A novel mass cytometry protocol optimized for immunophenotyping of low-frequency antigen-specific T cells.

Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for flow and mass cytometry. Our method can be used for primary cells of both mouse and human origin for the detection of low-frequency T-cell response using a dual-barcoding system for individual samples and conditions. First, live-cell barcoding was performed using anti-CD45 antibodies prior to an in vitro T-cell stimulation assay. Second, to discriminate between stimulation conditions and prevent cell loss, sample barcoding was combined with a commercial barcoding solution. This dual-barcoding approach is cell sparing and, therefore, particularly relevant for samples with low cell numbers. To further reduce cell loss and to increase debarcoding efficiency of multiplexed samples, we combined our dual-barcoding approach with a new centrifugation-free washing system by laminar flow (Curiox™). Finally, to demonstrate the benefits of our established protocol, we assayed virus-specific T-cell response in SARS-CoV-2-vaccinated and SARS-CoV-2-infected patients and compared with healthy non-exposed individuals by a high-parameter CyTOF analysis. We could reveal a heterogeneity of phenotypes among responding CD4, CD8, and gd-T cells following antigen-specific stimulations. Our protocol allows to assay antigen-specific responses of minute populations of T cells to virus-derived peptides, allergens, or other antigens from the same donor sample, in order to investigate qualitative and quantitative differences.

Extracellular vesicles as a platform to study cell-surface membrane proteins.

Producing intact recombinant membrane proteins for structural studies is an inherently challenging task due to their requirement for a cell-lipid environment. Most of the procedures developed involve isolating the protein by solubilization with detergent and further reconstitutions into artificial membranes. These procedures are highly time consuming and suffer from further drawbacks, including low yields and high cost. We describe here an alternative method for rapidly obtaining recombinant cell-surface membrane proteins displayed on extracellular vesicles (EVs) derived from cells in culture. Interaction between these membrane proteins and ligands can be analyzed directly on EVs. Moreover, EVs can also be used for protein structure determination or immunization purposes.

IFNγ producing CD8+ T cells modified to resist major immune checkpoints induce regression of MHC class I-deficient melanomas.

Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8+ T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8+ T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8+ T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8+ T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8+ T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8+ T cells to inhibition both by PD-1/PDL-1 engagement and by TGFß1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients.

Control of CD8 T cell proliferation and terminal differentiation by active STAT5 and CDKN2A/CDKN2B.

CD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and long-term replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to γc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16(INK) (4A) transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16(INK) (4A) or p19(ARF) expression (as shown using T cells from CDKN2A(-/-) mice) but was associated with expression of transcripts encoding p15(INK) (4B) , another protein involved in senescence induction. We conclude that T-cell-receptor- and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16(INK) (4A) and p19(ARF) .

Active STAT5 regulates T-bet and eomesodermin expression in CD8 T cells and imprints a T-bet-dependent Tc1 program with repressed IL-6/TGF-ß1 signaling.

In adoptive therapy, CD8 T cells expressing active STAT5 (STAT5CA) transcription factors were found to be superior to unmanipulated counterparts in long-term persistence, capacity to infiltrate autochthonous mouse melanomas, thrive in their microenvironment, and induce their regression. However, the molecular mechanisms sustaining these properties were undefined. In this study, we report that STAT5CA induced sustained expression of genes controlling tissue homing, cytolytic granule composition, type 1 CD8 cytotoxic T cell-associated effector molecules granzyme B(+), IFN-γ(+), TNF-α(+), and CCL3(+), but not IL-2, and transcription factors T-bet and eomesodermin (Eomes). Chromatin immunoprecipitation sequencing analyses identified the genes possessing regulatory regions to which STAT5 bound in long-term in vivo maintained STAT5CA-expressing CD8 T cells. This analysis identified 34% of the genes differentially expressed between STAT5CA-expressing and nonexpressing effector T cells as direct STAT5CA target genes, including those encoding T-bet, Eomes, and granzyme B. Additionally, genes encoding the IL-6R and TGFbRII subunits were stably repressed, resulting in dampened IL-17-producing CD8 T cell polarization in response to IL-6 and TGF-ß1. The absence of T-bet did not affect STAT5CA-driven accumulation of the T cells in tissue or their granzyme B expression but restored IL-2 secretion and IL-6R and TGFbRII expression and signaling, as illustrated by IL-17 induction. Therefore, concerted STAT5/T-bet/Eomes regulation controls homing, long-term maintenance, recall responses, and resistance to polarization towards IL-17-producing CD8 T cells while maintaining expression of an efficient type 1 CD8 cytotoxic T cell program (granzyme B(+), IFN-γ(+)).

Immunosuppression in inflammatory melanoma: can it be resisted by adoptively transferred T cells?

Discovery of tumor antigen (TA) recognized by autologous T cells (TCs) in patients with melanoma has led to clinical protocols using either vaccination or adoptive transfer of TA-specific TCs. However, efficacy of these treatments has been hampered by inhibitory effects exerted on tumor-infiltrating TCs by tumor-intrinsic mediators or by recruitment of immunosuppressive cells. A mouse model of autochthonous melanoma recapitulates some aspects of inflammatory melanoma development in patients. These include a systemic Th2-/Th17-oriented chronic inflammation, recruitment of immunosuppressive myeloid cells and acquisition by tumor-infiltrating TCs of an 'exhausted' phenotype characterized by expression of multiple inhibitory receptors including programmed death-1, also expressed on patients' melanoma-infiltrating TCs. Rather than using extracellular blocking reagents to inhibitory surface molecules on TCs, we sought to dampen negative signaling exerted on them. Adoptively transferred TCs presenting increased cytokine receptor signaling due to expression of an active Stat5 transcription factor were efficient at inducing melanoma regression in the preclinical melanoma model. These transferred TCs thrived and retained expression of effector molecules in the melanoma microenvironment, defining a protocol endowing TCs with the ability to resist melanoma-induced immunosuppression.

Activated STAT5 promotes long-lived cytotoxic CD8+ T cells that induce regression of autochthonous melanoma.

Immunotherapy based on adoptive transfer of tumor antigen-specific CD8(+) T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8(+) effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytoma- or melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti-IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8(+) TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs.