Anti-tumor efficacy of a combination therapy with PD-L1 targeted alpha therapy and adoptive cell transfer of PD-1 deficient melanoma-specific human T-lymphocytes.

The optimization of adoptive transfer approaches of anti-tumor T cells requires both the functional improvement of the injected T cells and the modulation of the tumor microenvironment, favoring the recruitment of these T cells and their activation. We have recently shown the therapeutic benefit of two approaches tested individually in a melanoma model wich were on one hand the adoptive transfer of specific T cells deficient for the expression of the inhibitory receptor PD-1, and on the other hand PD-L1 targeted alpha therapy (TAT). In this study, we sought to investigate the efficacy of these two therapies combined, compared to each monotherapy, in order to evaluate the synergy between these two approaches, in the same melanoma model. Here we used melanoma-specific T-cell clones, previously validated for the edition of PDCD1 gene and with previously demonstrated superior anti-tumor activity than their wild-type counterparts, after adoptive transfer in NSG mice engrafted with PD-L1 expressing human melanoma tumors. We also used a previously validated TAT approach, using a 213Bi-anti-human-PD-L1 mAb, alone or in combination with adoptive cell transfer, in the same mouse model. We confirmed previous results obtained with each monotherapy and documented the safety and the superior ability of a combination between the adoptive transfer of PD-1 deficient T cells and TAT targeting PD-L1 to control the growth of melanoma tumors in NSG mice. This study provides the first proof-of-concept of the efficacy of a combination therapy using TAT, adoptive cell transfer and genomic editing of IC-coding genes.

Immunodominant CD8 T cell response to Epstein-Barr virus.

Epstein-Barr virus (EBV) provides one of the most informative systems for analysing cytotoxic T lymphocyte responses in humans. The viral infection and its persistence are the results of an alternation of lytic and latent phases that are controlled by the immune response. Using a transient COS transfection assay that permits semi-quantitative estimation of CD8 T cell responses against a large number of HLA/viral protein combinations, we analyzed responses to EBV within a large number of polyclonal T cell lines. This allowed a rapid identification of major epitopes and the demonstration that EBV-specificT cells were mainly directed against a restricted set of immunodominant epitopes, primarily generated during the early lytic cycle. Knowledge of the antigen specificity of CDB T cell responses against EBV should help generate cytotoxic T cell lines to this herpesvirus, and more generally to study the molecular basis of immunodominance.

Molecular regulation of CC-chemokine receptor 3 expression in human T helper 2 cells.

In developing T helper 1 (Th1) and Th2 cells the acquisition of effector function is intimately connected with the acquisition of new migratory capacities, as exemplified by differential expression of chemokine receptors. This study investigates the molecular mechanisms responsible for Th2-restricted expression of the CC-chemokine receptor 3 (CCR3). The minimal promoter in T cells was identified in the -149 base pair (bp) upstream sequence that contains a positive regulatory element. A strong negative element was also localized in the flanking intronic sequence. The study further investigates the role of chromatin remodeling in the regulation of this Th2-specific gene. Drugs that affect the chromatin structure facilitate CCR3 expression in T cells. Furthermore, in differentiating Th2 cells, selected regions are associated with acetylated-H3 histones and become more accessible to DNase I. These results suggest that in Th2 cells both cytokine production and migratory capacity are regulated through a similar mechanism involving chromatin remodeling.

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.

Regulation of inhibitory and activating killer-cell Ig-like receptor expression occurs in T cells after termination of TCR rearrangements.

A small fraction of T cells expresses killer-cell Ig-like receptors (KIR), a family of MHC class I-specific receptors that can modulate TCR-dependent activation of effector functions. Although KIR(+) cells are enriched within Ag-experienced T cell subsets, the precise relationships between KIR(+) and KIR(-) T cells and the stage of KIR induction on these lymphocytes remain unclear. In this study, we compared KIR(-) and KIR(+) alphabeta T cell clones, sorted by means of the CD158b (KIR2DL2/KIR2DL3/KIR2DS2) specific mAb GL183. We isolated several pairs of CD158b(+) and CD158b(-) alphabeta T cell clones sharing identical productive and nonproductive TCR transcripts. We showed that expression of functional KIR on T cells is regulated after termination of TCR rearrangements. Transcriptional regulation of KIR genes was documented in multiple T cell clones generated from the same donor, and the presence of KIR transcripts was also detected in KIR(-) T cells. These results document a complex regulation of KIR expression in T cells at both pre and posttranscriptional levels, under the control of yet undefined signals provided in vivo.

A global appraisal of immunodominant CD8 T cell responses to Epstein-Barr virus and cytomegalovirus by bulk screening.

Knowledge of the immunodominant responses to Epstein-Barr Virus (EBV) and human cytomegalovirus (HCMV) should help to generate cytotoxic T cell lines to these herpesviruses. Here we report on the analysis of CD8 T cell responses to EBV and HCMV in the blood of kidney transplant recipients undergoing viral reactivation (n = 16) and in healthy virus carriers (n = 10). We used a transient COS transfection assay that permits semi-quantitative estimation of CD8+ T cell responses against a larger number of HLA/viral protein combinations within polyclonal T cell lines and thus allows a rapid identification of major epitopes. From the comparison of these responses to those that we previously described in the synovial fluid of patients suffering from various forms of chronic arthritis (n = 32), it appears that EBV-specific T cells are mainly directed against a restricted set of immunodominant epitopes, primarily generated during the early lytic cycle and that both IE1 and pp65 are targets of the anti-HCMV response. We suggest that this method could be generally applied to the rapid identification of immunodominant T cell epitopes in viral and tumor immunity, and could help selecting HLA-peptide complexes that could be used to detect and sort specific T cell populations.

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.

Frequent enrichment for CD8 T cells reactive against common herpes viruses in chronic inflammatory lesions: towards a reassessment of the physiopathological significance of T cell clonal expansions found in autoimmune inflammatory processes.

We recently evidenced a dramatic enrichment for T cells reactive against Epstein-Barr virus (EBV) within inflamed joints of two rheumatoid arthritis patients. To assess the generality of this phenomenon and its relevance to autoimmunity, we studied the responses of CD8 T cells from patients with either acute or chronic inflammatory diseases (rheumatoid arthritis: n = 18, ankylosing spondylitis: n = 5, psoriatic arthritis: n = 4, Reiter's syndrome: n = 3, arthrosis: n = 2, uveitis: n = 2, multiple sclerosis: n = 2, encephalitis: n = 1) against viral proteins derived from EBV and another common herpes virus, human cytomegalovirus (CMV). T cell responses against EBV and/or CMV epitopes were frequently observed within CD8 T cells derived from chronic inflammatory lesions, irrespective of their location (knee, eye, brain) and autoimmune features. In most cases, CD8 T cells derived from affected organs yielded stronger anti-viral T cell responses than CD8 T cells derived from patients' PBL, even in chronic inflammatory diseases devoid of autoimmune features or induced by defined bacterial agents. Taken together, these results suggest that the presence of virus-specific T cells within inflamed lesions of patients suffering from autoimmune diseases is a general phenomenon associated with chronic inflammation rather than the initiating cause of the autoimmune process. Since this phenomenon was sometimes associated with long-term T repertoire biases within inflamed lesions, the physiopathological significance of T cell clonal expansions found in a recurrent fashion within chronically inflamed autoimmune lesions should be interpreted with caution.

EBV gene expression not altered in rheumatoid synovia despite the presence of EBV antigen-specific T cell clones.

T cells infiltrating the rheumatoid arthritis (RA) joint are oligoclonal, implicating an Ag-driven process, but the putative joint-specific Ags remain elusive. Here we examine expression of selected EBV genes in RA synovia and find no abnormal expression in RA. DNA of CMV and EBV was detectable by PCR in the synovial tissue of RA. RNA of several latent and lytic EBV genes was also detectable. However, there were no differences in EBV gene expression in synovial tissues or peripheral blood when comparing RA with osteoarthritis, Gulf War syndrome, and other disease controls. RA synovia with highly expanded CD8 T cell clones reactive with defined EBV peptide Ags presented by HLA class I alleles lacked evidence of abnormal mRNA expression for the relevant EBV Ag (BZLF1) or lacked amplifiable mRNA (BMLF1). Thus, local production of EBV Ags in synovial tissues may not be the cause of the accumulation of T cell clones specific for these Ags. Instead, APCs loaded with processed EBV peptides may migrate to the synovium. Alternatively, EBV-specific T cells clones may be generated in other tissues and then migrate to synovia, perhaps due to cross-reactive joint-specific Ags or because of expression of homing receptors.

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.

T cell response to Epstein-Barr virus transactivators in chronic rheumatoid arthritis.

Rheumatoid arthritis is a multistep disorder associated with autoimmune features of yet unknown etiology. Implication of viruses such as Epstein-Barr virus (EBV) in rheumatoid arthritis pathogenesis has been suspected on the basis of several indirect observations, but thus far, a direct link between EBV and rheumatoid arthritis has not been provided. Here we show that a large fraction of T cells infiltrating affected joints from a patient with chronic rheumatoid arthritis recognizes two EBV transactivators (BZLF1 and BMLF1) in a major histocompatibility complex-restricted fashion. Responses to these EBV antigens by synovial lymphocytes from several other chronic rheumatoid arthritis patients were readily detectable. Thus these results suggest a direct contribution of EBV to chronic rheumatoid arthritis pathogenesis. They also demonstrate for the first time the occurrence of T cell responses against EBV transactivating factors, which might be central in the control of virus reactivation.

The choice between alternative IIIb and IIIc exons of the FGFR-3 gene is not strictly tissue-specific.

An essential feature of fibroblast growth factor receptors (FGFR) is the existence of multiple possibilities for 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. We show here that the alternative splicing choice between IIIb and IIIc exons of the FGFR-3 is not strictly tissue-specific: epithelial cells show exclusively IIIb transcripts while fibroblastic cells show a mixture of IIIb and IIIc transcripts. This is in contrast with the strictly exclusive alternative choice between IIIb or IIIc exons of the FGFR-2 gene: epithelial cells make only the IIIb choice while fibroblastic cells make only the IIIc choice.

Oncoprotein fos activation in epithelial-cells induces an epitheliomesenchymal conversion and changes the receptor encoded by the fgfr-2 messenger-RNA from k-sam to bek.

Activation of c-Fos, by using an inducible c-Fos estrogen receptor fusion protein, triggers the epitheliofibroblastoid cell conversion of mouse mammary epithelial cells. We show that this change in phenotype is accompanied by a definitive switch of the fibroblast growth factor receptor 2 from K-SAM to BEK. This splicing switch occurs a few hours after estrogen stimulation. Our data suggest that Fos proteins could be important in modulating the FGFR-2 splicing choice. Moreover, these observations reinforce previous evidence that the BEK/K-SAM choice is strictly tissue-specific: the K-SAM exon is expressed exclusively in epithelial cells, the BEK exon in cells of the fibroblastic type.