Combination vaccine based on citrullinated vimentin and enolase peptides induces potent CD4-mediated anti-tumor responses

BackgroundStress-induced post-translational modifications occur during autophagy and can result in generation of new epitopes and immune recognition. One such modification is the conversion of arginine to citrulline by peptidylarginine deiminase enzymes.MethodsWe used Human leukocyte antigen (HLA) transgenic mouse models to assess the immunogenicity of citrullinated peptide vaccine by cytokine Enzyme linked immunosorbant spot (ELISpot) assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma and ID8 ovarian models expressing either constitutive or interferon-gamma (IFNγ) inducible Major Histocompatibility Complex (MHC) class II (MHC-II) as represented by most human tumors. To determine the importance of CD4 T cells in tumor therapy, we analyzed the immune cell infiltrate into murine tumors using flow cytometry and performed therapy studies in the presence of CD4 and CD8 T cell depletion. We assessed the T cell repertoire to citrullinated peptides in ovarian cancer patients and healthy donors using flow cytometry.ResultsThe combination of citrullinated vimentin and enolase peptides (Modi-1) stimulated strong CD4 T cell responses in mice. Responses resulted in a potent anti-tumor therapy against established tumors and generated immunological memory which protected against tumor rechallenge. Depletion of CD4, but not CD8 T cells, abrogated the primary anti-tumor response as well as the memory response to tumor rechallenge. This was further reinforced by successful tumor regression being associated with an increase in tumor-infiltrating CD4 T cells and a reduction in tumor-associated myeloid suppressor cells. The anti-tumor response also relied on direct CD4 T cell recognition as only tumors expressing MHC-II were rejected. A comparison of different Toll-like receptor (TLR)-stimulating adjuvants showed that Modi-1 induced strong Th1 responses when combined with granulocyte-macrophage colony-stimulating factor (GMCSF), TLR9/TLR4, TLR9, TLR3, TLR1/2 and TLR7 agonists. Direct linkage of the TLR1/2 agonist to the peptides allowed the vaccine dose to be reduced by 10-fold to 100-fold without loss of anti-tumor activity. Furthermore, a CD4 Th1 response to the citrullinated peptides was seen in ovarian cancer patients.ConclusionsModi-1 citrullinated peptide vaccine induces potent CD4-mediated anti-tumor responses in mouse models and a CD4 T cell repertoire is present in ovarian cancer patients to the citrullinated peptides suggesting that Modi-1 could be an effective vaccine for ovarian cancer patients.

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Homocitrullination of lysine residues mediated by myeloid-derived suppressor cells in the tumor environment is a target for cancer immunotherapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001910 ◽

2021 ◽

Vol 9 (7) ◽

pp. e001910

Author(s):

Katherine W Cook ◽

Wei Xue ◽

Peter Symonds ◽

Ian Daniels ◽

Mohamed Gijon ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Therapy ◽

Suppressor Cells ◽

Cd4 T Cell ◽

Cell Repertoire ◽

Mhc Ii ◽

Patients With Cancer ◽

Healthy Donors ◽

Therapy Studies

BackgroundHomocitrullination is the post-translational modification of lysine that is recognized by T cells.MethodsThis study identified homocitrullinated peptides from aldolase, enolase, cytokeratin and binding immunoglobulin protein and used human leukocyte antigen (HLA) transgenic mice to assess immunogenicity by enzyme-linked immunosorbent spot assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma expressing constitutive or interferon γ (IFNγ)-inducible major histocompatibility complex class II (MHC-II) as represented by most human tumors. To determine the mechanism behind the therapy, immune cell infiltrates were analyzed using flow cytometry and therapy studies in the presence of myeloperoxidase (MPO) inhibitor and T-cell depletion performed. We assessed the T-cell repertoire to homocitrullinated peptides in patients with cancer and healthy donors using flow cytometry.ResultsHomocitrulline (Hcit) peptide vaccination stimulated strong CD4 T-cell responses and induced significant antitumor therapy in an established tumor model. The antitumor response was dependent on CD4 T cells and the effect was driven mainly via direct tumor recognition, as responses were only observed if the tumors were induced to express MHC-II. In vitro proliferation assays show that healthy donors and patients with cancer have an oligoclonal CD4 T-cell repertoire recognizing homocitrullinated peptides. Inhibition of cyanate generation, which mediates homocitrullination, by MPO inhibition reduced tumor therapy by the vaccine induced T cells (p=0.0018). Analysis of the tumor microenvironment (TME) suggested that myeloid-derived suppressor cells (MDSCs) were a potential source of MPO. The selected B16 melanoma model showed MDSC infiltration and was appropriate to see if the Hcit vaccine could overcome the immunosuppression associated with MDSCs. The vaccine was very effective (90% survival) as the induced CD4 T cells directly targeted the homocitrullinated tumor and likely reversed the immunosuppressive environment.ConclusionWe propose that MPO, potentially produced by MDSCs, catalyzes the buildup of cyanate in the TME which diffuses into tumor cells causing homocitrullination of cytoplasmic proteins which are degraded and, in the presence of IFNγ, presented by MHC-II for direct CD4 T-cell recognition. Homocitrullinated proteins are a new target for cancer vaccines and may be particularly effective against tumors containing high levels of MPO expressing MDSCs.

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High prevalence of low affinity peptide–MHC II tetramer–negative effectors during polyclonal CD4+ T cell responses

Journal of Experimental Medicine ◽

10.1084/jem.20101574 ◽

2011 ◽

Vol 208 (1) ◽

pp. 81-90 ◽

Cited By ~ 103

Author(s):

Joseph J. Sabatino ◽

Jun Huang ◽

Cheng Zhu ◽

Brian D. Evavold

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Lymphocytic Choriomeningitis ◽

Cd4 T Cell ◽

Cell Repertoire ◽

Mhc Ii ◽

High Affinity ◽

Effector Cytokines ◽

Affinity Peptide

T cell affinity for antigen initiates adaptive immunity. However, the contribution of low affinity cells to a response is unknown as it has not been possible to assess the entire affinity range of a polyclonal T cell repertoire. In this study, we used a highly sensitive two-dimensional binding assay to identify low affinity cells in polyclonal autoreactive and pathogen-reactive CD4+ T cell populations specific for myelin oligodendrocyte glycoprotein (MOG) and lymphocytic choriomeningitis virus (LCMV) antigens, respectively. Low affinity CD4+ T cells, below detection with peptide–major histocompatibility complex class II tetramers, were at least as frequent as high affinity responders and contributed significant effector cytokines in both primary antigen–specific responses. We further demonstrated that MOG- and LCMV-specific CD4+ T cells possessed similarly broad ranges in their affinities (>100-fold wide), only differing in the frequencies of low and high affinity cells. Thus, low as well as high affinity CD4+ T cells are critical effectors in autoimmune and pathogen-specific responses.

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186 Distinct immune signatures predicting clinical response to PD-1 blockade therapy in gynecological cancers revealed by high-dimensional immune profiling

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0186 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A200-A200

Author(s):

Yuki Muroyama ◽

Sasikanth Manne ◽

Alexandar Huang ◽

Divij Mathew ◽

...

Keyword(s):

Ovarian Cancer ◽

T Cells ◽

Endometrial Cancer ◽

T Cell ◽

Clinical Response ◽

Cancer Patients ◽

T Cell Response ◽

High Dimensional ◽

Uterine Endometrial Cancer ◽

Immune Profiling

BackgroundAlthough immune checkpoint blockade revolutionized cancer therapy, response rates have been mixed in gynecological malignancies. While uterine endometrial cancer with high microsatellite instability (MSIHI) and high tumor mutational burden (TMB) respond robustly to checkpoint blockade, high-grade serous ovarian cancer (HGSOC) with low TMB respond modestly. Currently, there has been no known immune signature or T cell phenotype that predicts clinical response in gynecological tumors.MethodsTo dissect the immune landscape and T cell phenotypes in gynecological cancer patients receiving PD-1 blockade, we used high-dimensional cytometry (flow cytometry and mass cytometry (CyTOF)). We performed longitudinal deep immune profiling of PBMC from patients with recurrent uterine endometrial cancer receiving single-arm nivolumab, and HSGOC patients receiving neoadjuvant nivolumab plus platinum-based chemotherapy prior to debulking surgery.ResultsChemotherapy-resistant MSI-H uterine cancer patients treated with nivolumab had a proliferative T cell response 2–4 weeks post PD-1 blockade, consistent with responses seen in high TMB melanoma and lung cancer. The responding Ki67+ CD8 T cell population was largely CD45RAloCD27hi or CD45RAloCD27lo and highly expressed PD1, CTLA-4, and CD39, consistent with the phenotype of exhausted T cells (TEX). These exhausted-like cells are enriched in responders, whereas early expansion Tregs are enriched in non-responders. Unlike patients with uterine endometrial cancer, patients with TMBlo ovarian cancer did not have a clear proliferative CD8 T cell response after neoadjuvant nivolumab plus chemotherapy treatment, suggesting systemic immune suppression. At baseline, ovarian cancer without recurrence have more terminally differentiated effector-like CD8 T cells, and patients with recurrence have more naive-like cells. Thus, both high and low TMB gynecological tumors have distinct immune landscapes associated with clinical response. Additionally, in MSI-H uterine endometrial cancer patients, the length of time between the prior chemotherapy and the initiation of immunotherapy was negatively correlated with T cell reinvigoration post immunotherapy and clinical response. This suggests the importance of optimize therapeutic timing to maximize the therapeutic efficacy when combining immunotherapy and chemotherapy.ConclusionsCollectively, our immune profiling revealed the distinct immune signatures associated with clinical response to PD-1 blockade in gynecological cancers. Our results also suggest that TMBhi inflamed versus TMBlo cold tumor microenvironment, and timing of chemo/immunotherapy could impact differentiation and functions of T cells.Ethics ApprovalThe study was approved by MSKCC Ethics Board, approval number 17–180 and 17–182.

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Deciphering and predicting CD4+ T cell immunodominance of influenza virus hemagglutinin

Journal of Experimental Medicine ◽

10.1084/jem.20200206 ◽

2020 ◽

Vol 217 (10) ◽

Cited By ~ 4

Author(s):

Antonino Cassotta ◽

Philipp Paparoditis ◽

Roger Geiger ◽

Ramgopal R. Mettu ◽

Samuel J. Landry ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antigen Processing ◽

Ex Vivo ◽

Influenza Pandemic ◽

Cd4 T Cell ◽

Cytotoxic T Cell ◽

Cell Repertoire ◽

Th Cells ◽

Cytotoxic T Cell Responses

The importance of CD4+ T helper (Th) cells is well appreciated in view of their essential role in the elicitation of antibody and cytotoxic T cell responses. However, the mechanisms that determine the selection of immunodominant epitopes within complex protein antigens remain elusive. Here, we used ex vivo stimulation of memory T cells and screening of naive and memory T cell libraries, combined with T cell cloning and TCR sequencing, to dissect the human naive and memory CD4+ T cell repertoire against the influenza pandemic H1 hemagglutinin (H1-HA). We found that naive CD4+ T cells have a broad repertoire, being able to recognize naturally processed as well as cryptic peptides spanning the whole H1-HA sequence. In contrast, memory Th cells were primarily directed against just a few immunodominant peptides that were readily detected by mass spectrometry–based MHC-II peptidomics and predicted by structural accessibility analysis. Collectively, these findings reveal the presence of a broad repertoire of naive T cells specific for cryptic H1-HA peptides and demonstrate that antigen processing represents a major constraint determining immunodominance.

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Characterization of T cell repertoire of blood, tumor, and ascites in ovarian cancer patients using next generation sequencing

OncoImmunology ◽

10.1080/2162402x.2015.1030561 ◽

2015 ◽

Vol 4 (11) ◽

pp. e1030561 ◽

Cited By ~ 34

Author(s):

Miran Jang ◽

Poh-Yin Yew ◽

Kosei Hasegawa ◽

Yuji Ikeda ◽

Keiichi Fujiwara ◽

...

Keyword(s):

Ovarian Cancer ◽

Next Generation Sequencing ◽

T Cell ◽

Cancer Patients ◽

T Cell Repertoire ◽

Next Generation ◽

Cell Repertoire ◽

Generation Sequencing

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Telomerase and CD4 T Cell Immunity in Cancer

Cancers ◽

10.3390/cancers12061687 ◽

2020 ◽

Vol 12 (6) ◽

pp. 1687

Author(s):

Magalie Dosset ◽

Andrea Castro ◽

Hannah Carter ◽

Maurizio Zanetti

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Critical Role ◽

Tumor Formation ◽

T Cell Immunity ◽

Cd4 T Cell ◽

Current Status ◽

Mhc Ii ◽

Cell Immunity

Telomerase reverse transcriptase (TERT) is a conserved self-tumor antigen which is overexpressed in most tumors and plays a critical role in tumor formation and progression. As such, TERT is an antigen of great relevance to develop widely applicable immunotherapies. CD4 T cells play a major role in the anti-cancer response alone or with other effector cells such as CD8 T cells and NK cells. To date, efforts have been made to identify TERT peptides capable of stimulating CD4 T cells that are also able to bind diverse MHC-II alleles to ease immune status monitoring and immunotherapies. Here, we review the current status of TERT biology, TERT/MHC-II immunobiology, and past and current vaccine clinical trials. We propose that monitoring CD4 T cell immunity against TERT is a simple and direct way to assess immune surveillance in cancer patients and a new way to predict the response to immune checkpoint inhibitors (ICPi). Finally, we present the initial results of a systematic discovery of TERT peptides able to bind the most common HLA Class II alleles worldwide and show that the repertoire of MHC-II TERT peptides is wider than currently appreciated.

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The critical role of CD4+ T cells in PD-1 blockade against MHC-II–expressing tumors such as classic Hodgkin lymphoma

Blood Advances ◽

10.1182/bloodadvances.2020002098 ◽

2020 ◽

Vol 4 (17) ◽

pp. 4069-4082

Author(s):

Joji Nagasaki ◽

Yosuke Togashi ◽

Takeaki Sugawara ◽

Makiko Itami ◽

Nobuhiko Yamauchi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Critical Role ◽

Cd4 T Cell ◽

Antitumor Effects ◽

Cell Axis ◽

Mhc I ◽

Mhc Ii

Abstract Classic Hodgkin lymphoma (cHL) responds markedly to PD-1 blockade therapy, and the clinical responses are reportedly dependent on expression of major histocompatibility complex class II (MHC-II). This dependence is different from other solid tumors, in which the MHC class I (MHC-I)/CD8+ T-cell axis plays a critical role. In this study, we investigated the role of the MHC-II/CD4+ T-cell axis in the antitumor effect of PD-1 blockade on cHL. In cHL, MHC-I expression was frequently lost, but MHC-II expression was maintained. CD4+ T cells highly infiltrated the tumor microenvironment of MHC-II–expressing cHL, regardless of MHC-I expression status. Consequently, CD4+ T-cell, but not CD8+ T-cell, infiltration was a good prognostic factor in cHL, and PD-1 blockade showed antitumor efficacy against MHC-II–expressing cHL associated with CD4+ T-cell infiltration. Murine lymphoma and solid tumor models revealed the critical role of antitumor effects mediated by CD4+ T cells: an anti-PD-1 monoclonal antibody exerted antitumor effects on MHC-I−MHC-II+ tumors but not on MHC-I−MHC-II− tumors, in a cytotoxic CD4+ T-cell–dependent manner. Furthermore, LAG-3, which reportedly binds to MHC-II, was highly expressed by tumor-infiltrating CD4+ T cells in MHC-II–expressing tumors. Therefore, the combination of LAG-3 blockade with PD-1 blockade showed a far stronger antitumor immunity compared with either treatment alone. We propose that PD-1 blockade therapies have antitumor effects on MHC-II–expressing tumors such as cHL that are mediated by cytotoxic CD4+ T cells and that LAG-3 could be a candidate for combination therapy with PD-1 blockade.

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Second Class Minors

Journal of Experimental Medicine ◽

10.1084/jem.20021961 ◽

2003 ◽

Vol 197 (3) ◽

pp. 375-385 ◽

Cited By ~ 25

Author(s):

Hiroeki Sahara ◽

Nilabh Shastri

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

T Cell Responses ◽

Cd4 T Cell ◽

Interleukin 4 ◽

General Strategy ◽

Mhc Ii ◽

Cell Responses ◽

Antigen Presenting

CD4 T cells regulate immune responses that cause chronic graft rejection and graft versus host disease but their target antigens remain virtually unknown. We developed a new method to identify CD4 T cell–stimulating antigens. LacZ-inducible CD4 T cells were used as a probe to detect their cognate peptide/MHC II ligand generated in dendritic cells fed with Escherichia coli expressing a library of target cell genes. The murine H46 locus on chromosome 7 was thus found to encode the interleukin 4–induced IL4i1 gene. The IL4i1 precursor contains the HAFVEAIPELQGHV peptide which is presented by Ab major histocompatibility complex class II molecule via an endogenous pathway in professional antigen presenting cells. Both allelic peptides bind Ab and a single alanine to methionine substitution at p2 defines nonself. These results reveal novel features of H loci that regulate CD4 T cell responses as well as provide a general strategy for identifying elusive antigens that elicit CD4 T cell responses to tumors or self-tissues in autoimmunity.

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Effect of anti-CTLA-4 antibody treatment on T-cell repertoire evolution in treated cancer patients.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.3020 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 3020-3020

Author(s):

Edward Cha ◽

Yafei Hou ◽

Mark Klinger ◽

Craig Cummings ◽

Malek Faham ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cancer Patients ◽

Multiple Time ◽

T Cell Repertoire ◽

Cell Repertoire ◽

Antibody Treatment ◽

Tcr Repertoire ◽

Tcr Diversity ◽

Morisita Index

3020 Background: CTL-associated antigen 4 (CTLA-4) is an immune checkpoint expressed by T cells. While treatment with anti-CTLA-4 antibody can induce clinical responses in advanced cancer patients, its effects on the breadth of the T cell response is unknown. Methods: We used a sequencing-based method, LymphoSIGHT, to assess T cell repertoire diversity in 46 patients with metastatic castration resistant prostate cancer or metastatic melanoma. Peripheral blood mononuclear cells were obtained from patients prior to and during treatment with anti-CTLA-4 antibody. mRNA was amplified using locus-specific primer sets for T cell receptor (TCR) beta, and the amplified product was sequenced. Sequence reads were used to quantitate absolute TCR frequencies using standardized clonotype determination algorithms with normalization by spiked reference TCR sequences. Following clonotype quantitation, repertoire differences between serial samples were assessed by the Morisita index, a statistical measure of population dispersion. Results: 97 paired samples were assessed, of which 46 (47%) had increases and 22 (23%) had decreases in TCR diversity by more than 2-fold. By comparison, none of 9 untreated sample pairs underwent more than a 2-fold change in diversity (P = 0.005, Fisher’s exact test, two tailed). TCR repertoire differences between monthly samples were markedly higher than for time-matched controls. After the first treatment, median Morisita index between samples was 0.197 for treated samples versus 0.039 for untreated (P = 0.0005, Mann-Whitney U test). The median number of clones that significantly changed in abundance was 421 for treated versus 45 for controls. In patients with multiple time points, this rapid clonotype evolution continued through treatment. Despite this global turnover in repertoire, a subset of high frequency clones, including CMV-specific T cells, remained relatively constant over the course of the study. Conclusions: CTLA-4 blockade increases the global rate of T cell clonotype turnover and influences TCR diversity. This evolution of the TCR repertoire may reflect a mechanism by which CTLA-4 blockade enhances tumor-specific T cells over time.

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Tumor Antigen-Dependent and Tumor Antigen-Independent Activation of Antitumor Activity in T Cells by a Bispecific Antibody-Modified Tumor Vaccine

Clinical and Developmental Immunology ◽

10.1155/2010/423781 ◽

2010 ◽

Vol 2010 ◽

pp. 1-12 ◽

Cited By ~ 2

Author(s):

Philippe Fournier ◽

Volker Schirrmacher

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Cancer Patients ◽

Tumor Antigen ◽

Immune Escape ◽

Tumor Vaccine ◽

Cell Repertoire ◽

Tumor Immune Escape

New approaches of therapeutic cancer vaccination are needed to improve the antitumor activity of T cells from cancer patients. We studied over the last years the activation of human T cells for tumor attack. To this end, we combined the personalized therapeutic tumor vaccine ATV-NDV—which is obtained by isolation, shortin vitroculture, irradiation, and infection of patient's tumor cells by Newcastle Disease Virus (NDV)—with bispecific antibodies (bsAbs) binding to this vaccine and introducing anti-CD3 (signal 1) and anti-CD28 (signal 2) antibody activities. This vaccine called ATV-NDV/bsAb showed the unique ability to reactivate a preexisting potentially anergized antitumor memory T cell repertoire. But it also activated naive T cells to have antitumor propertiesin vitroandin vivo. This innovative concept of direct activation of cancer patients' T cells via cognate and noncognate interactions provides potential for inducing strong antitumor activities aiming at overriding T cell anergy and tumor immune escape mechanisms.

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