Anne-Marie Schmitt-Verhulst

Lymphoid Cell Surface Interaction Structures Detected Using Cytolysis‐Inhibiting Monoclonal Antibodies

We screened monoclonal antibodies obtained by xenogeneic immunization for their capacity to inhibit T cell-mediated cytolysis. These antibodies fell into two classes according to the cell structures they recognized, of 30-35 K and 94-180 K apparent molecular weight, respectively. The main features of these structures and of their interaction with the corresponding antibodies were reviewed. The inhibition of cytolysis by these antibodies was shown to occur mainly at the effector cell level, at the recognition stage of cytolysis, and to depend on the nature of target cells, effector cells, and link between these cells. T cell functions other than cytolysis were also inhibited by some of these antibodies. We considered various possible mechanisms to account for the inhibition of cytolysis by these mAb. We favor an hypothesis based on inhibition by these mAb of lymphoid cell surface interaction structures. This hypothesis was discussed within the general framework of cell interaction structures in immunological and non-immunological experimental systems.

Major Histocompatibility Complex Restricted Cell-Mediated Immunity

Publisher Summary The major histocompatibility complex (MHC) has been found to code for cell surface antigens involved in allograft rejection. This genetic region has also been shown to control immune response potential in a number of animal species. In the mouse, the MHC, known as the H-2 complex, has been divided into four major regions: K, I , S, and D. The K and D regions determine the strong serologically detectable transplantation antigens of the mouse, which are important for tissue graft rejection. The chapter also discusses the different ways in which the respective antigenic determinants (e.g., virus, hapten, or minor antigen) can be presented in association with H-2-coded products on the cell surface. Moreover, the chapter also discusses CML model. One advantage that the chemically modified CML model has over the viral and weak transplantation antigen models is the potential for investigating the antigenic contribution made by the modifying agent to the fine specificity of the T-cell recognition. The fact that some of the H-2-restricted syngeneic CML models are under the control of H-2-linked Ir genes as well as being restricted with K and D region products, suggests multiple functional roles for H-2-coded gene products in cell-mediated immunity. Finally, the chapter concludes that MHC products may be simultaneously important for recognition by T lymphocytes and for the H-2-restricted structures which they recognize the K, I, or D region products associated with the infectious agent.

Direct evidence for chromosomal inversion during T-cell receptor beta-gene rearrangements.

A germline T-cell receptor variable region (Vβ) gene segment (Vβ14) has been mapped 10 kilobases to the 3′ side of the constant region (Cβ2) gene. The Vβ14 gene segment is in an inverted transcriptional polarity relative to the diversity-region (Dβ) and joining-region (Jβ) gene segments and the Cβ genes. Analyses of a T-cell clone (J6.19), which has productively rearranged the Vβ14 gene segment, indicate that the productive Vβ-Dβ-Jβ rearrangement and its reciprocal flank recombination product are linked and located at either border of a chromosomal inversion. These data demonstrate for the first time a linkage between mammalian V and C genes and verify that a functional T-cell receptor Vβ gene can be constructed through a chromosomal inversion.

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.

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 TGFβ1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients.

Granzyme B-tdTomato, a new probe to visualize cytolytic effector cell activation

An important function of the immune system consists in eliminating infected or transformed cells. Naive CD8 T lymphocytes differentiate in peripheral lymphoid organs following a first antigen contact. There they acquire the different constituents of the cytolytic machinery and become cytolytic T lymphocytes (CTLs), before migration to the tissues where they meet their specific target. Target cell killing is mediated by the release of granules expressing the Lamp-1 marker [1] and containing effector proteins including perforin [2, 3] and granzymes (granzyme A (GZMA) and B (GZMB) being the main proteases). Effective target cell lysis depends on many factors; so deciphering the mechanisms involved is important, in particular to palliate the failings of the immune system during tumor development. Transient labeling of acidic granules with Lysotracker has elegantly been used to analyze kinetics of granule polarization in CTL/target conjugates. Intracellular staining of fixed and permeabilized cells has allowed elucidation of important steps of CTL granule movements, fusion and degranulation [4–6]. In order to develop a fluorescent probe that would stably label the contents of cytolytic granules in living cells, we designed a construct encoding a fusion protein composed of an N-terminal GZMB, a 12 amino-acid linker and a C-terminal tdTomato (tdTom) (excitation: 554 nM, emission: 581 nm, stable at the acidic pH of the granules (pKa 4.7) [7], GZMB-tdTom). This was inserted in the retroviral expression vector MSCV-IRESHuCD2t (Supporting Information Fig. 1). We first transduced a T-cell hybridoma (HybT) and obtained stable expression of GZMB-tdTom in granules co-expressing GZMB and Lamp-1 (Supporting Information Fig. 2–5). Immunoblots revealed the fusion protein GZMB-tdTom at 85 kDa and tdTom at 55 kDa MW, as expected (Supporting Information Fig. 4). GZMB enzymatic activity could be detected in GZMB-tdTom-HybT cells, albeit at a low level as compared with that in CTLs (Supporting Information Fig. 5D). Whether this results from incomplete processing of the protein in HybT cells requires further investigation (Supporting Information Fig. 5D). To address more physiological conditions, we transduced normal CD8 CTLs with the GZMB-tdTom construct (Supporting Information Fig. 6). As observed by confocal microscopy, the GZMB-tdTom fusion protein was localized in granules (Fig. 1A). Co-localization between GZMB-tdTom, Lamp-1 and GZMB was observed in granules of CTLs alone (Fig. 1B-i) in CTL/antigenic target conjugates (Fig. 1B-ii) that had re-localized the red granules to the cell–cell contact zone, and in conjugates of CTLs with targets presenting control peptide (Fig. 1B-iii). Redistribution of granules to the CTL/ target contact zone was observed in 80% of antigen-specific versus 5% of nonspecific conjugates (Supporting Information Fig. 7B). TdTom-transduced cells expressed red tdTom protein spread throughout the cytoplasm (Fig. 1B-iv) and similarly to untransduced CTLs (Supporting Information Fig. 7A) relocalized GZMB-containing granules expressing Lamp-1 to the CTL/target contact zone (Fig. 1B-iv). Mathematical analyses showed that GZMB-tdTom colocalized with Lamp-1 and GZMB (Pearson’s Rr coefficient around 0.55) whereas tdTom did not show any colocalization (Rr 0.1) (Supporting Information Fig. 7C). Following TCR/antigen engagement, calcium flux and PKC activation are important signals for gene activation and granule migration to the CTL/target contact zone preceding degranulation [4, 8]. CTLs preloaded with Fluo-4 were used to monitor by video microscopy the Ca fluxes and the redistribution of GZMB-tdTom-containing granules. When GZMB-tdTom-transduced P14-TCR CTLs faced a specific target, an attachment signal preceded a rapid Ca flux (10–20 s) and granule translocation to the contact zone occurring at various times (20–480 s) (Fig. 1C-i and ii, Supporting Information Fig. 7D, Video 1). No significant signal was observed when the CTLs were facing control targets (Fig. 1C-iii and iv, Video 2). These kinetics are in agreement with published studies using CTL clones [6], [9]. We used the Lamp-1 exposure method to assess CTL degranulation in response to antigenic stimulation and to observe the fate of GZMB-tdTom during that process. GZMB-tdTom-transduced P14-TCR CTLs exposed Lamp-1 in response to gp33loaded RMA-S, the extent of degranulation being dependent on peptide concentration (Fig. 2A). The percent of GZMB-tdTom fluorescent CTLs markedly decreased (from 20% for non-stimulated or controlpeptide stimulated CTLs to 13% for CTLs activated with 10 6 M gp33-loaded RMA-S), with a level of GZMB-tdTom fluorescence much lower in Lamp-1–positive (MRFI 422 (MRFI, mean relative fluorescence intensity)) as compared to Lamp-1–negative (607) CTLs. GZMB expression as measured on fixed and permeabilized cells were also reduced (about 50%) in the antigen-

Functional Relationships of Lymphocyte Membrane Structures Probed with Cytolysis and/or Proliferation-Inhibiting H35-27.9 and H35-89.9 Monoclonal Antibodies

The mechanism(s) of T lymphocyte “functions” such as cytolysis (1–4) or proliferation involve those cell surface structures that insure specific recognition and may involve other cell surface structures as well. Detection of these may be via the use of monoclonal antibodies (mAb) selected for their ability to inhibit lymphocyte functions. Indeed, anti-Lyt-2 mAb have been extensively studied as to their inhibitory effect on mouse T cell-mediated cytolysis, with repeated suggestions that Lyt-2 itself may be related to the T cell specific receptor (5–10). We have developed a range of xenogeneic rat anti-mouse mAb selected for their ability to inhibit T cell-mediated cycolysis (11,12). Three of them will be used in the present report: H35-17.2 mAb, which is most probably an anti- Lyt-2 mAb, as an experimental counterpoint to the two other mAb; H35-27.9 mAb, which differs from an anti-Lyt-2 mAb at least by the tissue distribution of the structures it recognizes; and H35-89.9 mAb, which immunoprecipitates from lymphoid cell surfaces two polypeptides of 180K and 94K molecular weight.

Abstract A083: Molecular profiling of CD8 T cells from autochthonous melanoma identifies Maf as driver of T cell exhaustion

T-cells infiltrating neoplasms express surface molecules typical of chronically virus-stimulated T-cells, often termed “exhausted” T-cells. We analyzed the transcriptome of “exhausted” CD8 T-cells infiltrating autochthonous melanomas compared to those of naive and acutely stimulated CD8 T-cells. In spite of strong similarities between transcriptional signatures of tumor- and virally-induced exhausted CD8 T-cells, notable differences appeared. Among transcriptional regulators, Nr4a2 was expressed in both exhaustions, whereas Maf was highly over-expressed only in tumor-exhausted T-cells. Anti-tumor CD8 T-cells transduced to express Maf showed dampened anti-tumor activity upon adoptive transfer, whereas Nr2a4 over-expression was without effect. Maf-expressing CD8 T-cells showed unaltered homeostasis but failed to accumulate in tumor-bearing hosts and developed defective anti-tumor secondary responses. We also found that Maf expression in CD8 T cells induced part of the transcriptional program associated with tumor-induced exhaustion. We further identified TGFβ and IL-6 as main inducers of Maf expression in CD8 T-cells and showed that Maf-deleted tumor specific CD8 T-cells were much more potent to restrain tumor growth in vivo. Therefore the melanoma microenvironment contributes to skewing of CD8 T-cell differentiation programs, in part by TGFβ/IL-6 mediated induction of Maf. Citation Format: Gregory Verdeil, Marilyn Giordano, Anne-Marie Schmitt-Verhulst, Daniel Speiser, Claire Imbratta. Molecular profiling of CD8 T cells from autochthonous melanoma identifies Maf as driver of T cell exhaustion [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A083.

Unleashing antitumor T-cell activation without ensuing autoimmunity: the case for A20-deletion in adoptive CD8+ T-cell therapy

Mechanisms controlling immune reactivity prevent excessive inflammation and autoimmunity, but generally dampen antitumor activity. We recently showed that adoptively transferred antitumor CD8+ T cells harboring a deletion of A20/Tnfaip3, a molecule controlling NF-κB activation, possessed heightened antitumor activity in vivo. The boosted immunity of A20-deleted CD8+ T cells correlated with a heightened capacity to produce IFNγ and TNFα while expressing reduced levels of the immune checkpoint molecule PD-1.

GENERATION OF TNP-SPECIFIC H-2-RESTRICTED MURINE CYTOTOXIC CELLS AS A FUNCTION OF TNP-CELL SURFACE PRESENTATION

Publisher Summary This chapter highlights the generation of trinitrophenyl (TNP) specific H-2-restricted murine cytotoxic cells as a function of TNP-cell surface presentation. The generation of the cytotoxic effectors sensitized by the addition of trinitrobenzene sulfonate-modified cells on TNP proteins is dependent on the presence of both T lymphocytes and a population of glass-adherent, radio resistant cells. The chemically modified H-2-restricted CML model may not depend on actual covalent conjugation of the TNP group to H-2-coded cell surface products and, therefore, could be similar to the H-2 -restricted viral and minor transplantation antigen models. These findings could also raise the possibility that H-2 -restricted cytotoxic immune reactions involving autoimmunity may be generated by soluble components associated with autologous cells. The analysis of the antigenicity and the immunogenicity of TNP-presenting cells as a target or stimulator for TNP-specific, H-2-restricted T-cell mediated cytotoxicity is summarized as a function of the mode of presentation of the TNP moiety on the cell surface.