Therapeutic activity of GARP:TGF-ß1 blockade in murine primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by the clonal expansion of myeloid cells, notably megakaryocytes (MKs), and an aberrant cytokine production leading to bone marrow (BM) fibrosis and insufficiency. Current treatment options are limited. TGF-ß1, a profibrotic and immunosuppressive cytokine, is involved in PMF pathogenesis. While all cell types secrete inactive, latent TGF-ß1, only a few activate the cytokine via cell type-specific mechanisms. The cellular source of the active TGF-ß1 implicated in PMF is not known. Transmembrane protein GARP binds and activates latent TGF-ß1 on the surface of regulatory T lymphocytes (Tregs) and MKs or platelets. Here, we found an increased expression of GARP in the BM and spleen of mice with PMF and tested the therapeutic potential of a monoclonal antibody (mAb) that blocks TGF-ß1 activation by GARP-expressing cells. GARP:TGF-ß1 blockade reduced not only fibrosis but also the clonal expansion of transformed cells. Using mice carrying a genetic deletion of Garp in either Tregs or MKs, we found that the therapeutic effects of GARP:TGF-ß1 blockade in PMF imply targeting GARP on Tregs. These therapeutic effects, accompanied by increased IFN-γ signals in the spleen, were lost upon CD8 T-cell depletion. Our results suggest that the selective blockade of TGF-ß1 activation by GARP-expressing Tregs increases a CD8 T-cell-mediated immune reaction that limits transformed cell expansion, providing a novel approach that could be tested to treat patients with myeloproliferative neoplasms.

IRF4 impedes human CD8 T cell function and promotes cell proliferation and PD-1 expression.

Human CD8 tumor-infiltrating lymphocytes (TILs) with impaired effector functions and PD-1 expression are categorized as exhausted. However, the exhaustion-like features reported in TILs might stem from their activation rather than the consequence of T cell exhaustion itself. Using CRISPR-Cas9 and lentiviral overexpression in CD8 T cells from non-cancerous donors, we show that the T cell receptor (TCR)-induced transcription factor interferon regulatory factor 4 (IRF4) promotes cell proliferation and PD-1 expression and hampers effector functions and expression of nuclear factor κB (NF-κB)-regulated genes. While CD8 TILs with impaired interferon γ (IFNγ) production exhibit activation markers IRF4 and CD137 and exhaustion markers thymocyte selection associated high mobility group box (TOX) and PD-1, activated T cells in patients with COVID-19 do not demonstrate elevated levels of TOX and PD-1. These results confirm that IRF4+ TILs are exhausted rather than solely activated. Our study indicates, however, that PD-1 expression, low IFNγ production, and active cycling in TILs are all influenced by IRF4 upregulation after T cell activation.

Immunosuppressive low-density neutrophils in the blood of cancer patients display a mature phenotype.

The presence of human neutrophils in the tumor microenvironment is strongly correlated to poor overall survival. Most previous studies have focused on the immunosuppressive capacities of low-density neutrophils (LDN), also referred to as granulocytic myeloid-derived suppressor cells, which are elevated in number in the blood of many cancer patients. We observed two types of LDN in the blood of lung cancer and ovarian carcinoma patients: CD45high LDN, which suppressed T-cell proliferation and displayed mature morphology, and CD45low LDN, which were immature and non-suppressive. We simultaneously evaluated the classical normal-density neutrophils (NDN) and, when available, tumor-associated neutrophils. We observed that NDN from cancer patients suppressed T-cell proliferation, and NDN from healthy donors did not, despite few transcriptomic differences. Hence, the immunosuppression mediated by neutrophils in the blood of cancer patients is not dependent on the cells' density but rather on their maturity.

HELIOS-expressing human CD8 T cells exhibit limited effector functions.

Introduction: The transcription factor HELIOS is primarily known for its expression in CD4 regulatory T cells, both in humans and mice. In mice, HELIOS is found in exhausted CD8 T cells. However, information on human HELIOS+ CD8 T cells is limited and conflicting. Methods: In this study, we characterized by flow cytometry and transcriptomic analyses human HELIOS+ CD8 T cells. Results: These T cells primarily consist of memory cells and constitute approximately 21% of blood CD8 T cells. In comparison with memory HELIOS- T-BEThigh CD8 T cells that displayed robust effector functions, the memory HELIOS+ T-BEThigh CD8 T cells produce lower amounts of IFN-γ and TNF-α and have a lower cytotoxic potential. We wondered if these cells participate in the immune response against viral antigens, but did not find HELIOS+ cells among CD8 T cells recognizing CMV peptides presented by HLA-A2 and HLA-B7. However, we found HELIOS+ CD8 T cells that recognize a CMV peptide presented by MHC class Ib molecule HLA-E. Additionally, a portion of HELIOS+ CD8 T cells is characterized by the expression of CD161, often used as a surface marker for identifying TC17 cells. These CD8 T cells express TH17/TC17-related genes encoding RORgt, RORa, PLZF, and CCL20. Discussion: Our findings emphasize that HELIOS is expressed across various CD8 T cell populations, highlighting its significance beyond its role as a transcription factor for Treg or exhausted murine CD8 T cells. The significance of the connection between HELIOS and HLA-E restriction is yet to be understood.

MDSC in Mice and Men: Mechanisms of Immunosuppression in Cancer.

Myeloid-derived suppressor cells (MDSCs) expand during pathological conditions in both humans and mice and their presence is linked to poor clinical outcomes for cancer patients. Studying MDSC immunosuppression is restricted by MDSCs' rarity, short lifespan, heterogeneity, poor viability after freezing and the lack of MDSC-specific markers. In this review, we will compare identification and isolation strategies for human and murine MDSCs. We will also assess what direct and indirect immunosuppressive mechanisms have been attributed to MDSCs. While some immunosuppressive mechanisms are well-documented in mice, e.g., generation of ROS, direct evidence is still lacking in humans. In future, bulk or single-cell genomics could elucidate which phenotypic and functional phenotypes MDSCs adopt in particular microenvironments and help to identify potential targets for therapy.

Reverse immunology: From peptide sequence to tumor-killing human T-cell clones.

Recent advances in next generation sequencing expanded the availability of tumor mutanome data that list the mutations present in cancer cells. Mutated proteins are an interesting source of neoantigens that can be used to specifically target tumor cells in the context of immunotherapy. However, identifying new antigenic peptides from mutated proteins remains challenging. In this chapter, we present Reverse Immunology as an approach to identify potential antigens from any given polypeptide sequence. First, we explain the rationale behind the identification of candidate HLA-binding peptides through mass spectrometry or in silico approaches. Then, we describe the isolation of low-frequency T-cell precursors specific for the candidate peptides using peptide-HLA multimers. Finally, we discuss validation steps leading to the identification of a T-cell clone recognizing tumor cells that endogenously process the candidate peptide. We also present approaches to study the impact of the proteasome complex on candidate peptide processing.

Protocol to assess the suppression of T-cell proliferation by human MDSC.

Inhibition of T-cell proliferation is the most common approach to assess human myeloid-derived suppressor cell (MDSC) functions. However, diverse methodologies hinder the comparison of results obtained in different laboratories. In this chapter, we present a T-cell proliferation assay procedure based on allogeneic MDSC and T-cells that is potentially suitable to multi-center studies. The T-cells are isolated from non-cancerous donors and frozen for later use in different research groups. We observed that pure thawed T-cells showed poor proliferative capacities. To retain proliferation, T-cell-autologous mature dendritic cells are supplemented after thawing. MDSC are isolated from clinical samples and represent the sole variant between assays. Flow cytometry is used to assess T-cell proliferation by the dilution of a tracking dye.