Production of IL2 and induction of IL2 receptor: two independent and dissociable events during allogenic stimulation in vitro in man

Donors from HLA genotyped families are extremely useful for immunogenetic studies. Analysis of the in vitro allogenic response of lymphocytes from HLA recombinant siblings can dissect the respective role of HLA class I and class II antigens for the lymphocyte activation. Using lymphocytes from such donors, we have demonstrated that IL2 production is triggered by HLA class II disparities (but not by HLA class I), HLA class I disparities can prime the cytotoxic T lymphocytes precursors inducing the IL2 receptor on the specific clones which will proliferate in presence of IL2 either from endogenous origin (in the case of HLA class II disparities) or from experimentally added IL2 in the culture.     

Interleukin-15 is a major regulator of the cell-microenvironment interactions in human renal homeostasis

Experiments in IL-15^?/? and IL-15Rα^?/? mice show that intra-renal IL-15, through IL-15Rα behaves as an epithelial survival factor. Recent data highlight new functions of IL-15 in renal homeostasis mediated by IL-15Rγ (CD132). Indeed, in CD132+ renal epithelial tubular cells IL-15 preserves E-cadherin expression inhibiting epithelial-mesenchymal transition (EMT). By contrast, during allograft rejection, the increased intra-graft IL-15 expression favors tubular destruction facilitating the intraepithelial recruitment of CD8 T cells expressing the E-cadherin ligand CD103. In renal cancer, loss of CD132 by epithelial cells defines a tumoral microenvironment where IL-15 triggers E-cadherin down-regulation and EMT. Finally, in CD132+ renal cancer stem cells IL-15 induces the generation of non-tumorigenic epithelial cells sensitive to cytotoxic drugs. These findings are discussed in the light of IL-15-based immunotherapy for renal cancer.     

Anticancer immunotherapy by CTLA-4 blockade: obligatory contribution of IL-2 receptors and negative prognostic impact of soluble CD25

The cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibody ipilimumab induces immune-mediated long-term control of metastatic melanoma in a fraction of patients. Although ipilimumab undoubtedly exerts its therapeutic effects via immunostimulation, thus far clinically useful, immunologically relevant biomarkers that predict treatment efficiency have been elusive. Here, we show that neutralization of IL-2 or blocking the α and β subunits of the IL-2 receptor (CD25 and CD122, respectively) abolished the antitumor effects and the accompanying improvement of the ratio of intratumoral T effector versus regulatory cells (Tregs), which were otherwise induced by CTLA-4 blockade in preclinical mouse models. CTLA-4 blockade led to the reduction of a suppressive CD4⁺ T cell subset expressing Lag3, ICOS, IL-10 and Egr2 with a concomitant rise in IL-2-producing effector cells that lost FoxP3 expression and accumulated in regressing tumors. While recombinant IL-2 improved the therapeutic efficacy of CTLA-4 blockade, the decoy IL-2 receptor α (IL-2Rα, sCD25) inhibited the anticancer effects of CTLA-4 blockade. In 262 metastatic melanoma patients receiving ipilimumab, baseline serum concentrations of sCD25 represented an independent indicator of overall survival, with high levels predicting resistance to therapy. Altogether, these results unravel a role for IL-2 and IL-2 receptors in the anticancer activity of CTLA-4 blockade. Importantly, our study provides the first immunologically relevant biomarker, namely elevated serum sCD25, that predicts resistance to CTLA-4 blockade in patients with melanoma.     

Tumor-associated macrophages (TAMs) form an interconnected cellular supportive network in anaplastic thyroid carcinoma

Background

A relationship between the increased density of tumor-associated macrophages (TAMs) and decreased survival was recently reported in thyroid cancer patients. Among these tumors, anaplastic thyroid cancer (ATC) is one of the most aggressive solid tumors in humans. TAMs (type M2) have been recognized as promoting tumor growth. The purpose of our study was to analyze with immunohistochemistry the presence of TAMs in a series of 27 ATC.

Methodology/Principal Findings

Several macrophages markers such as NADPH oxidase complex NOX2-p22phox, CD163 and CD 68 were used. Immunostainings showed that TAMs represent more than 50% of nucleated cells in all ATCs. Moreover, these markers allowed the identification of elongated thin ramified cytoplasmic extensions, bestowing a “microglia-like” appearance on these cells which we termed “Ramified TAMs” (RTAMs). In contrast, cancer cells were totally negative. Cellular stroma was highly simplified since apart from cancer cells and blood vessels, RTAMs were the only other cellular component. RTAMs were evenly distributed and intermingled with cancer cells, and were in direct contact with other RTAMs via their ramifications. Moreover, RTAMs displayed strong immunostaining for connexin Cx43. Long chains of interconnected RTAMs arose from perivascular clusters and were dispersed within the tumor parenchyma. When expressed, the glucose transporter Glut1 was found in RTAMs and blood vessels, but rarely in cancer cells.

Conclusion

ATCs display a very dense network of interconnected RTAMs in direct contact with intermingled cancer cells. To our knowledge this is the first time that such a network is described in a malignant tumor. This network was found in all our studied cases and appeared specific to ATC, since it was not found in differentiated thyroid cancers specimens. Taken together, these results suggest that RTAMs network is directly related to the aggressiveness of the disease via metabolic and trophic functions which remain to be determined.     

Human CD5 protects circulating tumor antigen-specific CTL from tumor-mediated activation-induced cell death

We previously characterized several tumor-specific T cell clones from PBL and tumor-infiltrating lymphocytes of a lung cancer patient with identical TCR rearrangements and similar lytic potential, but with different antitumor response. A role of the TCR inhibitory molecule CD5 to impair reactivity of peripheral T cells against the tumor was found to be involved in this process. In this report, we demonstrate that CD5 also controls the susceptibility of specific T cells to activation-induced cell death (AICD) triggered by the tumor. Using a panel of tumor-infiltrating lymphocytes and PBL-derived clones expressing different levels of CD5, our results indicate that T lymphocyte AICD in response to the cognate tumor is inversely proportional to the surface expression level of CD5. They also suggest a direct involvement of CD5 in this process, as revealed by an increase in tumor-mediated T lymphocyte AICD following neutralization of the molecule with specific mAb. Mechanistically, our data indicate that down-regulation of FasL expression and subsequent inhibition of caspase-8 activation are involved in CD5-induced T cell survival. These results provide evidence for a role of CD5 in the fate of peripheral tumor-specific T cells and further suggest its contribution to regulate the extension of CTL response against tumor.     

Interleukin-15 plays a central role in human kidney physiology and cancer through the γc signaling pathway

The ability of Interleukin-15 (IL-15) to activate many immune antitumor mechanisms renders the cytokine a good candidate for the therapy of solid tumors, particularly renal cell carcinoma. Although IL-15 is being currently used in clinical trials, the function of the cytokine on kidney's components has not been extensively studied; we thus investigated the role of IL-15 on normal and tumor renal epithelial cells. Herein, we analyzed the expression and the biological functions of IL-15 in normal renal proximal tubuli (RPTEC) and in their neoplastic counterparts, the renal clear cell carcinomas (RCC). This study shows that RPTEC express a functional heterotrimeric IL-15Rαβγc complex whose stimulation with physiologic concentrations of rhIL-15 is sufficient to inhibit epithelial mesenchymal transition (EMT) commitment preserving E-cadherin expression. Indeed, IL-15 is not only a survival factor for epithelial cells, but it can also preserve the renal epithelial phenotype through the γc-signaling pathway, demonstrating that the cytokine possess a wide range of action in epithelial homeostasis. In contrast, in RCC in vitro and in vivo studies reveal a defect in the expression of γc-receptor and JAK3 associated kinase, which strongly impacts IL-15 signaling. Indeed, in the absence of the γc/JAK3 couple we demonstrate the assembly of an unprecedented functional high affinity IL-15Rαβ heterodimer, that in response to physiologic concentrations of IL-15, triggers an unbalanced signal causing the down-regulation of the tumor suppressor gene E-cadherin, favoring RCC EMT process. Remarkably, the rescue of IL-15/γc-dependent signaling (STAT5), by co-transfecting γc and JAK3 in RCC, inhibits EMT reversion. In conclusion, these data highlight the central role of IL-15 and γc-receptor signaling in renal homeostasis through the control of E-cadherin expression and preservation of epithelial phenotype both in RPTEC (up-regulation) and RCC (down-regulation).     

Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function

Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21(waf/CIP1). Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway.     

Generation of CTL recognizing an HLA-A*0201-restricted epitope shared by MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 tumor antigens: implication in a broad-spectrum tumor immunotherapy

MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 are expressed in a significant proportion of primary and metastatic tumors of various histological types and are targets of tumor Ag-specific CTL. Individual MAGE-A expression varies from one tumor type to the other but, overall, the large majority of tumors expresses at least one MAGE-A Ag. Therefore, targeting epitopes shared by all MAGE-A Ags would be of interest in immunotherapy against a broad spectrum of cancers. In the present study, we describe a heteroclitic MAGE-A peptide (p248V9) that induces CTL in vivo in HLA-A*0201 transgenic HHD mice and in vitro in healthy donors. These CTL are able to recognize two low HLA-A*0201 affinity peptides differing at their C-terminal position and derived from MAGE-A2, -A3, -A4, -A6, -A10, and -A12 (p248G9) and MAGE-A1 (p248D9). Interestingly, p248V9-specific CTL respond to endogenous MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 in an HLA-A*0201-restricted manner and recognize human HLA-A*0201(+)MAGE-A(+) tumor cells of various histological origin. Therefore, this heteroclitic peptide may be considered as a potent candidate for a broad-spectrum tumor vaccination.     

Allogeneic T cell activation triggering by MHC class I antigens

The role of MHC-encoded class I molecules in allogeneic activation and proliferation of human T lymphocytes was investigated. The study was performed by using primary mixed culture of lymphocytes from MHC recombinant siblings identical for MHC class II Ag (DR, DP, DQ) and displaying MHC class I disparity. The results indicate that such allogeneic combination is sufficient to trigger early activation steps within responder T cells without promoting a significant proliferation. After MHC class I allosensitization, a significant proportion of cells entered the cell cycle (G0----G1). The stimulatory potential of MHC class I Ag was further stressed by the specific induction on responder cells of IL-2R (22% T cell activation Ag positive). Under the same experimental conditions, transferrin receptor expression and IL-2 activity were not detectable. This is consistent with the low T cell proliferation. Exogenous rIL-1 did not improve IL-2 production and the subsequent T cell proliferation indicating that these two events were not associated with a defective accessory cell function involving IL-1 release. MHC class I disparity can also prime precursor CTL to differentiate into IL-2-dependent functional MHC restricted cytotoxic T cells. Conversely IFN-gamma had no effect. Addition to the culture of W6/32, a mAb specifically directed against a monomorphic determinant on human class I HLA-A, -B, and -C Ag was able to block all these activation events. These data clearly indicate a role of HLA class I Ag involvement in the early events triggering allogeneic T cell activation.