Roles of Chemokine Receptors in T Cell Accmulation in Omental Tumors

The omentum is an adipose tissue in the peritoneal cavity that contains immune aggregates termed milky spots. Functionally similar to conventional lymphoid tissues, these specialized leukocyte clusters harbor effector and suppressor cells, mount immune responses to antigens, and sustain peritoneal homeostasis. However, the pre-existing leukocytes fail to protect the tissue from tumor colonization. In fact, the omentum is frequently involved in peritoneal metastasis, during which the balance between effector and suppressor immune cells is compromised with the latter gaining dominance. Regulatory T cells (Tregs) represent a major immunosuppressive population in the omentum and rapidly accumulate after tumor implantation. The increased Treg abundance may be attributed to the local proliferation of pre-existing Tregs, peripheral Treg conversion, and/or cell recruitment from the circulation. Using T cell receptor (TCR) repertoire analysis, we show that tumor progression in the omentum does not promote Treg clonal expansion but leads to the influx of small Treg clonotypes. Naive CD4+ T cells poorly differentiate to Tregs in tumor-bearing omenta, suggesting a minimal contribution of peripheral conversion to the enlargement of the omental Treg pool. Parabiosis experiments reveal that circulating Tregs robustly migrate to the omentum as tumors progress, wherein they metabolically adapt to the environment and acquire the transcriptional signature of adipose-resident Tregs. We then find that CXCR3 is essential for Treg recruitment to omental tumors, and that CD4-specific CXCR3 ablation abrogates Treg accumulation and enhances tumor control. Interestingly, CXCR3 also supports effector CD8+ T cell responses, which mediate tumor control. Without this chemokine receptor, CD8+ T cells fail to undergo proper effector differentiation and maintain their numerical abundance in omental tumors. Therefore, our data reveal the mechanisms for the accumulation of Tregs and CD8+ T cells in omental tumors and uncover the pivotal roles of CXCR3 in this process

Opposing tumor-cell-intrinsic and -extrinsic roles of the IRF1 transcription factor in antitumor immunity

Type I interferon (IFN-I) and IFN-γ foster antitumor immunity by facilitating T cell responses. Paradoxically, IFNs may promote T cell exhaustion by activating immune checkpoints. The downstream regulators of these disparate responses are incompletely understood. Here, we describe how interferon regulatory factor 1 (IRF1) orchestrates these opposing effects of IFNs. IRF1 expression in tumor cells blocks Toll-like receptor- and IFN-I-dependent host antitumor immunity by preventing interferon-stimulated gene (ISG) and effector programs in immune cells. In contrast, expression of IRF1 in the host is required for antitumor immunity. Mechanistically, IRF1 binds distinctly or together with STAT1 at promoters of immunosuppressive but not immunostimulatory ISGs in tumor cells. Overexpression of programmed cell death ligand 1 (PD-L1) in Irf1-/- tumors only partially restores tumor growth, suggesting multifactorial effects of IRF1 on antitumor immunity. Thus, we identify that IRF1 expression in tumor cells opposes host IFN-I- and IRF1-dependent antitumor immunity to facilitate immune escape and tumor growth

The role of residual antigen and of the alarmin IL-33 in shaping the antiviral immune response

The aims of the thesis were threefold: Firstly, we were interested in studying the potency of residual LCMV antigen-depots in transiently CD4 T cell depleted mice for the induction of an antiviral B cell response. Secondly, we studied whether a heterologous arenavirus vector prime-boost was beneficial for the establishment of an effective tumor-specific CD8 T cell response. Thirdly, we aimed to define the role of the IL-33 ST2 axis for the formation of an anti-viral CD8 T cell response in the context of a chronic viral infection

Deciphering T-cell exhaustion in the tumor microenvironment: paving the way for innovative solid tumor therapies

In solid tumors, the tumor microenvironment (TME) is a complex mix of tumor, immune, stromal cells, fibroblasts, and the extracellular matrix. Cytotoxic T lymphocytes (CTLs) constitute a fraction of immune cells that may infiltrate into the TME. The primary function of these T-cells is to detect and eliminate tumor cells. However, due to the immunosuppressive factors present in the TME primarily mediated by Myeloid-Derived Suppressor Cells (MDSCs), Tumor associated macrophages (TAMs), Cancer Associated Fibroblasts (CAFs) as well as the tumor cells themselves, T-cells fail to differentiate into effector cells or become dysfunctional and are unable to eliminate the tumor. In addition, chronic antigen stimulation within the TME also leads to a phenomenon, first identified in chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, where the T-cells become exhausted and lose their effector functions. Exhausted T-cells (Tex) are characterized by the presence of remarkably conserved inhibitory receptors, transcription and signaling factors and the downregulation of key effector molecules. Tex cells have been identified in various malignancies, including melanoma, colorectal and hepatocellular cancers. Recent studies have indicated novel strategies to reverse T-cell exhaustion. These include checkpoint inhibitor blockade targeting programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (Tim-3), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), or combinations of different immune checkpoint therapies (ICTs) or combination of ICTs with cytokine co-stimulation. In this review, we discuss aspects of T-cell dysfunction within the TME with a focus on T-cell exhaustion. We believe that gaining insight into the mechanisms of T-cell exhaustion within the TME of human solid tumors will pave the way for developing therapeutic strategies to target and potentially re-invigorate exhausted T-cells in cancer

Metabolic reprogramming in the tumor microenvironment: unleashing T cell stemness for enhanced cancer immunotherapy

T cells play a pivotal role in the immune system by distinguishing between various harmful pathogens and cancerous cells within the human body and initiating an immune response. Within the tumor microenvironment (TME), immune effector T cells encounter both immunosuppressive cells and factors that hinder their functionality. Additionally, they endure robust and persistent antigenic stimulation, often leading to exhaustion and apoptosis. However, the stemness of T cells, characterized by their ability to survive and self-renew over extended periods, represents a primary target in immune checkpoint therapies such as anti-PD-1 therapy. T cell stemness encompasses specific memory T cell subsets and progenitor-exhausted T cells with stem cell-like properties. Therefore, understanding the impact of the TME on T cell stemness, including factors like K+, lactate, and H+, holds significant importance and can facilitate the mitigation of terminal T-cell depletion, the identification of potential resilient biomarkers or therapeutic targets resistant to immune checkpoint therapies, and ultimately lead to sustained anti-tumor effects. Thus, it offers a novel perspective for advancing tumor immunotherapy

Dissecting the immune environment in glioblastoma

Glioblastoma (GBM) is the most common primary brain tumour in adults. Unfortunately, it is also the most malignant, conferring a median survival of only 6 months, increasing to 14-24 months with maximal therapy. Despite much effort, there have been relatively little effective advances made for the treatment of GBM in the last decade. Immunotherapy, especially checkpoint inhibition, has revolutionised the management of some previously untreatable cancers, such as metastatic melanoma. However, such treatments have yet to be shown to have any significant effect for the treatment of GBM. This is in part due to the low tumour mutational burden and poor immunogenicity of this immune ‘cold’ tumour. In this thesis I show that there are potential neoantigen encoding mutations, as well as a wide range of cancer testis antigens, that are not expressed or minimally expressed in GBM. Treatment of U87MG and GBM primary cell lines with the DNA methlytransferase inhibitor decitabine, leads to an increase in the expression of these immunogenic antigens due to hypomethylation. This increase in antigen expression leads to increased T cell recognition, activation and killing in a major histocompatibility complex – T cell receptor (TCR) dependent fashion. Further experiments are planned to translate these in vitro findings into an in vivo mouse model with a view to establishing a clinical trial. Secondly, I also present single cell TCR sequencing data performed on primary and recurrent GBM samples from 13 patients. I show that the TCR diversity is much reduced in the tumour samples with many more expanded T cell clones. I further show that testing of the expanded TCR reveals some of these T cell clones are tumour reactive, but they are not the most significantly expanded clones. Further work is being undertaken to further characterise the tumour specific TCR identified in more detail as well as interrogate the clonal TCR seen in other patients

T cells in the microenvironment of solid pediatric tumors: the case of neuroblastoma

Neuroblastoma (NB) is an immunologically “cold” tumor with poor or no inflamed substrates as most of solid pediatric tumors (SPT). Consistent data indicate that NB tumor microenvironment (TME) is dominated by myeloid cells, with little (but variable) T cell infiltration. The obstacles to lymphocyte infiltration and to their anti-tumor activity are due to different tumor immune evasion strategies, including loss of HLA Class I molecules, high expression of immune checkpoint molecular ligands leading to exhaustion of T effector (and NK) cells, induction of T regulatory, myeloid and stromal cells and secretion of immunosuppressive mediators. In odds with adult solid tumors, NB displays weak immunogenicity caused by intrinsic low mutational burden and scant expression of neoepitopes in the context of MHC-class I antigens which, in turn, are particularly poorly expressed on NB cells, thus inducing low anti-tumor T cell responses. In addition, NB is generated from embryonal cells and is the result of transcriptional abnormalities and not of the accumulation of genetic mutations over time, thus further explaining the low immunogenicity. The poor expression of immunogenic molecules on tumor cells is associated with the high production of immunosuppressive factors which further downregulate lymphocyte infiltration and activity, thus explaining the limited efficacy of new drugs in NB, as immune checkpoint inhibitors. This review is focused on examining the role of T effector and regulatory cells infiltrating TME of NB, taking into account their repertoire, phenotype, function, plasticity and, importantly, predictive value for defining novel targets for therapy

A single-cell multi-omic approach to the analysis of T cell differentiation

This thesis aims to investigate T cell differentiation through the bioinformatic analysis of single-cell multi-omic data. T cells are an important part of the adaptive immune system, involved in the immune response to infections and cancer. Single-cell technologies have advanced to the point where multiple modes of data, such as gene and protein expression, can be assayed on the same cells. Greater understanding of T cell differentiation pathways at the single-cell level can help in the design of immunotherapies to treat cancer and autoimmune disease. The thesis begins by presenting a multi-omic workflow that combines scRNA-seq and T cell receptor (TCR) sequence extraction. The principles developed for this workflow were applied to investigate T cell differentiation in two scenarios. The first scenario was an application of single-cell multi-omics to the study of CD8+ T cell peripheral tolerance mechanisms in a mouse model. This work demonstrated that tolerance is a distinct differentiation program to functional effector responses, and T cells progressively commit to the tolerised state over the first 60hrs post exposure to triggering antigen. A gene signature for the tolerised state was identified, containing genes uniquely upregulated in tolerised cells. Quiescent and Proliferating clusters were found in tolerised cells, indicating that a proportion of cells exit cell cycle within each division. The second scenario was an investigation of the differentiation of CD4+ CAR T cells in vivo, and the evolution of a lymphoma derived from these cells. Three cell types, proliferating, cytotoxic and resting, were observed within the malignant CAR T-cells, and these types were also observed within non-malignant CAR T and endogenous CD4+ T cells. The lymphoma was characterised by expression of the NF-κB transcription factor in all three cell types, while each cell type had differing expression levels for several other known oncogenes. This thesis has contributed to the understanding of T cell differentiation in tolerance and CAR T therapy, and has helped meet the challenge of increasingly large and complex single- cell datasets through the development of bioinformatic workflows to integrate samples from multiple patients and sequencing technologies, and integrate gene, protein, TCR sequence, cell division count and somatic mutation data at the single-cell level

Characterization of CD8+ T-CELL populations of the human peripheral blood

Tese de doutoramento (Registo), Univ. de Lisboa, nº 28, 2009, (Immunologie), Université René Descartes - Paris V, 2009Following antigenic challenge, naïve CD8+ T lymphocytes undergo severalchanges, including the expression of cell-surface molecules. In humans, theassociation of CCR7, CD45RA, CD27 and CD28 is widely used to discriminate areproducible set of functionally different subpopulations of CD8+ T cells. However,the prevailing data concerning the description of these subsets remainsfragmentary, since a multitude of studies used a different and limited set of surfacemarkers. Hence, some CD8+ T-cell subsets are still not clearly established,especially within the CCR7 CD45RA+ and CCR7 CD45R0+ compartments, andthe correspondent differential roles and lineage relationships remain undisclosed.The present study aims to define a predictable and precise correlationbetween particular cell surface markers and CD8+ T-cell functional properties. Weassociated CCR7, CD45RA, CD27 and CD28 expression levels to subdivide CD8+T cells into fourteen different cell types. These populations were further isolatedand gene expression of 18 genes was assessed, simultaneously, in single-cells bya novel multiplex RT-PCR method we developed. Our results demonstrate that thedifferent subpopulations display distinct and characteristic gene co-expressionpatterns, reproducible between donors. CD45RA expression is required to definethe naïve subset, but does not discriminate functionally different populations ofprimed cells. In contrast, gene expression profiles of CCR7-CD8+ T cells correlatesignificantly to CD27 expression levels and CD27/CD28 co-expression, and ahierarchy of activation stages could be established as follows: naïve < CD27high <CD27+CD28+ < CD28+CD27 < CD27+CD28 < CD27 CD28 . Surprisingly, wefound that CD45RA+ and CD45RA cells of each of these subsets had the samegene expression patterns at both qualitative and quantitative level. Importantly, weidentified minor subsets displaying characteristics of recent activation that could befound in both CD45RA+ and CD45RA compartments. These findings stronglysuggest that differentiation of naïve CD8+ T cells into effectors does notnecessarily imply CD45RA downregulation. Furthermore, they describe novelCD8+ T cell subsets and establish a correlation between surface phenotype andcell function, which helped to identify homogeneous populations