CD4 Helper Research Articles

Assessment of HIV-1 integration in tissues and subsets across infection stages.

The integration of HIV DNA into the host genome contributes to lifelong infection in most individuals. Few studies have examined integration in lymphoid tissue, where HIV predominantly persists before and after antiretroviral treatment (ART). Of particular interest is whether integration site distributions differ between infection stages with paired blood and tissue comparisons. Here, we profiled HIV integration site distributions in sorted memory, tissue-resident, and/or follicular helper CD4+ T cell subsets from paired blood and lymphoid tissue samples from acute, chronic, and ART-treated individuals. We observed minor differences in the frequency of nonintronic and nondistal intergenic sites, varying with tissue and residency phenotypes during ART. Genomic and epigenetic annotations were generally similar. Clonal expansion of cells marked by identical integration sites was detected, with increased detection in chronic and ART-treated individuals. However, overlap between or within CD4+ T cell subsets or tissue compartments was only observed in 8 unique sites of the 3540 sites studied. Together, these findings suggest that shared integration sites between blood and tissue may, depending on the tissue site, be the exception rather than the rule and indicate that additional studies are necessary to fully understand the heterogeneity of tissue-sequestered HIV reservoirs.

Persistent STAT5 activation reprograms the epigenetic landscape in CD4+ T cells to drive polyfunctionality and antitumor immunity.

The presence of polyfunctional CD4+ T cells is often associated with favorable antitumor immunity. We report here that persistent activation of signal transducer and activator of transcription 5 (STAT5) in tumor-specific CD4+ T cells drives the development of polyfunctional T cells. We showed that ectopic expression of a constitutively active form of murine STAT5A (CASTAT5) enabled tumor-specific CD4+ T cells to undergo robust expansion, infiltrate tumors vigorously, and elicit antitumor CD8+ T cell responses in a CD4+ T cell adoptive transfer model system. Integrated epigenomic and transcriptomic analysis revealed that CASTAT5 induced genome-wide chromatin remodeling in CD4+ T cells and established a distinct epigenetic and transcriptional landscape. Single-cell RNA sequencing analysis further identified a subset of CASTAT5-transduced CD4+ T cells with a molecular signature indicative of progenitor polyfunctional T cells. The therapeutic significance of CASTAT5 came from our finding that adoptive transfer of T cells engineered to coexpress CD19-targeting chimeric antigen receptor (CAR) and CASTAT5 gave rise to polyfunctional CD4+ CAR T cells in a mouse B cell lymphoma model. The optimal therapeutic outcome was obtained when both CD4+ and CD8+ CAR T cells were transduced with CASTAT5, indicating that CASTAT5 facilitates productive CD4 help to CD8+ T cells. Furthermore, we provide evidence that CASTAT5 is functional in primary human CD4+ T cells, underscoring its potential clinical relevance. Our results implicate STAT5 as a valid candidate for T cell engineering to generate polyfunctional, exhaustion-resistant, and tumor-tropic antitumor CD4+ T cells to potentiate adoptive T cell therapy for cancer.

Evidence of potent humoral immune activity in COVID-19-infected kidney transplant recipients.

Whether kidney transplant recipients are capable of mounting an effective anti‐severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) adaptive immune response despite chronic immunosuppression is unknown and has important implications for therapy. Herein, we analyzed peripheral blood cell surface and intracellular cytokine phenotyping by flow cytometry along with serum antibody testing in 18 kidney transplant recipients with active coronavirus disease 2019 (COVID‐19) infection and 36 matched, transplanted controls without COVID‐19. We observed significantly fewer total lymphocytes and fewer circulating memory CD4+ and CD8+ T cells in the COVID‐19 subjects. We also showed fewer anergic and senescent CD8+ T cells in COVID‐19 individuals, but no differences in exhausted CD8+ T cells, nor in any of these CD4+ T cell subsets between groups. We also observed greater frequencies of activated B cells in the COVID‐19 patients. Sixteen of 18 COVID‐19 subjects tested for anti‐SARS‐CoV‐2 serum antibodies showed positive immunoglobulin M or immunoglobulin G titers. Additional analyses showed no significant correlation among immune phenotypes and degrees of COVID‐19 disease severity. Our findings indicate that immunosuppressed kidney transplant recipients admitted to the hospital with acute COVID‐19 infection can mount SARS‐CoV‐2‐reactive adaptive immune responses. The findings raise the possibility that empiric reductions in immunosuppressive therapy for all kidney transplant recipients with active COVID‐19 may not be required.

IL-21 in Homeostasis of Resident Memory and Exhausted CD8 T Cells during Persistent Infection.

CD4 T cells guide the development of CD8 T cells into memory by elaborating mitogenic and differentiation factors and by licensing professional antigen-presenting cells. CD4 T cells also act to stave off CD8 T cell dysfunction during repetitive antigen stimulation in persistent infection and cancer by mitigating generation of exhausted T cells (TEX). CD4 T cell help is also required for establishing and maintaining tissue-resident memory T cells (TRM), the nonrecirculating memory T cell subset parked in nonlymphoid tissues to provide frontline defense against reinvading pathogens. Interleukin (IL)-21 is the signature cytokine secreted by follicular helper CD4 T cells (TFH) to drive B cell expansion and differentiation in germinal centers to mount high-affinity, isotype class-switched antibodies. In several infection models, IL-21 has been identified as the CD4 T help needed for formation and survival of TRM and TEX. In this review, we will explore the different memory subsets of CD8 T cells in persistent infections, the metabolic profiles associated with each, and evidence documenting the importance of CD4 T cell-derived IL-21 in regulating CD8 TRM and TEX development, homeostasis, and function.

Clinical significance of assessing the subpopulation composition of blood lymphocytes in patients with infectious corneal lesions in high-risk keratoplasty

The purpose is to study the shifts in the subpopulation composition of blood lymphocytes in patients with infectious corneal lesions, depending on the number of keratoplasties (KP) undergone by the patient. Material and methods . We examined 53 patients (aged 18 to 79) with infectious keratitis, divided into three groups, depending on the number of KP: group I (n = 27) with no KP; groups II (n = 12) who underwent one KP; group III (n = 14) who has two or more KPs. The control group consisted of 33 apparently healthy donors. The relative and absolute content of subpopulations of T lymphocytes (CD3+), T helper cells (CD3+CD4+CD8-), T cytotoxic cells (CD3+CD4-CD8+), double positive T lymphocytes (CD3+CD4+CD8+), NK cells (CD16+CD56+), and B lymphocytes (CD19+) was determined using flow cytometry, the CD4+/CD8+ index was calculated. Results. The analysis of the total population of T lymphocytes and the CD3+CD4+ subpopulation showed a significant increase in the absolute number of T lymphocytes in group II (CD3+: 1.77 ± 0.10 × 10 9 /l; T helpers: 1.02 ± 0.09 × 10 9 /l) as compared to group I (1.44 ± 0.06 × 10 9 /l and 0.85 ± 0.03 × 10 9 /l, respectively). In group III, there was a substantial significant increase in the relative amount of CD3+CD4+(50.1 ± 1.9 %) as compared to groups without KP (I: 43.2 ± 1.5 %) and one surgery group (II: 43.3 ± 1.8 %), which led to a significant increase in the ratio of CD4+/CD8+ in group III (2.16 ± 0.19). In the blood of all patients (group I: 0.020 ± 0.003 × 10 9 /l; II: 0.030 ± 0.002 10 9 /l; III: 0.030 ± 0.001 10 9 /l) a significant increase in the absolute number of double positive T lymphocytes (CD3+CD4+CD8+) as compared to the control (0.009 ± 0.007 10 9 /l) was revealed. Conclusion. The changes revealed in the cellular component of the immune status, common for all patients were characterized exclusively by shifts in the T link of immunity. Individual analysis of the nature of shifts of T helper cells and CD4+/CD8+ going upward from the norm allowed us to qualitatively identify group III (with two or more KPs). The obtained data are important for the development of the criteria for predicting high-risk keratoplasty and confirm that immunosuppression is needed.

PD-L1 expression is associated with tumor infiltrating lymphocytes that predict response to NACT in squamous cell cervical cancer.

Cancer immunotherapy has significantly improved the management of many malignancies in recent years. Although cervical cancer is the second most common women's cancer in the world, there are still few information about the role of checkpoint inhibitors in this neoplasm, especially in the neoadjuvant setting. In the present study, we retrieved 38 consecutive patients with squamous cell cervical cancer who underwent platinum-based neoadjuvant chemotherapy (NACT) followed by radical surgery. Pre-therapy biopsies were evaluated for the presence of tumor-infiltrating lymphocytes (TILs), including T (both cytotoxic CD8+ and helper CD4+) and B lymphocytes, macrophages, natural-killer cells, and eosinophils. Immunohistochemistry was performed to characterize the inflammatory cells and to evaluate programmed death-ligand 1 (PD-L1) expression on both neoplastic and inflammatory cells. We divided our study population in three groups using three cut-offs (< 10%, 10-40%, >40%), for both TILs and PD-L1 evaluation. Pathological response to NACT was obtained from the histological reports of the post-therapy surgical specimens. We observed that all cases showed stromal TILs, with a predominance of CD3+/CD4+ T helper cells, thus supporting the strong immunogenic potential of cervical cancer. The vast majority of neoplasms expressed PD-L1: 100% on immune cells and 92% on tumor cells. Firstly, we noticed that the percentage of neoplastic cells PD-L1+ was positively associated with high TIL percentage (p = 0.0073) and with increased PD-L1 expression on inflammatory cells (p = 0.0297). Secondly, we observed a significant correlation between both the percentage (p = 0.0105) of TILs and the expression of PD-L1 (p = 0.01045) on inflammatory cells and pathological response to NACT. These results suggest that cervical cancer could be a good target for immunotherapy, also in the neoadjuvant setting. Furthermore, PD-L1 expression was significantly associated with stromal TILs that interestingly may predict pathological response to NACT.

Interleukin-2 (IL-2) Interacts With IL-2 Receptor Beta (IL-2Rβ): Its Potential to Enhance the Proliferation of CD4+ T Lymphocytes in Flounder (Paralichthys olivaceus).

Interleukin-2 (IL-2) is an important immunomodulatory cytokine that primarily promotes the activation, proliferation, and differentiation of CD4+ T helper subsets and CD4+ T regulatory cells. In our previous studies, IL-2 and IL-2 receptor beta (IL-2Rβ) genes of flounder (Paralichthys olivaceus) were cloned, and IL-2Rβ molecules expressed on both B and T lymphocytes were identified. In the present study, the interaction of flounder IL-2 (fIL-2) with the IL-2 receptor beta (fIL-2Rβ) was investigated. The proportion of CD4+ T lymphocytes and IL-2Rβ+ cells were detected both in vivo and in vitro. Firstly, the binding of recombinant flounder IL-2 protein (rfIL-2) and rfIL-2Rβ was verified by pull-down assay and enzyme-linked immunosorbent assay. Indirect immunofluorescence assay showed that rfIL-2 enhanced the proliferation of CD4+ and IL-2Rβ+ cells in the gill and spleen. Furthermore, CD4-1+, CD4-2+ T lymphocytes and IL-2Rβ+ cells were significantly upregulated in cultured peripheral blood lymphocytes (PBLs) with addition of rfIL-2, as shown by Flow cytometry. The related genes were examined by Q-PCR in cultured PBLs with added rfIL-2. The results showed that the IL-2–IL-2R interaction induced upregulated expression of T lymphocyte surface makers, Th1-related cytokines or transcription factors, and critical genes of the IL-2 signaling pathway. In addition, these IL-2–elicited biological functions and immune responses were downregulated by blocked with anti–rfIL-2Rβ and anti–rfIL-2 Abs, showing that IL-2Rβ plays an indispensable role in IL-2 elicited biological function. Our results demonstrated that the interaction between IL-2 and IL-2Rβ showed its potential to enhance the proliferation of CD4+ T lymphocytes in flounder. As found in mammals, a Th1-mediated mechanism regulated by this interaction exists in teleost.

Distinct features between HLA-DR+ and HLA-DR- PD-1hi CXCR5- T peripheral helper cells in seropositive rheumatoid arthritis.

PD-1hi CXCR5- T peripheral helper (Tph) cells are newly identified pathogenic CD4 helper T cells in RA. We evaluated the usefulness of Tph cell subsets as biomarkers of RA. RA patients who visited our rheumatology department between May 2015 and September 2017 and met the 2010 ACR/EULAR classification criteria were included. We compared the correlation of DAS28-ESR between Tph cell subsets and 40 immune cell subsets. We also explored which subsets reflected the chronological changes in the disease activity after treatment. Thirty-four seropositive RA patients, 11 seronegative RA patients and 34 healthy controls were included. Tph cell subsets that correlated with the DAS28-ESR were HLA-DR+ Tph cells (rs = 0.50, P = 0.002), HLA-DR- Tph cells (rs = 0.39, P = 0.03) and Tph1 cells (rs = 0.41, P = 0.02). Among the other 40 immune cell subsets, HLA-DR+ Th1-17 cells (rs = 0.38, P = 0.03), activated B cells (rs = -0.35, P = 0.04), plasma cells (rs = 0.43, P = 0.01) and CD14++ CD16+ monocytes (rs = 0.36, P = 0.04) correlated, but not strongly as HLA-DR+ Tph cells. However, MTX treatment reduced the proportion of HLA-DR+ Tph cells independently of the disease activity. In contrast, HLA-DR- Tph cells accurately reflected the change in the DAS28-ESR during MTX treatment. HLA-DR+ Tph cells were decreased with MTX treatment, independent of the disease activity, while HLA-DR- Tph cells reflected the disease activity accurately during the treatment.

Preliminary indications that recombinant human IL-16 attracts and stimulates lymphocytes of the amphibian, Xenopus laevis implying an ancestral role for CD4 as a cytokine receptor

The D1 domain of the CD4 co-receptor interacts with MHC class II during Helper CD4+ Th-cell activation and effector function in all gnathostomes but the sequence and structure of this region are not well conserved through phylogeny. Conversely, the proximal D4 domain of CD4 is the binding site of the cytokine IL-16 and is highly conserved, allowing for promiscuous binding of IL-16 to CD4 between disparate gnathostomes. We report here that recombinant human IL-16 (rhIL-16) bound to Xenopus lymphocytes to allow separation on a magnetic column. Incubation with rhIL-16 resulted in an increased expression of MHC class II mRNA by Xenopus CD8− cells more than by CD8+ cells. An in vivo assay demonstrated that rhIL-16 can recruit lymphocytes of Xenopus frogs. Our data suggest that a subset of Xenopus laevis lymphocytes express a CD4 homolog on their surface that is capable of binding IL-16. These results imply that CD4 most likely arose from a primordial cytokine receptor.

Abstract 920: Characterization of the orthotopic MMTV PyMT murine mammary carcinoma model following radiation and immune checkpoint blockade

Abstract Radiotherapy (RT) is a highly utilized clinical treatment approach for certain types of breast cancer. The use of RT, in combination with checkpoint blockade, to prime and activate the immune system is an area of intensive research. Developing preclinical models for breast cancer research with well characterized tumor immune profiles is highly sought after. MMTV PyMT was originally created as a transgenic mouse model that we have optimized as an orthotopic transplantable model. The baseline immune profile shows an equal distribution of CD8+ and CD4+ T cells, with considerable presence of NK cells. MMTV PyMT tumors have a large myeloid population represented predominantly by granulocytic myeloid-derived suppressor cells and M1 tumor-associated macrophages. In this transplantable model, we tested anti-tumor responses to RT and immunomodulatory agents to determine if the model is more in line with an immunosuppressive “cold” tumor or an immuno-responsive “warm” tumor. Tumor-bearing mice were treated with checkpoint blockade antibodies against PD 1, PD L1, or CTLA 4 alone or in combination with focal RT (SARRP; Xstrahl). Single agent checkpoint inhibitors showed minimal responses while single agent focal RT proved to be highly effective, producing dose dependent tumor regressions at all dosage levels tested (5, 10 &amp; 20Gy). The combination of low dose focal RT (5Gy) with anti CTLA 4 (10mg/kg) resulted in one partial regression and stagnate tumor growth. To examine combination effects on the immune response, we examined infiltration of immune cell types in the tumor using flow cytometry at 2 time points. No remarkable changes occurred in any treatment group, at either time point. Moreover, positive expression of PD 1 remained low, both with and without treatment (&amp;lt;36% of tumor-derived CD8+ T cells). In most cases, ex vivo TIL stimulation with PMA and Ionomycin failed to induce cytokine expression (IFNγ, TNFα, and IL 2) in above 10% of T and NK cell subsets. IL-2 production was increased in several subsets from mice treated with radiation and/or anti mCTLA 4. However, no additive effect was observed in the combined group. Finally, the frequency of polyfunctional helper CD4+ T cells producing both IL 2 and TNFα correlated with in vivo efficacy response, regardless of treatment suggesting that activated helper T cells may be important for the anti-tumor response. In the MMTV PyMT transplant model, early assessment of immune cell populations were predictive for the lack of in vivo efficacy with checkpoint blockade. Interestingly, the modulation in cytokines (IL 2 and TNFα) that was produced by the combination of focal RT with or without anti mCTLA 4 treatment was indicative of an efficacious therapy. Citation Format: Erin E. Trachet, Derrik Germain Germain, Sumithra Urs, David Draper, Maryland Rosenfeld Franklin. Characterization of the orthotopic MMTV PyMT murine mammary carcinoma model following radiation and immune checkpoint blockade [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 920.

Abstract 6622: The identification of extranodal NK/T cell lymphoma-associated antigen for helper T cell-based immunotherapy

Abstract Extranodal natural killer/T-cell lymphoma, nasal type (NKTL) is a fatal destructive tumor that develops around the nasopharyngeal and nasal/paranasal areas. Although this disease was originally considered to be endemic to East Asia, Central and South America and was extremely rare in Europe and the United States, the incidence of NKTL has increased during the last several decades in countries where it was previously not epidemic. The combination of irradiation and chemotherapies such as SMILE and MPVIC-P are effective. However, these therapies cause severe and sometimes fatal adverse effects indicating that a novel strategy to treat NKTL is required. The positive results of clinical trials using immune checkpoint inhibitors in solid tumors proved that immune cells are capable of eliminating tumor. However, autoimmune diseases are inevitable adverse events in this type of therapy, which non-specifically activates T cells. Thus, the identification of epitope peptides in tumor has re-emerged as an attractive concept. I will describe our study to identify NKTL-derived T cell epitopes as a basis of cancer vaccine. The expression of c-Met and its ligand HGF plays a critical role in cell proliferation and is involved in numerous malignancies. Because c-Met expression and its role in NKTL remain unclear, we first studied the expression and function of c-Met in NKTL cells. As a result, we found that HGF and c-Met were expressed and contributed to the proliferation of cells in NK/T-cell lymphoma. Next, the helper epitopes were identified from c-Met protein using computer-based algorism. The identified epitope peptides could elicit peptide-specific helper CD4 T cells (HTLs), which showed antitumor responses in vitro. The addition of c-Met inhibitor suppressed TGF-β production from tumor followed by the augmentation of HTL responses. In summary, we identified novel c-Met HTL epitopes that can elicit effective antitumor responses against tumors expressing c-Met. Our results provide the rationale of combining c-Met targeting therapy and immunotherapy for NKTCLs and epithelial tumors. Citation Format: Takumi Kumai, Ryusuke Hayashi, Tatsuya Hayashi, Hiroya Kobayashi, Yasuaki Harabuchi. The identification of extranodal NK/T cell lymphoma-associated antigen for helper T cell-based immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6622.

Abstract 6528: Anti-PD-1 induces the endocytosis of the co-receptor from the surface of T-cells: Nivolumab is more effective than Pembrolizumab

Abstract Immune checkpoint blockade (ICB) has revolutionized the treatment of cancer. Present models show that anti-PD-1 operates simply by blocking co-receptor binding to its ligand PD-L1. Surprisingly, no previous studies have assessed whether ICB also induces the internalization or endocytosis of PD-1 from the cell surface. Many, but not all receptors, undergo endocytosis upon antibody or ligand binding. In this study, PD-1 internalization was assessed on activated human T cells by flow cytometry and fluorescence microscopy. Immunoprecipitation and western blotting were performed to assess the destiny of PD-1 after internalization. Our results report that anti-PD-1 induces the rapid internalization of PD-1 from the surface of T-cells in a manner comparable to CD28. A substantial minority of receptors were also resistant to antibody-induced endocytosis. Further, Nivolumab was considerably more effective than Pembrolizumab in inducing the endocytosis of PD-1 in primary human T-cells. This difference was the result of several factors where Nivolumab bound to a greater number of PD-1 molecules on the surface of T-cells than Pembrolizumab, indicating that the co-receptor might exist with different conformations on activated cells. Moreover, Nivolumab induced more rapid endocytosis (5.5 versus 3.6% per minute) and a greater percentage of PD-1 molecules than Pembrolizumab (70% versus 50%). The relative effect of anti-PD-1 molecules on internalization was seen equally on activated CD4 helper and CD8+ effector/memory T-cells. The internalization and the degradation of PD-1 were partially blocked by the inhibition of clathrin-mediated endocytosis and proteasome activity. Our studies show for the first time that ICB involves a second level of modulation involving the internalization of PD-1 from the surface of T-cells, an event which will contribute to the efficacy of ICB. Citation Format: Elham Ben Saad, Andres Oroya, Christopher E. Rudd. Anti-PD-1 induces the endocytosis of the co-receptor from the surface of T-cells: Nivolumab is more effective than Pembrolizumab [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6528.

Abstract 939: Evaluating anti-PD-1/4-1BB combination therapy: In vivo efficacy with humanized double knock-in mice

Abstract The transmembrane protein receptor 4-1BB is a member of the tumor necrosis factor (TNF) receptor super family and is involved in the co-stimulation of immune cells. 4-1BB is expressed in activated CD4 helper T cells, B cells, natural killer cells, natural killer T cells, dendritic cells, and activated endothelium. Most regulatory T cells (Tregs) also express 4-1BB, but it remains unclear that whether agonistic antibody treatment exerts pro- or anti-suppressive effect on these cells. 4-1BB enhances tumor rejection because it is upregulated on T cells following activation as its engagement increases T cell proliferation and pro-inflammatory cytokine production. Despite of the toxicity of 4-1BB-targeting drugs associated with overt immune activation, new therapeutic modalities using 4-1BB targeting aptamers, as well as combination therapies with other immuno-modulatory and traditional anti-cancer treatments, have revived excitement for the use of 4-1BB agonists in the clinic. Biocytogen has developed a double humanized B-hPD-1/h4-1BB mouse model to unlock the tremendous potential of combination therapy involving 4-1BB agonists and anti-PD-1 antibodies. In such model, mouse PD-1 gene exon 2, and 4-1BB gene exons 2-7 were replaced with their human counterparts. Murine colon cancer MC38 cells, humanized PD-L1 in MC38 cells, humanized PD-L1 in murine melanoma B16F10 cells, and murine lymphoma EL4 cells were subcutaneously implanted into homozygous B-hPD-1/h4-1BB mice. Combination treatment of Urelumab (anti-4-1BB) and Pembrolizumab (anti-PD-1) has shown enhanced inhibitory effects compared to single antibody treatments using the B-hPD-1/h4-1BB mice models. Based on these findings, it could be concluded that the B-hPD-1/h4-1BB double-humanized mouse model is a validated tool for in vivo evaluation of hPD-1 and h4-1BB antibody combination therapy. Citation Format: Tian Gan, Xiaofei Zhou, Yuting Hu, Chengzhang Shang, Gary Ng. Evaluating anti-PD-1/4-1BB combination therapy: In vivo efficacy with humanized double knock-in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 939.

Abstract 589: Immune biomarker profiling and safety evaluation response to 4-1BB antibody (Urelumab) in humanized 4-1BB mice

Abstract 4-1BB (CD137) belongs to the TNFR superfamily and is expressed by activated CD4 helper T cells, B cells, natural killer cells, natural killer T cells, dendritic cells and Treg cells. 4-1BB is a potential therapeutic target as it enhances tumor rejection by upregulating T cells following activation and its engagement increases T cell proliferation and pro-inflammatory cytokine production. However, the liver toxicity associated with over immune activation has obstructed the clinical development of 4-1BB targeting therapies. To address the issue, Biocytogen has developed humanized B-h4-1BB mice as an efficient platform of both in vivo MOA and safety studies of 4-1BB antibody. In B-h4-1BB mice, murine colon cancer MC38 cells were subcutaneously implanted. Biomarkers, such as, mCD45, mCD3, mCD4, mCD8, mNK, mFoxp3, mCD11b, and etc., were profiled after the treatment with an 4-1BB antibody (Urelumab). FACS, IHC, and IF data showed that Urelumab have increased the ratio of CD8+ T cell, but on the other hand decreased Treg cell, the PD-1 and Tim3 (T cell exhausted marker) upon treatment. Toxicity have also been with the treatment of Urelumab using this model. The blood chemistry assays showed that the high dose Urelumab had toxicity effects compared with the control group and WT C57BL/6 mice. Based on these findings, we conclude that the B-h4-1BB humanized mouse model is a powerful platform for both MOA (biomarker) discovery and safety evaluation of 4-1BB antibody. Citation Format: Jie Xiang, Dirui Li, Hao Yang, Gary Ng, Yuelei Shen. Immune biomarker profiling and safety evaluation response to 4-1BB antibody (Urelumab) in humanized 4-1BB mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 589.

Proteome-Wide Zika Virus CD4 T Cell Epitope and HLA Restriction Determination.

Zika virus (ZIKV) is a mosquito-borne pathogen that caused an epidemic in 2015-2016. ZIKV-specific T cell responses are functional in animal infection models, and helper CD4 T cells promote avid Abs in the vaccine context. The small volumes of blood available from field research limit the determination of T cell epitopes for complex microbes such as ZIKV. The goal of this project was efficient determination of human ZIKV CD4 T cell epitopes at the whole proteome scale, including validation of reactivity to whole pathogen, using small blood samples from convalescent time points when T cell response magnitude may have waned. Polyclonal enrichment of candidate ZIKV-specific CD4 T cells used cell-associated virus, documenting that T cells in downstream peptide analyses also recognize whole virus after Ag processing. Sequential query of bulk ZIKV-reactive CD4 T cells with pooled/single ZIKV peptides and molecularly defined APC allowed precision epitope and HLA restriction assignments across the ZIKV proteome and enabled discovery of numerous novel ZIKV CD4 T cell epitopes. The research workflow is useful for the study of emerging infectious diseases with a very limited human blood sample availability.

CD4 Deficiency Causes Poliomyelitis and Axonal Blebbing in Murine Coronavirus-Induced Neuroinflammation.

Mouse hepatitis virus (MHV) is a murine betacoronavirus (m-CoV) that causes a wide range of diseases in mice and rats, including hepatitis, enteritis, respiratory diseases, and encephalomyelitis in the central nervous system (CNS). MHV infection in mice provides an efficient cause-effect experimental model to understand the mechanisms of direct virus-induced neural-cell damage leading to demyelination and axonal loss, which are pathological features of multiple sclerosis (MS), the most common disabling neurological disease in young adults. Infiltration of T lymphocytes, activation of microglia, and their interplay are the primary pathophysiological events leading to disruption of the myelin sheath in MS. However, there is emerging evidence supporting gray matter involvement and degeneration in MS. The investigation of T cell function in the pathogenesis of deep gray matter damage is necessary. Here, we employed RSA59 (an isogenic recombinant strain of MHV-A59)-induced experimental neuroinflammation model to compare the disease in CD4-/- mice with that in CD4+/+ mice at days 5, 10, 15, and 30 postinfection (p.i.). Viral titer estimation, nucleocapsid gene amplification, and viral antinucleocapsid staining confirmed enhanced replication of the virions in the absence of functional CD4+ T cells in the brain. Histopathological analyses showed elevated susceptibility of CD4-/- mice to axonal degeneration in the CNS, with augmented progression of acute poliomyelitis and dorsal root ganglionic inflammation rarely observed in CD4+/+ mice. Depletion of CD4+ T cells showed unique pathological bulbar vacuolation in the brain parenchyma of infected mice with persistent CD11b+ microglia/macrophages in the inflamed regions on day 30 p.i. In summary, the current study suggests that CD4+ T cells are critical for controlling acute-stage poliomyelitis (gray matter inflammation), chronic axonal degeneration, and inflammatory demyelination due to loss of protective antiviral host immunity.IMPORTANCE The current trend in CNS disease biology is to attempt to understand the neural-cell-immune interaction to investigate the underlying mechanism of neuroinflammation, rather than focusing on peripheral immune activation. Most studies in MS are targeted toward understanding the involvement of CNS white matter. However, the importance of gray matter damage has become critical in understanding the long-term progressive neurological disorder. Our study highlights the importance of CD4+ T cells in safeguarding neurons against axonal blebbing and poliomyelitis from murine betacoronavirus-induced neuroinflammation. Current knowledge of the mechanisms that lead to gray matter damage in MS is limited, because the most widely used animal model, experimental autoimmune encephalomyelitis (EAE), does not present this aspect of the disease. Our results, therefore, add to the existing limited knowledge in the field. We also show that the microglia, though important for the initiation of neuroinflammation, cannot establish a protective host immune response without the help of CD4+ T cells.

Efficacy of Serology Testing in Predicting Reinfection in Patients With SARS-CoV-2.

In many parts of the United States, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases have reached peak infection rates, prompting administrators to create protocols to resume elective cases. As elective procedures and surgeries get scheduled, ambulatory surgery centers (ASCs) must implement some form of widespread testing in order to ensure the safety of both the ASC staff and the patients being seen. The US Centers for Disease Control and Prevention (CDC) recently announced the approval of new serological testing for SARS-CoV-2, a test that can indicate the presence of IgM and IgG antibodies in the serum against viral particles. However, the possibility for reinfection raises questions about the utility of this new serological test, as the presence of IgG may not correspond to long-term immunity. SARS-CoV-2 has been known to form escape mutations, which may correspond to a reduction in immunoglobulin binding capacity. Patients who develop more robust immune responses with formation of memory CD8+ T-cells and helper CD4+ T-cells will be the most equipped if exposed to the virus, but, unfortunately, the serology test will not help us in distinguishing those individuals. Given the inherent disadvantages of serological testing, antibody testing alone should not be used when deciding patient care and should be combined with polymerase chain reaction testing.

Abstract PR06: Tumor-infiltrating B cells colocalize with CD4 T effector cells within tertiary lymphoid structures to present antigen and educate the antitumor immune response in human primary tumors

Abstract Despite the success of checkpoint blockade immunotherapies, i.e., anti-PD1, only 20% of patients produce a durable response to these treatments. Thus, a need exists to develop additional therapeutic strategies to treat these patients, which includes evaluation of other tumor-infiltrating immune cells that could further augment the CD8+ and CD4+ T-cell response. Tumor-infiltrating B cells (TIL-B) represent a possible target for immunotherapy due to their predominance in the tumor microenvironment and crucial role in the immune response. In fact, current evidence suggests an antitumor role for TIL-Bs. Specifically, detection of TIL-Bs within tertiary lymphoid structures (TLS) correlates with both increased survival in patients with solid tumors and enhanced response to anti-PD1 immunotherapy. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer by presenting tumor antigens to CD4 TILs within TLS. Utilizing single-cell RNAseq and high-level flow cytometry, we observed increased numbers of activated TIL-Bs in the primary tumors of head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) patients. We further assessed the location of TIL-Bs within the TME and determined that they predominantly sit adjacent to CD4+ tumor-infiltrating lymphocytes (TILs) within TLS. Thus, we generated an antigen presentation assay in vitro, and we observed increased CD4+ TIL responses when TIL-Bs presented autologous tumor antigens. Further, there was a differential impact on CD4+ TIL phenotype; specifically, if TIL-Bs were activated (CD27+), the CD4+ TILs were T helper (anti-tumor) CD4+ T cells and if the TIL-Bs were nonactivated (CD27-), the CD4+ TILs were T regulatory cells (protumor). These data suggest that TIL-Bs influence the phenotype and function of CD4+ TILs in patient tumors within TLS. Ultimately, results from this study will increase effective targeting of TIL-Bs within patient primary tumors. Citation Format: Ayana T. Ruffin, Anthony R. Cillo, Sheryl Kunning, Robert L. Ferris, Dario A.A. Vignali, Tullia C. Bruno. Tumor-infiltrating B cells colocalize with CD4 T effector cells within tertiary lymphoid structures to present antigen and educate the antitumor immune response in human primary tumors [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr PR06.

T cells expressing the lupus susceptibility allele Pbx1d enhance autoimmunity and atherosclerosis in dyslipidemic mice.

Patients with systemic lupus erythematosus (SLE) present a high incidence of atherosclerosis, which contributes significantly to morbidity and mortality in this autoimmune disease. An impaired balance between regulatory (Treg) and follicular helper (Tfh) CD4+ T cells is shared by both diseases. However, whether there are common mechanisms of CD4+ T cell dysregulation between SLE and atherosclerosis remains unclear. Pre-B cell leukemia transcription factor 1 isoform d (Pbx1d) is a lupus susceptibility gene that regulates Tfh cell expansion and Treg cell homeostasis. Here, we investigated the role of T cells overexpressing Pbx1d in low-density lipoprotein receptor-deficient (Ldlr-/-) mice fed with a high-fat diet, an experimental model for atherosclerosis. Pbx1d-transgenic T cells exacerbated some phenotypes of atherosclerosis, which were associated with higher autoantibody production, increased Tfh cell frequency, and impaired Treg cell regulation, in Ldlr-/- mice as compared with control T cells. In addition, we showed that dyslipidemia and Pbx1d-transgenic expression independently impaired the differentiation and function of Treg cells in vitro, suggesting a gene/environment additive effect. Thus, our results suggest that the combination of Pbx1d expression in T cells and dyslipidemia exacerbates both atherosclerosis and autoimmunity, at least in part through a dysregulation of Treg cell homeostasis.

High expression of the checkpoint molecule PD-1 on regulatory and helper CD4+ T cells in metastatic breast cancer patients with poor prognosis

Background: Recurrence and metastasis are common in patients of breast cancer even after chemotherapy and radiotherapy. This recurrence may occur due to the emergence of immunoregulatory T (Treg) cells and the immune checkpoint molecules such as programmed cell death protein 1 (PD-1) which are known to suppress anti-tumor immunity. Aim: The aim of this study is to investigate the numbers of Treg cells and the expression of PD-1 molecule on T cells in the peripheral blood of breast cancer patients at different stages (II, III, and IV) compared to healthy individuals. Materials and Methods: Blood samples were collected from early diagnosed metastatic cancer patients before surgery. Leucocytes were stained with different antibodies including CD4, CD25, CD127, and PD1, and then were analyzed by multiparametric flow cytometry. Results: The results showed that the relative and absolute numbers of Treg cells were significantly increased in breast cancer patients. Interestingly, we found a correlation between the numbers and cancer progression from stages II to III and IV, as compared with healthy individuals. Furthermore, significant increases in PD-1 expression were observed on Treg cells and CD4+ T cells in cancer patients as compared to healthy controls. This profile of PD-1 expression was also correlated with the poor prognosis of the patients. Conclusion: The results provide a better understanding of the immunomodulatory role of Treg cells and PD-1 expression in breast cancer patients who are the target for immunotherapy using checkpoint inhibitors.