bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of IRE1 RNase in dendritic cells curtails antitumor adaptive immunity in melanoma.
Authors: Osorio Fabiola , Flores Felipe , Fernandez Dominique
Published: January 2023

The unfolded protein response (UPR) sensor IRE1 (inositol-requiring enzyme 1 alpha) and its target, the transcription factor XBP1s (X-box binding protein 1, spliced) critically regulates the function of dendritic cell (DC) subtypes. However, the contribution of the IRE1/XBP1s axis in DC subsets to the initiation of antitumor immunity is not entirely understood. In this work, using reporter mice we found that DCs, in particular type 1 conventional DCs (cDC1s), are major targets of IRE1 RNase activity in melanoma tumors. Deletion of XBP1s in DCs resulted in augmented tumor growth, impaired effector T cell responses and marked accumulation of TIM-3+ CD8+ T cells with a terminal exhausted profile. Transcriptomic studies further revealed that XBP1s deletion in tumor cDC1s induced the loss of major proteostasis core genes and elicited mRNA degradation via regulated IRE1-dependent decay (RIDD), which accounted for the dysregulated T cell immunity in melanoma. Thus, our work demonstrates that a strict regulatory circuit involving IRE1 RNase and XBP1s in DCs ensures optimal antitumor T cell immunity in melanoma models.

Supported by HHMI international research scholar grant # 55008744 FONDECYT (National Fund for Scientific and Technological Development) #1200793 CONICYT-PFCHA/DoctoradoNacional/2017-21170366
CD95/Fas-ligation and resting NK or IL-2 activated NK (LAK) attack limit NK-92 efficacy.
Authors: Navarrete-Galvan Lydia T , Guglielmo Michael , Amaya Judith Cruz , Merica Rebecca , Smith-Gagen Julie , Lombardi Vincent C , Hudig Dorothy
Published: January 2023

Adoptively transferred NK-92 cells and genetically modified variants must undergo irradiation to prevent proliferation, since NK-92 cells are themselves tumors. This malignant quality also makes NK-92 cells susceptible to attack by patients’ NK cells. Here, we investigated the effect of gamma-irradiation and/or Fas-ligation on NK-92 killing against K562 tumor cells, as well as the effect of irradiation on the susceptibility of NK-92 cells to attack by primary NK cells. To evaluate killing, we used a 51Cr-release assay with multiple effector to target (E:T) K562 tumor cell ratios at 4- and 8-hour time points, 10 Gy irradiation and IgM antibody ligation of Fas. (K562 cells lack detectable Fas). NK-92 cells, maintained in 1000 U/ml of recombinant IL-2, retained high cytotoxic activity immediately after irradiation, but lost >50% activity one day after 10 Gy irradiation. NK-92 cells were highly resistant to death initiated by CD95-ligation, whether irradiated or not. However, the surviving NK-92 cells suffered loss of cytotoxicity following Fas-ligation. This loss of cytotoxicity was more pronounced when Fas-ligation and irradiation were combined. Irradiation also made NK-92 cells more sensitive to attack by resting NK cells and IL-2 activated NK (LAK) cells. These findings demonstrate that Fas-ligation and irradiation are detrimental to NK-92 functionality and support the recommendation that genetic removal of CD95/Fas may increase cytotoxicity by NK-92 cells and potentially, other cell lines used for adoptive immunotherapies.

This research was supported in part by a UNR Foundation Award, an NIH P30 GM110767 for the Cytometry Center, a Nevada Undergraduate Research Award, and by the Nevada INBRE program (NIH GM103440) for research scholarship and supplies that helped support the project.
Characterization of the Tumor Immune Microenvironment in Soft Tissue Sarcoma Patients Undergoing Surgery
Authors: Cruz Sylvia M , Judge Sean J , Darrow Morgan A , Perry Lauren M , Farley Lauren E , Iranpur Khurshid R , Dunai Cordelia , Chen Shuai , Thorpe Steven W , Canter Robert J
Published: January 2023

Tumor infiltrating lymphocytes (TILs) have been shown to predict survival in soft tissue sarcomas (STS), but the specific contribution of natural killer (NK) and CD8+ T cells to outcomes is undefined. Therefore, we sought to characterize the extent of NK and CD8+ T cell infiltration in STS.

Prospectively, we evaluated 15 patients using fresh tumor from surgery for flow cytometric analysis. Retrospectively, we evaluated archived tumor tissue from 90 STS patients by immunohistochemistry (IHC) for CD3, CD8, CD45RO, NKp46, TIGIT, MHC-I, and p53. We analyzed metastasis-free survival (MFS) and overall survival (OS) by Kaplan-Meier method and log-rank test.

By flow cytometry, we observed significant variability in CD45+ leukocytes in the STS TME (mean 29±24% of total live cells) with low percentages of tumor-infiltrating CD3-CD56+ NK cells (1.7±1.9% of total live cells and 5.3±3.0% of live CD45+ cells) and CD8+ T cells (1.6±1.6% of total live cells and 29.6±30.5% of live CD45+ cells). By IHC, NK and T cell infiltrates were low (median H score 0, range 0–66.5 and 2.7, range 0–110, respectively). We confirmed a positive correlation between CD8+ T cell infiltration and significantly improved OS (P<0.05) and a trend for improved MFS. We also observed a trend for improved OS among patients with higher NKp46 scores (P=0.07). MHC-I expression positively correlated with both T and NK cell infiltration (P<0.05), whereas TIGIT expression positively correlated with T cell infiltration (P<0.05), but not NK infiltration.

Infiltration of NK and CD8+ T cells is overall low in STS patients undergoing surgery but associated with superior OS. Further characterization of the immune infiltrate in STS may yield better biomarkers of prognosis and immune targeting.

Supported by the following grants: NIH/NCI 1R03CA252793 NIH/NCI T32 CA251007-01
Tumor resident memory CD8 T cell formation and concomitant tumor immunity is CD40L dependent and CD4 independent.
Authors: Gough Michael J , Kramer Gwen , Bambina Shelly , Alice Alejandro , Blair Tiffany , Duhen Thomas , Duhen Rebekka , Crittenden Marka R
Published: January 2023

The implantation of cancer cell lines into immunocompetent mice acts as a vaccination event, resulting in T cell immunity that impacts both tumor progression and the response to subsequent therapy. We have previously shown that blocking immunity at tumor implantation using anti-CD40L antibodies blocks development of an intratumoral antigen-specific CD8 T cell population with tissue resident memory (Trm) phenotypes, and also limits responsiveness to radiation therapy and immunotherapy combinations. Using pancreatic tumors derived from Pdx1-Cre and Pdx1-Cre /R26LSL-LSIY mice crossed with KrasLSL-G12D/+ Tp53LSL-R172H/+ mice, we explored the role of CD4 T cells in providing CD40-CD40L help to generate Trm and conventional T central memory CD8 T cells (Tcm) cells in the tumor and systemically. Using depleting antibodies, we identified that Trm cells in the tumor could form independently of CD4 T cells but were lost following CD40L blockade. By contrast, systemically circulating Tcm cells that could re-expand after antigen-specific challenge were dependent on both CD4 T cells and CD40-CD40L interactions. Notably, concomitant immunity generated by tumor implantation that can prevent tumor growth on the opposite flank could also occur independently of CD4 T cells, but was lost on CD40L blockade. Systemic vaccination with Listeria monocytogenes expressing SIY efficiently induced the formation of SIY-specific Tcm cells but did not impact tumor implantation. These data suggest that tumor implantation generates Trm cells independently of CD4 T cells, and that these cells, not Tcm cells, sustain concomitant immunity. These data may impact immune therapies designed to control metastatic seeding in patients.

Supported by grants from NIH (R01CA182311, R01CA244142, R01CA208644).
TGF-β-dependent Lymphoid Tissue Residency of Stem-like T cells Limits the Response to Tumor Vaccine
Authors: Zhang Nu , Li Guo , Wang Liwen , Srinivasan Saranya , Ma Chaoyu , Liao Wei , Liu Yong , Mishra Shruti , Zhang Xin , Qiu Yuanzheng , Lu Qianjin
Published: January 2023

Stem-like CD8+ T cells represent the key subset responding to multiple tumor immunotherapies, including tumor vaccination. However, the signals that control the differentiation of stem-like T cells are not entirely known. Most previous investigations on stem-like T cells are focused on tumor infiltrating T cells (TIL). The behavior of stem-like T cells in other tissues remains to be elucidated. Tissue-resident memory T cells (TRM) are often defined as a non-circulating T cell population residing in non-lymphoid tissues. TILs carrying TRM features are associated with better tumor control. Here, we found that stem-like CD8+ T cells differentiated into TRMs in a TGF-β and tumor antigen dependent manner almost exclusively in tumor draining lymph node (TDLN). TDLN-resident stem-like T cells were negatively associated with the response to tumor vaccine. In other words, after tumor vaccine, TDLN stem-like T cells transiently lost TRM features, differentiated into migratory effectors and exerted tumor control.

This work is supported by NIH grants AI125701 and AI139721, Cancer Research Institute CLIP program and American Cancer Society grant RSG-18-222-01-LIB to N.Z. Data generated in the Flow Cytometry Shared Resource Facility were supported by the University of Texas Health Science Center at San Antonio (UTHSCSA), NIH/NCI grant P30 CA054174-20 (Clinical and Translational Research Center [CTRC] at UTHSCSA), and UL1 TR001120 (Clinical and Translational Science Award).
Pre-assembled universal SNAP-CARs show potent anti-tumor function
Authors: Ruffo Elisa , Kvorjak Michael , Adams Eric , Lohmueller Jason
Published: January 2023

Universal chimeric antigen receptors (CARs) are engineered T cell receptors that instead of directly binding to an antigen, recognize one or more adaptor molecules that bind to a target antigen. Universal CARs are of high clinical interest due to their tunable activity by antibody adaptor dose and the capabilities of targeting multiple antigens on the same cancer or different cancers by combining with different adaptors. We previously developed a unique universal CAR, “SNAP-CAR,” that carries out a chemical reaction to covalently fuse to antibody adaptors with a benzylguanine tag. Our previous experiments showed potent SNAP-CAR function with co-administered antibody adaptor. We now expand on these findings evaluating the function of the SNAP-CAR when pre-assembled with the adaptor prior to therapy. Primary human SNAP-CAR T cells were pre-incubated with different amounts of antibody adaptor, showing a titratable level of receptor assembly. When challenged with antigen positive target cells, SNAP-CAR T cells showed potent activation and killing activity that correlated with the amount of preloaded adaptor in a dose-dependent manner. The activity of pre-assembled SNAP-CAR T cells was then tested in vivo in a human tumor xenograft NSG mouse model targeting the HER2 antigen. A single dose of preloaded SNAP-CAR T cells showed significant tumor regression that could be further enhanced by additional injections of the preloaded SNAP-CAR T cells. Administration of adaptor along with preloaded SNAP-CAR T cells further improved tumor regression. In conclusion, preloading of SNAP-CAR T cells prior to therapeutic administration can deliver a potent pulse of anti-tumor activity with the capability of further programming by additional adaptor.

Supported by NIH: R01GM142007-01A1
Sex hormones more than sex chromosomal complement predict sex differences in influenza vaccine-induced immunity and protection in mice
Authors: Seddu Kumba , Dhakal Santosh , Ganesan Abhinaya , Jacobsen Henning , Klein Sabra L
Published: January 2023

Following influenza vaccination, adult female mice have greater quantity and quality of antiviral antibodies than males, which is a correlate of protection against live influenza virus challenge. Whether sex steroid hormone concentrations, sex chromosome complement, or both underlie greater immunity and protection following vaccination has not been determined. To dissect the relative contribution of steroid hormones and chromosome complement, we used the Four Core Genotype (FCG) mouse model on a C57BL/6 background to evaluate how gonadal sex (i.e., testes or ovaries) and sex chromosome complement (i.e., XX or XY) can independently or together contribute to sex differences in influenza immunity. Adult FCG mice were vaccinated intramuscularly with inactivated mouse-adapted A/California/04/09 (ma2009 H1N1). Blood samples were collected at several time points post vaccination to measure total IgG and neutralizing antibodies. All mice were challenged intranasally with live ma2009 H1N1 drift variant virus and used to either measure lung virus replication kinetics or disease pathogenesis. Gonadal females (i.e., XXF and XYF), regardless of chromosome complement, produced greater antibody responses and had less pulmonary virus replication than gonadal males (i.e., XYM and XXM). Gonadal sex and sex chromosomal complement intersected to impact disease severity following challenge, in which among gonadal females, XX females experienced less morbidity than XY females. In contrast, there was no effect of chromosome complement among gonadal males. Our data suggests that sex steroid hormones more than sex chromosome complement underlie sex differences in influenza vaccine-induced immunity and protection.

Financial support provided by NIH/OWRH/NIA U54AG062333
Improvement of the aging-related reduced efficacy of the H1N1 pandemic influenza vaccine by the complex of poly-γ-glutamic acid and alum
Authors: Kim Jaemoo , Jihyun Yang , Haryoung Poo
Published: January 2023

Highly contagious respiratory diseases caused by viral infections, like influenza are a constantly emerging threat, particularly among the elderly. Vaccination is one of the most effective strategy for preventing influenza, but the aged-related reduced efficacy of the influenza vaccine remains an unresolved problem. Here, we demonstrate that the aging-related reduced efficacy of the influenza vaccine can be improved by the complex of poly-γ-glutamic acid (γ-PGA) and alum called PGA/Alum, a new adjuvant capable of inducing innate and adaptive immune responses in aged mice. PGA/Alum induced activation of dendritic cells isolated from spleen of aged mice, with increases of expression of co-stimulatory molecules and production of pro-inflammatory cytokines. Although aged mice were observed decrease of T and B cell population and weaken efficacy of pandemic H1N1 influenza vaccine than young mice, PGA/Alum recovered the vaccine efficacy in aged mice. Notably, the vaccine-PGA/Alum-immunized aged mice increased hemagglutination-inhibition titers, serum-neutralization titers, cytotoxic T lymphocyte activity, and generation of multifunctional CD4+ T lymphocytes. Taken together, these results suggest that PGA/Alum could be a potent vaccine adjuvant that improves the aging-related reduced efficacy of the influenza vaccine, thereby resulting in effective prevention against influenza virus infection in the elderly.

This research was funded by a grant from KRIBB Research Initiative Program (KGM9942112).
Immunization with a mucosal, post-fusion F/G protein-based polyanhydride nanovaccine protects against BRSV infection in neonatal calves
Authors: Maina Teresia , Grego Elizabeth , Narasimhan Balaji , Sacco Randy E , McGill Jodi L
Published: January 2023

Human respiratory syncytial virus (HRSV) is a leading cause of severe acute lower respiratory tract (LRT) disease in young children worldwide. Currently, there is no approved vaccine for HRSV. Bovine RSV (BRSV) is closely related to HRSV and is the cause of severe acute LRT disease in young cattle. A neonatal calf model presents an opportunity to test HRSV vaccines and study a naturally susceptible host-pathogen interaction similar to HRSV infection. We tested the efficacy of a polyanhydride nanoparticle-vaccine (nanovaccine) encapsulating the post-fusion F and G proteins from BRSV and CpG adjuvant in neonatal calves with maternally-derived antibodies (MDA). A prime-boost immunization of neonatal calves with the BRSV post-fusion F/G CpG nanovaccine induced clinical protection in the presence of MDA. Vaccinated calves developed moderate or no clinical symptoms and were mostly protected from virus-associated lung pathology compared to unvaccinated controls. Calves that received a mucosal/systemic nanovaccine immunization had complete virological protection against BRSV infection in the lungs, which correlated with BRSV-specific immune responses in the bronchoalveolar lavage. Vaccination with the nanovaccine induced anamnestic BRSV-specific IgA titers in the lungs and systemic T cell responses with BRSV-specific CD4+ T cell proliferation and antigen-specific IL-17 and IFNγ secretion. Overall, the BRSV post-fusion F/G CpG nanovaccine is a promising candidate for further studies to protect neonatal calves and infants from RSV infection.
Polyfunctionality of T cell immunity in pre-immune and naïve individuals after COVID-19 mRNA vaccination
Authors: Moraes Vanessa S , Ross Ted M
Published: January 2023

Humans make SARS-CoV-2-specific antibodies, CD4+ T cells, and CD8+ T cells in response to infection. Neutralizing antibodies are associated with protective immunity against secondary infection and are the mechanism that can provide truly sterilizing immunity. While not necessarily providing sterilizing protection, T cell responses are critical for limiting disease severity and can limit the danger of antibody escape variants by providing for rapid clearance. Thus, studying antibody, memory B cell, CD4+ T cell, and CD8+ T cell memory to SARS-CoV-2 in an integrated manner is important for understanding mechanisms and durability of protective immunity against COVID-19. Here we add SARS-CoV-2-specific memory CD4+ T cells and CD8+ T cells to those efforts with a particular interest in assessing whether pre-existing cross-reactive T cell memory impacts vaccine-generated immunity. We monitored T cell responses in 70 healthy individuals vaccinated with Pfizer–BioNTech mRNA vaccine over 9 months. We found that vaccination induced the development of a sustained spike-specific CD4+ and CD8+ T cell response which persisted for at least 6 months. Vaccine-generated spike-specific memory CD4+ T cells were comparable in quantity and quality to convalescent cases 6 months post-boost, including the frequency of T follicular helper cells and IFNγ-producing cells. Subjects with pre-existing cross-reactive CD4+ T cell memory had increased CD4+ T cell and antibody responses to the vaccine, demonstrating the biological relevance of SARS-CoV-2–cross-reactive CD4+ T cells. These data show that vaccination elicits an immunologically competent and long-lived SARS-CoV-2-specific T cell population in naïve and pre-immune individuals.

Supported by National Institute of Allergy and Infectious Diseases (NIAID), a component of the U.S. National Institutes of Health (NIH), Department of Health and Human Services, contract 75N93019C00052
Clonal structure and epitope specificity of Ad5-nCoV vaccine-induced T cell response to SARS-CoV-2
Authors: Sheetikov Savely , Khmelevskaya Alexandra A , Zornikova Ksenia V , Zvyagin Ivan V , Shomuradova Alina S , Afanasiev Andrei , Dzutseva Vitalina , Efimov Grigory A
Published: January 2023

As a part of phase 3 clinical trial of a single-dose adenoviral COVID-19 vaccine, we evaluated the dynamics and clonal structure of T cell response to the SARS-CoV-2 Spike protein.

T cell response measured in 50 donors by IFNg ELISpot after Spike stimulation peaked on the 14th day (d14) after vaccination. Flow cytometry demonstrated that the response was skewed to CD4+ cells. To isolate Spike-specific T cells we performed short-term T cell expansion of d14 samples (n=17 donors) with the recombinant Spike, restimulated and FACS sorted IFNg+ cells.

T cell receptor (TCR) beta chain sequencing identified from 36 to 629 Spike-specific clones (median 186) per individual. CD4+ response was more clonal than CD8+ (median 116 and 17 respectively) but CD8+ occupied a larger share of the total TCR repertoire (median 0.25% for CD4+ and 0.52% for CD8+) as a result of the larger average clone size at d14 (3.4×10−5 for CD8+ vs. 6.6×10−6 for CD4+). Both the number and total share of Spike-specific clones in the TCR repertoire decreased at day 28 and further at day 180. At 6 months a median of 1 (0 to 18) of the initial Spike-specific clones were detectable in the peripheral blood. This number increased to 39 (9 to 105) as a result of T cell expansion.

For 14 donors we also performed rapid expansion of T cells sampled at d14 with 11 Spike-epitopes (9 MHC I and 2 MHC II) followed by sorting of activated cells and TCR sequencing. We identified on average 41 unique epitope-specific clones (2 to 141). They were mostly undetectable in the peripheral blood at 6 months (0 to 8 clones), but their numbers increased to the median of 7 clones (0 to 35) after cultivation with Spike.

Overall this demonstrates the induction of polyclonal and stable T cell response by a single injection of adenoviral vaccine.

The work was supported by the Russian Science Foundation grant 20-15-00395.
Antibody-dependent enhancement of SARS-CoV-2 spike binding to human ACE2 receptor by virus neutralizing antibodies
Authors: Magazine Nicholas J , Guan Yongjun , Yu Lei , Carossino Mariano , Zhang Tianyi , McGee Michael C , Huang Weishan
Published: January 2023

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated disease COVID-19 has caused over 275 million cases of illness and over 5 million deaths as of December 2021. The spike glycoproteins expressed on surface of the SARS-CoV-2 particle are the primary target of both vaccination and monoclonal antibody therapies. Widely reported evidence suggests that a subset of both naturally produced and synthetic antibodies (Abs) enhance infection, a phenomenon known as antibody-dependent enhancement (ADE). Recently, it has been demonstrated that the mechanism by which some Abs may elicit ADE is by enhancing the binding affinity of SARS-CoV-2 spike to its ligand - the human ACE2 receptor. Using in vitro spike-ACE2 binding assays, we identified two Abs which demonstrated ADE for spike/human ACE2 binding. Surprisingly, these binding-enhancing antibodies sufficiently neutralized SARS-CoV-2 pseudo-virus infection in human ACE2-knockin 293T cells, as well as live virus infection in Vero-E6 (non-human primate cell line) and Calu-3 (human cell line) cells in vitro. Furthermore, these receptor-binding enhancing antibodies significantly reduce but not enhance signs of SARS-CoV-2 infection in human ACE2-knockin mice, including viral replication and tissue pathology. Taken together, our data suggest a discrepancy between results of in vitro spike/receptor binding assay and virus neutralization assays for SARS-CoV-2 infection. Enhanced spike binding to host receptors is not a reliable measure of screening for ADE antibodies, but may be a mechanism by which virus neutralization is achieved in nature.

This work was supported in part by grants from the National Institutes of Health (R01 AI151139, R56 AI146226 and P20 GM130555). W. Huang also received research support from MegaRobo Technologies Corporation.
Differences between autoantibodies induced by SARS-CoV-2 infection and Pfizer-BioNTech SARS-CoV-2 vaccination
Authors: Geanes Eric S , Bradley Todd
Published: January 2023

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can induce severe disease and lead to hospitalization and/or death in some subpopulations of patients. The underlying mechanisms of disease severity between individuals remain unclear. Recently, autoantibodies have been detected after severe SARS-CoV-2 infection, including anti-idiotypic IgM antibodies targeting ACE2, the receptor for SARS-CoV-2. In this study, we examined autoantibody differences within hospitalized patients with severe SARS-CoV-2 infection, individuals with previous SARS-CoV-2 infection (seropositive), and individuals with no previous exposure to SARS-CoV-2 (seronegative). An autoantibody multiplex panel was run on serum collected from seropositive and seronegative individuals before vaccination and at 7 weeks after two doses of vaccine. Anti-ACE2 antibodies were measured for these samples in addition to serum collected from hospitalized individuals with severe SARS-CoV-2 infection. Severe SARS-CoV-2 infection resulted in elevated anti-ACE2 IgG, IgA, and IgM antibodies compared to all vaccine groups. Seropositive samples resulted in significantly higher anti-ACE2 IgG after vaccination, and significantly higher IgA and IgM antibodies before vaccination compared to seronegative samples. ACE2 inhibition was measured for samples with measurable anti-ACE2 IgG to determine if anti-ACE2 autoantibodies were affecting ACE2 function. The findings of this study elucidate severe SARS-CoV-2 infection results in higher IgG, IgA, and IgM anti-ACE2 antibodies compared to non-hospitalized SARS-CoV-2 infection and SARS-CoV-2 naïve individuals and in turn may provide future novel identifiers for SARS-CoV-2 disease severity.

Supported by funds provided by the Children’s Mercy Research Institute
Optimizing a multi cancer antigen plasmid-based vaccine using an in situ prediction model.
Authors: Cecil Denise L , Drovetto Nicholas , Doan Ivy , Rodmaker Erin , Corulli Lauren , Sei Shizuko , Disis Mary L
Published: January 2023

Little is known about optimization of a class II restricted plasmid-based vaccine targeting multiple cancer antigens. We set out to define parameters of an in situ prediction model in vaccine design for optimal immunogenicity. Using previously defined immunogenic class II epitopes from five cancer antigens, we evaluated the effects of using linkers between antigens and varying the antigen order. The construct with linkers was superior to the construct without linkers. The mice receiving the vaccine with linkers developed a greater magnitude (p<0.0001), incidence and breadth IFN-g immune response. To determine the optimal antigen order, the sequences of all possible permutations were generated. Each sequence was evaluated in four publicly available algorithms to assess potential generation of immunity and physiochemical properties of the fusion protein. Of all parameters assessed, only predicted class II immunogenicity, in vivo half-life and percent of epitopes in helical secondary structure differed considerably among the constructs. We evaluated three constructs with similarly high class II immunogenicity; one with (A) long in vivo half-life and high percent in helices, one with (B) short half-life and high percent in helices or one with (C) short half-life and low percent in helices. Construct A generated a higher magnitude IFN-g response than Construct B or C (p<0.006 for all). While there was 100% incidence of an immune response to all constructs, the breadth of the response for Construct A was greater than the other two constructs. Class II immunogenicity, in vivo half-life and the percent of the protein in a helical secondary structure can be used to discern the most immunogenic multi-antigen plasmid vaccine construct.
CD45RA expressing lymphocytes inhibit the outgrowth of Epstein-Barr Virus Specific T-cells from Patients with EBV+ Lymphoma
Authors: Sharma Sandhya , Woods Mae , Mehta Naren , Rooney Cliona
Published: January 2023

EBV antigen specific T-cells (EBVSTs) are difficult to expand from patients with EBV-positive lymphoma, perhaps because they circulate with low frequency or are rendered dysfunctional by the suppressive lymphoma microenvironment. As a result, we were unable to produce EBVSTs from ~25% of EBV+ lymphoma patients in our clinical trial (NCT01555892). To enrich memory T-cells prior to EBVST generation, we depleted the CD45RA+ subset of PBMCs, that includes naïve T-cells (TN), NK-cells, T-stem cells (TSCM) and terminally differentiated T-cells (TEMRA). CD45RA depletion (RAD) enhanced EBVST proliferation, antigen-specificity, polyfunctionality, cytotoxicity and tumor clearance in a murine model, and most importantly, enabled the outgrowth of EBVSTs from patient PBMCs that had previously failed to respond. This observation led to our hypothesis that bystander CD45RA+  cells inhibit the outgrowth of memory EBV antigen specific T-cells.

TCR-β chain sequencing showed that the clonotypes with greatest expansion in RAD-EBVSTs failed to expand or expanded poorly in EBVSTs from whole PBMCs (W-EBSVTs), reflecting a more selective clonal expansion in RAD-EBVSTs. TCR repertoire of W-EBVSTs was more similar to that of EBVSTs derived from CD45RA+ PBMCs that lacked detectable EBV specificity and to that of the original PBMCs. Subsets add back to RAD-PBMCs suggested that the CD3+ CD45RA+ subset inhibits the reactivation and clonal expansion of EBV-Ag specific memory T-cells. This may represent a physiological strategy to prevent the oligoclonal outgrowth of inappropriate memory T-cells during primary infection. This approach has been translated into our ongoing clinical trials using NCT01555892 and NCT04288726 using EBVST against lymphoma.

This work was funded and supported by CPRIT RP160283- Baylor College of Medicine Comprehensive Cancer Training Program, NIH-NCI P50 CA126752, American Society of Gene and Cell Therapy (ASGCT) Career Development Award, and Tessa Therapeutics.
Pre-existing biochemical entities predict severity of cytokine release syndrome in B-cell lymphoma patients treated with CD19-directed CAR T-cells
Authors: Gupta Neetu , Jalota Akansha , Hershberger Courtney E , Patel Manishkumar S , Mian Agrima , Faruqi Aiman , Khademi Gholamreza , Rotroff Daniel M , Hill Brian T
Published: January 2023

Potentially severe immune-related adverse events (irAEs) develop in relapsed/refractory large B cell lymphoma (R/R LBCL) patients during treatment with anti-CD19 CAR T-cell therapy. Therefore, prediction and better management of toxicities is critically required to improve patient outcomes. Cytokine release syndrome (CRS), one of the most notable acute irAEs, is the result of an inflammatory response of activated CAR T-cells and myeloid cells. Interestingly, high levels of glucose and low levels of certain amino acids are associated with pro-inflammatory immune activation in infectious and autoimmune diseases, but such associations have not been investigated in CAR T-cell therapy. Therefore, we employed mass spectrometry-based untargeted and targeted metabolomics analysis to identify pre-treatment host biological metabolites that predict rapid onset and severe CRS in 41 R/R LBCL patients treated with Axicabtagene Ciloleucel or Tisagenlecleucel. Analysis of plasma metabolites revealed significant associations of CRS with several metabolic classes, including carbohydrates, lipid, amino acids, dipeptides and nucleotides. Interestingly, high levels of the carbohydrates, glucose, mannose and 1,5-anhydroglucitol were observed in patients who experienced more rapid onset of CRS. In contrast, patients with greater severity of CRS had lower plasma abundance of the amino acids, glycine, proline and glutamate. Our data suggest that specific pre-existing plasma metabolites have the potential to serve as predictive biomarkers of CRS in patients undergoing anti-CD19 CAR T-cell therapy, and may be used for risk stratification and clinical management of irAEs in advance of treatment.
Promoted checkpoint inhibitor-based tumor immunotherapy by small-molecule prodrug hydrogelator
Authors: Xu Dongqing , Wan Fengyi
Published: January 2023

Cancer immunotherapy with immune checkpoint inhibitors (ICIs) has shown great promise in harnessing host immune system to combat cancer. That said, only a portion of patients has benefit directly from the anti–programmed cell death protein 1 (anti-PD1) therapy, whereas the others with an immunosuppressive tumor microenvironment (TME) achieve poor clinical outcomes. Hence, promoting an immune-stimulating TME that favors tumor-infiltrating lymphocytes is an important means to maximize the cancer immunotherapeutic potentials. Small-molecule prodrugs conferred intratumoral delivery of ICI agents have emerged as novel local delivery approaches to enhance antitumor immunity for cancer therapy. Our recent study revealed a combined chemoimmunotherapy could serve as a reservoir for extended release of DNA-damaging agent camptothecin and ICI agent anti-PD1 antibody, thus eliciting robust and durable systemic antitumor immunity in mice. The underlying mechanisms on how this carrier-free therapeutic system boosts immune sensing of immunogenic tumors and priming of CD8+ T cells against tumor antigens and the impact of microbiota will be discussed. Together, these results provide important insights into the use of small-molecule prodrugs as both chemotherapeutic and carrier, to awaken and enhance antitumor immune system for improved cancer therapy.

Supported by NIH grant R01CA244350
Effect of Standard of Care Treatment on Tumor Membrane Vesicle Vaccine for Triple-Negative Breast Cancer
Authors: Monterroza Lenore , Munoz Luis , Bommireddy Ramireddy , Samaranayake Sarah , Pack Christopher , Selvaraj Periasamy
Published: January 2023

Metastatic triple-negative breast cancer (TNBC) has a poor prognosis primarily due to metastatic potential and resistance to currently available therapies. Recent studies suggest that TNBC may be a viable target for immunotherapy due to the presence of high levels of infiltrating lymphocytes. However, many TNBC patients do not respond due to high intra-tumoral and inter-patient heterogeneity. Utilizing a novel protein transfer method, we generate a personalized vaccine based on tumor membrane vesicles (TMVs) derived from whole tumor tissues modified with glycolipid-anchored forms of the immunostimulatory molecules (GPI-ISMs) B7-1 and IL-12. In prior studies we observed that simultaneous delivery of TMVs along with biological adjuvants enhanced CD8 T cell immunity. Herein, we investigated the impact of standard-of-care (SOC) chemotherapy on the resulting quality of the TMV vaccine. Mice were injected with 4T1 TNBC cells subcutaneously and treated with three rounds of SOC drugs starting at day 15. Chemotherapies affected tumor growth to varying degrees. Mice were euthanized and tumors were harvested for TMV preparation. Results show that depending on the treatment, the yield and particle size of TMVs was impacted. While SOC drugs affected some surface markers on TMVs, expression of CD24 and CD11b was unchanged. Further, there was reduced uptake of TMVs by dendritic cells prepared from cisplatin and 5-fluorouracil treated tumors. GPI-ISMs were equally incorporated by protein transfer onto TMVs despite SOC treatments. In summary, our studies in the 4T1 model show that a TMV vaccine can be successfully prepared after chemotherapy, but the impact on TMV yield and size justifies further investigation.

Supported by R01 CA202763
Extracellular CIRP Induces Novel Nectin-2+ (CD112+) Neutrophils to Promote Th1 Differentiation in Sepsis
Authors: Murata Kensuke , Murao Atsushi , Aziz Monowar , Wang Ping
Published: January 2023

Neutrophil heterogeneity represents different subtypes, states, phenotypes, and functionality of neutrophils implicated in sepsis pathobiology. Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern that promotes inflammation and alters neutrophil phenotype and function through TLR4. Nectin-2 or CD112 is an Ig-like superfamily member. CD112 serves as the ligand for DNAM-1 (CD226), which induces Th1 differentiation in naive CD4+ T cells. Th1 cells produce IFN-γ to fuel inflammation. CD112 is expressed mainly on APCs, but its expression in neutrophils is unknown. We hypothesize that eCIRP induces CD112 expression in neutrophils, promoting Th1 differentiation in sepsis. Incubation of neutrophils with recombinant murine (rm)CIRP significantly increased the gene and protein expression of CD112 in neutrophils. Anti-TLR4 Ab-treated neutrophils significantly decreased CD112+ neutrophils compared with controls upon rmCIRP stimulation. After 4 h of rmCIRP injection in mice, CD112+ neutrophils were significantly increased in the blood and spleen. At 20 h after cecal ligation and puncture–induced sepsis, CD112+ neutrophils were also significantly increased. Blood and splenic CD112+ neutrophils in septic CIRP−/− mice were much lower than in septic wild-type mice. Coculture of naive CD4 T cells with rmCIRP-treated (CD112+) neutrophils significantly increased IFN-γ-producing Th1 cells compared with coculture with PBS-treated neutrophils. CD112 Ab significantly attenuated Th1 differentiation induced by rmCIRP-treated neutrophils. Thus, eCIRP increases CD112 expression in neutrophils via TLR4 to promote Th1 differentiation in sepsis. Targeting eCIRP may attenuate sepsis by reducing Th1-promoting CD112+ neutrophils.
TBK1 Isoform Inhibits Grass Carp Reovirus Infection by Targeting the Degradation of Viral Nonstructural Proteins NS80 and NS38
Authors: Zhang Jie , Chang Ming Xian
Published: January 2023

TANK-binding kinase 1 (TBK1) undergoes alternative splicing, and the previously reported TBK1 isoforms are negative regulators of RIG-I–like receptor–mediated type I IFN production. Although a study has suggested that grass carp TBK1 has an opposite effect at high- and low-titer of grass carp reovirus (GCRV) infection, the functions of grass carp TBK1 isoforms in GCRV infection remain unclear. In this study, we show that a TBK1 isoform from grass carp (Ctenopharyngodon idellus) named as gcTBK1_tv3, which has a 1-aa difference with zebrafish TBK1_tv3, inhibits the replication and infection of GCRV both at high and low titers of infection in C. idellus kidney cells. gcTBK1_tv3 can colocalize and interact with the NS80 and NS38 proteins of GCRV. Furthermore, gcTBK1_tv3 specifically degrades the NS80 and NS38 proteins of GCRV through the ubiquitin-proteasome pathway. Mechanistically, gcTBK1_tv3 promotes the degradation of NS80 or NS38 for K48-linked ubiquitination by targeting the Lys503 residue of NS80 or Lys328 residue of NS38, respectively, which ultimately impairs the production of cytoplasmic viral inclusion bodies and limits GCRV replication and infection. Taken together, our findings provide insight into the function of TBK1 isoform in the antiviral immune response and demonstrate that TBK1 isoform can target the nonstructural proteins of GCRV for impairing the formation of viral inclusion bodies.
A Non-Specific Cutaneous Sensitivity Which is Passively Transferable with the Serum to the Normal Human Skin
Authors: Walzer Matthew
Published: January 2023

Prausnitz and Küstner (1) demonstrated that the serum of a fish-sensitive individual with positive cutaneous reactions possessed the power of specifically and locally sensitizing normal skin. This finding has been shown to be generally applicable to atopic sensitivity to antigens and has served as a basis for the indirect method of testing for hypersensitiveness (2). In this technic, it has generally been accepted that when the reaction on the sensitized site is greater than that of the control test on normal skin, a specific sensitivity has been transferred with the injected serum.

In the course of studying more than 200 cases of atopic hypersensitiveness by the indirect method of testing, the writer has found 3 sera which, when injected into normal skins, induce a non-specific hypersensitiveness; that is, the resulting reaction in the sensitized site is greater than that obtained on a normal site with the same intracutaneous test, regardless of the substance with which the injected site is subsequently tested intracutaneously.
The Specificity of Precipitins for Serum
Authors: Wolfe Harold R.
Published: January 2023

It has been verified that a single series of injections of small quantities (2 to 3 cc.) of undiluted serum-protein into rabbits will yield an antiserum that will give “group” reactions but seldom “extra-group” reactions. Such antisera do not give settled precipitates under the conditions described and the flocculative titer is lower than the interfacial titer. Reinjection of such rabbits with minute quantities of a 2 per cent homologous antigen results in an antiserum but slightly different from the initial antiserum produced. The titer of the rabbit's blood falls considerably within a 2 months' period after the first injection-series or reinjections with dilute solutions. Reinjections with undiluted serum result in antisera whose reactions (a) are aspecific, (b) give much settled precipitation, (c) react more quickly than those resulting from one injectionseries or reinjection of 2 per cent solutions, (d) reach a maximal titer (after one hour) usually after 2 injection-series, which thereafter cannot be increased by further injections.
Studies in Experimental Hypersensitiveness in the Rhesus Monkey
Authors: Straus Henry W. , Coca Arthur F.
Published: January 2023

Summary and Conclusions

By an encircling incision in the arm of Rhesus monkeys and dissection of the skin downward toward the elbow in the manner of a cuff, an interruption of continuity of the skin was established and maintained by means of a plaster-of-Paris cast applied in a way to prevent contact of the limb with other parts of the body. In 3 of the monkeys, sensitivity to poison-ivy was established in one of the mutually isolated areas of skin, and in all of these the sensitivity was found to be limited to the treated area. These results indicate (a) that the sensitivity of contact-dermatitis develops locally in the epidermal cells; (b) that the sensitization of cutaneous areas distant from the site of the sensitizing application of an extract of poison-ivy is not due either to blood-borne anti-bodies or to transportation of the excitant through the internal body-fluids, but probably to a diffusion of the oily excitant through the oily substances normally present in the skin.
Serological Behavior of Heated Protein Mixtures
Authors: Nigg Clara
Published: January 2023

It may be concluded from tests with precipitin for either species component that, on heating a mixture of proteins from two animal species, aggregates are formed which consist of the two sorts of protein. However, the appearance of new specific properties in these aggregates could not be conclusively demonstrated.
Studies on the Physiology of Anaphylactic Shock
Authors: Hochwald Adolf , Rackemann Francis M.
Published: January 2023

Whereas both human serum and horse serum are somewhat toxic for the intestinal muscle of the rat, the response of the muscle of sensitized animals is at least in the case of horse serum significantly more marked than the response of the non-sensitized. As in the in vivo experiments, the time interval is of vital importance since marked reactions are observed only in those animals whose muscle is tested during the period between twelve and seventeen days after the last sensitizing dose. When horse serum is used, the contrast between the response of muscles from sensitized and from normal rats is striking and shows variations of at least one hundred fold.

Histamine induces reactions of the rat intestinal muscle which are quite inconstant and irregular. In some cases the muscle is resistant to histamine so that even a 1:1000 dilution of the drug is tolerated without reaction. The fact that the reaction to histamine results sometimes in a contraction and sometimes in a relaxation is interesting, if only because it is in some contrast to the reaction to acetylcholine which is always that of contraction.
The Interchangeability of the Complement Components of Different Animal Species
Authors: Rice Christine E. , Crowson C. N.
Published: January 2023

Titrations of the four major complement components in fresh sera from twelve mammalian and two avian species have been made using test reagents prepared from guinea-pig complement. All seven of the mammalian complements evincing hemolytic activity for sheep red-cells sensitized with rabbit hemolysin, were shown to contain all four components. The five mammalian complements without hemolytic effect on this system contained considerable amounts of C′1, variable amounts of C′3, but no demonstrable C′4. Small amounts of C′2 could be detected in three of these five “non-hemolytic” complements when heated guinea-pig serum was added to ensure an adequate excess of the heat-stable components, particularly of C′4. Owing possibly to species incompatibility, the guinea-pig test reagents failed to detect any of the four components in chicken or turkey sera which initially had exhibited some hemolytic activity, a presumable indication of the presence of all four.

Studies of the interchangeability of the four components in the complements of the various species showed only those of guinea pig, man, and hamster to be mutually compatible, those of the other species replacing each other partially. The C′1 component of the non-hemolytic complements could be substituted for C′1 of guinea pig and certain of the other hemolytic complements; their C′2 components were not found to be mutually interactive.

The role of inhibitory substances in reducing or obscuring the activities of components present in low concentration is considered, as is also the practical importance of differences in the lability of various complements in determining titer values of individual components. Fixation of complement due to interspecies antigen-antibody reactions is suggested as one possible source of anticomplementary effects.
Inhibition of the Growth of Viruses in the Chick Embryo by Aromatic Diamidines
Authors: Eaton Monroe D. , Levenson Charlotte G. , Perry Maude
Published: January 2023

Inhibitory effects of aromatic diamidines on the viruses of influenza, mumps, feline pneumonitis and meningopneumonitis in the allantoic sac of chick embryos are compared.

With pentamidine the order of activity may be stated as follows: feline pneumonitis > meningopneumonitis > mumps > influenza B > influenza A. Stilbamidine was inhibitory only to the mumps virus.

Growth of influenza viruses in de-embryonated allantoic sac preparations was slowed down by propamidine and pentamidine.

In mice the agents of feline pneumonitis and meningopneumonitis were inhibited to a slight degree, while results with influenza were negative.
Studies in Specific Hypersensitiveness
Authors: Cooke Robert A.
Published: January 2023

The cutaneous test is accepted today as a diagnostic procedure in the study of human hypersensitiveness. The marked immediate cutaneous reaction that can be confirmed repeatedly is the evidence of the hypersensitiveness of the skin. In the writer's article “On Constitutional Reactions,” in this issue of the Journal (page 000), it is shown by a table of cases that the typical cutaneous reaction is likewise indicative of clinical hypersensitiveness in at least 95 per cent of the cases reacting to the extracts of such airborne substances as pollens, powdered root of orris and animal danders.

Bronchial asthma was early recognized as the chief clinical manifestation of the hypersensitive state in human beings and many of the cases were readily diagnosed by testing with extracts of such substances as those mentioned above, easily conceived and long known clinically to be important excitants of an attack.