bluuebunny/crossref_metadata_2025 huggingface dataset:
Potent interaction between CMV reactivation and GVHD: Immunologic evidence for the blunting of CMV-driven immune reconstitution in the setting of GVHD in transplant patients
Authors: Suessmuth Yvonne , Betz Kayla , Yu Alison , Bratrude Brandi , Watkins Benjamin , Qayed Muna , Horan John , Kean Leslie S , Langston Amelia
Published: January 2023

Allogeneic hematopoietic cell transplantation (HCT) may be curative for patients with marrow and immune disorders, but acute graft-vs-host-disease (aGVHD) and infections cause significant morbidity and non-relapse mortality. We conducted a multicenter, double blind, placebo-controlled phase II trial of costimulation blockade with abatacept (Aba) combined with standard GVHD prophylaxis with a calcineurin inhibitor and methotrexate (CNI + MTX) following HLA matched unrelated donor transplant (n=140).

In order to assess whether Aba produces clinically relevant compromise of immune reconstitution, and whether this reconstitution is influenced during CMV reactivation, we longitudinally evaluated post-transplant whole blood samples with multiparameter flow cytometry using markers for CD3, CD4, CD8, CD197 and CD45RA to measure reconstitution of CD4 and CD8 T cell populations and their respective memory subsets over time.

We show here that post-transplant CMV reactivation induces a marked expansion of CD8 effector memory (EM) cells, which is similar in Aba vs placebo pts. We found that development of moderate (gr 2–4) or severe (gr 3–4) GVHD does not cause more CMV reactivation in patients, but patients with moderate GVHD show a blunted expansion of CD8 EM cells compared to those without GVHD. Remarkably, CD8 EM expansion was essentially absent among CMV reactivating patients with severe aGVHD.

Our results suggest that adding abatacept to CNI/MTX does not materially affect reconstitution of T cell immunity in the presence or absence of CMV reactivation, but aGVHD remains a major driver of compromised immune recovery after HCT.
Plasma cells are not necessary for regulatory B cells and preventing their differentiation markedly prolongs allograft survival
Authors: Ding Qing , Rothstein David
Published: January 2023

Plasma Cells produce Abs. Recently, PCs were reported to be the major source of B-cell IL-10, and absence of PCs worsened EAE, implying that PCs underlie “Breg” activity. To test the hypothesis in transplantation, we transplanted Bc islets into chemically diabetic B6 Blimp-1fl/flCD19-Cre+/− (PCKO) mice. Surprisingly, allograft survival (GS) was prolonged by ~42% in untreated PCKO vs. Cre-neg Ctl recipients (Mean GS: 27 vs. 19d; p<0.05). This prompted us to re-examine the EAE model where the regulatory role of PCs was first identified. Surprisingly, in our hands, the severity of EAE (MOG35–55 induced) was dramatically reduced in PCKO vs. Ctl mice (p<0.05). These unexpected results suggested that PCs are not essential for Breg activity, but might actually promote EAE and allograft rejection. These improved results do not appear to be mediated simply by loss of Ab production, since EAE is worse in the complete absence of B cells. To investigate the differences between our own and published results, we examined B cells from PCKO mice and found a 1.4-fold ↑ in IL-10+and >2-fold ↓ in IL-17+ B cells vs. Ctl mice (intracellular staining; flow cytometry). Moreover, CD4+T cells from PCKO mice exhibited a ~2.5× ↑ in IL-10 and ~2× ↓ in IL-17.

Therefore, we examined cytokine expression by splenic PCs in alloimmunized Blimp-1-YFP reporter mice. While PCs are enriched for IL-10 expression (42±3%), they are also highly enriched for IL-17 (50 ±9%).

Thus, PCs are not essential for Breg activity in either auto- or allo- immunity. We hypothesize preventing newly induced PCs may reduce immune responsiveness through the loss of potentially proinflammatory PCs. To further address these questions, we are now developing novel mice with PC-specific deletion of IL-10 or IL-17.
Recovery of innate lymphoid cells after allogeneic stem cell transplant: A single institution pilot study
Authors: Srinivasan Anand , Crawford David , Bajana Sandra , Yuen Carrie , Sun Xiao-Hong , Shah Rikin
Published: January 2023

The response of Innate Lymphoid Cells (ILCs) to conditioning regimens and subsequent recovery in the setting of allogeneic hematopoietic stem cell transplant (aHSCT) in pediatric patients is not known. The impact of this recovery pattern on graft versus host disease (GVHD) has not been prospectively studied. We aim to study the recovery of ILCs after aHSCT in children and discover its role in GVHD. Pediatric patients undergoing aHSCT were enrolled in a prospective translational study since May 2019. Patients blood were analysed by flow cytometry at following time points: prior to the conditioning regimen, weekly starting on day 0 until +30 (+/− 3 days), then +60, +90 and +180 days. Ten patients have been enrolled to date - demographics: median age 9.5 years; 4 malignant, 6 non-malignant; haploidentical donor (n=1), matched sibling (n=3), unrelated (n= 6). Graft source was bone marrow for all patients. Most common conditioning regimen was fludarabine, busulfan and rabbit antithymocyte globulin (40%). GVHD prophylaxis for most patients was tacrolimus with mini-methotrexate (90%). We saw substantial drop in number of ILCs in response to conditioning regimen in all patients, with nadir on +14 that plateaued through +30, followed by a spike on +60 and return to normal levels by +90. Similar trend was seen in type 1 ILCs (Lin− CD127+ CRTH2− CD117−) but showed a slower drop, reaching nadir on +21 with slow recovery by +60. Type 2 ILCs (Lin− CD127+ CRTH2+) and Type 3 ILCs (Lin− CD127+ CRTH2-ve CD117+) however showed a small increase on +7 compared to day 0 with both cell types recovering by +60. We showed that it is feasible to prospectively study the recovery of ILCs after aHSCT. Continued enrollment will allow us to evaluate the correlation between ILCs and GVHD.
Pancreatic and colon cancer stem cells escape NK cell effector function via PCNA–NKp44 interaction
Authors: Malaer Joseph D. , Mathew Porunelloor A.
Published: January 2023

Natural killer (NK) cells play an important role in the innate immune response against cancer. Unlike B or T lymphocytes, NK cells do not require prior sensitization and can immediately respond to target cells. NK cell function depends on a balance of signals transmitted from activating and inhibitory receptors interacting with ligands on the surface of cells. Cancer cells may escape NK-mediated responses by expressing inhibitory ligands. Proliferating cell nuclear antigen (PCNA), in association MHC I, forms the inhibitory ligand for NKp44. Cancer stem cells (CSC), a unique subset of tumor cells, possess a stem-cell-like phenotype and are thought to facilitate metastasis by escaping NK cell effector function. CSC can be identified by a variety of markers such as surface co-expression of CD44 and CD133. In both Panc-1 and HCT 116 cell lines, surface PCNA is associated with co-expression of CD44 and CD133. Triple positive (PCNA+CD44+CD133+) cells have increased CSC transcription factor (NANOG, SOX2, and Oct-4) expression compared to double positive (PCNA−CD44+CD133+) or negative cells. Additionally, blocking the PCNA–NKp44 interaction alters IFN-g secretion and increases specific lysis of cancer cells by NK cells. Taken together, these data suggest that surface co-expression of PCNA, CD44, and CD133 are markers of pancreatic and colon CSC and blocking the PCNA–NKp44 interaction may provide an immunotherapeutic approach to target pancreatic and colon CSC and prevent metastasis.
Post-translational modification of cytoskeleton regulates anti-tumor cytotoxicity in γδ T cells
Authors: Hu Yi
Published: January 2023

γδ T cells based immunotherapy has emerged as a new treatment strategy for malignant tumors. The underlying cytotoxic mechanism against cancer cells involves surface receptor recognition molecules such as LFA-1, NKG2D, TRAIL, CD16, CD107a, and FasL, as well as cytokines such TNFα, IFNγ, perforin, and granzyme B. Previously, it has been shown that the cytoskeleton plays pivotal roles in T cell immunity. Nevertheless, the role of cytoskeleton in γδ T cell anti-tumor immunity remains largely unknown.

In our work, we attempted to elucidate the functions of cytoskeleton in regulating the antitumor cytotoxicity of γδ T cells. We discovered that the acetylation of tubulin-α is positively correlated with γδ T cells cytotoxicity. While the down-regulation of tubulin-α acetylation in γδ T cells leads to decreased cancer cells killing efficacy, up-regulation could potentiate it through the expression of cytotoxic related molecules such as NKG2D, IFN-γ, CD16, and suppression of PD-1 molecule. Moreover, we observed that the acetylation of tubulin-α in γδ T cells drastically differs after challenged by MDA-MB-231, SkBr3, and MCF7 breast cancer cells, respectively, implying diverse γδ T cell immune responses towards these cell types. This result was further supported by the discrepancies in γδ T cell in vitro killing efficacy.

Together, our work will provide important scientific references to further understand the cytoskeletal function in γδ T cell anti-tumor immunity.
Two-color flow cytometric detection of serial cell-mediated cytotoxicity by natural killer (NK) lymphocytes
Authors: Navarrete-Galvan Lydia T , Guglielmo Michael , Smith-Gagen Julie , Merica Rebecca , Lombardi Vincent C , Hudig Dorothy
Published: January 2023

Serial killers are much more productive than one-hit wonders, especially when it comes to killing cancer. Natural killer lymphocytes are significant cells in many anti-cancer immunotherapies, such as monoclonal antibody based- and adoptive cell transfer therapies. However, current data show that less than 30% of peripheral blood NK cells are capable of sequentially killing more than one cell, i.e., serial killing. These serial killers are not only capable of killing up to 6 target cancer cells each, but are also reported to deliver lytic hits at a faster rate and with more killing power than their counterpart NK cells. Current methods to identify serial killer NK cells, such as time-lapse cinematography, are laborious, expensive, and require highly skilled expertise and equipment. In order to enrich and grow serial NK cells, an inexpensive, simple assay is needed to detect and isolate them from blood for subsequent in vitro growth. Here we developed a flow cytometric method to identify serial killers via 2-color detection of the cell surface marker CD107a, expressed on the NK cell surface following killing. The NK-92 cell line was incubated with target Raji B leukemia cells for 2-hours, then labeled with one color, AF647, anti-CD107a mAb. Cells were then re-incubated for an additional 4-hours and subsequently labeled with a second color, PE, anti-CD107a mAb. Serial killers labeled double positive for both colors at 6 hours. We found that NK-92s are capable of killing at least 14 target Raji cells each and about 18% of the NK-92s can be identified as serial killers via two-color CD107a mAb labeling.
Role of the KLRG1 pathway during the immune response to cancer
Authors: Tata Angela , Vivier Eric , Brossay Laurent
Published: January 2023

Immunotherapy drugs targeting T cells have been successfully developed for a variety of different cancers. In contrast, comparably less work has been done on targeting natural killer (NK) cells for immunotherapy. NK cells are intriguing targets for a number of different reasons, including their cytotoxic abilities and relatively lower autoimmunity risks. Thus, treatments that target NK cells, either alone or in conjunction with T cells, should be explored for the development of new treatments. One of the most well-known immunotherapy targets, programmed death-1 (PD-1) has recently been implicated in modulating NK cell function in cancer and demonstrates that targeting NK cells is an important aspect of cancer treatment. In addition to PD-1, there are many checkpoint molecules that may be capable of impacting NK cell function. One such target is KLRG1, a well conserved C-type lectin inhibitory receptor expressed on NK cells, which contains an ITIM in its cytoplasmic domain. To determine the role of KLRG1, and expand on the role of PD-1, as potential NK cell checkpoints, we developed a series of mice deficient for KLRG1 and PD-1, and took advantage of the NK sensitive cancer model B16. Following administration of B16-E-cadherin, we found that KLRG1 deficient animals had significantly fewer tumors in the lung tissue, compared to wild type controls. In addition, B16-E-cadherin resulted in a higher number of tumors in wild-type mice, compared to the parental B16 cell line. Altogether, these data suggest that KLRG1 is a checkpoint target candidate.
Therapeutic potential of follicular helper T cells for melanoma treatment
Authors: Nurieva Roza I , Liu Zhongshan , Zhao Yang-Zhi , Divenko Margarita , Alekseev Andrei , Sahoo Anupama
Published: January 2023

Clinical relapse and metastases are the major causes of death in melanoma. Currently, adoptive T cell therapy using tumor-infiltrating lymphocytes rich in cytotoxic CD8+ T cells (CTLs) is one of the promising approaches that helps overcome this major challenge in melanoma; however, CTL cell transfer alone does not improve clinical response, suggesting a supportive role of helper CD4+ T cells. There is increasing evidence on the potential contribution of tumor-infiltrated CD4+ T follicular helper (Tfh) cells and intratumoral lymphoid structure formation in tumor eradication; however to date, the role of Tfh cells in anti-tumor immunity has not been clearly elucidated. Remarkably, using a murine melanoma model, we found that the percentage of intratumoral Tfh cells negatively correlates with melanoma growth. In addition, we observed enhanced tumor growth in Bcl6 T-cell conditional knockout mice, which lack Tfh cells as compared to wild-type mice, suggesting that Tfh cells might play a protective role in tumor growth and could serve as a marker for tumor regression. Importantly, in support of mouse studies, in melanoma patients the number of Tfh cells correlate with the rate of patient survival. Importantly, adoptive transfer of antigen specific Tfh cells into tumor bearing mice results in increased number of intratumoral CTLs, as well as in an efficient tumor eradication, indicating the essential role of Tfh cells in promoting CTL expansion and anti-tumor reactivity. Our results thus, for the first time, indicate the therapeutic potential of Tfh cells in promoting CTL-mediated anti-tumor immunity against melanoma and provide the basis for potential usage of these cells to improve current immunotherapy approaches.
STINGing the tumor microenvironment to promote therapeutic tertiary lymphoid organogenesis
Authors: Chelvanambi Manoj , Taylor Jennifer , Storkus Walter
Published: January 2023

Classical tertiary lymphoid structures (TLS) are non-encapsulated aggregates of B cells, T cells and dendritic cells (DCs) that form around high endothelial venules (HEVs) in tissue sites affected by chronic inflammation. Homeostatic chemokines/cytokines CCL19, CCL21, lymphotoxin α, lymphotoxin β, IL-36γ, type I interferons, CXCL9 and CXCL10 have collectively been implicated in secondary lymphoid organogenesis as well as in formation of TLSs. As these factors can be produced by appropriately activated dendritic cells (DC), we posit that conditioning of intratumoral DCs/stromal cells can lead to TLS formation, improved immune cell infiltration and slowed tumor growth. We report that STING activation in bone marrow derived CD11c+ DCs leads to the production of various TLS forming factors through a STING-TBK1-IRF3 signaling cascade. In vivo, B16.F10 bearing mice treated intratumorally with a human/murine STING agonist, ML-RR-S2-CDA, showed slowed tumor growth dependent on CD8+ T cells. Immunofluorescent imaging of treated tumors showed increased aggregation of CD11c+ DCs with CD3+ T cells around PNAd+/MAdCAM1+ HEVs as early as 5 days post-treatment. These changes occurred in association with evidence for normalization of the tumor vasculature based on enhanced PDGFRβ+ pericyte coverage of CD31+ VECs. Future studies will analyze how the presence of TLS shapes the evolving local (tumor) vs. systemic therapeutic anti-tumor T cell repertoire.
Therapeutic vascular normalization to promote tumor-associated tertiary lymphoid structures
Authors: Filderman Jessica , Storkus Walter
Published: January 2023

Tertiary lymphoid structures (TLS) are non-encapsulated immune cell aggregates that form at sites of chronic inflammation. Recent studies have shown that the presence of TLS in human tumors indicates positive clinical outcome. However, many tumors have poorly organized and leaky vasculature that impedes entry of immune effector cells into tumors and consequently TLS formation. Recently, studies have shown that low doses of antiangiogenic agents normalize tumor vasculature, leading us to hypothesize that treating tumors with low doses of vascular normalizing (VN) therapies will improve immune cell infiltration and TLS formation within the tumor microenvironment (TME). To test this hypothesis, tumor-bearing mice were treated intratumorally with VN agents. RNA was isolated from digested tumors and transcript levels of TLS-promoting factors and markers of inflammation/immune cell infiltration were measured and compared to PBS treated controls. Changes in tumor vasculature were evaluated using immunofluorescence microscopy. Additionally, primary cultures of murine T cells and DCs and the BPR melanoma cell line were treated in vitro with VN agents. We observed that the VN agents dasatinib, STING agonist, bevacizumab, and agonist anti-TNFR1 antibody each induced global changes in the TME that are potentially supportive of immune cell infiltration and TLS formation. In vitro, dasatinib induced DCs and BPR melanoma cells to express higher levels of co-stimulatory receptors, MHC class I and II, and TLS-promoting chemokines. Treatment with a STING agonist was able to enhance T cell activation, while treatment with dasatinib inhibited T cell activation.
Exosomal PD-L1 expression as non-invasive biomarker for immune checkpoint inhibitors in non-small cell lung cancer
Authors: Gunasekaran Muthukumar , Russo Alessandro , Cardona Andrés F , de Miguel Perez Diego , Lapidus Rena , Cooper Brandon , Kaushal Sunjay , Adamo Vincenzo , Rolfo Christian
Published: January 2023

The development of immune checkpoint inhibitors (ICIs) significantly improved survival outcomes of non-small cell lung cancer (NSCLC) patients compared to chemotherapy. However, identification of predictive exosomal biomarker to reduce the invasive biopsy as well as to understand the efficacy of ICIs in NSCLC patients are unexplored. In this investigation, we demonstrate for the first time exosomal PD-L1 expression as predictive biomarker (liquid biopsy) for immune checkpoint blockage with PD-1/PD-L1 inhibitors in advanced/metastatic NSCLC.

Plasma samples from NSCLC patients (n=25) with matched paired samples at baseline and after 8 weeks of PD-1 blockade (Nivolumab, pembrolizumab and atezolizumab) were utilized for exosomal PD-L1 analysis by immunoblot. No statistically significant differences were reported for PD-L1 changes between pretreatment samples and at 8 weeks in both progressive patients (p=0.3783) and non-progressing patients (p=0.8066). Interestingly, when considering PD-L1 changes at 8 weeks between patients with progressive disease and partial response/stable disease (non-progressors), a trend towards PD-L1 reduction was observed in the non-progressors group, albeit this difference was not statistically significant (p=0.1941) due to the small sample size. Interestingly, dynamic changes during treatment of exosomal PD-L1 expression, assessed as exosomal PD-L1 ratio between pretreated samples and after 8 weeks of treatment, predicted the outcome of ICI treated patients in terms of both PFS and OS.

In conclusion, reduced exosomal PD-L1 expression at 8 weeks observed in non-progressors suggesting that exosomal PD-L1 might be used as non-invasive serum marker for ICIs treatment in NSCLC patients.
Mechanism of Heterodimeric IL-15 in tumor reduction: Induction of myeloid-lymphoid cell interactions leads to IFN-γ dependent lymphocyte recruitment by dendritic cells into tumors
Authors: Pavlakis George N , Bergamaschi Cristina , Stellas Dimitri , Karaliota Sevasti , Nagy Bethany , Stravokefalou Vasiliki , Dimas Konstantinos , Jensen Shawn , Fox Bernard A. , Felber Barbara K.
Published: January 2023

Background

hetIL-15 is evaluated in on-going immunotherapy clinical trials. We studied the mechanism of hetIL-15 antitumor function in several mouse models (MC38, TC-1, 4T1 and EO771).

Methods

We evaluated tumor immune infiltrate by flow cytometry, multi-color immunohistochemistry (IHC), transcriptomics and proteomics.

Results

hetIL-15 treatment delayed primary tumor growth, induced tumor regression and reduced metastases. Flow cytometry and IHC showed increased intratumoral NK and CD8+ T cell infiltration. Tumor-resident NK and CD8+ T cells were activated with enhanced IFN-γ production, increased proliferation (Ki67+), expression of survival factors (Bcl-2) and cytotoxic potential (Granzyme B+). hetIL-15 therapy resulted in increased IFN-γ and XCL1 levels and enhanced tumor accumulation of conventional type 1 dendritic cells (cDC1) expressing XCR1, IRF-8 and CD103. Production of CXCL9 and CXCL10 chemokines by tumor infiltrating cDC1 was boosted in an IFN-γ-dependent manner. Increased frequency of circulating NK and CD8+ T cells expressing CXCR3 was found, suggesting their migration toward tumors following the CXCL9/10 chemokine gradient.

Conclusions

hetIL-15 inhibits tumor growth by triggering lymphocyte-myeloid cell interactions modifying the tumor chemokine and cytokine milieu. hetIL-15 causes induction of XCL1 by lymphocytes, inducing DC intratumoral number, subsequent IFN-γ dependent CXCL9/10 production by DC and increased attraction of effector lymphocytes.
Investigating microbiome-based anti-tumor immune modulation using patient-derived mouse models
Authors: Fessler Jessica , Matson Vyara , Gajewski Thomas
Published: January 2023

Growing data supports that the gut microbiota can impact cancer immunotherapy. We previously found certain bacterial taxa associated with response to immune checkpoint blockade. These correlations warrant deeper mechanistic studies using tractable mouse models to establish the role of the microbiota in determining immunotherapy efficacy.

To understand how the microbiota affects anti-tumor immunity we generated patient-based microbiota models. Germ-free mice were colonized using patient feces, bred, and subsequent generations studied using transplantable tumor models. We focused on two representative patient-based models: one from a responder (R) to immunotherapy and one from a non-responder (NR).

Mice established from the R demonstrated greater benefit from anti-PD-L1 therapy compared to NR colonized mice and had superior CD8+ T cell responses with greater accumulation of tumor-specific CD8+ T cells and increased proliferation. In addition, compared to NR-derived mice, R-derived mice had increased intra-tumoral CD103+ dendritic cells (DCs), as well as increased BATF3-lineage DCs in the mesenteric lymph nodes and spleen. In agreement with a stronger anti-tumor immune response, R mice had higher circulating levels of IFN-γ, IL-12p70, and CXCL5 compared to NR mice.

To identify potential systemic signals originating from the gut microbiome and capable of altering global immune populations we profiled serum using an unbiased metabolomics-based approach. We found distinct metabolite profiles in the R- and NR-derived mice, suggesting potential bacteria-derived or -modified metabolite modulating immune function. These data support a role for the gut microbiome determining host sensitivity to cancer immunotherapy.
miR-26a as a novel potential therapeutic via inhibit CD38 translation and may overcome datatumumab resistance in multiple myeloma(MM)
Authors: Zhao Jianjun , Hu Yi , Liu Huimin , Fang Chuanfeng , Li Chen , Xhyliu Fjorela , Bodo Juraj , Dysert Hayley , Habermehl Gabriel , Russell Benjamin , Li Wenjun , Chappell Marcia , Jiang Xiaofeng , Ondrejka Sarah , Hsi Eric , Maciejewski Jaroslaw P , Yi Qing , Anderson Kenneth C , Munshi Nikhil C , Ao Geyou , Valent Jason , Lin Jianhong
Published: January 2023

Daratumumab is the first CD38 antibody drug for MM patients therapy. CD38 remains at a low level during daratumumab treatment and shows a sustained high quality response. In daratumumab resistant MM cells, CD38 expression was found either totally restored or at even higher levels. Nevertheless, a strategy to pressure the MM cells to maintain low CD-38 expression may have clinical benefit. miR-26a is usually downregulated in cancer and has been show to have potential clinical use. Here, we investigated miR-26a function in MM and tested single-wall carbon nanotube delivery of miR-26a in vitro and in vivo. GSE16558 dataset analysis showed miR-26a was downregulated in patient MM cells compared with plasma cells from healthy donors. miR-26a overexpression inhibited proliferation, repressed migration, and induced apoptosis in MM cell lines. To identify the targets of miR-26a, RPMI8226-V-miR-26-GFP and RPMI8226-V-GFP cells were cultured using Stable isotope labeling by amino acids in cell culture (SILAC) medium followed by mass spectrometry analysis. CD38 protein was found downregulated in MM cells overexpressing miR-26a and subsequently confirmed to be a direct target of miR-26a by reporter assay and immunoblotting. In addition, when overexpressed, miR-26a had a synergistic effect with bortezomib (BTZ) or melphalan (MEL) treatment. To determine whether the inhibition of MM was due to inhibited CD38, we knocked down CD38 in MM cells and duplicated the MM inhibition observed with exogenous expression of miR-26a, whereas restoration of CD38 overcame the inhibition of miR-26a in MM cells. Furthermore, miR-26a inhibited CD38 expression provoked cell apoptosis and inhibited cell proliferation in human MM xenograft mouse models.
Inhibigens, personal neoantigens that drive suppressive T cell responses, abrogate protection of therapeutic anti-tumor vaccines
Authors: DeVault Victoria L , Starobinets Hanna , Adhikari Sanmit , Singh Simran , Rinaldi Stephanie , Classon Brendan , Flechtner Jessica B. , Lam Hubert
Published: January 2023

Therapeutic cancer vaccine efficacy is reliant on tumor-specific neoantigens. Empirical methods to identify relevant neoantigens may overcome poor predictive values of current in silico approaches. The ATLAS™ platform is a personalized bioassay that uses high-throughput screening of autologous APCs and T cells against a patient’s mutanome to identify neoantigens that are characterized as stimulatory or inhibitory (inhibigens) based on changes in T cell cytokine secretion. In a B16F10 murine model, therapeutic vaccination with ATLAS-identified neoantigens resulted in arrested tumor growth with durable immune responses that protected mice from re-challenge. Mice that were vaccinated with an inhibigen and neoantigen combination had significantly abrogated tumor protection relative to controls. IFNγ ELISpot analysis revealed that inhibigen vaccination suppressed all antigen-specific immune responses, suggesting that inhibigens can be immunodominant. Inhibigen vaccinated mice had multiple tumor microenvironment changes including reduced T cell infiltration, altered CD8+/CD4+ T cell ratios and increased inhibitory marker expression relative to controls. These results indicate that the presence of inhibigens in an otherwise protective vaccine can alter the tumor microenvironment and abolish T cell-mediated protection. Epitope mapping of neoantigens and inhibigens and tetramer design for differential gene signature analysis of inhibigen-specific T cells is being performed. These data suggest that identification and exclusion of inhibigens from cancer vaccines is critical to prevent unintended pro-tumor responses.
Momordica charantia and Cisplatin Treatment Increase the Cytotoxic Effects on Acute Leukemia Cells
Authors: Valenzuela Hector F , Teller Taylor
Published: January 2023

Chemotherapy was designed to target specific proteins by either upregulating or downregulating specific cellular pathways to induce apoptosis and stop cell proliferation of cancerous cells. However, a patient’s cancer cells are known to mutate and potentially develop resistance the cytotoxic mechanisms of these chemotherapy drugs. In addition, high dosages of chemotherapy drugs results in harmful side effects to the patients. As a result, researchers have been searching for ways to reduce drug dosages but maintain chemotherapeutic drugs killing efficiency. Bitter Melon (Momordica charantia) extract is considered a potential anti-cancer agent due to its antitumor and anti-inflammatory properties. The objective of this study is to examine the anti-cancer effects of bitter melon extract (BME) and to show an increased cytotoxicity of acute T-cell leukemia (Jurkat) treated with both cisplatin and BME. The results have shown that BME, when introduced at a 1% concentration, decreased the viability of Jurkat cells by an average of 12% (P-value 0.02). A high dosage treatment of Cisplatin (10μM) and a 1.5% concentration of BME decreased cell viability by 75% percent (P ≤ 0.05) when compared to the control group. At a low dose treatment of cisplatin (5μM), a 1.5% concentration of BME decreased the cell viability by 58.2% (P-value = 0.005). Our results indicate that a combination of these two compounds may help reduce to the need to use high dosages of cisplatin.
Uptake and activation profile of murine dendritic cells in response to stimulation with functionalized iron oxide nanoparticles
Authors: Golshan Aneesa , Hui George
Published: January 2023

Subunit vaccines typically have better safety profiles than live attenuated or killed whole-cell vaccines, but are often less immunogenic and less efficacious when deployed alone. Thus, immunological adjuvants are frequently formulated with subunit vaccines to improve efficacy. However, few vaccine adjuvants are FDA-approved; and for many adjuvants their mechanisms of action are poorly understood. We previously explored the use of iron oxide (IO) nanoparticles (NPs) in subunit vaccine delivery and show that the IO NPs also possess adjuvant-like qualities, eliminating the need for additional adjuvants in the vaccine formulation. This study further dissects the mode of action of IO NPs as immunological adjuvants by examining the relationship of particle size and uptake on the activation profile of murine bone marrow derived dendritic cells (BMDCs) in response to in vitro stimulation with functionalized IO NPs. Using IO NPs of sizes ranging 5 – 30 nm, we showed by flow cytometry immunophenotyping that only IOs of 20nm and 30nm could induce an activated BMDC subset. The level of NP uptake by BMDCs, as measured by Prussian Blue staining, also varied with particle size, with IO of 5nm being the most efficiently internalized. Intriguingly, the levels of BMDC activation did not correlate with the level of IO NP uptake. We hypothesize only IO NPs taken up via selective endocytic pathways will activate BMDCs. The production of cytokine/chemokines by these BMDCs as further characterizations of the adjuvant-like profiles of IO NPs in relation to particle size and cellular uptake is currently being examined. These studies begin to dissect the mode of action of IO NPs as a self-adjuvanted vaccine delivery system on the innate-adaptive immune interface.
Promotion of cross-presenting DC expansion in humanized mice and antigen-specific immunity in murine models of vaccination and cancer by Albumin-Flt3L
Authors: Lam Brandon Krishna , Lin John , Kung Yu Jui , Tan Marietta , Yang Andrew , Wu T.C. , Hung Chien-Fu
Published: January 2023

The ability to engender CD8+ T cell antitumor immunity is indispensable to the success of essentially all cancer treatment strategies. Because tumor (TM) cells do not efficiently process and present immunostimulatory epitopes to CD8+ T cells, cross-presenting dendritic cells (cDC1s) are often required to elicit potent cytotoxic T cell responses. Flt3L is a cytokine known for its ability to expand cDC1s, but is limited therapeutically due to short half-life and global distribution in vivo. To overcome this, we have generated a novel fusion protein of Albumin and Flt3L (Alb-Flt3L), as albumin has a long half-life and selective trafficking to the lymph node (LN) and TM. We demonstrate that Alb-Flt3L potently expands cDC1s in a Batf3-dependent fashion, exhibits long half-life, and accumulates in the LN and TM. Since the phenotype of human and mouse DCs differ, CD34+ stem cell humanized NSG mice were used to assess the immunomodulatory ability of Alb-Flt3L on human DCs. 20-color cytometry and single cell RNA-seq reveals that Alb-Flt3L potently expands human DCs while enhancing a pro-inflammatory transcriptional profile. To gauge the adjuvant ability of Alb-Flt3L, vaccination models using either OVA or HPV16 E7 proteins were performed and revealed that Alb-Flt3L potently enhances antigen-specific T and B cell responses. Finally, in multiple murine models of cancer, Alb-Flt3L + standard-of-care chemoradiation is able to promote tumor control and neoantigen-specific cytotoxic T cells by tetramer staining. The breadth and depth of the TCR repertoire was also investigated using TCRβ chain sequencing. In conclusion, Alb-Flt3L serves as a simple, yet potent strategy to induce DC expansion, antigen-specific immunity and tumor control.
Anti-tumor efficacy and immune responses induced by a novel therapeutic HPV-specific gorilla adenovirus off-the-shelf immunotherapeutic (PRGN-2009)
Authors: Pellom Samuel Troy , Rumfield Claire Smalley , Morillon Yves Maurice , Roller Nicholas , Brough Douglas , Sabzevari Helen , Greiner Jack , Schlom Jeffrey , Jochems Caroline
Published: January 2023

There are about 44,000 cases of Human Papilloma Virus (HPV) associated cancer each year in the US, most commonly caused by HPV 16 and 18. Prophylactic vaccines have been successful at preventing healthy patients from acquiring HPV infections via the induction of HPV specific antibodies. To treat established HPV-associated malignancies, however, the continued development of new therapies is necessary. One promising new modality is HPV immunotherapeutics, which target the HPV 16 and 18 oncoproteins E6 and E7 with the goal to activate antigen specific cytotoxic T cells. Here, we evaluated PRGN-2009, a novel gorilla adenovirus GAd HPV off-the-shelf immunotherapeutic containing multiple CTL agonist epitopes of E6 and E7 to enhance the immune responses. Three weekly administrations with PRGN-2009 in the TC-1 mouse model led to decreases in tumor volume and weight, and significant increases in HPV16 E6-specific splenocytes evaluated by IFNg ELISPOT. PRGN-2009 also increased the tumor-infiltrating CD8 and CD4 T cells, as well as multi-functional (IFNg+, GzmB+) CD8 T cells in the tumor microenvironment (TME). Additionally, PRGN-2009 was tested in SiHa, a human HPV16+ cervical tumor, in the NSG β2m−/− PBMC humanized mouse model. Weekly PRGN-2009 administrations led to a reduction in tumor and a trending increase in CD8 and CD4 T cells in the tumor; IHC confirmed the increase of CD8 T cells into the TME. These studies provide the first evaluation of the GAd HPV off-the-shelf immunotherapeutic PRGN-2009 and its therapeutic impact against HPV-associated cancers.
Activation Induced Cytidine Deaminase and the Evolution of Antigen Receptor Immunogenetics
Authors: Criscitiello Michael F
Published: January 2023

All jawed vertebrates share fundamental components of the adaptive immune system. Cartilaginous fish are the most divergent jawed vertebrate group relative to mammals and use a major histocompatibility complex, B cells, and T cells. RAG mediates the rearrangement of gene segments to create both B and T cell receptors. B cell receptors further undergo diversification after antigen exposure when activated B cells extensively alter their rearranged genes to enhance affinity to antigen. This receptor modification is mediated by the enzyme activation-induced cytidine deaminase (AID). Remarkably, AID also catalyzes receptor modifications in alpha chain of TCR within shark thymus. Thus, the ancestor to modern vertebrates may have used an AID-like enzyme for lymphocyte receptor development, a mechanism lost in later vertebrates because of its potential for breaking down self-tolerance in mature lymphocytes. At the other end of the vertebrate tree, the repertoire of Bos taurus antibodies is characterized by a subset of heavy chains with variable domains VH encoding ultralong complementarity determining region CDR3. These ultralong CDR3 range from forty to over seventy amino acids in length and form a unique beta-ribbon “stalk” and disulfide bonded “knob”. Deep sequence analysis of the B. taurus heavy chain repertoire unveiled ultralong CDR3s were products of rearrangement between the longest VH and DH segments, IGHV1-1 and DH8. Both germline and somatic genetic factors and processes appear to be involved in diversification of this structurally unusual bovine ultralong CDR3 repertoire. These unheralded uses of AID will be discussed in the broader context of immunogenetics evolution.
Interaction between biological sex and early life adversity on gastrointestinal (GI) immune development
Authors: Fardisi Mahsa , Thelen Kyan , Rajput Mrigendra , Moeser Adam J
Published: January 2023

Early postnatal life represents a critical window of GI immune development when lifelong immune function is shaped. Yet, little is known on how early life events impact long-term immune development in animals and people. Based on our previous research, we investigated how biological sex and early weaning (EW) stress interact to shape long-term GI immune development in the pig. Female (F), male intact (MI) and male castrated (MC) piglets (Yorkshire-cross) were weaned at 17 d (EW) or at 26 d of age (Later weaning; LW). At 77 d of age, tissue samples were harvested for RNA-seq, qPCR, and immunoassays. Serum cytokine and ileal mucosal IgG responses were measured following oral and intramuscular vaccination for Lawsonia intracellularis (LI) and PCV2, respectively. Compared with LW pigs, EW MC and MI pigs had reduced body weight gain (P<0.05). The impact of biological sex and EW were observed in vaccine responses. Serum from F showed higher PCV2-specific IgG titers and LI-antibody levels compared with MC and MI pigs. EW F had higher IgG titers in response to vaccination compared with LW F. In contrast, EW MC had suppressed antibody titers compared with LW MC. Pathway analysis of RNA-seq data from ileal mucosa revealed enrichment of genes associated with increased in immune-related pathways (e.g. inflammation) in EW F. In contrast, EW MC exhibited enrichment for genes associated with a down-regulation of immune response (e.g. immune cell migration). Together, these studies showed that biological sex and EW have a major impact on GI immune development in the pig. An understanding of the role and underlying sex-specific mechanisms driving altered immune development in EW pigs could reveal new targets for enhancing early and long-term immune development.
Innate-like CD8 T cells in the teleost olfactory-CNS axis express NK cell markers and mount rapid polyclonal responses to viral antigens
Authors: Salinas Irene , Das Pankoj Kumar , Magadan Susana
Published: January 2023

T lymphocytes can perform innate immune functions and display tissue specificity. Wheres innate T cells have been extensively studied in mammals, little is known about their presence and function in other vertebrates. Previous work in our laboratory identified a subset of TCRalpha beta T cells that rapidly infiltrate the olfactory organ of rainbow trout upon nasal vaccination. This rapid response suggested that these lymphocytes play important functions in mucosal innate immune responses. Here we report that CD8 T cells rapidly infiltrate the olfactory organ of rainbow trout not only in response to nasal viral immunization but also in response to other nasal microbial immunizations including killed and live bacteria. Nasal delivery of PAMPS such as LPS or poly I:C did not elicit these responses. Further phenotyping of these cells using qPCR and FISH staining indicate that these innate lymphocytes are NK T cells since they express the teleost NK cell markers NCCRP1 and NITR2. Repertoire analyses of infiltrating CD8 T cells showed a public but polyclonal response with 14 amino acid long CDR3 in response to the virus. Our findings represent the first characterization of NK T cells in early vertebrates and underscore the importance of NK T cells at defending the mucosal barriers of vertebrate animals.
The Association between Colostral density and immunoglubulin concentrations of Dairy Cattle
Authors: Ha Seungmin , Lee Sangjin , Park Yeiju , Hur Taiyoung , Son Junkyu , Kim Euntae , Kim Donghyeon , Lee Jihwan , Sung Hyunhoon , Kim Myounghoo
Published: January 2023

The colostrum contains diverse and numerous immunoglobulins. The neonatal calf obtains majority of passive immunity from colostrum, which especially has huge effects on calf health. Historically, the amount of immunoglobulins in colostrum has been predicted through the density of the colostrum, but this correlation has not yet been clearly determined. In this study, we determined the correlation between the density and the immunoglobulin concentration, the difference between Holstein and Jersey cattle, and the impact by parity number. Densities were measured with Cruise densimeter. The concentration of immunoglobulins was assayed by ELISA. The concentration of total immunoglobulin G (IgG) and antibody against foot-and-mouth disease virus (FMDV) tend to increase following the density of the colostrum. However, the level of antibody against rota-corona virus was not positively correlated with the density. Regarding species and parity effects, interestingly, Holstein cows had significantly higher values in total IgG and FMDV antibody than Jersey cows. Multiparous dairy cows produced colostrums with higher concentrations of total IgG, FMDV antibody than primiparous dairy cows.
GLYCOSYLATION SITE OCCUPANCY OF THE HEMAGGLUTININ GLOBULAR HEAD IS IMPLICATED IN THE IMMUNE RESPONSE TO INFLUENZA A VIRUS
Authors: Alymova Irina , Cipollo John , An Yanming , Kamal Ram , Contessa Joseph , Gansebom Shane , York Ian
Published: January 2023

Objective

N-linked glycosylation of the hemagglutinin (HA) affects host immune responses to influenza A virus (IAV). This study determines the significance of N-linked glycosylation site occupancy of the HA globular head in the generation of effective antibody responses to IAV in mice.

Methods

We grew A(H3N2)2013 IAV in the presence of an inhibitor of N-linked glycosylation, NGI-1, which reduces glycosylation in mammalian cells by targeting the oligosaccharyltransferase. Mass spectrometry-based analysis of this virus revealed reduction of glycosylation in six out of twelve HA N-linked glycosylation sites, compared to virus grown without the inhibitor. The globular head of HA was affected by NGI-1 treatment. The ability of NGI-1-treated HA to generate immune responses was assessed by immunization of naïve mice with two 10 μg HA doses followed by measurement of serum antibody titers by hemagglutination inhibition and ELISA tests.

Results

Reduced occupancy of N-linked glycosylation sites IAV HA globular head correlated with a significant reduction in cross-strain antibody responses. Serum antibody titers directed toward recombinant HAs from A(H3N2) IAVs of 2003–2013 were from 2 to 4 times lower in mice immunized with HA from NGI-1-treated virus than in the mice immunized with HA from the fully glycosylated virus. Similarly, titers of the antibodies that are correlated with protection against influenza disease, measured by hemagglutination inhibition assays, were significantly lower in mice immunized with NGI-treated virus than those immunized with fully-glycosylated virus.

Conclusion

Glycosylation site occupancy of the HA globular head affects the immune response to IAV.
Expression of chemokines and cytokines in influenza A and B patients have a significant correlation to the levels of serum miRNAs
Authors: Othumpangat Sreekumar , William Lindsley G , Beezhold Donald H , Kashon Michael L , Burrell Carmen N , Mubareka Samira , Noti John D
Published: January 2023

Several host cytokines display differential expression in blood plasma and serum levels during influenza virus infection. Influenza may induce a T helper 2 polarized immune response leading to the activation of cytotoxic CD8+ T cells followed by expression of interleukins (IL). MiRNAs are remarkably stable and are key regulators of mRNA transcripts for proteins. MiRNAs are involved in the regulation of influenza virus replication in many cell types. During the 2015–16 and 2016–17 influenza season, we collected blood samples from patients infected with influenza to identify the role of serum miRNAs and cytokines. In influenza B patients, 76 miRNAs were differentially expressed compared to healthy subjects (p < 0.05), while 26 exosome miRNAs were differentially expressed in influenza A patients (p < 0.05). Of these miRNAs, 11 were expressed in both influenza A and B patients. The miRNAs that were differentially regulated between influenza A and B patients included miRNA-21, miR-191-5p, miR-324-5p and miR-199a-5p. MiR-21 is reported to target IP-10. Multiplex analysis of chemokines and cytokines showed that expression of IP-10 was highest in both A and B patients. In addition, influenza A patients showed increased expression of IFNα-2, GM-CSF, IL-13, IL-17, IL-1β, IL-6 and TNF-α, while influenza B patients showed only increase in IL-1α. MiR-150-5p expression showed a high correlation with MCP-1. MiR-133a-3p potentially targets both IL-17A and IL-1b and IL-15 potentially is targeted by miR-885 and miR-122, indicating that some cytokines were targeted by more than one miRNA. Our study shows that specific circulating miRNAs may be expressed by infected lung cells and that these miRNAs may be involved in the inflammatory responses to influenza.
Dietary cholesterol affects the pathogenesis of influenza A virus infection in mice
Authors: Louie Allison Y , Wetzel Lisa , Diaz Katiria Soto , Dray Evan , McKim Daniel , Steelman Andrew J
Published: January 2023

Influenza is one of the most prevalent and devastating infectious diseases in the world. But it is unclear how host factors may function to increase disease susceptibility or severity. Here, we sought to determine the role of dietary cholesterol in modulating the immune response or altering viral activity during influenza A infection. A pilot experiment indicated mice fed a 2% cholesterol diet prior to inoculation with mouse-adapted human influenza A virus (Puerto Rico/8/1934 H1N1; 1 HAU) exhibit greater morbidity compared to controls fed an energy density-matched diet. To confirm these data, 5-week-old C57BL/6 mice were fed either standard rodent diet or matched diet with 2% cholesterol for 5 weeks, then inoculated intranasally with either saline or flu (0.7 HAU). The effect of diet on circulating leukocytes was determined by flow cytometry. Serum cholesterol was measured by ELISA. Viral load and pathology were quantified by RTqPCR and immunohistochemistry. Finally, serum virus-neutralizing antibody levels were evaluated by haemagglutination inhibition. As observed previously, infected cholesterol-fed mice exhibited greater weight loss than diet matched controls. Total plasma cholesterol did not differ between diet groups. Infection increased the percentage of circulating granulocytes and decreased that of B cells at day 4 post-infection (p.i.). In contrast, diet did not affect circulating leukocytes. The amount of viral RNA at day 8 p.i. as well as serum neutralizing antibody levels were not affected by diet. Taken together, these data suggest dietary cholesterol affects the pathogenesis of influenza infection but prompt further study into better understanding the mechanism.