgM levels modestly increased after BCMA-CARTx compared to a small but significant reduction after CD19/20-CARTx that remained persistently lower at one year compared to baseline. IgG levels remained similar when comparing between groups at each time point, but IgG levels significantly decreased after CD19/20-CARTx.

CD19+ B cell counts had distinct kinetics in BCMA- and CD19/20-CARTx recipients (Fig.2d-g; Supplementary Fig.3a,b; Supplementary Table 3). Both groups had similarly low CD19+ B cell counts prior to CARTx. In BCMA-CARTx recipients, there was a significant increase in CD19+ B cell counts over time post-CARTx, with the majority of recipients achieving a B cell count ≥20 cells/µL by six months (70%) and 1 year (82%). In contrast, CD19/20-CARTx recipients had a significant decrease in CD19+ B cell count after CARTx, with a minority having a CD19+ B cell count ≥20 cells/µL at six months (17%) but recovery to pre-CARTx levels at one year (39%). Naïve B cells (CD19+CD27–IgD+) had a similar pattern. Memory B cells (unswitched, CD19+CD27–IgMhiIgDlo and class switched, CD19+CD27+IgD–) were low pre-CARTx in both groups with further decline after CD19/20-CARTx but some recovery after BCMA-CARTx. Bone marrow CD138+ plasma cells in BCMA-CARTx recipients showed sharp declines after treatment (Fig.2j). Overall, these patterns are congruent with the expected effects of CARTx by target and are consistent with the findings of CAR-T cell persistence. Finally, CD4+ and CD8+ T cell counts were similar and stable by CARTx target and time point (Fig.2h,i).

Kinetics of pathogen-specific antibody titers before and after CARTx
We tested serum for IgG to 12 vaccine-preventable pathogens at each time point. First, we compared baseline antibody titers among 100 adults and 22 CAYA prior to CD19, CD20, CD22, or CD19/22-targeted CARTx. We observed that the youngest individuals (0-8 years old) had lower antibody titers and frequency of seroprotection for many pathogens compared to older individuals although with some differences, such as higher antibodies for hepatitis viruses compared to adults (Supplementary Fig.4a-l; Supplementary Fig.5a-l). Compared to population-based seroprevalence studies26,48–51, BCMA-CARTx recipients had a lower prevalence of pre-CARTx seroprotection, while CD19/20-CARTx recipients had a similar prevalence of seroprotection for most pathogens (Supplementary Fig.6).

Next, we evaluated changes in pathogen-specific antibody titers over time in the CARTx cohort with a pre-CARTx sample (n=128; median days before CARTx, 7 days; interquartile range [IQR], 5-12) and at least one post-CARTx sample at approximately six months (n=110; median days after CARTx, 193 days; IQR 175-217) and/or one year (n=91; median, 376 days; IQR, 354-407) after CARTx (Fig.3a-l; Supplementary Table 4). At baseline, BCMA-CARTx recipients had significantly lower pathogen-specific antibody titers for most pathogens compared to CD19/20-CARTx. BCMA-CARTx recipients showed a general pattern of increases in antibody titers by one-year post-CARTx for several pathogens, which was contrary to our original hypothesis. After CD19/20-CARTx, pathogen-specific IgG levels were stable over time, consistent with our hypothesis. However, pathogen-specific antibody titers at one year remained lower for most pathogens in the BCMA-CARTx cohort compared to the CD19/20 CARTx cohort. Similar patterns were observed when comparing the proportion of participants with antibody titers considered to be seroprotective (Supplementary Fig.7a-l; Supplementary Table 5). Seroprotection to pathogens corresponding to live-attenuated vaccines was particularly low in BCMA-CARTx recipients (Supplementary Fig.8a,b).

To better appreciate the pattern of gains, losses, or stability in seroprotective antibody titers, we generated divergent stacked bar charts (Supplementary Fig.9a-d; Supplementary Fig.10a-d). These visually depict that there was more change in seroprotection status after BCMA-CARTx compared to CD19/20-CARTx. To further summarize these data, we analyzed net gains or losses in the total number of pathogens with seroprotection per participant from pre- to post-CARTx (Fig.4a,b). Compared to CD19/20-CARTx recipients, BCMA-CARTx recipients were more likely to both gain and lose seroprotective levels of pathogen-specific antibodies compared to pre-CARTx levels, both at six months and one year but had a higher net gain of protective immunity by one year post-CARTx.

Variables associated with seroprotective pathogen-specific antibody titers before and after CARTx
We created a summary measure of pathogen-specific immunity by computing the percentage of pathogens with seroprotective antibodies per participant and time point (Fig.4c). Across time points, the median percentages for this metric ranged from 67%-75% for CD19/20 CARTx recipients and 42%-50% for BCMA CARTx recipients. We computed GEE models comparing the composite outcome between and within groups over time, adjusted for baseline and time-dependent clinical and immunologic variables (Fig.4d; Supplementary Table 6,7). This demonstrated that the proportion of pathogens with seroprotective antibody titers was stable over time after CD19/20-CARTx and modestly increased after BCMA-CARTx (although the increase did not reach statistical significance). BCMA-CARTx recipients had a lower proportion of pathogens with seroprotective antibody titers at all time points compared to CD19/20-CARTx recipients, consistent with the individual-level antibody results. The model-predicted differences translate to approximately two to three fewer pathogens with seroprotection for the BCMA group compared to the CD19/20 group at each time point.

We identified other variables associated with a higher proportion of seroprotective antibody titers from the adjusted models, including higher total IgG, higher total IgA, white race, and receipt of IGRT within 8-16 weeks before sampling (Supplementary Table 7). CD19+ B cell counts and receipt of relevant vaccines during the study period were not associated with the proportion of seroprotective antibody titers. Antibody kinetics restricted to data points without a preceding relevant vaccine administration are depicted in Supplementary Fig.11a-l.

Comprehensive serological profiling using VirScan to further explore the broader impact of CARTx on antigen-specific humoral immunity
In addition to individual assays to test antibody titers for vaccine-preventable pathogens, we tested the same samples using VirScan to comprehensively analyze antigen-specific antibody profiles. The VirScan assay contains peptides derived from most known human viruses (244 viral species; 1,361 strains), 62 bacterial species (72 strains), 22 parasite and yeast species, and 86 allergens. The median number of “epitope hits” per participant across all time points was 132 (IQR, 72-200) across a median of 55 (IQR, 33-76) species. Among the CD19/20 cohort of adults and CD19, CD22, and CD19/22 CAYA cohort at baseline pre-CARTx, two to three times more epitope hits were observed in adults compared to children <18 years old (Fig.5a). Consistent with results for vaccine preventable pathogen-specific IgG, BCMA-CARTx recipients demonstrated significantly fewer epitope hits compared to CD19/20 recipients at each measured time point (Fig.5b). Both groups had relatively stable epitope hits over time despite CARTx, and these findings were confirmed in an adjusted GEE model (Supplementary Tables 8,9). A history of prior HCT or prior CARTx was significantly associated with fewer epitope hits.

We also assessed measures of diversity including richness (number of different targets represented) and evenness (relative abundance of epitopes from each target). We first estimated Shannon’s Evenness Index, a measure of the evenness of epitopes of different targets distributed within a sample. This metric was stable over time in both cohorts but significantly higher in BCMA-CARTx recipients, reflecting depletion of epitopes across targets after BCMA-CARTx (Fig.5c). In contrast, Simpson’s Index, a combined measure of evenness and richness, was similar between BCMA- and CD19/20-CARTx recipients and stable over time (Fig.5d). Together, this indicates more richness in the CD19-CARTx cohort due to a higher number of targets with epitope counts >1.

Humoral immune response to routine vaccines after CARTx
We next evaluated the immunogenicity of vaccines administered per standard of care52 in 72 participants comprised of a subset of 59 participants from the pre/post-CARTx cohort and an additional 13 participants prospectively enrolled after CARTx (Fig.1; Supplementary Table 10). Sixty participants received CD19/CD20-CARTx and 12 received BCMA-CARTx. Demographic and clinical characteristics were similar to the overall cohort (Supplementary Table 11). The median number of evaluable unique vaccines administered per participant was five (IQR, 5-6; CD19/20 median, 6; IQR, 5-6 vs. BCMA median, 5; IQR, 3-5), targeting a median of six pathogens (IQR, 5-8) with a median of two doses per vaccine (IQR, 1-3; CD19/20 median, 2; IQR, 1-3 vs. BCMA median, 1; IQR, 1-3). Time to first vaccination was a median of 8.6 months (IQR, 7.1-13.6; CD19/20 median, 8.7; IQR, 7.1-13.6 vs. BCMA median, 8.2; IQR, 6.8-12.3) post-CARTx (Supplementary Fig.12a,b). Details pertaining to the number of participants receiving each dose per pathogen, timing relative to CARTx, and type of pneumococcal vaccines are in Supplementary Tables 12,13.

We evaluated pre-vaccine immunoglobulins, B cell subsets, and T cell subsets, stratified by receipt of CD19/CD20 or BCMA-CARTx (Supplementary Fig.13a-e). Prior to vaccination, IgA was generally lower, and CD19+ B cell counts were generally higher, in BCMA- compared to CD19/20-CARTx recipients, although these differences were not statistically significant. IgM, IgG and CD4+ T cell count were similar between CARTx groups.

Pathogen-specific antibody titers prior to and after each relevant vaccine dose are shown in Fig.6a-l with significance testing shown in Supplementary Table 14. Overall, the majority of testable pathogens showed no significant change in titers after a single vaccine dose, but significant increases were observed after ≥2 doses for 33% of tested pathogens in the CD19/20 cohort and 75% of tested pathogens in the BCMA cohort. These data are similarly displayed based on the proportion with seroprotective titers in Supplementary Fig.14a-l. Pre-vaccine seroprotective pathogen-specific antibody titers were present for 201 of 357 (56%) total antibody tests in the CD19/20 cohort and 27 of 54 (50%) tests in the BCMA cohort.

To estimate and compare seroresponse rates following each vaccine dose, as well as to better understand the clinical utility of vaccination and factors associated with a response, we evaluated vaccine response in participants with pre-vaccine antibody titers below the threshold for seroprotection. Due to relatively high levels of seroprotection for some pathogens, only a subset of participants and pathogens were eligible for this analysis. Demographics and clinical characteristics for those included in the seroresponse analysis were largely similar to those for participants in the vaccine cohort that were excluded from this analysis (Supplementary Table 15). In this subgroup of 58 participants (48 CD19/20, 10 BCMA) with a median of 3 (IQR 2-4) evaluable pathogens per participant, we estimated the cumulative incidence of seroresponse, defined as a ≥2-fold increase in antibody titers after vaccination. Thirty seven participants (30 CD19/20, 7 BCMA) had a seroresponse for at least one pathogen. The overall cumulative incidence of a seroresponse was approximately 50% or higher for H. influenzae, C. diphtheriae, hepatitis B virus, and S. pneumoniae after ≥2 vaccine doses (Fig.6m-q; Supplementary Table 16). BCMA-CARTx recipients had earlier and greater overall seroresponse rates compared to CD19/20-CARTx recipients (Fig.6m-q), although the relatively low number of participants preclude definitive conclusions.

We used Cox regression models to further evaluate correlates of vaccine responsiveness. In adjusted models, receipt of BCMA-CARTx (adjusted hazard ratio [aHR], 2.53; 95% CI,1.05-6.08; p=0.04) and higher CD19+ B cell count (aHR per log increase, 2.16; 95% CI, 1.44-3.23; p<0.001) were significantly associated with increased seroresponse, when averaged across pathogens (Table 2). CD19+ B cell count ≥20 cells/µL (indicative of B cell recovery) was a pre-specified threshold for analysis and similarly associated with a greater rate of seroresponse (aHR, 3.72; 95% CI, 1.72-8.01; p<0.001; Table 2). To further explore the impact of CD19+ B cell count on individual vaccine responses, we created cumulative incidence curves of seroresponse stratified by CD19+ B cell count, which showed a general pattern of greater seroresponse in participants with ≥20 cells/µL (indicative of B cell recovery) across evaluable pathogens (Supplementary Fig.15a-e; Supplementary Fig.16a-c). Notably, vaccine responses were also observed in participants with low (<20 cells/μL) or no CD19+ B cell counts (Supplementary Tables 17,18). For example, seroresponses after 2 doses of vaccination in individuals with CD19+ B cell counts <20 cells/μL ranged from 17%-50%, depending on the pathogen (Supplementary Table 18).

S. pneumoniae functional and serotype specific responses to vaccination
To evaluate whether vaccine seroresponse based on total S. pneumoniae IgG titers correlates with functional humoral immunity and serotype-specific responses, we assessed pneumococcal opsonophagocytic activity and serotype-specific IgG levels among twelve participants who met the seroresponse criterion and had paired pre-vaccine and post-dose 2 serum samples available for testing. Responses were tested for the 13 serotypes included in the PCV13 vaccine. Among this subgroup, a ≥4-fold increase in functional opsonophagocytic activity was detectable for at least one serotype for 10 of the 12 participants and to a median of 8 serotypes (IQR, 5-9) in these 10 participants (Supplementary Fig.17a). Serotype-specific IgG testing demonstrated a ≥4-fold increase in IgG for at least one serotype in 9 of 12 participants and to a median of 5 serotypes (IQR, 4-9) in these 9 participants (Supplementary Fig.17b). A moderate correlation was observed between fold change in opsonization and IgG (ρ=0.65 aggregated across all serotypes, and ranging from ρ=0.36 to 0.87 for individual serotypes).

Methods
Study approval
All participants provided written informed consent prior to inclusion in the study in accordance with the Declaration of Helsinki. The investigation was approved by the Fred Hutch Institutional Review Board.

Study design and participants
Pediatric and adult participants were enrolled as part of a prospective observational study of individuals with an underlying B cell lineage malignancy with planned treatment with chimeric antigen receptor T cell therapy (CARTx). Individuals were recruited from the Fred Hutchinson Cancer Center (Fred Hutch) or Seattle Children’s Hospital (SCH). All individuals who received commercial or investigational CARTx for a B cell or plasma cell malignancy were approached for enrollment. Informed consent was obtained from all participants. Investigational study protocols for participants enrolled in this study included the following at SCH (NCT02028455, NCT03244306, NCT03330691, NCT03338972, and NCT03684889) and Fred Hutch (NCT01865617, NCT02706392, NCT03103971, NCT02706405, NCT02631044, NCT03277729, NCT03105336, NCT02614066, NCT03331198, NCT03575351, NCT03502577, NCT03338972, NCT02028455). Demographic and treatment details for participants were extracted from medical records and electronic databases. Sex and gender were not considered in the study design. Sex was determined based on self-report of participants as indicated in the electronic medical record. Gender was not collected as part of enrollment. Participants were compensated for each blood sample received.

Eligible individuals for pre/post-CARTx analyses included those in sustained remission from their underlying malignancy after CARTx, without new anti-tumor directed therapy or hematopoietic cell transplant (HCT) for their underlying malignancy prompting CARTx. Receipt of pre-planned maintenance therapy was not exclusionary. Follow-up samples were obtained at approximately six months and one-year post-CARTx. For inclusion in pre/post-CARTx analysis, a pre- and at least one post-CARTx sample 3 months from CARTx was required. A subset of pediatric participants with only pre-CARTx samples were included in descriptive results comparing baseline pre-CARTx data only.

We evaluated vaccine responses among participants eligible for the above analyses who received at least one vaccine and who had a preceding and ≥1 post-vaccine sample obtained for testing. This analysis also included additional participants prospectively enrolled after CARTx. Vaccinations after CARTx were administered according to the Fred Hutch Standard Practice Guideline52 at the discretion of health care providers. Briefly, killed or inactivated vaccinations were recommended in patients who were ≥6 months post-CARTx and off IVIG for ≥2 months, irrespective of immune reconstitution. Live vaccines were recommended in patients who were ≥12 months post-CARTx, off immunosuppression for ≥12 months, had not received IVIG for ≥8 months, and had a CD4+ T cell count >200 cells/mm3.

Samples obtained within eight weeks (<2 half-lives for circulating IgG) of immunoglobulin replacement therapy (IGRT) were excluded from all analyses to reduce contribution of supplemental IgG to the interpretation of pathogen-specific titer results26,63.

Samples and testing
Samples from all time points were blinded to clinical details and batch tested per participant to minimize batch effects. Serum was isolated from whole blood collected in red-top vacutainer tubes and stored at –80°C. Peripheral blood mononuclear cells (PBMCs) from whole blood were collected in acid citrate dextrose vacutainers. PBMCs were isolated by Ficoll-Histopaque gradient separation followed by centrifugation to obtain a mononuclear cell layer. PBMCs were washed, aliquoted in freezing medium of 90% fetal bovine serum and 10% DMSO before storage in liquid nitrogen.

Testing for serum total immunoglobulins and pathogen-specific IgG titers
Total IgG, total IgM, and total IgA in serum were tested via turbidometry at the University of Washington Department of Laboratory Medicine. In participants with multiple myeloma (MM), we estimated total IgG, IgM and IgA by subtracting the monoclonal component. Pathogen-specific total IgG or serotype specific-IgG was measured for hepatitis A, hepatitis B, poliovirus type 1 and 3, varicella zoster virus, measles, mumps, rubella, Haemophilus influenzae type B, Clostridium tetani, Corynebacterium diphtheriae, and Streptococcus pneumoniae using gold standard tests in Clinical Laboratory Improvement Amendments (CLIA)-certified reference laboratories as detailed in Supplementary Table 19. Immunoglobulin and pathogen-specific IgG values that were below the lower detection limit were assigned values of the lower detection limit divided by 2; values above the upper detection limit were assigned values of the upper detection limit.

Evaluation of functional antibodies against S. pneumoniae using a multiplex opsonophagocytosis assay (MOPA).
Functional anti-pneumococcal antibodies were assessed using a standardized multiplex opsonophagocytosis assay (MOPA) performed by Sunfire Biotechnologies. Participants were included if they had paired pre-vaccine and post-dose 2 serum samples and demonstrated a ≥2-fold increase (i.e., seroresponse) in total S. pneumoniae IgG antibodies (Mayo Clinic Laboratories); pre-vaccine seroprotection was not an exclusion criterion. The assay quantified opsonization indices (OIs) against the 13 PCV13 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F using antibiotic-resistant target strains and differentiated HL-60 phagocytes, following validated MOPA protocols developed by Nahm and colleagues64,65. Heat-inactivated sera were serially diluted and incubated with pneumococci, complement, and HL-60 cells; surviving bacteria were enumerated after incubation, and OIs were calculated as the reciprocal dilution achieving 50% killing.

Evaluation of B cell subsets, T cell subsets, and plasma cell
B and T cell subsets were immunophenotyped from whole blood PBMC samples. All monoclonal antibodies used for flow-cytometric immunophenotyping were commercially available, research-grade reagents with established specificity and prior validation by the manufacturers. Antibodies were selected based on standard use in immune-cell profiling panels and clones with well-characterized performance. Samples were thawed and incubated in FACS buffer (DPBS + 2% fetal bovine serum) with antibody cocktails. Cells were labeled with antibodies including combinations of anti-CD4 Alexa Fluor 488 (clone OKT4, BioLegend), anti-IgM PerCP-Cy5.5 (clone G20-127, BD), anti-CD8 APC-H7 (clone SK1, BD), anti-CD19 BV421 (clone HIB19, BD), anti-CD45 BV510 (clone HI30, BD), anti-CD3 BV605 (clone UCHT1, BioLegend), anti-CD14 BV711 (clone M0P-9, BD), anti-CD16 BV711 (clone 3G8, BD), anti-IgD BUV737 (clone IA6-2, BD), anti-CD27 PE-Cy7 (clone LG.7F9, Thermo Fisher Scientific) and fixable viability dye (FVD).

B cells were defined as CD19+CD3-expressing cells in the lymphocyte population (Supplementary Fig.18). The following B cell populations were delineated: CD27-IgD+ (naive B cells), CD27+IgD–IgM- (switched memory B cells), and CD27+IgD-IgM+ (unswitched memory B cells). Additionally, T cells were defined as CD19-CD3+ expressing cells in the lymphocyte population. CD4+ and CD8+ T cells were further delineated. Plasma cells in bone marrow were evaluated clinically by the University of Washington Immunohistochemistry Lab, with CD138 expression measured via staining with anti-CD138 (clone BA38) on a Bond III autostainer. Absolute B and T cell counts were calculated by multiplying proportions from flow cytometry by absolute lymphocyte counts from complete blood cell count results. Analyses were performed using FlowJo Software (version 10.10).

IgG profiling using a systematic epitope scanning method (VirScan)
We performed VirScan59 utilizing the expanded VirScan bacteriophage display library (Vir3)66 containing 115,753 200-mer oligonucleotides, encoding 56-residue peptide tiles with 28-residue overlaps, that together span the reference protein sequences of most known human viruses (244 viral species; 1,361 strains). Additionally, the Vir3 library contains peptides for 62 bacterial species (72 strains), 86 allergens, 22 parasite and yeast species, and a small assortment of other organisms with sequences sourced from the Immune Epitope Database (IEDB).66

We mixed phage library with a volume of serum containing 2 µg of total IgG per sample. In participants with IgG MM, we estimated 2µg total non-multiple myeloma IgG by subtracting the monoclonal component, estimated from the gamma region of serum protein electrophoresis. All samples were tested in two technical replicates. Serum antibodies were allowed to bind to phage overnight at 4°C on a rotator with continuous mixing. Antibody-phage complexes were immunoprecipitated using Dynabead Protein A and Protein G Magnetic Beads (ThermoFischer Scientific) and lysed to release the corresponding DNA insert sequences from the bound phage. Phage DNA inserts were amplified to attach appropriate adapter sequences and individual index sequences. Indexed amplimers were then quantified with fluorometry using the Qubit dsDNA High Sensitivity Assay Kit (ThermoFischer Scientific). Samples were then pooled in equal proportions, gel purified and sequenced on an Illumina NextSeq P2 (Illumina) Next Generation Sequencing (NGS) using a custom read primer. Raw NGS outputs were analyzed through phippery.67 Sequence reads were aligned, and oligonucleotides were deconvoluted into antigen epitope ‘hits’ (presence of antibody against an epitope) using a Z-score enrichment approach to detect and measure the relative enrichment of each epitope against a background of beads-only samples without added serum.66 An epitope hit was assigned when the z-score was calculated to be above a threshold of >7.0 standard units in both replicates. Multiple epitope hits sharing a seven amino acid overlap were considered to have the same antibody specificity and collapsed into a singular hit. The highest z- score of the collapsed hits was retained and all lower scores were filtered out.

Evaluation of CAR-T cell persistence
CAR-T cell persistence was quantified via quantitative PCR assays on cryopreserved PBMC samples from six months and one year post-CARTx time points. Samples chosen for analysis were those for which a commercial or investigational qPCR assay was available. Tests were developed and conducted by the Fred Hutch Immune Monitoring Core. DNA was extracted from PBMCs and used to amplify the following gene sequences, as relevant: Flap-EF1, Hum CD19, and 28BB. Continuous fluorescent measurement of the PCR products was performed during each cycle of amplification. Samples were run in parallel with a no template control, a known copy number plasmid positive control, and participants’ samples. All controls and samples were set up on a 96-well plate and run in triplicate. Following qPCR, the mean cycle threshold (CT) values obtained from the triplicate samples were used to calculate the copy number of targeted genes from the participants’ samples based on the previously established standards curves. Samples with an absolute copy number of greater than 10 target sequences / g input DNA were defined as positive.

Statistical Analyses & Reproducibility
We prespecified a targeted sample size of 130 CARTx recipients (50 adult and 50 pediatric CD19/20-CARTx recipients, and 30 adult BCMA-CARTx recipients). Based on this sample size, we computed the precision of seroprotection estimates within each group and the minimum detectable differences in seroprotection between groups, for the 6-month time-point post-CARTx, for a given pathogen. Wilson 95% confidence intervals (CI) were within +/-14% for the CD19/20 cohorts and within +/-18% for the BCMA cohort. Assuming a range of 70%-90% for the percentage of adult CD19/20-CARTx recipients with seroprotection at 6 months, our study design had 80% power for detection of clinically relevant differences in the prevalence of seroprotection when comparing either the pediatric CD19/20-CARTx cohort (minimum detectable relative risks [RR], 1.5-2.9) or the BCMA-CARTx cohort (minimum detectable RR, 1.6-2.2) to the adult CD19/20-CARTx cohort; these calculations were based on Fisher’s exact test, with a 2-sided type I error rate of 0.025 for each comparison to maintain an overall type I error rate of 0.05 using a Bonferroni correction. For example, if we assumed 80% of adult CD19-CARTx recipients have seroprotective IgG to measles 6-months post-CARTx, we would be powered to detect a 44% or lower prevalence of seroprotective IgG to measles after adult BCMA-CARTx (minimum RR of 1.8).

Laboratory values other than those reported as percentages were expressed as log (value + 1) to accommodate values <1 and right-skewed distributions. For analyses of kinetics of pathogen-specific immunity and VirScan metrics before and after CD19/20 and BCMA-targeted CARTx, we used the following generalized estimating equations (GEE) framework. Our primary exposure variables were CARTx target (CD19/20 versus BCMA) and time point (six months and one year post-CARTx versus pre-CARTx); we included these variables as well as the interactions between CARTx target and time point in all models and referred to this as base model one. Using the interaction terms, CARTx target comparisons were conducted for each time point, and time point comparisons were conducted within each CARTx target group. These models used an independence working correlation with robust standard errors. Pathogen-specific comparisons of antibody titer outcomes (using the normal distribution) and proportion of participants with seroprotective antibody titer outcomes (using the binomial distribution with logit link) were conducted using base model one; separate models were used for each pathogen. Due to sparse data or zero cells for the proportion with seroprotective antibodies among certain pathogens, alternative methods were used in select cases; chi square or Fisher’s exact tests were used to compare outcomes by CARTx target, and GEE models using subgroups were used to compare outcomes by time point. We did not adjust for multiple comparisons in these or any analyses; two-sided tests were used for all analyses. Pathogen-specific proportion of participants with seroprotective antibodies were also estimated for each group and time point using Wilson 95% confidence intervals (CI).

For the pre- and post-CARTx pathogen-specific immunity analyses, our primary outcome was a summary metric defined as the proportion of pathogens with seroprotective antibody levels, computed for each participant at each time point. To test for differences in this outcome by groups, we used logistic GEE models with the dependent variable specified as the number of pathogens with seroprotective antibody levels (successes) out of the number of pathogens measured (trials) per participant at each time point, with similar specifications as described above. Here, we added time-dependent variables for receipt of IGRT in the 8-16 weeks before each sample and number of pathogens with vaccination prior to each sample to the base model one described above and referred to this as base model two. Pre-specified candidate adjustment variables included age at CARTx (>60 years versus ≤60 years), biological sex, race (nonwhite versus white), ethnicity, number of lines of cancer therapy prior to CARTx, any prior HCT or CARTx, any B-cell targeting therapy in six months before CARTx, type of CARTx product (commercial versus investigational), and the following laboratory measures as time-dependent covariates: total IgG, IgA, IgM; CD19+ B cell count; CD4+ T cell count. Multivariable model selection used a modified purposeful variable selection procedure as follows.68 For each candidate adjustment variable, we first ran models that added the candidate adjustment variable to base model two. Continuous variables were evaluated for functional form and categorized according to clinical cut points if a linear relationship was not supported. When included in these models, adjustment variables that either led to a >20% change in any of the primary exposure model coefficients or had a p-value < 0.20 were then added to a preliminary multivariable model. Next, candidate adjustment variables that were neither significant at the 0.10 level, nor changed any other variable coefficients by >20% were sequentially removed from the preliminary model. Finally, we confirmed that none of the initially excluded adjustment variables were significant at the 0.10 level nor changed other variable coefficients by >20%, to arrive at the final multivariable model. Model estimates were presented as odds ratios (OR) with 95% confidence intervals (CI). For easier interpretation of the primary comparisons, we also presented model predicted proportions of pathogens with seroprotection within primary exposure subgroups, using marginal standardization.69 This method computes a weighted average of predicted probabilities to reflect the confounder distribution in the target population, which, in our case, was the total cohort eligible for this analysis.

VirScan analyses
Statistical methods were adapted from ecologic analyses employed in microbiome or other population analyses: (i) total epitope score: the sum of unique epitopes observed for all antigens in VirScan (ii) Simpson's Index70,71, and (iii) Shannon's Evenness metric72,73.

We used GEE to describe and compare means and mean changes in the evaluated VirScan metrics at pre-, 6 month and 1-year post-CARTx time points. The base model included primary explanatory variables, including CARTx type and sampling time points. Interaction terms between different time points (pre-CARTx, 6-months post-CARTx, 1-year post-CARTx) and the CARTx group were included in the models to estimate changes within each subgroup and to test whether these changes differed by subgroup. Additionally, the base model was adjusted for receipt of IGRT 8-16 weeks before sample collection. We also adjusted for other potential confounders in the models and explored associations of other biologically relevant variables with these outcomes. To aid parsimonious model building, we used purposeful variable selection to choose the optimal number of additional confounders to adjust for in each model.

Violin plots were generated to graphically represent the spectrum of viruses and epitope densities at pre- and post-CARTx time points for each CARTx group (CD19 vs. BCMA). Furthermore, we explored the relationship between age (discretized age group categories 0-8, 9-17, 18- 39, 40-59, 60-69, >= 70) and VirScan outcomes at baseline for those receiving only the CD19 CARTx therapy (100 adults, 22 pediatric participants).

Vaccine immunogenicity analyses
Initial descriptive vaccine response analyses included participants with ≥1 pre- and ≥1 post-vaccine dose sample, regardless of the sequential dose to which this corresponded; a sample collected before the first vaccine dose was not required. Baseline (pre-first dose of vaccine) immunoglobulins, B cell subsets, and T cell subsets were defined as those corresponding to the earliest vaccine administered per participant. These baseline values were compared by CARTx target group using Wilcoxon rank-sum tests. Pathogen-specific antibody titers were compared by CARTx target group and time point using similar GEE models as described for previous analyses. Due to sparse data, values for post dose ≥2 were combined and for some pathogens, simplified models with fewer parameters and/or small sample bias corrections were used.

To estimate the cumulative incidence of vaccine response overall and by select baseline factors, we focused on a clinically relevant group of participants with baseline titers below the defined threshold for seroprotection for at least one pathogen. Vaccine response was evaluated separately for each pathogen. Pathogens with too few participants meeting baseline criteria for inclusion were excluded from these analyses. Fold differences in pathogen-specific antibody titers following each vaccine dose were computed using the ratio of post-dose to baseline untransformed values; baseline values of zero were assigned a value of half the distance to the minimum non-zero value for purposes of these calculations. Cumulative incidence of vaccine response was computed using dose as the time variable. Participants not achieving the seroresponse endpoint, defined as a ≥2-fold increase in antibody titers, were censored after the last dose received. To test for associations between select baseline factors (CARTx target, time from CARTx to first vaccine dose, and baseline CD19+ B cell count) and hazard rate of vaccine response, we used marginal discrete time (dose) multivariate Cox models with robust standard errors to accommodate participants that contributed vaccine response data for ≥1 pathogen (multiple endpoints).74 This model was stratified by pathogen, allowing for separate baseline hazards for each pathogen. We tested for equal effects across pathogens and estimated an average effect across pathogens when there was not sufficient evidence to suggest unequal effects. We first ran univariable models for each factor. We considered two different parameterizations of baseline CD19+ B cell count: continuous on the log scale and a pre-specified binary indicator of ≥20 cells/µL vs <20 cells/µL, based on the use of this threshold in other studies. Due to limited number of events, multivariable models including a maximum of two independent variables were considered by selecting variables significant in univariable analysis. Separate models were developed for each of the two parameterizations of baseline CD19+ B cell count.

SAS, version 9.4 (SAS Institute, Cary, North Carolina); Stata, version 18.5 (StataCorp LLC, College Station, Texas); and R (version 4.3.1) were used for analyses.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This prospective and comprehensive study provides valuable insights into humoral immunity following CAR-T\ncell therapy, including novel data on vaccine responses. Given the substantial infectious morbidity, the study is\nboth timely and highly relevant. The authors highlight differences in immune recovery between CAR-T constructs\nand propose a B cell threshold that may inform vaccination strategies.\nOverall, the study is methodologically rigorous and well presented. Below are some comments aimed at clarifying\nkey aspects and enhancing the interpretation of the data:\n1. Vaccinated vs. unvaccinated patients\nThe rationale for vaccination eligibility remains somewhat unclear. Figure 1 indicates that vaccinated\npatients were a subset of those followed longitudinally for humoral responses, and that 46% had\nreceived at least one vaccine by one year (line 230). Could the authors elaborate on the vaccination\nstrategy at their center? While it is noted that clinical routine was followed, was this based on immune\nreconstitution markers such as B cell count, IgG levels, or CD4⁺ T cell counts?\n2. Impact of vaccination on seroprotection\nLines 231–232 state that vaccination had no apparent impact on antibody levels at one year. This could\nbe misleading. Many patients appear to have initiated vaccination relatively late and received only one\nor two doses by that time point. This important nuance should be made explicit to avoid\nmisinterpretation that vaccination is ineffective in CAR-T recipients.\n3. Differences in vaccine responses by CAR-T target\nThe manuscript explores vaccine responses in CD19 vs. BCMA CAR-T recipients. Could the observed\ndifferences be partially explained by variation in vaccine exposure? Supplementary Table 9 suggests\nthat BCMA recipients received fewer vaccine doses on average. Figure 6 further shows that few BCMA\npatients received more than two doses of any vaccine-\npreventable infection (VPI). These factors should be considered when comparing response rates between groups.\n4. Humoral improvement in BCMA recipients\nThe manuscript mentions a trend toward increased humoral responses in BCMA recipients at one year, though this was not\nstatistically significant when adjusting for vaccination and IVIG. Given the risk of confounding, this finding should be\npresented with more caution and not highlighted as a key result.\n5. B cell threshold for guiding vaccination\nIdentifying a B cell threshold predictive of vaccine response is clinically meaningful. However,\nrecommending a strict cutoff of 20 cells/μL may be overly rigid. Some patients may not reach this\nthreshold, and T cell–mediated responses, which were not assessed, could still provide benefit. This is\nespecially relevant for viral vaccines (e.g., RSV in high-risk populations), where delaying vaccination\nbased on B cell count alone could be problematic. The authors are encouraged to acknowledge these\nlimitations and interpret the threshold flexibly. Notably, some patients with B cell counts <20 cells/μL still\ndemonstrated humoral responses.\n6. Pneumococcal and VZV-vaccine assessments\nThe assay used to evaluate pneumococcal responses appears to measure aggregated IgG to 23 serotypes. While\nacceptable for assessing baseline immunity, it is suboptimal for evaluating vaccine-specific responses. Were responses\nassessed based on the serotypes included in the vaccines given? The distribution of PCV13/PCV15/PCV20 or PPSV23 is\nnot provided, making interpretation difficult. This limitation should be discussed.\nIt appears that a pan-VZV IgG assay was used to assess response to Shingrix? This assay is less specific than glycoprotein\nE based assays and may underestimate vaccine-induced responses.\nMinor Comments\n1. A recent study in Blood Cancer Journal assessed antibody responses post-CAR-T vaccination and identified IgA levels\nand B cell counts as predictors of response. This supports the current findings and may merit citation.\n2. Consider including cytopenias (e.g., n-ICAHT) in the Introduction, given their frequency post-CAR-T and relevance to\ninfection risk.", "reviewer_2": "The long-term effects of CAR T treatment on pre-existing pathogen-specific immunity are poorly understood. That while\ninfections are the main cause for non-relapse mortality in CAR T treated patients. This study is adding to this gap in\nknowledge by prospectively measuring the remaining/loss of humoral antibody responses to prior infections/vaccinations,\nprior to and after CD19/CD20 CAR T or BCMA CAR T treatment respectively, over time. Furthermore a (very small)\nsubgroup of patients has been vaccinated post CAR T treatment with certain vaccines. Also here humoral responses are\nmeasured. These data are new and thereby valuable.\nHowever we should be aware of the small numbers of patients included (the whole group seems quite big, however when\nyou look at the different vaccines, then only 2 or 4 BCMA CAR T treated patients are included in analyses for example, as\nshown in figure 6). Furthermore vaccines responses are only measured by quantitative humoral tests, no\nfunctional/qualitative humoral tests have been performed, also no cellular tests (quantitive or functional) have been\nperformed, which would of course have been of great value, due to the specific patient group depicted; patients with disease\nin their B cell compartment or plasma cell compartment, heavily pretreated, and now treated with lymphodepleting\nchemotherapy and thereafter CD19/CD20 (B cell depleting) CAR T or BCMA (plasma cell and part of B cell depleting)\ntherapy, whereby humoral responses might be expected to be low. But that is not the whole picture for vaccines responses.\nVaccine responses might be underestimated as a consequence. Also, there is no information in this paper about protection\nagainst (severe) infections after vaccination. Off course the groups are far too small to analyze this, however it is important to\ndiscuss that humoral levels is the only one factor of vaccine response that is measured.\nNevertheless the information that is analyzed in this paper is important and concerns also new information and was, up till\nnow, missing. It might contribute to current guidelines for supportive care in CAR T treatment. For example, in current\nguidelines post CAR T treatment expensive prophylactic measurements as immunoglobulin suppletion is advised by\ndifferent experts without good evidence (no randomized trials), while immunoglobulin suppletion might hamper vaccine\nresponses. But, even worse, in the current guidelines little is mentioned about vaccination yet, while this is normally a very\ngood strategy to prevent (severe course of) infections, also after CAR T treatment (see publications as: Improved outcome of\nCOVID-19 over time in patients treated with CAR T-cell therapy: Update of the European COVID-19 multicenter study on\nbehalf of the European Society for Blood and Marrow Transplantation (EBMT) Infectious Diseases Working Party (IDWP)\nand the European Hematology Association (EHA) Lymphoma Group. Spanjaart AM, et al. Leukemia. 2024 Sep;38(9):1985-\n1991). Sometimes even statements are made that vaccination might not work due to B cell depletion and plasma cell\ndepletion with CD19/CD20 CAR T and BCMA CAR T treatment, or that all memory of prior vaccinations is gone and the\nimmune system after BCMA CAR T treatment is ‘like a baby’; all memory is expected to be gone. These are potential\ndangerous statements possibly preventing doctors fromm treating their patients with appropriate vaccination strategies. This\npaper adds to this gap in knowledge.\nNotably, in this paper certainly not all humoral response is gone for example after BCMA CAR T treatment. That is a very\nimportant message. Over time memory after treatment is maintained for a part of the patients! and might even further recover\n(possibly with recovery of plasmacell/B cell compartments?). This is an example that this study describes very important\nknowledge which is missing and might adjust guidelines to more cautious statements and more nuanced advices.\nThe authors state the above part in their introduction: “In light of these uncertainties, patients often receive\nprophylactic immunoglobuline replacement therapy (IGRT) to address hypogammaglobulinemia, although this blood product is\nexpensive, has frequent side effects, can reduce vaccine efficacy, and is of unclear benefit in this population.\nAlternatively,\nvaccination after CARTx may be a durable and cost-effective tool to mitigate irAEs. Vaccine responses after CD19-CARTx\nare biologically plausible given observations that patients can respond to vaccines ≥6 months after rituximab, even in the\nabsence of measurable peripheral blood B cells”. (rules 98-104).\n-Please adjust in the discussion that this seems also true for CD19/CD20 and BCMA CAR T treatment, that even in the\nabsence of measurable B cells there are responses to vaccination (although in bigger part of patients with measurable B\ncells, as far as it concerns humoral responses).\n- And please discuss the valuable observation that BCMA CAR T treated patients are able to maintain antibody levels over\ntime (not all of them are preserved before CAR T treatment, however it is preserved, and might even improve over time.\nPlease discuss the results with more attention, and also the possible consequences for guidelines.\n-Please remove that a minimum level of B cells should be reached prior to vaccination. It is not according to yours and prior\ndata and the risk exists that this is implemented in guidelines, and patients that do not recover in their B cell counts will not\nbe vaccinated. This statement is not true. Especially when taken into account that in this study only humoral responses are\nmeasured. But also when looking at your data (20-50% with responses while low B cells!)\nOther comments:\n- Please change the title: “Influence of B cell or plasma cell-targeted CAR-T cell therapy on humoral immunity and vaccine\nimmunogenicity: The HICAR study;” because vaccine immunogenicity might implicate the humoral and cellular responses,\nin this study just humoral immunity quantitively is measured (also no qualitive/functional humoral tests have been\nperformed). The title does not cover the tests performed. Please adjust (HICAR stands for humuroral immunity in CARTx\ntreated patients? That covers the tests performed indeed. Therefore please remove vaccine immunogenicity\n- The infectious complications leading to non-relapse mortality after CAR T is mentioned, are these vaccine-\npreventable infections? What is the percentage of infections that is vaccine-preventable (besides from the fact that vaccination might\nhave less beneficial effects in this specific group of patients)? Please add to the introduction\n(it has been described that in the first months after CAR T treatment there are mainly bacterial infections (likely due to the\nlymphodepleting chemotherapy), later after CAR T treatment we do see a lot of viral infections (as covid and other airway\ninfections) and viral reactivations (CMV, HHV), sometimes even with letal\nconsequences, i.e. HHV6 encephalitis. However\nthese infections are not (yet) vaccine-preventable. It is important to add this knowledge to the paper. And alos of course the\ninfections that is vaccine-preventable)\n- There are multiple challenges with vaccination after CAR T; what kind of pathogens cause (severe) infections after CAR T\ntreatment; are they vaccine-preventable? What is the effect of vaccination? This paper measures the humoral responses,\nhowever depending on the vaccines there is also a cellular response. And is there any information about vaccines\neffectiveness? In the general population? In this specific population?\nFor Covid for example?\n- There are different types of vaccines used in this paper. Please discuss the different vaccines types and what to expect in\nthe general population and in this specific population. Humoral responses only? Cellular responses? Memory?\n- Are there controls? Because also in the general population the humoral response to vaccination is not 100% and is waning\nfor most vaccine-preventable infections. It would be helpful to add controls to the figures. So random time after vaccination a\nmeasurement (as is also the case for this patients before start of treatment the CAR T, although these patients have a B-cell\nand or plasma cell disease, and also prior treatments that hamper (the measurement of) memory responses. And also add\nage-matched controls to vaccinations after vaccinations in a control population. Aldo please elucidate this in the discussion.\n- Please describe in the discussion that the lack of B cells and or plasma cells due to disease and/or prior treatment effects\ndoes not necessarily mean that prior vaccines responses are completely gone. In the introduction (lines 110-111) there is\nstated that it is hypothesized that CD19 or CD20 CAR T treatment does not impact pre-existing antibody levels, however\nwhen there are no B-cells (due to underlying disease or due to prior treatment) then it is not possible to measure this\nresponse (though it might not be gone indeed). Please discuss.\n- “Conversely, we hypothesized that BCMA-CARTx targeting plasma cells would result in loss of pathogen-specific antibody\nlevels but have less impact on vaccine responses”. Why? Also here it might not be measurable, however not gone. BCMA is\npresent on plasma cells and B cells? Why the hypothesis that that has no impact on vaccine responses? Please explain?\n- “Here, we report the results of a prospective observational study evaluating the impact of CD19/20 and BCMA-targeted\nCARTx on multiple metrics of humoral and pathogen-specific immunity, as well as the role of routine vaccines as a means to\nmitigate humoral irAEs in CARTx recipients”. Please explain how to mitigate humoral irAEs? When there are no B-cells then\nwe do not measure humoral responses, with or without vaccinations. That might not mean that there are no effects on\nvaccination. Please adjust. More important is the risk for vaccine-preventable infections after vaccination for AE’s of course.\nPlease add this to the discussion and why these data are missing.\n- When the B-cell compartment and plasma cell compartment is recovered, then it might be possible to measure prior\nhumoral responses again. Off course the measurement before treatment with CAR T is also a starting point in this study,\nhowever please discuss that with recovery of B and T cells there might also be an effect due to measurement of effects of\nprior vaccination, earlier not possible to measure at humoral level due to B-cell and/or plasma cell depletion due to the\ndisease itself and prior treatments (patients in this study are heavily pretreated).\n- Use of Immunoglobulin suppletion in which patients?\n- Which vaccines were given to which patients (vaccination history) before start of CAR T treatment? Is it logical that younger\npatients have more protection against hepatitis viruses vs elder patients, and the opposite for the other pathogen-specific\nantibodies tested?\n- Where the MM patients pretreated with bispecific or trispecific antibodies targeting BCMA and CD3 (within the last year)\nbefore treatment with BCMA CAR T? This is important data to add, whil bispefics are used in high number of patients and\nwere authorized before CAR T treatment was for MM. It has the same plasma cell and mature B cell depletion effects as\nBCMA CAR T\n- “BCMA-CARTx recipients showed a general pattern of increases in antibody titers by one-year post-CARTx for several\npathogens, which was contrary to our original hypothesis” (rule 194-195); This is really important and new information (not\npriorly measured/published yet)!! Please give more attention to this finding.\n- “Consistent with results for vaccine preventable pathogen-specific IgG, BCMA-CARTx recipients demonstrated\nsignificantly fewer epitope hits compared to CD19/20 recipients at each measured time point (Figure 5B)”. (Rule 245-247)\nand “A history of prior HCT or prior CARTx was significantly associated with fewer epitope hits.” (rule 249); is this also\nantibody based? Is it maybe the same problem, that without / with less than normal B-cells and/or plasma cells antibodies it\ncannot adequately be measured?\n- “Due to relatively high levels of seroprotection for some pathogens, only a subset of participants and pathogens were\neligible for this analysis. In this subgroup of 58 participants (48 CD19/20, 10 BCMA) with a median of 3 (IQR 2-4) evaluable\npathogens per participant, we estimated the cumulative incidence of seroresponse, defined as a ≥2-fold increase in antibody\ntiters after vaccination. Thirty seven participants (30 CD19/20, 7 BCMA) had a seroresponse for at least one pathogen. The\noverall cumulative incidence of a seroresponse was approximately 50% or higher for H. influenzae, C. diphtheriae, hepatitis\nB virus, and S. pneumoniae after ≥ 2 vaccine doses (Figure 6B; Table S12)”. (rules 288-294); You have to be aware that by\nselecting only patients that were not able to mount seroprotective antibody levels, there might be a selection bias. You might\nselect the worst patients. And even they were able to mount higher levels of antibodies partly. Please discuss. Here the\npatients with the lowest protection level are selected, so the worst patients so to say\n- And: where these patients vaccinated in their past? What is their vaccination history?\n- “BCMA-CARTx recipients had earlier and greater overall seroresponse rates compared to CD19/20-CARTx recipients\n(Figure 6B)”; what might be the explanation?\n- “Overall, our results demonstrate that CD19/20-CARTx recipients have stable pathogen-specific humoral immunity after\ntreatment but may lack seroprotective antibodies for up to one-third of vaccine-preventable pathogens.” (rules 310-311);\nvaccination status?\n- “although this difference was not statistically significant after adjustment for CD19+ B cell count.” (rules 319); Why\nadjustments for B-cell counts? That means that higher number of B cell counts are responsible for higher absolute antibody\nlevels. That is well known off course. However if you are able to double your antibody level, and these antibodies might be\nqualitively of high level, then the number itself does not matter; it is also important if these antibodies are able to kill the\npathogen adequately. Especially when there is a memory (prior vaccination or infection) than the amount of antibody rise\nseems not that impressive, but they are earlier mature compared to exposure to a new pathogen/vaccine. Therefore please\nremove this.\n- “A pre-vaccine peripheral blood CD19+ B cell count ≥20 cells/μL was the primary variable associated with vaccine\nresponsiveness, supporting the use of B cell recovery as a key metric for routine vaccine initiation protocols after CARTx for\nB cell lineage malignancies. (rules 320-321); be carefull with this statement, because that antibody levels are low with low B\ncell counts is logical, but not the only or even a parameter that the vaccine works (for most vaccines). Please remove this\nsentence, it might be misleasing. Please elucidate in discussion about this topic, that we are missing functional humoral\nresponses, cellular responses and (really important) vaccine effectiveness in this B cell and/or plasma cell depleted patient\ngroup, meaning that, depending on the vaccine, it might not be wise to advice vaccination depending on antibody levels in B\ncell or plasma cell depleted patients.\n- “This also provides insights into the broader immunologic effects of CD19/20- targeted CARTx, and the potential utility (or\nlack thereof) for other indications such as certain autoimmune diseases despite depletion of the target B cell population.”\n(rule 327); Also here, be careful, because in auto-immune diseases also the T-cells seems to be very important for the\ndisease evolution, and despite that plasma cells (antibody titers) might be preserved, the immune reconstitution (or reset)\nmight be working with CD19/CD 20 CAR T treatment. So, there is more to say about the hypothesis why CAR T-cell\ntherapies are working in auto-immune diseases. The fact that plasma cells are preserved (and auto-antibodies therefore\nlikely too) is known, but clearly not the most important factor for working (although the groups treated in auto-immune\ndisease are still very small of course).\n- “Thus, we hypothesized that BCMA-CARTx recipients would have a loss of pathogen-specific IgG and antibody diversity.\nWe observed that these participants already had substantial pathogen-specific immune deficits prior to CARTx, likely due to\ntheir underlying plasma cell malignancy and prior anti-tumor therapies (median of 8 prior lines of therapy, and 75% had a\nprior HCT). After BCMA-CARTx, we observed a modest recovery of pathogen-specific immunity, which may have been in\npart due to use of IGRT in the 8-16 weeks prior to sampling and re-vaccination based on our finding that the increase was\nnot statistically significant after adjusting for these variables.” (rules 331-337); really small groups, and by further correcting\nfor factors you might expect that the results lose their significance. It is fair to note here that the groups are too small. That\nmight not be due to the factors you corrected for, but solely due to the low numbers (becoming even lower with adding\ncorrections).\n- “Individuals with B cell counts <20 cells/μL had a relatively low cumulative incidence of seroresponse of approximately 20-\n50% after ≥2 vaccine doses across different pathogens.” (rules 360-361); 20-50% is certainly not nothing! And then the factor\nthat there are vaccins that also work via cellular responses. What a positive finding that even in patients with really low B cell\ncounts 20-50% shows response! That is important to pronounce in a positive way!\n- “Together, our data demonstrate that CD19/20- and BCMA-CARTx recipients have ongoing immunologic deficits through\none year after CARTx, with greater deficits in BCMA-CARTx recipients. (rules 363-364); please add humoral deficits, also\nexplaining the lower counts of seroprotective levels\n- “and requiring a peripheral blood B cell count ≥20 cells/μL prior to (re)vaccinating after CARTx is a reasonable metric for\nguiding patient care.” (rules 365-366); please remove. As your data shows there is no lower limit of B cells, and taking into\naccount the missing in this study of cellular responses and vaccine effectiness, this is not a good advice. Please remove.\n- “Whether routine use of prophylactic IGRT is beneficial prior to and after CARTx remains an open question, but pediatric\nand BCMA-CARTx recipients appear to be most likely to benefit based on our findings.” (rules 370-372); Why? Please\nexplain further, and please remove the statement. It is not the topic of this paper and you did not do research for that question\nin this paper.\n- “The inclusion of a CAYA cohort allowed for unique baseline comparisons across age groups to inform the pathogen-\nspecific immune status of young individuals prior to CARTx, which has not been previously well described.” (rules 377-378);\nPlease add that a healthy control group is necessary to make real statements about age.\n- “Finally, there are few published data pertaining to vaccination responses, and\n- strategies to inform when to vaccinate CARTx recipients, aside from SARS-CoV-2.” (rules 379-380); what is your advice\nabout timing? I missed that in the paper.\n- “and BCMA-CARTx recipients had some albeit limited recovery of pathogen-specific humoral immunity over time.” (rule\n395); please change to: BCMA-CARTx recipients had some recovery of pathogen specific immunity over time. Please try to\ndescribe your results as objective as possible, try to avoid additions as albeit limited (defined as?). Please adjust.\n- Please add to figure 3 the significance values between different timepoints for BCMA respectively CD19/CD20 CAR Tx\n(between the orange boxes, and between the blue boxes, over time; paired analyses). Please add also the number of\npatients vaccinated for the specific disease per group (a minority of course, however, good to mention). Also please add the\n% of patients that was vaccinated in the past for the different groups (had the BCMA patients all received BMR for\nexamples?)\n- Figure 6: be very carefull with conclusions based on BCMA CARTx when there are just 2 or 4 patients vaccinated. Be\naware: there is an enormous bias; because why these patients are vaccinated and others are not? It is not a study to which\nevery patient (re)vaccination is offered after CAR T treatment, but the patients who are invited to vaccination are offered\nparticipation of this study. That is a big difference. Please discuss in discussion the small numbers and the bias in patients\nthat are vaccinated, to which the study is offered (and also discuss the ones you possibly miss (with no B-cells and/or t cells\nfor example? Are they not referred for vaccination (yet?) Only the best patients are vaccinated might be a huge bias risk)", "reviewer_3": "Study Summary:\nThe manuscript titled “Influence of B cell or plasma cell-targeted CAR-T cell therapy on humoral immunity and vaccine\nimmunogenicity: The HICAR study” presents a prospective observational study examining how CAR-T therapy targeting\nCD19/20 or BCMA affects humoral immunity and vaccine responsiveness. Using VirScan profiling, the authors analyze over\n150 patients and reveal that BCMA-CAR-T recipients exhibit broader deficits in pathogen-specific antibodies. A B cell\nthreshold of ≥20 CD19+ cells/μL is proposed as a predictor for vaccine response. The findings provide valuable insight\nabout immune reconstitution and guide post-CAR-T vaccination strategies.\nMajor Comments:\n1. Please clarify if the statistical analysis was adjusted for mutiple comparisons\n2. Patients with Mutiple Myeloma are inherently different from patients with Lymphoid malignancies. Relying solely on the\ntarget might oversimplify biological differences. Consider a stratified analysis if numbers allow.\n3. Please clarify how the B cell count threshold was chosen and if/how it has been validated.\nMinor comments:\n1. The study includes the CAYA population, which is novel; however, the study is underpowered to make any substantial\ninferences.\n2."}, "author_response": "Response: We agree with the Reviewer’s emphasis on vaccination strategy as a key detail for\nunderstanding observed responses. We added additional language in the Methods (lines 468-473)\ndescribing the recommended vaccination strategy and added a citation referencing the standard of\ncare guidelines at Fred Hutch Cancer Center generally followed by treating physicians.\n“Briefly, killed or inactivated vaccinations were recommended in patients who were ≥6 months post-\nCARTx and off IVIG for ≥2 months, irrespective of immune reconstitution. Live vaccines were\nrecommended in patients who were ≥12 months post-CARTx, off immunosuppression for ≥12\nmonths, had not received IVIG for ≥8 months, and had a CD4+ T cell count >200 cells/mm3.”\nAdditionally, we added a new table providing participant characteristics for those included in the\nvaccine cohort versus the pre/post-CARTx cohort only (Table S11), to better clarify participant\neligibility for vaccination.\n2. Impact of vaccination on seroprotection\nLines 231–232 state that vaccination had no apparent impact on antibody levels at one year. This could\nbe misleading. Many patients appear to have initiated vaccination relatively late and received only one\nor two doses by that time point. This important nuance should be made explicit to avoid\nmisinterpretation that vaccination is ineffective in CAR-T recipients.\nResponse: We agree with this point. To avoid confusion, we deleted the details about vaccine\nadministration and emphasis on the vaccine variable from the text. We added the details about the\nnumber of administered vaccines in the legend for Figures 3 and 4D. In response to comments from\nReviewer #2 below, we have additionally added Figure S11, which illustrates pre/post-CARTx\nantibody levels in the absence of a previous relevant vaccine during this study period, which shows\nsimilar results as Figure 3.\n3. Differences in vaccine responses by CAR-T target\nThe manuscript explores vaccine responses in CD19 vs. BCMA CAR-T recipients. Could the observed\ndifferences be partially explained by variation in vaccine exposure? Supplementary Table 9 suggests\nthat BCMA recipients received fewer vaccine doses on average. Figure 6 further shows that few BCMA\npatients received more than two doses of any vaccine-preventable infection (VPI). These factors should\nbe considered when comparing response rates between groups.\nResponse: Thank you for this comment. We agree that there were some differences in the number of\nvaccine doses administered between the two cohorts, with slightly fewer doses in the BCMA cohort.\nWe added to the results that the median and IQR of doses administered in the CD19/20 versus\nreceived between the two cohorts, our seroresponse analyses utilized discrete time-to-event\nmethodology with vaccine dose as the time variable and censoring at the last dose received. This\nensures that group comparisons are made following the same number of vaccine doses.\n4. Humoral improvement in BCMA recipients\nThe manuscript mentions a trend toward increased humoral responses in BCMA recipients at one year,\nthough this was not statistically significant when adjusting for vaccination and IVIG. Given the risk of\nconfounding, this finding should be presented with more caution and not highlighted as a key result.\nResponse: We agree with this suggestion to de-emphasize the modest overall increase in BCMA-CARTx\nrecipient pathogen-specific immunity.\nWe updated the description in the Key Points and Abstract as follows: “Pathogen-specific humoral\nimmunity did not significantly change after CD19-, CD20-, or BCMA-targeted CAR-T cell therapy\n(CARTx). However, seroprotective antibodies were absent for one-third of routine vaccine-\npreventable pathogens in CD19/CD20-CARTx recipients and half of vaccine-preventable pathogens\nin BCMA-CARTx recipients by one-year post-CARTx.”\n5. B cell threshold for guiding vaccination\nIdentifying a B cell threshold predictive of vaccine response is clinically meaningful. However,\nrecommending a strict cutoff of 20 cells/μL may be overly rigid. Some patients may not reach this\nthreshold, and T cell–mediated responses, which were not assessed, could still provide benefit. This is\nespecially relevant for viral vaccines (e.g., RSV in high-risk populations), where delaying vaccination\nbased on B cell count alone could be problematic. The authors are encouraged to acknowledge these\nlimitations and interpret the threshold flexibly. Notably, some patients with B cell counts <20 cells/μL still\ndemonstrated humoral responses.\nResponse: This is a helpful and important comment, and we adjusted our overall focus and\nrecommendations accordingly. We agree that while a cut-off of ≥20 cells/μL for starting vaccines may\nbe a useful clinical heuristic, this rule will not completely discriminate between individuals who will\nor will not respond to vaccination. Additionally, it does not consider T cell-mediated responses, which\nwe address in the discussion. We restructured relevant aspects of the Results and Discussion to\ndemonstrate that a higher CD19+ B cell count is associated with a greater likelihood of\nseroresponse. We provide results from an additional Cox regression model evaluating the\nassociation of CD19+ B cell count on a continuous scale (per log increase) with seroresponse\n10\n(Table 2). Higher CD19+ B cell count is significantly associated with seroresponse in this model,\nand we added this to the results. To further explore seroresponse by gradations of B cell counts, we\nadded cumulative incidence estimates of seroresponse by three categories of B cell counts (<1\ncell/μL, 1 to <20 cells/μL, and ≥20 cells/μL) for a subset of pathogens with adequate sample sizes in\nthese categories (Figure S16 and Table S18). These updates highlight the higher probability of\nseroresponse among those with B cell counts ≥20 cells/μL but also illustrate that seroresponses are\nobserved with <20 cells/μL, including <1 cell/μL. We updated the Discussion (Lines 395-399) to\nillustrate these points.\n6. Pneumococcal and VZV-vaccine assessments\nThe assay used to evaluate pneumococcal responses appears to measure aggregated IgG to 23\nserotypes. While acceptable for assessing baseline immunity, it is suboptimal for evaluating vaccine-\nspecific responses. Were responses assessed based on the serotypes included in the vaccines given?\nThe distribution of PCV13/PCV15/PCV20 or PPSV23 is not provided, making interpretation difficult.\nThis limitation should be discussed.\nIt appears that a pan-VZV IgG assay was used to assess response to Shingrix? This assay is less\nspecific than glycoprotein E based assays and may underestimate vaccine-induced responses.\nadministered for participants included in vaccine response analyses (Table S13). PCV13 was used\nin most participants and for most doses. We also conducted new comprehensive testing for IgG\nantibodies to 12 of the 13 S. pneumoniae serotypes included in PCV13 among 12 participants who\nhad evidence of a seroresponse (≥2-fold increase in total S. pneumoniae antibody titer) after 1-2\nvaccine doses and who had residual paired samples from pre-vaccine dose 1 and post-vaccine dose\n2. We present these results in Figure S17B, which demonstrates a ≥4-fold increase in S.\npneumoniae serotype-specific antibody titers for at least one of the 12 serotypes in 9 of 12\nparticipants. Among those 9 participants, the median number of serotypes with a ≥4-fold increase\nwas 5 (IQR, 4-9). In response to a suggestion from Reviewer 2, we also tested for functional antibody\nresponses in the same samples using a validated multiplex opsonophagocytosis assay (MOPA) for\nthe 13 serotypes included in PCV13 (Figure S17A). This demonstrated a ≥4-fold increase in\nfunctional opsonophagocytic activity for at least one serotype for 10 of the 12 participants. Among\nthose individuals, a median of 8 serotypes showed a ≥4-fold increase in functional opsonophagocytic\nactivity (IQR, 5-9). Fold-change in serotype-specific IgG levels and opsonophagocytic activity had\nmoderate overall correlation (ρ=0.65).\nTo determine whether the pan-VZV assay used in our study was a reasonable correlate for the\nmore specific glycoprotein E based assay, we conducted an additional limited analysis comparing\nour results to a validated glycoprotein E-specific assay developed by Dr. Jeff Cohen, NIAID (VZV\nLIPS Assay; PMID 24990909). Data for seven participants, depicted in the figure below, show\nstrong correlation (ρ = 0.64) between the pan-VZV IgG and gE LIPS assay. As such, we think it is\nreasonable to keep our results as presented, as gE-specific assays are not routinely available.\nThese results are provided here for reference but are not included in the manuscript. If the\nReviewer or Editors prefer that we add these data, we are happy to do so at their discretion.\nvaccination.\nLongitudinal measurements from seven participants (each panel) show anti-varicella-zoster virus (VZV)\nantibody levels measured by a pan-VZV IgG assay (blue) and a glycoprotein E (gE) luciferase\nimmunoprecipitation system (LIPS) assay (red) at three time points: pre-vaccination, after the first vaccine\ndose (Post-1-dose), and after the second vaccine dose (Post-2-dose). The y-axes represent the log -\n10\ntransformed values for each assay (left, log₁₀ Index Value for the pan-VZV IgG assay; right, log Light\nacross all time points (Spearman’s ρ = 0.64).\nMinor Comments\n1. A recent study in Blood Cancer Journal assessed antibody responses post-CAR-T vaccination and\nidentified IgA levels and B cell counts as predictors of response. This supports the current findings and\nmay merit citation.\nResponse: Thank you for bringing this recent publication to our attention. The results of this retrospective\nstudy are largely in agreement with our findings. We reference and discuss their findings in the\nDiscussion section (Lines 395-396).\n2. Consider including cytopenias (e.g., n-ICAHT) in the Introduction, given their frequency post-CAR-T and\nrelevance to infection risk.\ndetailing the immune related adverse effects of CARTx (Lines 83-84).\nREVIEWER #2:\nThe long-term effects of CAR T treatment on pre-existing pathogen-specific immunity are poorly understood.\nThat while infections are the main cause for non-relapse mortality in CAR T treated patients. This study is\nadding to this gap in knowledge by prospectively measuring the remaining/loss of humoral antibody responses\nto prior infections/vaccinations, prior to and after CD19/CD20 CAR T or BCMA CAR T treatment respectively,\nover time. Furthermore a (very small) subgroup of patients has been vaccinated post CAR T treatment with\ncertain vaccines. Also here humoral responses are measured. These data are new and thereby valuable.\nHowever we should be aware of the small numbers of patients included (the whole group seems quite big,\nhowever when you look at the different vaccines, then only 2 or 4 BCMA CAR T treated patients are included in\nanalyses for example, as shown in figure 6).\nResponse: We appreciate the Reviewer’s recognition of the importance of investigating humoral\nimmunity after CARTx and the gap in knowledge in the current literature. We acknowledge that for\nthe vaccine analyses, the number of participants in specific vaccine type subgroups is small, but our\nmodels incorporate data across multiple vaccine types, and we note that this is the largest\nprospective cohort to date examining vaccine responsiveness after CARTx to our knowledge.\nFurthermore vaccines responses are only measured by quantitative humoral tests, no functional/qualitative\nhumoral tests have been performed, also no cellular tests (quantitive or functional) have been performed,\nwhich would of course have been of great value, due to the specific patient group depicted; patients with\ndisease in their B cell compartment or plasma cell compartment, heavily pretreated, and now treated with\nlymphodepleting chemotherapy and thereafter CD19/CD20 (B cell depleting) CAR T or BCMA (plasma cell and\npart of B cell depleting) therapy, whereby humoral responses might be expected to be low. But that is not the\nwhole picture for vaccines responses. Vaccine responses might be underestimated as a consequence. Also,\nthere is no information in this paper about protection against (severe) infections after vaccination. Off course\nthe groups are far too small to analyze this, however it is important to discuss that humoral levels is the only\none factor of vaccine response that is measured.\nResponse: We agree with the Reviewer’s comments on the limits of quantitative humoral testing as only\na corollary to immune protection. To address this, we conducted additional testing to evaluate\nfunctional humoral responses to vaccination. For the pathogens in this study, there are few validated\nlaboratory assays for this purpose. Given the clinical relevance of S. pneumonia in general and in\nthis patient population, the availability of a validated assay to assess functionality of antibodies, and\nthe high frequency of vaccination for this pathogen in our cohort, we focused on functional humoral\nimmune testing for S. pneumoniae. To do this, we identified 12 participants who had evidence of a\nseroresponse (≥2-fold increase in total S. pneumoniae antibody titer) after 1-2 vaccine doses and\nwho had residual paired samples from pre-vaccine dose 1 and post-vaccine dose 2. We tested these\nsamples for functional antibody responses using a multiplex opsonophagocytosis assay (MOPA) for\nthe 13 serotypes included in PCV13. This demonstrated that most participants also showed\nfunctional responses to correlate with the IgG antibody responses (Figure S17A) and correlate\n(ρ=0.65) with newly generated data for serotype specific antibody responses in the same samples\n(Figure S17B). Further, recent data from a trial of S. pneumoniae vaccination in an\nimmunocompromised transplant cohort demonstrated similar correlation between functional and\nnon-functional humoral immunity based on similar assays (PMID 37338158). These results are\npresented on page 14, lines 327-338 and in the Discussion on page 16, lines 389-391. Methods\npertaining to the assays are on lines 504-514 and Table S19.\nWe additionally agree with the importance of vaccine-induced cellular immune responses in\nprotecting CARTx recipients from infection. These responses have been more comprehensively\nexamined in CARTx recipients receiving respiratory virus vaccines for SARS-CoV-2 and influenza in\n5\nwork by our group and by others, but the relevance and pathogen-specific assays for the vaccines\nin our paper are less clear. T cell specific testing is beyond the scope of this current work, which\nfocuses on humoral immunity overall. However, we discuss the importance of T cell responses in\nthe Discussion section (Lines 399, 431-432) and cite work done in this area.\nRegarding vaccine effectiveness for preventing against severe infections, it was unfortunately\ninfeasible to accurately capture data on relevant infections, as microbiologic documentation of\ninfections was not routinely available. As recognized by the Reviewer, this study would be\nsubstantially underpowered to make meaningful conclusions about vaccine effectiveness for\nclinically documented infections.\nNevertheless the information that is analyzed in this paper is important and concerns also new information and\nwas, up till now, missing. It might contribute to current guidelines for supportive care in CAR T treatment. For\nexample, in current guidelines post CAR T treatment expensive prophylactic measurements as\nimmunoglobulin suppletion is advised by different experts without good evidence (no randomized trials), while\nimmunoglobulin suppletion might hamper vaccine responses. But, even worse, in the current guidelines little is\nmentioned about vaccination yet, while this is normally a very good strategy to prevent (severe course of)\ninfections, also after CAR T treatment (see publications as: Improved outcome of COVID-19 over time in\npatients treated with CAR T-cell therapy: Update of the European COVID-19 multicenter study on behalf of the\nEuropean Society for Blood and Marrow Transplantation (EBMT) Infectious Diseases Working Party (IDWP)\nand the European Hematology Association (EHA) Lymphoma Group. Spanjaart AM, et al. Leukemia. 2024\nSep;38(9):1985-1991). Sometimes even statements are made that vaccination might not work due to B cell\ndepletion and plasma cell depletion with CD19/CD20 CAR T and BCMA CAR T treatment, or that all memory of\nprior vaccinations is gone and the immune system after BCMA CAR T treatment is ‘like a baby’; all memory is\nexpected to be gone. These are potential dangerous statements possibly preventing doctors fromm treating\ntheir patients with appropriate vaccination strategies. This paper adds to this gap in knowledge.\nNotably, in this paper certainly not all humoral response is gone for example after BCMA CAR T treatment.\nThat is a very important message. Over time memory after treatment is maintained for a part of the patients!\nand might even further recover (possibly with recovery of plasmacell/B cell compartments?). This is an\nexample that this study describes very important knowledge which is missing and might adjust guidelines to\nmore cautious statements and more nuanced advices.\nResponse: We agree with the Reviewer’s statements and appreciate the recognition of the need for\nincreasing the evidence base from which further guidelines can be drawn. We hope this study\nmeaningfully contributes to this body of evidence.\nThe authors state the above part in their introduction: “In light of these uncertainties, patients often receive\nprophylactic immunoglobuline replacement therapy (IGRT) to address hypogammaglobulinemia, although this\nblood product is expensive, has frequent side effects, can reduce vaccine efficacy, and is of unclear benefit in\nthis population. Alternatively, vaccination after CARTx may be a durable and cost-effective tool to mitigate\nirAEs. Vaccine responses after CD19-CARTx are biologically plausible given observations that patients can\nrespond to vaccines ≥6 months after rituximab, even in the absence of measurable peripheral blood B cells”.\n(rules 98-104).\n-Please adjust in the discussion that this seems also true for CD19/CD20 and BCMA CAR T treatment, that\neven in the absence of measurable B cells there are responses to vaccination (although in bigger part of\npatients with measurable B cells, as far as it concerns humoral responses).\nResponse: We agree that emphasizing that vaccine responses were observed even in individuals with\nundetectable B cells is an important finding. We added the following sentence in the introduction on\nlines 106-107:\n‘Similar observations have been made for CARTx recipients receiving SARS-CoV-2 and influenza\nvaccines’.\nanalysis stratified by B cell count (Figure S16) and altered our unadjusted and adjusted Cox\nregression models to evaluate seroresponse per log increase in CD19+ B cell count (Table 2). This\n10\nfinding was further expanded in the Discussion section (Lines 396-399) to emphasize this\nobservation.\n- And please discuss the valuable observation that BCMA CAR T treated patients are able to maintain antibody\nlevels over time (not all of them are preserved before CAR T treatment, however it is preserved, and might\neven improve over time. Please discuss the results with more attention, and also the possible consequences\nfor guidelines.\nResponse: We agree that this is an important finding. We have updated our Key Points and Abstract to\nstate that ‘Pathogen-specific humoral immunity did not significantly change after CD19-, CD20-, or\nBCMA-targeted CAR-T cell therapy (CARTx)’ while noting that there are residual deficits after\nCARTx. We have additionally included language in the Discussion emphasizing that we observed a\nmodest (although non-significant) increase in pathogen-specific immunity in recipients after BCMA-\nCARTx (Lines 365-366).\n-Please remove that a minimum level of B cells should be reached prior to vaccination. It is not according to\nyours and prior data and the risk exists that this is implemented in guidelines, and patients that do not recover\nin their B cell counts will not be vaccinated. This statement is not true. Especially when taken into account that\nin this study only humoral responses are measured. But also when looking at your data (20-50% with\nresponses while low B cells!)\nResponse: We agree with this suggestion. As per our response to a similar request from Reviewer 1, we\nagree that while a cut-off of ≥20 cells/μL for starting vaccines may be a useful clinical heuristic, this\nrule will not completely discriminate between individuals who will or will not respond to vaccination.\nAdditionally, it does not consider T cell-mediated responses, which we address in the discussion.\nWe restructured relevant aspects of the Results and Discussion to demonstrate that a higher CD19+\nB cell count is associated with a greater likelihood of seroresponse. We provide results from an\nadditional Cox regression model evaluating the association of CD19+ B cell count on a continuous\nscale (per log increase) with seroresponse (Table 2). Higher CD19+ B cell count is significantly\n10\nassociated with seroresponse in this model, and we added this to the results. To further explore\nseroresponse by gradations of B cell counts, we present cumulative incidence estimates of\nseroresponse by three categories of B cell counts (<1 cell/μL, 1 to <20 cells/μL, and ≥20 cells/μL) for\na subset of pathogens with adequate sample sizes in these categories (Figure S16 and Table S18).\nThese updates highlight the higher probability of seroresponse among those with B cell counts ≥20\ncells/μL but also illustrate that seroresponses are observed with <20 cells/μL, including <1 cell/μL.\nWe updated the Discussion (Lines 396-399) to illustrate these points.\nOther comments:\n- Please change the title: “Influence of B cell or plasma cell-targeted CAR-T cell therapy on humoral immunity\nand vaccine immunogenicity: The HICAR study;” because vaccine immunogenicity might implicate the humoral\nand cellular responses, in this study just humoral immunity quantitively is measured (also no\nqualitive/functional humoral tests have been performed). The title does not cover the tests performed. Please\nadjust (HICAR stands for humuroral immunity in CARTx treated patients? That covers the tests performed\nindeed. Therefore please remove vaccine immunogenicity\nResponse: This is a helpful comment. In response, we changed the title to “Influence of B cell-lineage\ntargeted CAR-T cell therapy on humoral immunity and vaccine-induced antibody response”. We\nhave removed the term immunogenicity in cases where we are measuring only quantitative humoral\nimmune response.\n- The infectious complications leading to non-relapse mortality after CAR T is mentioned, are these vaccine-\npreventable infections? What is the percentage of infections that is vaccine-preventable (besides from the fact\nthat vaccination might have less beneficial effects in this specific group of patients)? Please add to the\nintroduction\n(it has been described that in the first months after CAR T treatment there are mainly bacterial infections (likely\ndue to the lymphodepleting chemotherapy), later after CAR T treatment we do see a lot of viral infections (as\ncovid and other airway infections) and viral reactivations (CMV, HHV), sometimes even with letal\nconsequences, i.e. HHV6 encephalitis. However these infections are not (yet) vaccine-preventable. It is\nimportant to add this knowledge to the paper. And alos of course the\ninfections that is vaccine-preventable)\nResponse: We appreciation this suggestion. We have cited data from a recent study demonstrating that\napproximately 31% of infections after CARTx were vaccine-preventable (Lines 103-104).\n- There are multiple challenges with vaccination after CAR T; what kind of pathogens cause (severe)\ninfections after CAR T treatment; are they vaccine-preventable? What is the effect of vaccination? This paper\nmeasures the humoral responses, however depending on the vaccines there is also a cellular response. And is\nthere any information about vaccines effectiveness? In the general population? In this specific population?\nFor Covid for example?\nResponse: These are important questions that we believe illustrate a gap in knowledge in the field\noverall, which this study attempts to address. As mentioned above, ~31% of infections were\ndocumented as vaccine-preventable in a recent study. To the authors’ knowledge, there are no data\non vaccine effectiveness in this patient population, as the studies to date have been small studies of\nimmunogenicity. While cellular immune responses to vaccines are outside the scope of our current\nstudy as detailed above, cellular responses to vaccination have been documented in CARTx-\nrecipients after SARS-CoV-2 and influenza vaccination and are discussed and referenced on lines\n106-107 and 399.\n- There are different types of vaccines used in this paper. Please discuss the different vaccines types and what\nto expect in the general population and in this specific population. Humoral responses only? Cellular\nresponses? Memory?\nResponse: This is a helpful comment. In response, we added additional language detailing vaccine type\n(live/attenuated vs. inactivated/killed) to the Methods (Lines 469-473). We also added additional\ncomparisons, stratified by pathogens grouped by corresponding vaccine type (Figure S8); this\ndemonstrated that seroprotection among pathogens corresponding to live-attenuated vaccines were\nparticularly low in BCMA-CARTx recipients. Due to limited sample size for live vaccines in the\nvaccine response portion of the analysis, rigorous comparisons of vaccine response to\nlive/attenuated vs. other vaccines was not possible. However, of those who did receive\nlive/attenuated vaccines, most responded. Please see below for comparisons to the general\npopulation. Detailed description of the differential immunity to each vaccine type in healthy\npopulations is beyond the scope of this study.\n- Are there controls? Because also in the general population the humoral response to vaccination is not 100%\nand is waning for most vaccine-preventable infections. It would be helpful to add controls to the figures. So\nrandom time after vaccination a measurement (as is also the case for this patients before start of treatment the\nCAR T, although these patients have a B-cell and or plasma cell disease, and also prior treatments that\nhamper (the measurement of) memory responses. And also add age-matched controls to vaccinations after\nvaccinations in a control population. Aldo please elucidate this in the discussion.\nResponse: This is an important comment, as comparisons to the general population can help elucidate\nwhether the rates of humoral immunity observed are in-line with expected, or altered based on\nreceipt of CARTx. The study design did not allow for recruitment of a healthy control cohort, which\nwould be exceptionally challenging given that most of these vaccines are given during childhood or\nintermittently at specific intervals or ages. However, we added data in a new supplemental Figure\nS6 for comparisons to each pathogen based on large population-based seroprevalence studies. This\n8\ndemonstrates that for most pathogens, BCMA-CARTx recipients have lower pre-CARTx prevalence\nof seroprotection and CD19/20 CARTx recipients have more similar seroprotection prevalence\ncompared to US or global populations.\n- Please describe in the discussion that the lack of B cells and or plasma cells due to disease and/or prior\ntreatment effects does not necessarily mean that prior vaccines responses are completely gone. In the\nintroduction (lines 110-111) there is stated that it is hypothesized that CD19 or CD20 CAR T treatment does not\nimpact pre-existing antibody levels, however when there are no B-cells (due to underlying disease or due to\nprior treatment) then it is not possible to measure this response (though it might not be gone indeed). Please\ndiscuss.\nResponse: We agree with this comment, and we previously demonstrated recovery of vaccine-induced\nimmunity for SARS-CoV-2 or other viruses after CAR-T cell therapy. We added the following (and\nrelevant references) to the Introduction (Lines 95-96).\n‘Additionally, pathogen-specific B or T cells may recover after CAR-T cell therapy.’\n- “Conversely, we hypothesized that BCMA-CARTx targeting plasma cells would result in loss of pathogen-\nspecific IgG and antibody diversity”. Why? Also here it might not be measurable, however not gone. BCMA is\npresent on plasma cells and B cells? Why the hypothesis that that has no impact on vaccine responses? Please\nexplain?\nResponse: This is a critical mechanistic question for this study, and the Reviewer’s comment is helpful\nin emphasizing the need for clear explanation. Given the lack of expression of BCMA on naïve or\nmemory B cells, but high-level expression on predominantly antibody secreting plasma cells, we\nhypothesized that BCMA-CARTx may reduce pathogen-specific antibody production. However, due\nto not targeting the naïve B cell compartment and anticipated rapid recovery of naïve and memory B\ncells after lymphodepletion, we anticipated that BCMA-CARTx would have less interference on\nvaccine responses.\nWe added additional language and citations to Lines 95-96 clarifying this point.\n- “Here, we report the results of a prospective observational study evaluating the impact of CD19/20 and\nBCMA-targeted CARTx on multiple metrics of humoral and pathogen-specific immunity, as well as the role of\nroutine vaccines as a means to mitigate humoral irAEs in CARTx recipients”. Please explain how to mitigate\nhumoral irAEs? When there are no B-cells then we do not measure humoral responses, with or without\nvaccinations. That might not mean that there are no effects on vaccination. Please adjust. More important is\nthe risk for vaccine-preventable infections after vaccination for AE’s of course. Please add this to the\ndiscussion and why these data are missing.\nResponse: Thank you for this comment. As suggested, we adjusted this statement as shown in bold\nbelow:\n‘Here, we report the results of a prospective observational study evaluating the impact of CD19/20\nand BCMA-targeted CARTx on multiple metrics of humoral and pathogen-specific immunity, as well\nas the ability of routine vaccines to improve pathogen-specific immunity in CARTx recipients.’\nWe responded above to the question about vaccine preventable infection risk and effectiveness of\nvaccines in this population.\n- When the B-cell compartment and plasma cell compartment is recovered, then it might be possible to\nmeasure prior humoral responses again. Off course the measurement before treatment with CAR T is also a\nstarting point in this study, however please discuss that with recovery of B and T cells there might also be an\neffect due to measurement of effects of prior vaccination, earlier not possible to measure at humoral level due\n9\nto B-cell and/or plasma cell depletion due to the disease itself and prior treatments (patients in this study are\nheavily pretreated).\nResponse: Thank you for this comment. We added the following sentence and relevant references in\nthe introduction to address this point on lines 95-96:\n‘Additionally, pathogen-specific B or T cells may recover after CAR-T cell therapy, which may also\nimpact antibody titers26,32,33’.\nUnfortunately, due to enrollment structure, US vaccine schedule and the population being studied, it\nis not possible for us to know prior vaccine history in these patients. This is further discussed below.\nWe have added this to the limitations section of the Discussion (Line 422).\n- Use of Immunoglobulin suppletion in which patients?\nResponse: Data from samples obtained within eight weeks of receipt of immunoglobulin replacement\ntherapy (IGRT) were excluded from analyses. The proportion of CD19/CD20 participants who\nreceived IGRT in one year prior to the baseline, 6 month, and 12 month time points were 2% (median,\n6.4 months prior), 12% (median, 3.1 months prior, and 21% (median, 3.2 months prior), respectively.\nThe proportion of BCMA participants who received IGRT prior to the baseline, 6 month, and 12 month\ntime points were 4% (median, 3.8 months prior), 19% (median, 3.2 months prior), and 53% (median,\n3.5 months prior), respectively. These data were added as Table S1.\n- Which vaccines were given to which patients (vaccination history) before start of CAR T treatment? Is it\nlogical that younger patients have more protection against hepatitis viruses vs elder patients, and the opposite\nfor the other pathogen-specific antibodies tested?\nResponse: Pre-CARTx vaccination history was not available for participants enrolled in this study. We\nhave added this to the limitations section of the Discussion (Line 422). Inferences regarding\nexpected vaccination timing and exposure can be made based on the standard United States\nvaccine schedule (Murthy N, et al. Advisory Committee on Immunization Practices Recommended\nImmunization Schedule for Adults Aged 19 Years or Older - United States, 2024. MMWR Morb Mortal\nWkly Rep 2024;73:11–15.; Issa AN, et al. Advisory Committee on Immunization Practices\nRecommended Immunization Schedule for Children and Adolescents Aged 18 Years or Younger -\nUnited States, 2025. MMWR Morb Mortal Wkly Rep 2025;74:26–29). Given the expected infrequent\ninfection rates of Hepatitis A and B in the United States, pathogen-specific IgG levels are most likely\nto reflect vaccine exposure rather than prior natural infection. Given the timing of vaccine availability\n(Hepatitis A 1995, Hepatitis B 1982) and the median age of the studied cohort (62 years), it is logical\nthat most CARTx-recipients may have less Hepatitis virus immunity compared to younger studied\nparticipants. Other pathogen-specific antibodies tested are more likely to reflect a mixture of\nvaccination and natural infection and would be more likely to be higher in older participants. We have\nchosen not to directly address these details in the manuscript at this time, but the findings are evident\nfrom Figure S4 comparing antibody titers by age.\n- Where the MM patients pretreated with bispecific or trispecific antibodies targeting BCMA and CD3 (within the\nlast year) before treatment with BCMA CAR T? This is important data to add, whil bispefics are used in high\nnumber of patients and were authorized before CAR T treatment was for MM. It has the same plasma cell and\nmature B cell depletion effects as BCMA CAR T\nResponse: No participants with multiple myeloma who received BCMA-CARTx received BCMA/CD3\ntargeting bispecific or trispecific antibodies before or after CARTx. We added this as a footnote in\nTables 1 and S2.\n- “BCMA-CARTx recipients showed a general pattern of increases in antibody titers by one-year post-CARTx\n10\nfor several pathogens, which was contrary to our original hypothesis” (rule 194-195); This is really important\nand new information (not priorly measured/published yet)!! Please give more attention to this finding.\nResponse: We agree with the Reviewer’s interest in this finding as new and not previously demonstrated\nin the field. However, as detailed in Reviewer 1’s comment number 4, this finding was not statistically\nsignificant in GEE models of composite seroprotection scores. In this revision, we have tried to\nbalance the novelty of this finding, while emphasizing that additional investigation in the field is\nnecessary to clarify the timing and degree of recovery expected after BCMA-CARTx.\n- “Consistent with results for vaccine preventable pathogen-specific IgG, BCMA-CARTx recipients\ndemonstrated significantly fewer epitope hits compared to CD19/20 recipients at each measured time point\n(Figure 5B)”. (Rule 245-247) and “A history of prior HCT or prior CARTx was significantly associated with fewer\nepitope hits.” (rule 249); is this also antibody based? Is it maybe the same problem, that without / with less than\nnormal B-cells and/or plasma cells antibodies it cannot adequately be measured?\nResponse: Thank you for this question. For reference, VirScan is a phage immunoprecipitation\nsequencing method to profile the specificities of serum antibodies. Reduced B cell counts or function\nsecondary to prior HCT or CARTx therapy, and therefore reduced antibody production, would be\nexpected to impact VirScan results.\n- “Due to relatively high levels of seroprotection for some pathogens, only a subset of participants and\npathogens were eligible for this analysis. In this subgroup of 58 participants (48 CD19/20, 10 BCMA) with a\nmedian of 3 (IQR 2-4) evaluable pathogens per participant, we estimated the cumulative incidence of\nseroresponse, defined as a ≥2-fold increase in antibody titers after vaccination. Thirty seven participants (30\nCD19/20, 7 BCMA) had a seroresponse for at least one pathogen. The overall cumulative incidence of a\nseroresponse was approximately 50% or higher for H. influenzae, C. diphtheriae, hepatitis B virus, and S.\npneumoniae after ≥ 2 vaccine doses (Figure 6B; Table S12)”. (rules 288-294); You have to be aware that by\nselecting only patients that were not able to mount seroprotective antibody levels, there might be a selection\nbias. You might select the worst patients. And even they were able to mount higher levels of antibodies partly.\nPlease discuss. Here the patients with the lowest protection level are selected, so the worst patients so to say\nResponse: Thank you for raising this concern regarding selection bias for our analyses of seroresponse.\nWe chose to focus on participants lacking seroprotection pre-vaccination, as they may be the most\nclinically relevant group in which to demonstrate response to vaccination; those who already have\nseroprotective antibody levels may not require or benefit from vaccination. To evaluate for\ndemographic differences in these groups, we have added Table S15, which shows that participants\nincluded versus excluded from seroresponse analysis had overall similar clinical and demographic\ncharacteristics.\nHowever, we appreciate the point raised that the group we have selected may be biased towards\nparticipants with lower ability to respond to vaccination. Inherent in any outcome that is defined by a\nspecified fold-change increase is the requirement that the starting level allows for an increase in\nvalues. Because some of our study participants had pre-vaccination titers greater than the upper\ndetection limit/2, they cannot achieve a ≥2-fold increase in titers and therefore cannot be considered\nat risk for seroresponse, to be included in that analysis.\nIn response, to include all individuals in the vaccine cohort and reduce the chances of this type of\nselection bias, we added significance testing to compare pre- and post-vaccination log titers (Table\nincreases from pre-vaccine to post-dose ≥2, which is consistent with the seroresponse estimates\nfrom our original seroresponse analysis. We included the additional language (Lines 288-291) below\nto describe these findings:\n11\n“Overall, the majority of testable pathogens showed no significant change in titers after a single\nvaccine dose, but significant increases were observed after ≥2 doses for 33% of tested pathogens\nin the CD19/20 cohort and 75% of tested pathogens in the BCMA cohort.\"\nWe feel that this more global testing, in addition to our focused analysis of seroresponse among the\nclinically relevant group of participants that lacked seroprotection pre-vaccination, provides a more\ncomprehensive picture of humoral response to vaccination in our study population.\n- And: where these patients vaccinated in their past? What is their vaccination history?\nResponse: As detailed in response to the Reviewer’s comment above, we unfortunately are not able to\ndetermine the vaccine history of participants. We have noted this explicitly in the limitations section\nin the Discussion (Line 422).\n- “BCMA-CARTx recipients had earlier and greater overall seroresponse rates compared to CD19/20-CARTx\nrecipients (Figure 6B)”; what might be the explanation?\nResponse: Thank you for the emphasis on this question. We propose that the difference in seroresponse\nrates between recipients of each CARTx target are due to differences in CAR-T mediated effects on\nthe naïve B cell compartment. We have added additional language addressing this question in the\nDiscussion (Lines 386-387).\n- “Overall, our results demonstrate that CD19/20-CARTx recipients have stable pathogen-specific humoral\nimmunity after treatment but may lack seroprotective antibodies for up to one-third of vaccine-preventable\npathogens.” (rules 310-311); vaccination status?\nResponse: As detailed in response to the Reviewer’s comment above, we unfortunately are not able to\ndetermine the vaccine history of participants, and vaccination during the pre/post-CARTx portion of\nthe study did not have large impacts on antibody titers.\n- “although this difference was not statistically significant after adjustment for CD19+ B cell count.” (rules 319);\nWhy adjustments for B-cell counts? That means that higher number of B cell counts are responsible for higher\nabsolute antibody levels. That is well known off course. However if you are able to double your antibody level,\nand these antibodies might be qualitively of high level, then the number itself does not matter; it is also\nimportant if these antibodies are able to kill the pathogen adequately. Especially when there is a memory (prior\nvaccination or infection) than the amount of antibody rise seems not that impressive, but they are earlier\nmature compared to exposure to a new pathogen/vaccine. Therefore please remove this.\nResponse: Thank you for the question and suggestion. As requested, we removed this statement. We\nincluded B cell count as a variable in the adjusted model to evaluate the variables most strongly\nassociated with a seroresponse but agree that this statement is not needed here.\n- “A pre-vaccine peripheral blood CD19+ B cell count ≥20 cells/μL was the primary variable associated with\nvaccine responsiveness, supporting the use of B cell recovery as a key metric for routine vaccine initiation\nprotocols after CARTx for B cell lineage malignancies. (rules 320-321); be carefull with this statement, because\nthat antibody levels are low with low B cell counts is logical, but not the only or even a parameter that the\nvaccine works (for most vaccines). Please remove this sentence, it might be misleasing. Please elucidate in\ndiscussion about this topic, that we are missing functional humoral responses, cellular responses and (really\nimportant) vaccine effectiveness in this B cell and/or plasma cell depleted patient group, meaning that,\ndepending on the vaccine, it might not be wise to advice vaccination depending on antibody levels in B cell or\nplasma cell depleted patients.\nResponse: We appreciate the recommended clarification and have moderated our prior conclusions on\nusing a CD19+ B cell count ≥20 cells/μL as a cutoff, as detailed in prior comments. Additionally, we\nhave added additional data to this revision documenting development of functional humoral immune\n12\nresponses to S. pneumoniae following vaccination (Figure S17A). Our Discussion now also\naddresses that we are not testing cellular responses in this work on lines 431-432.\n- “This also provides insights into the broader immunologic effects of CD19/20- targeted CARTx, and the\npotential utility (or lack thereof) for other indications such as certain autoimmune diseases despite depletion of\nthe target B cell population.” (rule 327); Also here, be careful, because in auto-immune diseases also the T-\ncells seems to be very important for the disease evolution, and despite that plasma cells (antibody titers) might\nbe preserved, the immune reconstitution (or reset) might be working with CD19/CD 20 CAR T treatment. So,\nthere is more to say about the hypothesis why CAR T-cell therapies are working in auto-immune diseases. The\nfact that plasma cells are preserved (and auto-antibodies therefore likely too) is known, but clearly not the most\nimportant factor for working (although the groups treated in auto-immune disease are still very small of course).\nResponse: Thank you for this comment. As CARTx for auto-immune diseases was not part of this study,\nwe have removed this statement.\n- “Thus, we hypothesized that BCMA-CARTx recipients would have a loss of pathogen-specific IgG and\nantibody diversity. We observed that these participants already had substantial pathogen-specific immune\ndeficits prior to CARTx, likely due to their underlying plasma cell malignancy and prior anti-tumor therapies\n(median of 8 prior lines of therapy, and 75% had a prior HCT). After BCMA-CARTx, we observed a modest\nrecovery of pathogen-specific immunity, which may have been in part due to use of IGRT in the 8-16 weeks\nprior to sampling and re-vaccination based on our finding that the increase was not statistically significant after\nadjusting for these variables.” (rules 331-337); really small groups, and by further correcting for factors you\nmight expect that the results lose their significance. It is fair to note here that the groups are too small. That\nmight not be due to the factors you corrected for, but solely due to the low numbers (becoming even lower with\nadding corrections).\nResponse: This is an important caveat to our findings. As described by the Reviewer, these suppositions\nare based on a small number of CARTx recipients (n=28). However, we noted large changes in\nmodel estimates when we added variables for prior IGRT and vaccination to the multivariable model,\nindicating confounding effects, not just an increase in p-values which may relate to a small sample\nsize. Therefore, we feel it is likely that both IGRT use and prior vaccination may be explaining some\nof the increases in pathogen-specific immunity from pre- to post-CARTx in the BCMA cohort. We\nhave added additional language to the manuscript noting the small sample size and possibility of\nloss of significance due to small sample size (Line 368-369).\n“After BCMA-CARTx, we observed a modest but non-significant recovery of pathogen-specific\nimmunity, which may have been in part due to use of IGRT in the 8-16 weeks prior to sampling and\nre-vaccination, based on the attenuated change estimates and lack of statistical significance after\nadjusting for these variables. Notably, the small sample size restricts definitive conclusions.”\n- “Individuals with B cell counts <20 cells/μL had a relatively low cumulative incidence of seroresponse of approximately 20-\n50% after ≥2 vaccine doses across different pathogens.” (rules 360-361); 20-50% is certainly not nothing! And then the factor\nthat there are vaccins that also work via cellular responses. What a positive finding that even in patients with really low B cell\ncounts 20-50% shows response! That is important to pronounce in a positive way!\nResponse: This is a critical message to our paper, and we have added additional language in the\nDiscussion emphasizing that humoral and cellular immunity can be induced even with undetectable\nB cell counts (Line 396-399).\n- “Together, our data demonstrate that CD19/20- and BCMA-CARTx recipients have ongoing immunologic\ndeficits through one year after CARTx, with greater deficits in BCMA-CARTx recipients. (rules 363-364); please\nadd humoral deficits, also explaining the lower counts of seroprotective levels\nimmunity.\n- “and requiring a peripheral blood B cell count ≥20 cells/μL prior to (re)vaccinating after CARTx is a\nreasonable metric for guiding patient care.” (rules 365-366); please remove. As your data shows there is no\nlower limit of B cells, and taking into account the missing in this study of cellular responses and vaccine\neffectiness, this is not a good advice. Please remove.\nResponse: As detailed in response to previous Reviewer comments, this recommendation has been\nmoderated.\n- “Whether routine use of prophylactic IGRT is beneficial prior to and after CARTx remains an open question,\nbut pediatric and BCMA-CARTx recipients appear to be most likely to benefit based on our findings.” (rules\n370-372); Why? Please explain further, and please remove the statement. It is not the topic of this paper and\nyou did not do research for that question in this paper.\nResponse: This statement has been removed from the manuscript.\n- “The inclusion of a CAYA cohort allowed for unique baseline comparisons across age groups to inform the\npathogen-specific immune status of young individuals prior to CARTx, which has not been previously well\ndescribed.” (rules 377-378); Please add that a healthy control group is necessary to make real statements\nabout age.\nResponse: This is an important caveat in interpreting pathogen-specific immunity across age groups.\nWe have included additional language in this statement addressing the need for comparison to a\nhealthy cohort to make conclusions (Line 414).\n- “Finally, there are few published data pertaining to vaccination responses, and\n- strategies to inform when to vaccinate CARTx recipients, aside from SARS-CoV-2.” (rules 379-380); what is\nyour advice about timing? I missed that in the paper.\nResponse: We thank the Reviewer for this important clarifying comment. We do not make explicit\nrecommendations regarding timing post-CARTx of re-vaccination in this manuscript, as time from\nCARTx to first vaccine dose was not significantly associated with seroresponse in unadjusted Cox\nmodels (Table 2). We instead emphasize that B cell recovery is a more significant metric to predict\na humoral seroresponse.\n- “and BCMA-CARTx recipients had some albeit limited recovery of pathogen-specific humoral immunity over\ntime.” (rule 395); please change to: BCMA-CARTx recipients had some recovery of pathogen specific immunity\nover time. Please try to describe your results as objective as possible, try to avoid additions as albeit limited\n(defined as?). Please adjust.\nResponse: This statement has been adjusted in the manuscript, as recommended by the Reviewer.\n- Please add to figure 3 the significance values between different timepoints for BCMA respectively\nCD19/CD20 CAR Tx (between the orange boxes, and between the blue boxes, over time; paired analyses).\nResponse: This has been added to Figure 3.\nPlease add also the number of patients vaccinated for the specific disease per group (a minority of course,\nhowever, good to mention). Also please add the % of patients that was vaccinated in the past for the different\ngroups (had the BCMA patients all received BMR for examples?)\nResponse: As mentioned in our response to previous comments, we unfortunately do not have data on\nvaccination history. Regarding vaccination during our study, we felt this would be difficult to add to\nvaccinated during the longitudinal portion of the study in the Figure 3 legend and referred to a new\nsupplemental figure (Figure S11) that excludes data points following any prior vaccination during\nthe study follow up. Patterns were similar to those in Figure 3.\n- Figure 6: be very carefull with conclusions based on BCMA CARTx when there are just 2 or 4 patients\nvaccinated. Be aware: there is an enormous bias; because why these patients are vaccinated and others are\nnot? It is not a study to which every patient (re)vaccination is offered after CAR T treatment, but the patients\nwho are invited to vaccination are offered participation of this study. That is a big difference. Please discuss in\ndiscussion the small numbers and the bias in patients that are vaccinated, to which the study is offered (and\nalso discuss the ones you possibly miss (with no B-cells and/or t cells for example? Are they not referred for\nvaccination (yet?) Only the best patients are vaccinated might be a huge bias risk)\nResponse: We agree that our conclusions regarding response to vaccination in the BCMA cohort are\nlimited by the small sample size and have indicated this limitation in our discussion (lines 420). We\nappreciate the concern regarding selection bias related to the individuals included in the vaccine\ncohort. In response to a previous reviewer comment, we have added more details describing the\nrecommended vaccination strategy and added details and a citation in the Methods (line 468-473)\nreferencing the standard of care guidelines at Fred Hutch Cancer Center generally followed by\ntreating physicians as per below:\n“Briefly, killed or inactivated vaccinations were recommended in patients who were ≥6 months post-\nCARTx and off IVIG for ≥2 months, irrespective of immune reconstitution. Live vaccines were\nrecommended in patients who were ≥12 months post-CARTx, off immunosuppression for ≥12\nmonths, had not received IVIG for ≥8 months, and had a CD4+ T cell count >200 cells/mm3.”\nTo provide greater transparency regarding any potential selection biases for our vaccine cohort, we\nhave added a supplemental table comparing demographic and clinical characteristics between\nparticipants included and excluded from our vaccine cohort (Table S11). There were some minor\ndifferences in characteristics, such as the median B cell count at 6 months post-CARTx was higher\nin the vaccine cohort (0.6 log cells/µL) compared to those excluded from the vaccine cohort (0.2\n10\nlog cells/µL), although the vaccine cohort still had a wide range of B cell counts (0-3.3 log\n10 10\ncells/µL).\nREVIEWER #3:\nThe manuscript titled “Influence of B cell or plasma cell-targeted CAR-T cell therapy on humoral immunity and\nvaccine immunogenicity: The HICAR study” presents a prospective observational study examining how CAR-T\ntherapy targeting CD19/20 or BCMA affects humoral immunity and vaccine responsiveness. Using VirScan\nprofiling, the authors analyze over 150 patients and reveal that BCMA-CAR-T recipients exhibit broader deficits\nin pathogen-specific antibodies. A B cell threshold of ≥20 CD19+ cells/μL is proposed as a predictor for vaccine\nresponse. The findings provide valuable insight about immune reconstitution and guide post-CAR-T vaccination\nstrategies.\nMajor Comments:\n1. Please clarify if the statistical analysis was adjusted for mutiple comparisons\nResponse: We appreciate this comment. We did not adjust for multiple comparisons in any of our\nstatistical analyses, and we state this at the end of the first paragraph in the Statistical Analyses\nsection of the methods. For further clarity, we have revised our sentence from “We did not adjust for\nmultiple comparisons in these analyses” to “We did not adjust for multiple comparisons in these or\nany analyses.” (line 600).\n15\n2. Patients with Mutiple Myeloma are inherently different from patients with Lymphoid malignancies. Relying\nsolely on the target might oversimplify biological differences. Consider a stratified analysis if numbers allow.\nResponse: Thank you for this comment, and we agree that the biological differences by disease type\nare notable and important to account for. Because we stratify and/or adjust our analyses by target,\nwe are also accounting for these differences by comparing the patients with myeloma to those with\nlymphoid malignancies (myeloma patients only received BCMA-CARTx, and lymphoid malignancies\nonly received CD19- or CD20-CARTx). Further stratification for some analyses will be challenging\ndue to sample size. Notably, we also account for differences in prior treatment and immune function\nby considering relevant variables for adjustment as detailed in the Methods, including age, biological\nsex, race, ethnicity, number of lines of cancer therapy prior to CARTx, any prior HCT or CARTx, any\nB-cell targeting therapy in six months before CARTx, type of CARTx product (commercial versus\ninvestigational), and laboratory measures including total IgG, IgA, IgM; CD19+ B cell count; CD4+ T\ncell count.\n3. Please clarify how the B cell count threshold was chosen and if/how it has been validated.\nResponse: We thank the Reviewer for this clarifying question. The ≥20 cells/μL value was pre-specified\nas a practical marker of at least some evidence of B cell recovery based on prior clinical experience\nwith B cell-depleting therapies. Importantly, in addition to models using this threshold of CD19+ B cell\ncount, we have added a model that treats CD19⁺ B cell count as a continuous variable (per log₁₀\nincrease in cells/μL) and have shown that higher counts were associated with increased\nseroresponse (adjusted HR 2.16; 95% CI, 1.44–3.23; p<0.001; Table 2). We updated our\ninterpretation and discussion to indicate that ≥20 cells/μL is a heuristic that could be used to guide\ndecisions around readiness for some vaccines but that it is not a strict cutoff. Indeed, seroresponses\nwere also observed below this level (Table S18), and this does not account for the ability to make\ncellular immune responses. We have added text to the Methods, Results, and Discussion, as\ndescribed in responses to other Reviewer comments above, to reflect this framing.\nMinor comments:\n1. The study includes the CAYA population, which is novel; however, the study is underpowered to make\nany substantial inferences.\nResponse: We agree that the CAYA cohort is underpowered for inferential analyses and have clarified\nthat these data are presented descriptively to provide baseline context only. Due to subsequent HCT\nor IGRT use, few CAYA participants had eligible post-CARTx samples, precluding adequately\npowered longitudinal comparisons. We now state this explicitly in the Results and acknowledge it in\nthe Discussion as a limitation, noting that larger studies with age-matched healthy controls will be\nrequired to draw formal conclusions about post-CARTx humoral immunity in pediatric recipients.\n16"}, {"round": 2, "reviewer_comments": {"reviewer_1": "All of my previous comments have been thoroughly addressed. I have no further comments. The revised manuscript is\nexcellent.\n**Editorial note: this reviewer was also asked to comment on the authors response to the concerns of reviewer 3 who was\nunable to provide a response at this time.\nI have reviewed the comments raised by Reviewer 3 and the corresponding author responses. Overall, I find the responses\nto be clear, appropriate, and well justified.\nThe response to Comment 1 is acceptable. Regarding Comment 2, I fully agree with the authors’ reasoning and explanation.\nComment 3 raised an issue that I had also noted, and I think the authors have addressed this transparently and satisfactorily\nthrough the revisions made to the manuscript. Finally, the response to the minor comment is sufficient.\nOverall, I believe the authors have adequately addressed Reviewer 3’s concerns.", "reviewer_2": "I would like to thank the authors for addressing our reviewer questions so securely.\nIt is an impressive paper, with an important and timely topic.\nI have no further comments."}, "author_response": ""}]

Decision letter