Lack of MHC class II molecules favors CD8+ T-cell infiltration into tumors associated with an increased control of tumor growth.

Regulatory T-cells (Tregs) are crucial for the maintenance of immune tolerance and homeostasis as well as for preventing autoimmune diseases, but their impact on the survival of cancer patients remains controversial. In the TC-1 mouse model of human papillomavirus (HPV)-related carcinoma, we have previously demonstrated that the therapeutic efficacy of the CyaA-E7-vaccine, targeting the HPV-E7 antigen, progressively declines with tumor growth, in correlation with increased intratumoral recruitment of Tregs. In the present study, we demonstrated that these TC-1 tumor-infiltrating Tregs were highly activated, with increased expression of immunosuppressive molecules. Both intratumoral effector CD4+ T-cells (Teffs) and Tregs expressed high levels of PD-1, but anti-PD-1 antibody treatment did not impact the growth of the TC-1 tumor nor restore the therapeutic effect of the CyaA-E7 vaccine. To analyze the mechanisms by which Tregs are recruited to the tumor site, we used MHC-II KO mice with drastically reduced numbers of CD4+ effector T-cells. We demonstrated that these mice still had significant numbers of Tregs in their lymphoid organs which were recruited to the tumor. In MHC-II KO mice, the growth of the TC-1 tumor was delayed in correlation with a strong increase in the intratumoral recruitment of CD8+ T-cells. In addition, in mice that spontaneously rejected their tumors, the infiltration of E7-specific CD8+ T-cells was significantly higher than in MHC-II KO mice with a growing tumor. These results demonstrate that tumor-specific CD8+ T-cells can be efficiently activated and recruited in the absence of MHC class II molecules and of CD4+ T-cell help.

The T-cell receptor repertoire of tumor-infiltrating regulatory T lymphocytes is skewed toward public sequences.

The accumulation of CD4(+) T regulatory cells (Treg) in tumor tissue is a widely described phenomenon in mouse models and in human cancer patients. Understanding the mechanisms by which Treg migrate and accumulate in tumors is important because they strongly influence the potential efficacy of many immunotherapies. In this study, we used immunoscope technology to analyze the T-cell receptor (TCR) repertoire of tumor-infiltrating T cells in non-TCR transgenic mice. Both tumor-infiltrating Tregs and T effector cells (Teff) displayed sequence profiles in the CDR3 region that were characteristic of biased repertoires seen during clonal cell expansions, implying that strong T-cell responses have occurred within the tumor tissue. By comparing the TCR sequences of tumor-infiltrating Tregs, we obtained evidence of the presence of so-called public TCR sequences that are common to many individuals yet were tumor-specific in nature. Such comparisons also suggested that the Treg-Teff conversion process is not an active process at the tumor site or tumor-draining lymph nodes. Our findings strongly suggest that Treg infiltration of tumor tissue is followed by marked proliferation of a few dominant T-cell clones in the tumor.

The adjuvant effect of TLR agonists on CD4(+) effector T cells is under the indirect control of regulatory T cells.

TLR agonists have been suggested to directly impact Tregs, thereby enhancing or reversing their suppressive function. Here, in order to select TLR agonists leading to potent effector T-cell responses, while minimizing Treg inhibitory function, we used a model antigen, covalently linked to an inert delivery system, combined with a large panel of TLR agonists, for the immunization of mice with an attenuated/depleted or intact Treg subset. We observed that the negative modulation of effector CD4(+) T cells exerted by Tregs cannot be circumvented, whatever the TLR agonist used as adjuvant. To better understand the impact of TLR agonists on Tregs, we investigated (i) the TLR expression profile of highly purified CD4(+) Foxp3(+) Tregs, at steady state or subsequent to in vivo activation by TLR agonists and (ii) the Treg phenotype after in vivo and in vitro activation by TLR agonists. Our results demonstrate that TLR agonists, as single signal inducers, are not able to directly activate Tregs. The phenotypic Treg activation observed in vivo, following TLR administration, does not result from cross-talk with conventional T cells but is rather a consequence of the interaction with other immune cell type(s).

Interleukin-24: a molecule with potential anti-cancer activity and a cytokine in search of a function.

The gene encoding Melanoma-differentiation antigen-7 (MDA-7) was discovered more than 10 years ago. Its potential anti-cancer activity was surmised because its expression is inversely correlated with the cell proliferation status. Indeed adenoviral delivery of this gene proved to be efficient in killing several cancer cell lines and great strides have been made concerning its molecular ways of action. Later it was shown that mda7 encoded a secreted cytokine which belongs to the IL-10, class-II family of cytokines. We recently found that this molecule exerted apoptotic activity on stimulating but not on resting lymphocytes from a B cell leukaemia. This activity is distinct from that of intracellular MDA-7, and may pave the way for using the cytokine in cancers provided that they express the IL-24 Receptors; in this respect, melanomas are insensitive to the recombinant cytokine due to the lack of IL-24 receptors at their surface. In contrast to its anti-cancer activity, the immunological role of IL-24 is still unclear, with differences between mice and human. If however it is demonstrated that IL-24 can inhibit the function of STAT3 in normal lymphocytes as it is the case in leukemic cells, and given that STAT3 is needed for the differentiation of several lymphocyte subsets, this will give us hints as to the potential role of this cytokine in the immune system.

IL-24 induces apoptosis of chronic lymphocytic leukemia B cells engaged into the cell cycle through dephosphorylation of STAT3 and stabilization of p53 expression.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of long-lived monoclonal B cells mostly arrested at the G(0)/G(1) phase of the cell cycle. CLL cells strongly express intracellular melanoma differentiation-associated gene-7 (MDA7)/IL-24. However, adenovirus-delivered MDA7 was reported to be cytotoxic in several tumor cell lines. We report herein that rIL-24 alone had no effect; however, sequential incubation with rIL-2 and rIL-24 reduced thymidine incorporation by 50% and induced apoptosis of CLL cells in S and G(2)/M phases of the cell cycle, but not of normal adult blood or tonsil B cells. IL-24 stimulated STAT3 phosphorylation in IL-24R1-transfected cells but not in normal or CLL B cells. In contrast, IL-24 reversed the IL-2-induced phosphorylation of STAT3 in CLL, and this effect was neutralized by anti-IL-24 Ab. Phospho- (P)STAT3 inhibition induced by IL-24 was reversed by pervanadate, an inhibitor of tyrosine phosphatases. The addition of rIL-24 to IL-2-activated CLL B cells resulted in increases of transcription, protein synthesis. and phosphorylation of p53. The biological effects of IL-24 were reversed by the p53 inhibitor pifithrin-alpha and partly by the caspase inhibitor zvad. Troglitazone (a protein tyrosine phosphatase, PTP1B activator) phosphatase inhibited PSTAT3 and augmented p53 expression. PSTAT3 is a transcriptional repressor of p53, and therefore IL-24 induction of p53 secondary to PSTAT3 dephosphorylation may be sensed as a stress signal and promote apoptosis in cycling cells. This model explains why IL-24 can protect some resting/differentiated cells and be deleterious to proliferating cells.

Natural phosphorylation of CD5 in chronic lymphocytic leukemia B cells and analysis of CD5-regulated genes in a B cell line suggest a role for CD5 in malignant phenotype.

Chronic lymphocytic leukemia (CLL) results in the accumulation of B cells, presumably reflecting the selection of malignant cell precursors with Ag combined with complex alterations in protein activity. Repeated BCR stimulation of normal B cells leads to anergy and CD5 expression, both of which are features of CLL. Because CD5 is phosphorylated on tyrosine following BCR engagement and negatively regulates BCR signaling in normal B cells, we investigated its phosphorylation status and found it to be naturally phosphorylated on tyrosine but not on serine residues in CLL samples. To analyze the role of CD5, we established a B cell line in which CD5 is phosphorylated. Gene profiling of vector vs CD5-transfected B cells pointed out gene groups whose expression was enhanced: Apoptosis inhibitors (BCL2), NF-kappaB (RELB, BCL3), Wnt, TGFbeta, VEGF, MAPKs, Stats, cytokines, chemokines (IL-10, IL-10R, IL-2R, CCL-3, CCL-4, and CCR7), TLR-9, and the surface Ags CD52, CD54, CD70, and CD72. Most of these gene groups are strongly expressed in CLL B cells as compared with normal B cells. Unexpectedly, metabolic pathways, namely cholesterol synthesis and adipogenesis, are also enhanced by CD5. Conversely, CD5 inhibited genes involved in RNA splicing and processing, ribosome biogenesis, proteasome, and CD80 and CD86 Ags, whose expression is low in CLL. Comparison of CD5- vs tailless CD5-transfected cells further demonstrated the role of CD5 phosphorylation in the regulation of selected genes. These results support a model where CLL cells are chronically stimulated, leading to CD5 activation and cell survival. In addition to CD5 itself, we point to several CD5-induced genes as potential therapeutic targets.

Cyclosporin-A inhibits ERK phosphorylation in B cells by modulating the binding of Raf protein to Bcl2.

Extracellular signal-related kinase (ERK) signaling is regulated by sequential phosphorylation of upstream kinases including Raf. We report herein that ERK phosphorylation is inhibited by a short incubation with Cyclosporin-A (CsA) in anti-IgM activated Daudi B cells. As Bcl2, through its BH4 domain, was previously shown to bind both Calcineurin (Can) and Raf proteins, we hypothesized that CsA inhibited Can binding to Bcl2 allowing the latter to bind more Raf at the mitochondria thereby diverting it from activating the ERK cascade. In support of this less Bcl2 coprecipitated with Can heterodimer in total lysates of cells treated with CsA as compared to controls. In parallel, Raf1 was augmented in both the mitochondrial fractions of cells treated with CsA and in Bcl2 immunoprecipitates under CsA. Finally, introduction of a Bcl2 BH4 domain into Daudi cells augmented ERK phosphorylation in unstimulated cells and this augmentation was unsensitive to CsA. We therefore suggest that CsA indirectly inhibited ERK activation through sequestration of Raf1, at the mitochondria.