bluuebunny/crossref_metadata_2025 huggingface dataset:
The trophic stage of the opportunistic fungal pathogen Pneumocystis murina suppresses β-1,3-glucan-induced cytokine production, but is sufficient for generating protective CD4+ T cell and B cell responses (MPF2P.746)
Authors: Evans Heather , Garvy Beth
Published: January 2023

Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. The life cycle of Pneumocystis species consists of a trophic stage and ascus-like cysts. Our lab has published data demonstrating that cysts stimulate NF-κB translocation in alveolar macrophages, but the trophic form does not. Here, we demonstrate that bone marrow-derived dendritic cells pulsed with mixed P. murina express more IL-1β and induce greater CD4+ T cell interferon-γ production than bone marrow-derived dendritic cells pulsed with the trophic form alone. Additionally, our data indicate that the trophic form suppresses β-1,3-glucan-induced IL-1β production by bone marrow-derived dendritic cells. β-1,3-glucan is the predominant structural component in the cyst wall, but is not produced by the trophic form. Despite evidence of immunosuppression, our data demonstrate that the trophic form stimulates protective CD4+ T cell and B cell responses. CD4+ T cells transferred from BALB/c donor mice infected with the trophic form alone mediate clearance in RAG2KO host mice infected with either the trophic form or mixed P. murina. Infection with the trophic form generates protective B cell responses that mediate clearance following reinfection with either trophozoites or mixed P. murina. We propose that the trophic stage suppresses β-1,3-glucan-induced cytokine production, but is sufficient for stimulating protective responses in immunocompetent individuals.
Delayed viral clearance after respiratory tract infection results in prolonged inflammation in vitamin A deficient animals despite the presence of regulatory T cells (MUC1P.902)
Authors: Penkert Rhiannon , Surman Sherri , Sealy Robert , Jones Bart , Vogel Peter , Neale Geoff , Hurwitz Julia
Published: January 2023

Vitamin A deficiency (VAD) is a global health problem that persists in over half the countries in the world, affecting an estimated 250 million preschool children. VAD individuals suffer increased morbidity and mortality during enteric and respiratory infection, as vitamin A is an important regulator of immune system function. Previously, our lab has demonstrated that VAD mice have an impaired response to respiratory virus infection, exhibiting decreased IgA and CD8+ T cell responses to infection. Here we show that VAD mice fail to clear viral infection from the respiratory tract with the same efficiency as control vitamin A sufficient animals. We observed that prolonged viral presence can associate with an increased potential for T cell activation. As a result VAD animals experience a cytokine storm comprised of a mixed Th1/Th2 response including IFN-gamma, IL-4, IL-6 and IL-10 cytokines. Increased cytokine expression can be detected both on the transcriptional and protein level. Despite previous reports attributing VAD-related inflammation to a reduced frequency of regulatory T cells in the intestine, we observe no reduction in the number of FoxP3+ T cells in the respiratory tract of VAD animals compared to controls. Our results suggest that the increased inflammation observed during respiratory infection of VAD animals is due, at least in part, to poor IgA and CD8+ T cell responses and delayed viral clearance, and not exclusively to an absence of regulatory T cells.
Interleukin-10 is a critical regulator of white matter lesion containment following viral induced demyelination (MUC2P.940)
Authors: Puntambekar Shweta , Hinton David , Yin Xinghua , Savarin Carine , Bergmann Cornelia , Trapp Bruce , Stohlman Stephen
Published: January 2023

Formation of the astroglial scar in response to CNS injury is essential in limiting inflammation and damage to defined areas and initiating repair.The universal nature of this response suggests that defining mechanisms by which astrocytes segregate areas of tissue injury and promote repair is fundamental to confining demyelination.Neurotropic coronavirus induces an acute encephalomyelitis along with focal demyelination in which the initial lesions only increase slightly after the control of infection.Focal lesions are characterized by axonal sparing, myelin ingestion by microglia,and glial scars associated with hypertrophic astrocytes.Virus control is accelerated in mice lacking anti-inflammatory IL-10 resulting in limited initial demyelination.Despite ongoing viral control lesions fail to be confined and show sustained expansion providing a model of dysregulated white matter injury temporally remote from the acute CNS insult.Expanding IL-10-/- lesions are characterized by sustained microglial activation, partial loss of deactivated microglia and a lack of galectin-1 expressing astrocytes.Although IL-10 deficiency does not prevent astrocyte hypertrophy, astrocyte organization into mesh like structures at lesion borders is impaired. Formation of discrete foci of demyelination correlated with IL-10Rα expression on astrocytes in lesioned areas, highlighting a critical role for IL-10 signaling to astrocytes in limiting the expansion of the initial lesions into adjacent areas.
Human decidual CD8+ effector T cell responses in pregnancy (MUC2P.924)
Authors: van der Zwan Anita , Strominger Jack , Tilburgs Tamara
Published: January 2023

A unique challenge for maternal immune cells during pregnancy is to tolerate an allogeneic fetus, and yet to remain competent to respond to invading pathogens. CD8+ effector T cells play a key role in protection against virus infections and may directly recognize fetal MHC alloantigens expressed on invading fetal extravillous trophoblasts (EVT). HLA-C is the only polymorphic MHC molecule expressed by EVT and a potential ligand for the TCR of decidual CD8+ T cells (CD8+ dT). The number of differentiated, effector-memory (EM) CD8+ dT at the fetal-maternal interface was increased at least two-fold, while naïve CD8+ dT were nearly absent. These EM CD8+ dT had reduced expression of the cytotoxic molecule perforin when compared to peripheral blood EM CD8+ T cells (CD8+ pT). However, upon stimulation with anti-CD3/28, CD8+ dT increased expression of perforin, granzyme B and secreted IFN-γ and TNFα. Thus, CD8+ dT are not anergic and are able to mount a cytolytic response. Furthermore, after co-culture with EVT for 3 days the degranulation potential of CD8+ dT and their secretion of TNFα were inhibited. Interestingly, degranulation was inhibited only when CD8+ dT and EVT were obtained from the same pregnancy, not when EVT and CD8+ dT came from unmatched samples. These data may suggest that EVT inhibit CD8+ dT in an antigen-specific manner. Antigen specificity of CD8+ dT and factors that prevent a cytolytic CD8+ dT response to allogeneic fetal EVT are key topics for future research.
Memory B cells with gut-homing potential are generated by a prototype anti-enterotoxigenic E. coli vaccine given by intradermal immunization with LT(R192G) as an adjuvant (MUC3P.945)
Authors: Laird Renee , Shahabudin Sakina , Clarkson Kristen , Kaminski Robert , Savarino Stephen , Riddle Mark , Gutierrez Ramiro , Maciel Milton
Published: January 2023

Lymphocytes that express α4β7 integrin can home to the gut mucosa through interactions with MAdCAM-1 addressin on the intestinal endothelium. α4β7+ Ag-specific plasmocytes and memory B (BM) cells can be found after infection or experimental challenge with enteric pathogens as well as mucosal immunization, yet gut-homing B cells have not been reported in high frequencies in humans after parenteral immunization. Intradermal (ID) immunization is an attractive delivery route for enteric vaccines due to a potential connection between the skin and gut. Mucosal responses may be further enhanced by the addition of adjuvants such as LT(R192G), an attenuated form of the enterotoxigenic E. coli (ETEC) LT toxin. We analyzed BM cell generation in volunteers with two prototype vaccines based on the ETEC class 5a fimbriae tip adhesin CfaE with LT(R192G) by ID delivery. We observed both anti-CfaE and -LTB IgG and IgA BM cells, with a higher prevalence of Ag-specific IgG BM cells. Interestingly, anti-LTB IgA and anti-CfaE IgG BM cells were elicited at frequencies similar to those reported after ETEC infection, whereas anti-LTB IgG BM cells were found in higher frequencies after ID immunization compared to infection. The frequencies of anti-CfaE and -LTB IgG BM cells were similar in α4β7+ and α4β7- fractions, while the majority of anti-CfaE and -LTB IgA BM cells were α4β7-. These data suggest that the adjuvant LT(R192G) may be used to enhance mucosal BM responses following ID immunization.
Modeling mucosal immunity- gut microbiota interactions during Clostridium difficile infection (MUC3P.948)
Authors: Viladomiu Monica , Leber Andrew , Hontecillas Raquel , Abedi Vida , Hoops Stefan , Bassaganya-Riera Josep
Published: January 2023

Clostridium difficile is a facultative pathogen associated with antibiotic-induced infections which result in an exuberant inflammatory response, leading to nosocomial diarrhea, colitis and even death. We have previously reported novel evidence supporting a role of T cells in modulating host responses to C. difficile. Specifically, T cell alteration towards a pro-inflammatory phenotype due to lack of PPARγ resulted in increased disease symptoms and colonic pathology, which correlated with the abrogation of T cell regulatory (Treg) responses and shift towards a Th17-driven cellular response. To better understand the role of T cells during C. difficile infection from initiation through expansion to resolution, we built a computational model of the cellular host response to the bacterium. In line with our experimental findings, the model demonstrates a crucial role of Th17 effector and Treg responses, as well as an important contribution from commensal bacteria populations in the clearance of C. difficile and colonic pathology. Simulation results predict that proper clearance of infection with reduced damage to the epithelium is possible through a shift in the relative populations of Th17 and Treg cell subsets or through an accelerated regrowth of beneficial commensal strains. Our modeling provides novel insights on the mechanisms regulating C. difficile-associated immunopathology and show the path towards safer and more effective therapeutic interventions.
Targeted complement inhibition improves recovery in murine spinal cord injury model (THER2P.956)
Authors: Narang Aarti , Qiao Fei , Atkinson Carl , Kulik Liudmila , Holers V. , Yang Xiaofeng , Tomlinson Stephen
Published: January 2023

Spinal cord injury (SCI) initiates a cascade of events including ischemia, excitotoxicity and inflammation. Tissue injury exposes neoepitopes that elicit an innate immune response following their recognition by natural IgM. To investigate the role of natural IgMs in propagating SCI, we identified two IgM mAbs that bind to post-ischemic cell membranes.These mAbs B4 and C2 recognize annexin IV and a subset of phospholipids, respectively. Compared to wt mice, Rag1-/- mice recovered significantly better post SCI. However, reconstitution of Rag1-/- mice with B4 or C2 mAb restored injury to levels seen in wt animals. Further, we used peritoneal cavity hypotonic shock procedure to deplete B1a B cells (source of natural IgM), while keeping other components of the immune system intact.This resulted in reduced IgM and complement (C) deposition and protection from injury, indicating SCI is driven by the binding of specific natural IgM clones. Based on these data, we investigated a strategy to target a C inhibitor to the injured spinal cord. We constructed a single chain derived from B4IgM (B4scFv) and linked it to Crry, an inhibitor of C3 activation. This construct significantly improved locomotor recovery following SCI by both competing with pathogenic IgMs and inhibiting C. This study identifies pathophysiologically important epitopes expressed within the spinal cord after contusion injury, and describes a novel strategy for targeted C inhibition to reduce secondary injury after SCI.
JAK2 inhibition prevents NK-released IFNγ-mediated PD-L1 upregulation and enhances cetuximab mediated ADCC of HNC cells (TUM2P.1014)
Authors: Concha-Benavente Fernando , Ferris Robert
Published: January 2023

Programmed death ligand 1 (PD-L1) is an immunosuppressive molecule expressed by many cancer types, including a large proportion of head and neck cancers (HNC), and ligation of its receptor, programmed death 1 (PD-1), induces exhaustion of effector T cells, however its expression level and function on natural killer (NK) cells is not well characterized yet. It has been shown that interferon gamma (IFNγ) induces PD-L1 expression in many cancer types including glioblastoma, melanoma, lung and head and neck cancer (HNC). IFNγ signals through Janus Kinase 1/2 (JAK1/2) heterodimer complex and mediates signal transducer and activator of transcription 1 (STAT1) phosphorylation. Herein we demonstrate that IFNγ-mediated tumor cell PD-L1 expression is JAK2/STAT1 but not JAK1 dependent. Interestingly, NK cell activation through CD16 ligation of cetuximab coated tumor targets increased IFNγ release and PD-1 expression on NK cells. Therefore, blocking PD-L1/PD-1 axis is crucial in order to increase NK mediated antibody dependent cell cytotoxicity (ADCC). Herein, we demonstrate that specific JAK2 inhibition downregulated NK cell-derived IFNγ induced PD-L1 expression in tumor targets and enhanced cetuximab mediated ADCC. Our data suggest a crucial role for JAK2 in IFNγ mediated tumor cell PD-L1 upregulation. Importantly, JAK2 inhibition provides a logical strategy to increase tumor cell lysis through suppressing tumor cell PD-L1 expression in combination with anti-EGFR cetuximab therapy.
SLAMF6 signaling in murine chronic lymphocytic leukemia cells (TUM2P.1019)
Authors: Yigit Burcu , Chen Shihshih , Halibozek Peter , Arnason Jon , Chiorazzi Nicholas , Terhorst Cornelis
Published: January 2023

Chronic lymphocytic leukemia (CLL) is a circulating B cell tumor that initiates in the bone marrow and blood and spreads to the lymph nodes, liver and spleen. Disease pathogenesis is largely characterized by hyper-activation of B cell receptor (BCR) signaling and subsequent tumor accumulation in the secondary lymphoid organs. Targeting BCR downstream pathways with small inhibitor molecules, e.g. ibrutinib (an irreversible BTK inhibitor), shows great efficacy in the clinic. However, there is still a need for additional therapeutic targets for CLL, as patients eventually relapse. Because the homophilic cell surface receptor Slamf6 is highly expressed on CLL cells and plays a role in TFH - B cell interactions, the effects of anti-Slamf6 on the murine CLL cell line TCL1-192 was assessed. To this end SCID mice that had been transplanted with TCL1-192 received injections of mouse anti-Slamf6 (13G3) monoclonal antibody or isotype control. Injection of anti-Slamf6 significantly reduced tumor burden in the blood and spleen. This response is due to ADCC as well as to Slamf6 signaling as injections of F(ab’)2 fragments of anti-Slamf6 antibody results in down-regulation of proximal BCR components (pSyk, pBtk). Additionally, anti-Slamf6 resulted in a block in TCL1-192 tumor migration from the peritoneum to the blood and spleen. Overall, this study suggests that Slamf6 could be a potential new target for CLL therapy.
Identifying mechanisms of acquired resistance to immunotherapy (TUM2P.1025)
Authors: Jaiswal Ashvin , Ager Casey , Ai Midan , Bartkowiak Todd , Budhani Pratha , Curran Michael
Published: January 2023

Immunotherapy has demonstrated a remarkable potential to cure established cancer, but many tumors either relapse or fail to respond. The molecular mechanisms which drive these tumor relapses remain unknown. We hypothesize that tumors evolve resistance under immunotherapeutic pressure mediated by changes in gene expression which can be selected and propagated through serial passage and then identified and targeted. We are comparing untreated B16 melanoma tumors to those expanding despite treating with combinations checkpoint blockade (CTLA-4, PD-1, and PD-L1), 4-1BB-agonist antibody, adoptive transfer of TRP-1 specific CD4 T-cells, or growth in MHC-mismatched hosts. Through multiple in vivo passages, we have already selected a B16 melanoma tumor line that has evolved almost 100% resistance to combination blockade of CTLA-4, PD-1, and PD-L1, which could cure nearly 90% of tumor-bearing mice in the first cycle. After each passage under selective pressure of combination blockade, we have observed visible signs of increased angiogenesis correlating with enhanced therapy resistance. By determining the pathways engaged by these tumors to develop resistance to tumor-specific immunity, we hope to discover novel characteristics of the mammalian immune system as well as reveal new targets for immunotherapeutic intervention in melanoma patients.
Combination of anti-CD73 and agonistic anti-CD137 antibody therapy leads to tumor regression (TUM2P.1030)
Authors: chen siqi , Zhang Bin
Published: January 2023

Agonist antibodies (Ab) directed against costimulatory molecule CD137 (4-1BB) on the surface of antigen-primed T-lymphocytes are currently in various stages of pre-clinical and clinical development, though therapeutic benefit has been limited as single agents. Here, we examined the inhibitory role of CD73 on the antitumor efficacy of agonistic anti-CD137 Ab therapy. CD73-deificient mice treated with anti-CD137 Ab completely rejected CD73-nonexpressing B16-SIY melanoma growth when compared with wild-type mice, highlighting the importance of an inhibitory role for host-derived CD73. To explore the therapeutic relevance of these observations, we combined anti-CD137 and anti-CD73 Ab to treat mice with established B16-SIY melanoma. This combination therapy induced tumor regression associated with enhanced antitumor CD8+ T cell responses and reduced CD4+Foxp3+ regulatory T cells number and function. In contrast, neither anti-41BB nor anti-CD73 monotherapy is effective to control the growth of established melanoma. Our study demonstrates that CD73 blockade has critical implications for effective clinical targeting of CD137 and possibly other costimulatory molecules.
Ipilimumab reshapes T cell memory subsets in melanoma patients with clinical response (TUM2P.1006)
Authors: Moins-Teisserenc Helene , Felix Joana , Porcher Raphael , Pages Cecile , Sidina Irina , Cordeiro Debora , Maki Guitta , Viguier Manuelle , Maubec Eve , Toubert Antoine , Lebbe Celeste
Published: January 2023

CTLA-4 is the main negative regulator of T cell mediated antitumor immune responses. Treatment with Ipilimumab, a humanized anti-CTLA-4 mAb, was shown to provide increased survival in patients with melanoma. However, only a subset of patients benefit and it is therefore important to identify prognostic immunological correlates of clinical endpoints. 77 patients with stage III or IV melanoma were treated with ipilimumab every 3 weeks, during 9 weeks. Blood samples were collected at week 0, 3, 6, 9, 12. The clinical response was assessed at week 16. Lymphocyte phenotyping was performed on fresh blood samples. The median follow-up was 28 months with a median survival of 7 months, which was significantly improved when the absolute lymphocyte count (ALC) at the baseline was above 1000/uL. In addition ALC was predictive of disease control (DC) at week 16. Ipilimumab had a global effect on central, transitional and effector memory T cell subsets, with a marked increase of these populations between week 0 and 3 in patients with DC. In addition, higher proportions of Eomes+, Ki67+ and Granzyme B+ T cells were observed. These changes were associated with a significant decrease in the proportion of stem cell memory T cells (TSCM). These results suggest that Ipilimumab is able to reshape the memory subset at early time points and provide new insights into the mechanisms of memory T cell subset expansion with the potential involvement of TSCM.
Expression of IFNgR on tumor cells impact response to anti-CTLA-4 (TUM2P.1034)
Authors: Gao Jianjun , Shi Lewis , Xiong Liangwen , He Qiuming , Chen Jianfeng , Shen De-Yu , Sharma Padmanee
Published: January 2023

Blockade of T cell co-inhibitory receptor CTLA-4 with a monoclonal antibody, Ipilimumab (BMS), has led to augmented anti-tumor immune responses, clinical benefit, and FDA approval of Ipilimumab for the treatment of metastatic melanoma. Our published data indicate that anti-CTLA-4 treatment resulted in marked increase of IFN-gamma (IFNg) production by CD4 T cells expressing inducible costimulator (ICOS). Studies in mice have demonstrated the importance of IFNg for effective anti-tumor responses. We hypothesized that tumor cell expression of IFNg-receptor (IFNg-R) would be necessary for tumor regression mediated by anti-CTLA-4. To test our hypothesis, we conducted both in vitro and in vivo experiments with tumor cell lines containing knockdown of IFNg-R. In the presence of IFNg, proliferation of tumor cells was inhibited; however, tumor cells lacking IFNgR were resistant to IFNg and were able to proliferate. In addition, mice inoculated with tumor cells lacking IFNgR were treated with anti-CTLA-4 and found to have impaired tumor rejection and higher mortality as compared to mice bearing tumor cells with intact IFNgR. Our data strongly suggest that intact IFNg-R expression and signaling on tumor cells is important for anti-CTLA-4-mediated anti-tumor responses. Our data point to a potential mechanistic pathway to explain why some patients may respond to therapy while others do not and we’re evaluating tumor samples from patients treated with anti-CTLA-4 to test this concept.
Identification of miRNA-338-5P as an effector of the tumor suppressor activity of immunized anticancer bioactive peptide-3 (TUM2P.1036)
Authors: Su Xiulan , Zhang Jialing , Yu Lan , Xing Zhiwei , Liang Junqing , A Nanda
Published: January 2023

Gastric cancer remains one of the leading causes of global cancer mortality. Cancer stem cells (CSC) have been studied in many solid tumors, including gastric cancer and they are promising for the development of anticancer drugs that can target all CSC subsets within a tumor to prevent recurrence. Previously, we identified a novel anti-cancer bioactive peptide (ACBP), which isolated and purified from goat liver following immunization with human gastric cancer protein extract, and designated as ACBP-3. ACBP-3 inhibited cell proliferation and clone formation of gastric cancer stem cells (CSCs). In present study, we investigated the mechanism of ACBP-3 inhibition of gastric CSCs in vitro and in vivo. We employed a microRNA array and screened for the genes of which had increased expression by ACBP-3 in CD44 positive gastric carcinoma cell lines MKN45 and MKN74. MiRNA-338-5p showed the greatest difference in the expression level. In vivo, ACBP-3 alone significantly inhibited MKN45 tumor growth in a xenograft nude mouse model. The tumor tissues were harvested after treatment, and ACBP-3 treatment induced expression of miRNA-338-5p, Bac, and Bim as quantified by RT-PCR and western blot. Overexpression of miRNA-338-5p reduced cell proliferation and induced apoptosis in both MKN45 and MKN74 cell lines in vitro. We concluded that miRNA-338-5p is the effector of tumor suppressor activity of ACBP-3 in gastric cancer stem cells. ACBP-3 could be a potential anti-cancer agent.
Mantle cell lymphoma associated sphingosine-1 phosphate inhibits natural killer T cell mediated antitumor responses (TUM2P.1042)
Authors: Webb Tonya , Kimball Amy , Sun Wenji
Published: January 2023

The incidence of lymphoma in the US is rising. While chemoimmunotherapy causes remissions in mantle cell lymphoma (MCL), responses are not durable and most patients relapse. New strategies that focus on eliminating tumor-associated immunosuppressive factors are needed to improve patient outcomes. One of the earliest pathways to be activated during an immune response is CD1d-mediated presentation of lipid antigens to natural killer T (NKT) cells. We have found that MCL patients have a significant reduction in circulating NKT cell number and function. We hypothesized that MCL-associated sphingosine-1 phosphate (S1P) can down regulate CD1d-dependent NKT cell responses to MCL. We found that inhibition of S1P signaling in MCL cell lines led to increased CD1d-mediated NKT cell activation, as assessed by cytokine production and cytotoxicity. Furthermore, knockdown of sphingosine kinase 1 in MCL cell lines resulted in the presentation of an activating endogenous antigen. These results demonstrate that S1P regulates CD1d-mediated antigen processing and presentation to NKT cells by altering the endogenous repertoire of lipid antigens and suggest that targeting this pathway could be an important immunotherapeutic strategy.
Novel RORgamma agonists enhance anti-tumor activity of adoptive T cell therapy (TUM2P.1010)
Authors: Carter Laura , Moisan Jacques , Majchrzak Kinga , Hu Xia , Morgan Rodney , Liu Xikui , Demock Kellie , Wang Yahong , Lesch Chuck , Sanchez Brian , Bousley Dick , Taylor Clarke , Van Huis Chad , Skalitzky Don , Aicher Tom , Toogood Peter , Zou Weiping , Paulos Chrystal , Glick Gary
Published: January 2023

Adoptive cell transfer (ACT) therapy using tumor infiltrating lymphocytes (TILs) or T cells modified to express chimeric antigen receptors is a promising anti-cancer approach. Evidence suggests that Th17 or Tc17 cells display superior anti-tumor activity over unpolarized T cells commonly used in ACT clinical trials. As an approach to improve the potency and persistence of ACT, synthetic RORɣ agonists were designed to bolster the generation of type 17 cells. In vitro stimulation in the presence of RORɣ agonists modulates expression of costimulatory and coinhibitory molecules and increases IL-17A, IL-22 and GM-CSF production while maintaining IFNɣ production. Additional investigation revealed that treating tumor-specific Th0, Th17, Tc0 or Tc17 T cells with a RORɣt agonist in vitro significantly enhanced their ability to regress large and established EG.7 lymphoma and B16F10 melanoma tumors in vivo. Animals receiving RORɣ agonist-treated T cells had increased frequency of transferred cells, elevated IL-17A levels and decreased PD-1 expression in tumors and spleen compared to control animals. Previous studies have also shown that systemic delivery of an RORɣ agonist can also enhance anti-tumor immune response in syngeneic pre-clinical tumor models. By enhancing cytokine/chemokine production, promoting survival as well as decreasing PD-1 expression, RORɣ agonist molecules provide an effective means to enhance the potency of adoptively transferred T cell products.
Combination immunotherapy utilizing influenza infection and PD-1 blockade reduces lung melanoma tumor growth (TUM2P.1011)
Authors: Zloza Andrew , Broucek Joseph , Redondo Raquel , Zayas Janet , Lusciks Jevgenijs , Dolubizno Hubert , Grin Alisa , Kohlhapp Frederick , Kaufman Howard
Published: January 2023

Emerging epidemiological studies report increased cancer occurrence in patients with chronic viral infections, even when such infections are non-oncogenic and not in the same tissue as the cancer. In our recent studies, we observed decreased anti-tumor immune responses in the context of concomitant infection. Specifically, we discovered that anti-melanoma (gp100) CD8+ T cell responses in mice challenged with melanoma are shunted from the tumor site (skin) to the site of influenza infection (lung), leading to faster tumor growth and hastened host death. We observed similar findings with multiple tumor and virus combinations, including patient-derived melanomas in humanized mice infected with HIV. Based on this infection-mediated shunting of anti-tumor CD8+ T cells to the infection site, and towards developing related therapeutic interventions, we sought to determine whether an infection in a particular tissue would improve anti-tumor responses against a tumor also in that same tissue. We discovered that influenza infection within the lungs resulted in 51% fewer tumor foci within the lungs compared to no infection. Tumor foci were further reduced with systemic PD-1 blockade (more than 70% fewer tumor foci compared to no infection or PD-1 blockade alone). Future studies will further dissect the mechanisms underlying these findings in the setting of viral infection. We ultimately aim to translate these findings to combination cancer immunotherapies utilizing viral infections.
Interleukin-7 receptor contributes to increase of wound-healing migration as well as Invasion in the prostate cancer (TUM3P.1049)
Authors: Seol Min , Kim Jin-Hee , Oh Keunhee , Sim Ji Hyun , Lee Sun-Kyung , Shin Young-Kyoung , Kim Woo-jong , Lee Dong-Sup , Han Ilkyu , Ku Ja-Lok , Kim Hang-Rae
Published: January 2023

IL-7Rα plays a critical role in the development and maintenance of immune cells but no studies suggest a verification of a key factor of bone metastasis. To investigate the role of IL-7Rα on bone metastases from prostate cancer, we evaluate the expression of IL-7Rα, wound healing migration, invasion, and zymography on the prostate cancer cells after IL-7 treatment. As results, the expression of IL-7Rα in PC-3 cells was higher than in other metastatic cell lines. Not only wound-healing but also invasion was increased after IL-7 treatment at the PC-3 cells. And, MMP2, MMP9 was also increased in PC-3 cells upon IL-7 stimulation. Moreover, the invasiveness of cells overexpressing IL-7Rα (PC-3IL-7Rα OE) was significantly increased compared to control cells. Furthermore, in order to elucidate the role of IL-7Rα in bone metastasis, PC-3IL-7Rα OE cells were administered into NSG mouse by intra-cardiac injection. Metastasis into the bone was markedly increased when PC-3IL-7Rα OE were injected compared with control vector expressed PC-3 cells. In conclusion, migration and invasion were increase in PC-3 cells after IL-7 treatment. Metastasis into bone was enhanced by PC-3IL-7Rα OE, suggesting IL-7Rα might be involved in migration as well as invasion in the bone metastatic prostate cancer. Acknowledgement: This research was supported by National Research Foundation of Korea (2014M3A7B4052194 for H Kim and 2014R1A6A3A01058743 for J Kim)
Potential signaling molecules involved in the progression of metastatic prostate, breast and colorectal cancer (TUM3P.1051)
Authors: Ejaeidi Ahmed , Cruse Julius , Lewis Robert , Craft Barbara , Puniky Louis
Published: January 2023

Metastases and their complications lead to death in up to 90% of cancer patients. Hematogenous metastasis is associated with loss of cell adhesion, migration in the blood (as individual circulating tumor cells, (CTC) or clusters), adhesion to the target organ and proliferation to form metastases. The present investigation was designed to demonstrate key molecules in the sera of metastatic cancer patients that induce ERK 1/2 and Survivin protein expression. We showed that CTCs grown in media supplemented with patient sera express the potential to form colonies and spheres followed by senescence. The cultured CTCs overexpressed ERK and Survivin, which reflects their aggressiveness. We demonstrated by immunofluorescence and western blot, with confirmation by qRT-PCR (in progress), that prostate, breast and colorectal cancer-derived cell lines cultured in media supplemented with sera from patients with CTCs, induced expression of ERK 1/2 and Survivin proteins. Using the MTT assay, we demonstrated that cancer-derived cell lines treated with sera from CTC positive patients showed accelerated proliferation compared to controls. It is anticipated that correlation with clinical data might reveal future potential targets for adjuvant therapy to minimize the risk of metastasis.
Identification and characterization of novel immunosensitizing therapeutics for melanoma (VAC3P.1060)
Authors: Tsai Alexander , Davila Eduardo
Published: January 2023

Recent breakthroughs in the field of immunotherapy have revolutionized the treatment of melanoma patients. Specifically, immune checkpoint blockade of CTLA-4 and PD-1 have prolonged overall survival in a subset of melanoma patients. Despite dramatic and durable results in some cases, low overall response rates to these therapies leave the majority of melanoma patients without effective immunotherapeutic treatment options. In order to identify immunotherapies which might offer improved response rates with the potential to be rapidly translated to the clinic, we developed a high-throughput screen and tested over 2000 compounds, many of which are FDA-approved for various malignancies or other diseases. The screen revealed distinct families of multikinase inhibitors and DNA-damage repair inhibitors as novel melanoma immunosensitizing agents. We show that these two classes of drugs can reduce the expression of various immunosuppressive molecules (e.g. PD-L1, CD155) on the cell surface of a wide variety of genetically distinct human melanoma cell lines. Notably, the drugs also increased the expression of HLA and melanoma antigens in most cases. Importantly, preliminary data suggests that drug treatment sensitizes melanoma cells to T cell mediated cytotoxicity. Further preclinical studies are underway to verify drug targets, identify mechanisms of action, and establish in vivo anti-tumor activity.
Immune mechanisms associated with enhanced influenza A virus disease versus cross-protection in vaccinated pigs. (VAC4P.1064)
Authors: Hughes Holly , Olson Zahra , Gauger Phillip , Vincent Amy , Brockmeier Susan , Loving Crystal
Published: January 2023

Vaccine associated enhanced respiratory disease (VAERD) has been described in pigs vaccinated with whole-inactivated influenza virus (WIV) following infection with heterologous influenza A virus (IAV). WIV vaccination elicits production of cross-reactive, non-neutralizing antibody to the challenge IAV strain suggesting circulating immune complexes and/or antibody dependent cell-mediated cytotoxicity could contribute to pathology. Moreover, pathological findings in VAERD lungs include significant lymphocytic infiltration and increased levels of proinflammatory cytokines. In order to further delineate the immune mechanism associated with VAERD versus partial-protection we defined the lymphocyte populations infiltrating the lungs following challenge, as well as level of complement activation. Post-challenge, IAV-specific IFNγ secreting cells in the lungs were increased in pigs with VAERD compared to non-vaccinated, challenged controls. CD8 T cells and NK cells were significantly higher in the lungs of pigs protected from homologous challenge, suggesting these cells may play a role in protection. Interestingly, pigs with VAERD had a significant increase in memory T cells that was not observed in pigs without VAERD. Additionally, complement activation was significantly higher in the lungs of pigs experiencing VAERD. These data further describe the lymphocytic pathology associated with VAERD and suggest a role for complement activation in the development of VAERD disease pathology.
Autoimmunity-predisposing PTPN22 C1858T variant associates with diminished human CD4 T cell responses to seasonal influenza vaccination (VAC4P.1112)
Authors: Crabtree Juliet , Tien Shelly , Ferment Valerie , Guan Weihua , Peterson Erik
Published: January 2023

PRR-induced Type I IFN is necessary for optimal host responses to antiviral immunization, including CD4 T cell expansion and neutralizing antibody production. The magnitude of T and B cell vaccine responses correlates with protection against influenza virus infection. PTPN22 promotes TI IFN production after PRR engagement on myeloid cells. The autoimmune disease-associated C1858T variant of PTPN22 encodes an R620W substitution (PTPN22-W), which is a reduced function variant for TI IFN production. Since PTPN22-W carriers show reduced capacity for PRR-driven TI IFN production, we tested the hypothesis that PTPN22-W carriers will have diminished immune response to the influenza vaccine. Healthy PTPN22-W carriers (n=18) and age-matched controls were immunized with the 2013-2014 trivalent inactivated influenza vaccine, and immune responses were assessed. Carriers exhibited significantly decreased induction of influenza-specific CD4 T cells expressing TNFα, IL-2, and IFNγ at 2 weeks after immunization. Moreover, carriers show a trend toward impaired induction of neut. antibody titers post vaccination. We did not observe differences between carriers and controls between selected markers of the early interferon transcriptional response, possibly because of inadequate power to detect a partial phenotype. Nevertheless, defects in T cell immunity strongly suggest that PTPN22-W carriage predisposes to defective capacity for mounting protective immune responses to influenza vaccination.
Recombinant outer membrane protein: a potential candidate for Th17 based vaccine against Klebsiella pneumonia (VAC4P.1066)
Authors: Chen Kong , Kolls Jay
Published: January 2023

Bacterial pneumonia is a leading cause of mortality and one major pathogen associated with this disease is Klebsiella pneumoniae (KP). Recently, the emergence of antibiotic resistant strains demands an effective vaccine against these bacteria. We have previously shown that intranasal immunization with heat-killed KP or crude outer membrane proteins (OMPs) isolated from KP induced antigen specific Th17 responses and these Th17 cells conferred serotype independent protection against various clinical isolates of K. pneumoniae including the recently described multidrug resistant New Delhi Metallo-beta-lactamase-1 strain. To develop a clinically relevant vaccine, we cloned individual OMPs from K. pneumoniae and successfully expressed OmpX in the BL21 E. coli. The purified recombinant OmpX was recognized by KP immune serum by direct ELISA and also recognized by Th17 cells from KP immunized mice. In vivo, intranasal immunization of the purified OmpX induced robust mucosal Th17 responses and left IL-17 producing gamma-delta T cells unaffected. In combination with a mucosal adjuvant, both intranasal and intramuscular immunization provided serotype independent protection against live bacterial challenge. Taken together, these data demonstrated that OMPs serves as an excellent candidate for the development of clinical relevant vaccines against K. penumoniae especially the newly emerged antibiotic-resistant strains that impose great threat to the public health.
Improving immunity against mycobacteria by targeting iNKT cells in a partially humanized mouse model (VAC4P.1103)
Authors: Carreno Leandro , Batista-Gonzalez Ana , Prados-Rosales Rafael , Sharma Kharkwal Shalu , Yuan Weiming , Porcelli Steven
Published: January 2023

Invariant Natural Killer T cells (iNKT cells) are a specialized group of T lymphocytes which modulate many aspects of immune responses. They co-express an invariant T cell receptor (TCR) which recognizes glycolipid antigens bound to the CD1d molecule. Although mouse and human CD1d are relatively conserved, there are differences in the abundance and responses of human and mouse iNKT cells, which may correlate with differences in CD1d function. Thus, the study of iNKT cell responses and their role in disease models requires a more “humanized” model to enable more accurate translation of results to human therapy. Here, by using a human CD1d knock-in mouse, which displays similar frequency and phenotype in their iNKT cells to humans, we have vaccinated with different strains of potential tuberculos (TB) vaccine candidates carrying different glycolipid iNKT cell ligands. Our results shown that hCD1d-KI iNKT cells are able to enhance TB-specific T cell responses. We have also identified novel glycolipds with an enhanced ability to induce multifunctional T cells. Our results suggest that human CD1d knock-in mice are a valuable tool to study iNKT cell-based therapies.
CD20 peptides elicit a protective anti-tumor immune response in vivo (VAC5P.1126)
Authors: TEILLAUD JEAN-LUC , Riller Quentin , Siberil Sophie
Published: January 2023

We have investigated whether human CD20 (huCD20)-derived peptides are able to elicit a protective anti-tumor immune response in vivo. Based on in silico prediction and in vitro binding to recombinant MHC molecules (Kb, Db, and I-Ab), we first defined candidate peptides able to bind to various MHC molecules in a restricted manner (8-mers restricted to Kb, 9-mers restricted to Db, 15-mers and 20-mers restricted to I-Ab). Naive C57Bl/6 mice were then immunized s.c. with 7 different mixtures of these peptides in presence of adjuvants (CFA/IFA, Montanide ISA 51VG, CpG) and subsequently challenged with EL4-huCD20 tumor cells. Interestingly, a longer survival could be achieved in mice immunized with 5 of the 7 huCD20-derived peptide mixtures. In addition, MHC-peptide fluorescent pentamers (generated using peptides with the highest capacity of binding to MHC molecules) allowed the detection of anti-CD20 specific CD8+ T cells in one of the groups of immunized mice. Overall, we conclude that it is possible to define huCD20-derived peptides that elicit a T-cell response against EL4-huCD20 tumor cells. We suggest that these peptides could be the targets of the adaptive anti-tumor T cell response triggered by anti-CD20 treatment that we previously reported using a mouse model in which immune competent C57Bl/6 mice were i.v. injected with human CD20+ tumor cells and then treated with an anti-huCD20 mAb (1,2). 1. Abès et al., Blood, 2010 2. Deligne et al., Leukemia, 2014
Safety and immunogenicity of the Ad35/AERAS-402 tuberculosis vaccine in a phase 2 dose-finding clinical trial in BCG-vaccinated healthy infants (VAC5P.1119)
Authors: Hokey David , Tameris Michele , Henson Dawn , Dheenadhayalan Veerabadran , Blatner Gretta , McClain Bruce , Walker Robert , Hatherill Mark , Nduba Videlis , Sacarlal Jahit , Gray Glenda , Hendriks Jenny , Pau Maria , Douoguih Macaya , Sadoff Jerald , Barker Lewellys
Published: January 2023

Despite widespread use of BCG, tuberculosis (TB) remains a global threat, with 9 million new cases and 1.5 million deaths reported in 2013. Current vaccine candidates in clinical trials are designed to replace BCG or boost the variable efficacy observed with BCG. One of these is AERAS-402, a replication-deficient Ad35 vaccine encoding a fusion protein of the M. tuberculosis antigens 85A, 85B, and TB10.4. This vaccine has been tested in adults, infants, HIV+ adults and patients on or following treatment for TB and shown to be safe and immunogenic. A phase 2 clinical trial of two doses of AERAS-402 was conducted in healthy infants in South Africa, Kenya, and Mozambique. The original study design consisted of a dose-finding phase followed by an efficacy phase at the selected dose. The trial design was modified to include a third dose of AERAS-402. AERAS-402 was well-tolerated at all dose levels and up to three doses. Despite the addition of a third dose, the predefined immunogenicity target was not met and the efficacy portion of the trial was removed. The third dose failed to increase immune responses, which consisted of a dominant polyfunctional (IFN-γ, IL-2, TNF) and bifunctional (IFN-γ, TNF) CD8 response and a lower magnitude polyfunctional and bifunctional (IL-2, TNF) CD4 responses. These responses were considerably lower than those observed in adults. Alternative methods to enhance responses to AERAS-402, such as heterologous prime-boost approaches, are warranted.
CTLA4-CD28 chimeric gene modification of T cells enhances therapeutic efficacy of donor lymphocyte infusion for hematological malignancy (VAC5P.1122)
Authors: Park Hyung-Bae , Lee Ji Eun , Lee Sang , Eom Hyeon , Choi Kyungho
Published: January 2023

Donor lymphocyte infusion (DLI) has been used as an effective therapeutic regimen for the relapsed hematologic malignancies, such as chronic myelocytic leukemia, after allogenic bone marrow transplantation. However, therapeutic efficacy of DLI for other types of leukemia including acute lymphocytic leukemia (ALL) has been limited so far. Thus, we tested if increasing the reactivity of donor T cells by gene modification could enhance therapeutic effect of DLI in a murine model of ALL. For this purpose, we introduced CTLA4-CD28 chimeric gene (CTC28), in which the intracellular signaling domain of CTLA4 was replaced with the CD28 signaling domain, into donor T cells and evaluated the graft-versus-tumor (GVT) effect in an EL4 tumor-bearing mixed bone marrow chimera model. The modification significantly enhanced GVT effect of donor T cells. This effect was correlated with increased expansion of CD8 T cells and elimination of CD8 T cells abolished this effect. GVT effect and CD8 T cell expansion were dependent on the CTC28-modification of CD4 T cells, emphasizing the role of CD4 help in this therapeutic effect. Modified CD4 T cells not only expanded more, but also showed enhanced functional responsiveness. Modified T cell-derived IL-2 was indispensable for the GVT effect, although not required for expansion of the T cells. Thus, combined genetic modification of CD4 and CD8 donor T cells with CTC28 can be a promising strategy for enhancing therapeutic efficacy of DLI for ALL.
A novel pro-apoptotic function for Epstein-Barr virus LMP2A: increasing B cell sensitivity to Fas-mediated apoptosis via Lyn and Syk kinases (VIR1P.1136)
Authors: Swanson-Mungerson Michelle , Incrocci Ryan , Hussain Samira
Published: January 2023

Latent Membrane Protein 2A (LMP2A) of Epstein-Barr virus (EBV) protects B cells from many pro-apoptotic signals. LMP2A is expressed in EBV-infected B cells in the germinal center, which is a site of significant Fas-mediated apoptosis. Given that B cell receptor (BCR) stimulation protects B cells from Fas-mediated apoptosis and since LMP2A acts as a BCR mimic, we hypothesized that LMP2A would protect B cells from Fas-induced apoptosis. To test this hypothesis, LMP2A-negative and -positive B cell lines were exposed to a Fas-specific monoclonal antibody and apoptosis was assessed by Annexin V staining and PARP cleavage. Surprisingly, LMP2A-expressing B cell lines demonstrate an increased sensitivity to Fas-mediated apoptosis, due to a LMP2A-dependent enhancement in Fas expression. B cell lines harboring LMP2A mutations in either the Lyn or Syk binding site do not show an LMP2A-dependent increase in Fas levels and/or Fas-mediated apoptosis, indicating that LMP2A requires these two kinases to mediate these pro-apoptotic effects. Furthermore, experiments demonstrate that LMP2A enhances Fas-mediated apoptosis in human B cell lines in the presence of additional EBV latency proteins, indicating that LMP2A promotes Fas-dependent apoptosis in the natural context of the virus. These findings are the first to demonstrate a pro-apoptotic function of LMP2A and have implications for both EBV latency and EBV-associated malignancies.
Defining the role of CD4 T cells during acute Theiler's murine encephalomyelitis virus infection (VIR1P.1141)
Authors: Huseby April , Jin Fang , Johnson Aaron
Published: January 2023

Theiler’s murine encephalomyelitis virus (TMEV) infection of C57BL/6 mice has served as a model of acute neuroinflammation. Immune cells have been shown to be both pathogenic and protective during the acute stage of TMEV infection. Our lab has previously investigated the role for CD8 T cells directly interacting with virally infected neurons using confocal microscopy. Through this work, we demonstrated polarized immunological synapse formation in the CD8 T cell toward the virally infected neuron. In a subsequent study using confocal microscopy, we observed that CD4 T cells are also in tight apposition to virally infected neurons. This observation prompts an analysis to define the interaction between the CD4 T cell and the virally infected neuron. Defining the CD4 subset is a crucial first step to assessing their potential protective effector mechanisms. Future studies will investigate the role for Kupfer synapses or non-Kupfer type synapses statically and subsequent studies conducted with two-photon laser scanning microscopy (2PLSM) will allow us to measure the cell dynamics and motility, in vivo, of the immune components that contribute to the protection or pathogenesis during neuroinflammation.