Emmanuel Scotet

The interplay between the duration of TCR and cytokine signaling determines T cell polarization

Development of Th1 and Th2 effector lymphocytes is driven primarily by IL‐12 or IL‐4, but is also influenced by the strength of antigenic stimulation. However, the mechanism by which TCR signaling contributes to T cell polarization remains elusive. We show that in the presence of IL‐12 a short TCR stimulation can lead to efficient Th1 polarization and IL‐12 exerts its effect when present during, as well as after, TCR signaling. In contrast, Th2 polarization requires a prolonged TCR stimulation and IL‐4 is effective only when present during the period of TCR triggering. The simultaneous stimulation by TCR and IL‐4 is required to induce demethylation of IL‐4 and IL‐13 genes that accompanies the stochastic generation of Th2 cells producing either or both cytokines. Thus, the duration of TCR stimulation represents a crucial parameter that influences the response to polarizing cytokines and the acquisition of T cell effector functions.

Early Triggering of Exclusive IFN-γ Responses of Human Vγ9Vδ2 T Cells by TLR-Activated Myeloid and Plasmacytoid Dendritic Cells

γδ T cells, a major innate-like T cell subset, are thought to play in vivo an important role in innate and adaptive immune responses to various infection agents like parasites, bacteria, or viruses but the mechanisms contributing to this immune process remain ill defined. Owing to their ability to recognize a broad set of microbial molecular patterns, TLRs represent a major innate pathway through which pathogens induce dendritic cells (DC) maturation and acquisition of immunostimulatory functions. In this study, we studied the effects of various TLR ligands on the activation of human Vγ9Vδ2 T cells, a main human γδ PBL subset, which has been recently involved in the licensing of mycobacteria-infected DC. Both TLR3 and TLR4, but not TLR2 ligands, induced a rapid, strong, and exclusive IFN-γ production by Vγ9Vδ2 T cells. This γδ subset contributed to a large extent to the overall PBL IFN-γ response induced after short-term TLR stimulation of human PBMC. Importantly, this phenomenon primarily depended on type I IFN, but not IL-12, produced by monocytic DC upon TLR engagement. Vγ9Vδ2 T cells were similarly activated by plasmacytoid DC upon TLR8/9 activation or Yellow Fever virus infection. Moreover TLR-induced Vγ9Vδ2 IFN-γ noncytolytic response led to efficient DC polarization into IL-12p70-producing cells. Our results support an adjuvant role played by Vγ9Vδ2 T cells along microbial infections through a particular cross-talk with pathogen-associated molecular patterns-activated DC. Moreover they provide new insights into the mechanisms underlying functional activation of this unique peripheral innate-like T cell subset during viral infections.

Optimizing anti-CD3 affinity for effective T cell targeting against tumor cells

Bispecific antibodies binding to the TCR/CD3 complex and to a tumor‐associated surface molecule can be used to target cytotoxic T lymphocytes against tumor cells. We reasoned that high‐affinity binding to CD3 may reduce the efficiency of T cell stimulation and target the bispecific reagent to T cells rather than to tumor cells in vivo. We therefore mutated a bispecific single‐chain antibody (BscAb) directed to human CD3 and EpCAM to generate variants that bind to CD3 with higher or lower affinity. When compared to the wild‐type molecule, a mutant with increased binding to CD3 showed lower capacity to target T cells against an EpCAM+ tumour. In contrast, mutants with decreased binding to CD3, in spite of rapid dissociation, efficiently triggered T cell activation and cytotoxicity, especially when present on tumor cells at low copy number. These results are consistent with the TCR serial triggering model and suggest that BscAb with extremely low affinityfor the TCR‐CD3 complex could be exploited therapeutically because of their preferential localization to tumor cells.

IL-21-Mediated Potentiation of Antitumor Cytolytic and Proinflammatory Responses of Human Vγ9Vδ2 T Cells for Adoptive Immunotherapy

Vγ9Vδ2 T lymphocytes are a major human γδ T cell subset that react against a wide array of tumor cells, through recognition of phosphorylated isoprenoid pathway metabolites called phosphoantigens. Immunotherapeutic protocols targeting Vγ9Vδ2 T cells have yielded promising, yet limited, signs of antitumor efficacy. To improve these approaches, we analyzed the effects on γδ T cells of IL-21, a cytokine known to enhance proliferation and effector functions of CD8+ T cells and NK cells. IL-21 induced limited division of phosphoantigen-stimulated Vγ9Vδ2 T cells, but did not modulate their sustained expansion induced by exogenous IL-2. Vγ9Vδ2 T cells expanded in the presence of IL-21 and IL-2 showed enhanced antitumor cytolytic responses, associated with increased expression of CD56 and several lytic molecules, and increased tumor-induced degranulation capacity. IL-21 plus IL-2-expanded Vγ9Vδ2 T cells expressed higher levels of inhibitory receptors (e.g., ILT2 and NKG2A) and lower levels of the costimulatory molecule NKG2D. Importantly, these changes were rapidly and reversibly induced after short-term culture with IL-21. Finally, IL-21 irreversibly enhanced the proinflammatory Th1 polarization of expanded Vγ9Vδ2 T cells when added at the beginning of the culture. These data suggest a new role played by IL-21 in the cytotoxic and Th1 programming of precommitted Ag-stimulated γδ T cells. On a more applied standpoint, IL-21 could be combined to IL-2 to enhance γδ T cell-mediated antitumor responses, and thus represents a promising way to optimize immunotherapies targeting this cell subset.

1 Frameshifting as a Novel Mechanism to Generate a Cryptic Cytotoxic T Lymphocyte Epitope Derived from Human Interleukin 10

Recent data indicate that some cytotoxic T cells (CTLs) recognize so-called cryptic epitopes, encoded by nonprimary open reading frame (ORF) sequences or other nonclassical expression pathways. We describe here a novel mechanism leading to generation of a cryptic CTL epitope. We isolated from the synovial fluid of a patient suffering from a Reiters syndrome an autoreactive T cell clone that recognized cellular IL-10 in the HLA-B*2705 context. The minimal IL-10 sequence corresponding to nucleotides 379–408 was shown to activate this clone, upon cotransfection into COS cells with the DNA encoding HLA-B*2705, but the synthetic peptide deduced from this sequence did not stimulate the clone. Using a site-directed mutagenesis approach, we found that this clone recognized a transframe epitope generated by an internal +1 frameshifting in the IL-10 sequence and so derived partly from ORF1, partly from ORF2. We defined that +1 frameshifting was induced by a specific heptamer sequence. These observations illustrate the variety of mechanisms leading to generation of cryptic epitopes and suggest that frameshifting in normal cellular genes may be more common than expected.

Exon III splicing switch of fibroblast growth factor (FGF) receptor-2 and -3 can be induced by FGF-1 or FGF-2

An essential feature of fibroblast growth factor receptors (FGFRs) is the existence of multiple possibilities of alternative splicing. One of these concerns sequences of the mRNA coding for the C-terminal half of Ig domain 3 which corresponds to a part of the ligand-binding site: two alternative exons, IIIb and IIIc, encode the C-terminal half of Ig domain 3. The IIIb/IIIc choice in the FGFR-2 and FGFR-3 is strictly tissue-specific, the IIIb exon being expressed exclusively in epithelial cells. We describe here a reversible switch from IIIb to IIIc for FGFR-2 and FGFR-3 under the influence of exogenous and endogenous FGF-1 or FGF-2. We observed that FGF-induced FGF receptor exon switching (i) occurred as early as 1 h after exposure to FGF (ii) was receptor-mediated (iii) was dependent on cell confluency and showed a link with the cell cycle (iv) was correlated with a reversible loss of epithelial properties. These results support a role for FGF in the regulation of expression of alternatively spliced FGFR mRNA.

Overexpression of vascular endothelial growth factor induces cell transformation in cooperation with fibroblast growth factor 2.

Vascular endothelial growth factor (VEGF) is a family of homodimeric proteins produced from a single gene by alternative splicing of the VEGF transcript. VEGF induces in vivo angiogenesis and vascular permeability. We have recently demonstrated that VEGF is an autocrine growth factor for retinal pigment epithelial (RPE) cells. To further understand the role of VEGF, we overexpressed VEGF in rat RPE cells. The transfected cells exhibited a growth advantage in vitro and an increased response to the mitogenic effect of fibroblasts growth factor-2 (FGF-2), and formed colonies in soft agar upon FGF-2 addition. Moreover, analysis of FGF-receptors evidenced a dramatic increase in FGFR-1 mRNA and protein level, supporting the hypothesis that this receptor mediates the transforming effect of FGF-2. These results reveal that the oncogenic role of VEGF is exerted through a cross regulation between VEGF and FGF signal transduction pathways.

Frequent recognition of BCRF1, a late lytic cycle protein of Epstein-Barr virus, in the HLA-B*2705 context: evidence for a TAP-independent processing.

Using a transient COS transfection assay, allowing a rapid estimation of the dominant CD8+ T cell responses against a large number of HLA/viral protein combinations within polyclonal cell lines, we searched for HLA‐B*2705‐restricted CD8 T cell responses to Epstein‐Barr virus (EBV) within T cell samples enriched for EBV‐reactive cells. Among the 18 EBV proteins tested, only 2, the latent protein EBNA3A and the late lytic protein BCRF1 (viral IL‐10), appeared dominant in the B27 context, as they triggered significant TNF and cytolytic responses in some donors. We provide evidence that the B27/BCRF1 epitope (RRLVVTLQC) is located in the signal sequence and that it can be presented in a TAP‐independent manner. Using B27/BCRF1 monomeric complexes coated on immunomagnetic beads, we sorted out BCRF1‐specific CD8 T cells from 8 of 15 HLA‐B27+ donors. This is, to our knowledge, the first demonstration of a recognition of BCRF1, suggesting that some immune control against EBV exists even during the late stage of the lytic cycle. This result also strengthens the unusual ability of HLA‐B*2705 to present peptide in a TAP‐independent manner.

Impact on early outcomes and immune reconstitution of high-dose post-transplant cyclophosphamide vs anti-thymocyte globulin after reduced intensity conditioning peripheral blood stem cell allogeneic transplantation

We have compared prospectively the outcome and immune reconstitution of patients receiving either post-transplant cyclophosphamide (PTCY) (n = 30) or anti-thymocyte globulin ATG (n = 15) as Graft-versus-host disease (GVHD) prophylaxis after reduced-intensity conditioning (RIC) allogeneic peripheral blood stem cell (PBSC) transplantation (allo-SCT). The outcome and immune reconstitution of patients receiving either of these two regimens were compared prospectively. This study allowed also to investigate the impact of PTCY between haplo-identical vs matched donors and of clofarabine as part of the RIC regimen. The γ/δ T-cells, α/β T-cells (CD8+ and CD4+), NK T-cells, NK cells, B-cells, Tregs and monocytes were analyzed by flow cytometry from a total of 583 samples. In the PTCY group significant delayed platelets recovery, higher CD3+ donor chimerism, higher HHV-6 and lower EBV reactivations were observed. Early survival advantage for CD4+ T-cells, Tregs and α/β T-cells was documented in the PTCY group while it was the case for α/β T-cells, NK cells and monocytes in the ATG group. Higher counts of NK and monocytes were observed at days +30 and/or day+60 in the ATG group. Both results were retained even in the case of mismatched donors. However, higher percentages of CD4+ T-cells, α/β T-cells and Tregs were observed with haplo-identical donors in the PTCY group. Finally, clofarabine was responsible for early survival advantage of NK T-cells in the PTCY group while it abrogated the early survival advantage of γ/δ T-cells in the ATG group. In conclusion, there are marked differences in the immunological effects of ATG vs PTCY as GVHD prophylaxis for RIC PBSC allo-SCT.

CD277 an Immune Regulator of T Cell Function and Tumor Cell Recognition

Molecules involved in tumor cell recognition are potentially important targets in cancer therapeutics. Therefore, research on the identification of tumor cell antigens as well as, immune-regulatory molecules is essential. The study of new co-stimulatory molecules has attracted special interest due to, their role in activation and/or inhibition of effector function of immune cells. In this chapter, we focused on the study of CD277 and its putative counterreceptor, and their possible role in activation or inhibition of immune cells and recognition of cancer cells. CD277 has been involved in immune cells regulation, because of its role in activating or inhibiting effector, cytokine secretion and cytotoxic functions, depending on the activating conditions of these cells. Moreover, the fact that the stimulation of tumor cell lines with antiCD277 leave to a better recognition and killing by ┛├ and ┙┚ T cells lead us to postulate that these molecules is involved also in tumor recognition. We will discuss different aspects of CD277 and its counter-receptor in this chapter. Classically, the immune response has been divided into innate and adaptive immunity, with distinct properties. The innate immune response uses a small number of receptors that detect a limited set of conserved antigens. Induction of the adaptive immune response involves antigen presenting cells (APC) and T cells (Borghesi & Milcarek, 2007). The interaction between the T cell and the APC is the pivotal step that controls a series of events, including T cell activation, cell division and effector differentiation (Zhu & Chen, 2009). However, recognition of the processed antigenic peptide coupled to Major