Danila Valmori

Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming

The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8+ T cell responses, through in vivo cross-priming, has remained unclear. In this article, we report that repeated vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG ODN 7909, a potent stimulator of B cells and T helper type 1 (Th1)-type immunity, resulted in the early induction of specific integrated CD4+ Th cells and antibody responses in most vaccinated patients, followed by the development of later CD8+ T cell responses in a fraction of them. The correlation between antibody and T cell responses, together with the ability of vaccine-induced antibodies to promote in vitro cross-presentation of NY-ESO-1 by dendritic cells to vaccine-induced CD8+ T cells, indicated that elicitation of NY-ESO-1-specific CD8+ T cell responses by cross-priming in vivo was associated with the induction of adequate levels of specific antibodies. Together, our data provide clear evidence of in vivo cross-priming of specific cytotoxic T lymphocytes by a recombinant tumor antigen vaccine, underline the importance of specific antibody induction for the cross-priming to occur, and support the use of this type of formulation for the further development of efficient cancer vaccines.

Rapamycin-Mediated Enrichment of T Cells with Regulatory Activity in Stimulated CD4+ T Cell Cultures Is Not Due to the Selective Expansion of Naturally Occurring Regulatory T Cells but to the Induction of Regulatory Functions in Conventional CD4+ T Cells

Rapamycin is an immunosuppressive drug currently used in different clinical settings. Although the capacity of rapamycin to inhibit the mammalian target of rapamycin serine/threonine protein kinase and therefore T cell cycle progression is well known, its effects are complex and not completely understood. It has been reported recently that TCR-mediated stimulation of murine CD4+ T cells in the presence of rapamycin results in increased proportions of CD4+ T cells with suppressive functions, suggesting that the drug may also exert its immunosuppressive activity by promoting the selective expansion of naturally occurring CD4+ regulatory T cells (Treg). In this study, we show that stimulation of human circulating CD4+ T cells in the presence of rapamycin results indeed in highly increased suppressor activity. By assessing the effect of rapamycin on the growth of nonregulatory and Treg populations of defined differentiation stages purified ex vivo from circulating CD4+ T cells, we could demonstrate that this phenomenon is not due to a selective expansion of naturally occurring Tregs, but to the capacity of rapamycin to induce, upon TCR-mediated stimulation, suppressor functions in conventional CD4+ T cells. This condition, however, is temporary and reversible as it is dependent upon the continuous presence of rapamycin.

IL-1β and IL-2 convert human Treg into TH17 cells

Natural CD4(+)CD25(+) regulatory T cells (Treg) and interleukin 17 (IL-17)-producing T helper cells (T(H)17) carry out opposite functions, the former maintaining self-tolerance and the latter being involved in inflammation and autoimmunity. We report here that stimulation of human Natural Treg under T(H)17 polarizing conditions in the presence of IL-2 converts them into T(H)17 cells. Conversion of Tregs into T(H)17 cells occurs both from natural naïve Tregs (NnTregs) and, to a higher extent, from memory Tregs (MTregs). Among antigen presenting cells, fresh monocytes activated by microbial stimuli were the most efficient inducers of T(H)17 cells from Tregs. Conversion of Treg into T(H)17 cells was induced by IL-1beta and involved down-regulation of the Treg lineage transcription factor FOXP3 and suppressive functions. Our results indicate that, under inflammatory conditions, in the presence of IL-2, Treg can be converted into pro-inflammatory T(H)17 cells and establish a functional link between inflammation and autoimmunity.

Efficacy of Levo-1-Methyl Tryptophan and Dextro-1-Methyl Tryptophan in Reversing Indoleamine-2,3-Dioxygenase-Mediated Arrest of T-Cell Proliferation in Human Epithelial Ovarian Cancer

It has been reported that levo-1-methyl tryptophan (L-1MT) can block indoleamine-2,3-dioxygenase (IDO) expressed by human dendritic cells (DC), whereas dextro-1-methyl tryptophan (D-1MT) is inefficient. However, whether L-1MT or D-1MT can efficiently reverse IDO-induced arrest of human T-cell proliferation has not been clarified. Here, we show a marked immunosuppressive effect of IDO derived from INDO-transfected 293 cell, IDO+ ovarian cancer cells, and monocyte-derived DCs on CD4+ Th1 cells, CD8+ T cells, and natural killer cells derived from peripheral blood, ascites, and tumors of ovarian cancer patients. We found that, whereas L-1MT and D/L-1MT can restore proliferation of tumor-derived and peripheral blood T-cell subsets, D-1MT does not effectively restore IDO-induced arrest of T-cell proliferation. Although D-1MT inhibited kynurenine production at high concentrations, L-1MT was more effective in abrogating kynurenine generation and tryptophan depletion, whereas tryptophan was completely depleted by IDO even in the presence of high amounts of D-1MT. Together, the results indicate that, whereas the generation of tryptophan metabolites (kynurenines) by IDO is important in mediating suppression of T-cell proliferation, the degree to which tryptophan depletion is restored by 1MT is also critical in overcoming IDO-induced arrest of T-cell proliferation.

Cross-Presentation of HLA Class I Epitopes from Exogenous NY-ESO-1 Polypeptides by Nonprofessional APCs

NY-ESO-1, a germ cell Ag often detected in tumor tissues, frequently elicits Ab and CD8+ T cell responses in cancer patients. Overlapping long peptides spanning the NY-ESO-1 sequence have been used to map HLA class I-restricted epitopes recognized by NY-ESO-1-specific CD8+ T lymphocytes. To address the antigenicity of long peptides, we analyzed two synthetic 30-mer peptides from NY-ESO-1, polypeptides 80–109 and 145–174, for their capacity to be processed by APCs and to stimulate CD8+ T cells. By incubating APCs with polypeptides at different temperatures or in the presence of protease inhibitors, we found that NY-ESO-1 polypeptides were rapidly internalized by B cells, T2 cells, or PBLs and submitted to cellular proteolytic action to yield nonamer epitopes presented by HLA class I. Polypeptides were also immunogenic in vitro and stimulated the expansion of CD8+ T cells against naturally processed NY-ESO-1 epitopes in the context of three different HLA class I alleles. Polypeptides can thus serve as exogenous Ags that are cross-presented on HLA class I without requiring the action of professional APCs. These findings support innovative vaccination strategies using NY-ESO-1 polypeptides that would circumvent current limitations of HLA class I peptide vaccination, i.e., HLA eligibility criteria and knowledge of epitope, while allowing for facilitated immunogenicity in the presence of helper epitopes.

Consensus nomenclature for CD8+ T cell phenotypes in cancer

Whereas preclinical investigations and clinical studies have established that CD8+ T cells can profoundly affect cancer progression, the underlying mechanisms are still elusive. Challenging the prevalent view that the beneficial effect of CD8+ T cells in cancer is solely attributable to their cytotoxic activity, several reports have indicated that the ability of CD8+ T cells to promote tumor regression is dependent on their cytokine secretion profile and their ability to self-renew. Evidence has also shown that the tumor microenvironment can disarm CD8+ T cell immunity, leading to the emergence of dysfunctional CD8+ T cells. The existence of different types of CD8+ T cells in cancer calls for a more precise definition of the CD8+ T cell immune phenotypes in cancer and the abandonment of the generic terms “pro-tumor” and “antitumor.” Based on recent studies investigating the functions of CD8+ T cells in cancer, we here propose some guidelines to precisely define the functional states of CD8+ T cells in cancer.

Human Memory Helios− FOXP3+ Regulatory T Cells (Tregs) Encompass Induced Tregs That Express Aiolos and Respond to IL-1β by Downregulating Their Suppressor Functions

FOXP3+ regulatory T cells (Tregs) are critical regulators of self-tolerance and immune homeostasis. In mice and humans, two subsets of FOXP3+ Tregs have been defined based on their differential expression of Helios, a transcription factor of the Ikaros family. Whereas the origin, specificity, and differential function of the two subsets are as yet a source of controversy, their characterization thus far has been limited by the absence of surface markers to distinguish them. In this article, we show that human memory Helios+ and Helios− Tregs are phenotypically distinct and can be separated ex vivo based on their differential expression of IL-1RI, which is restricted to Helios− Tregs, in combination with CCR7. The two populations isolated using this strategy are distinct with respect to the expression of other Ikaros family members. Namely, whereas Eos, which has been reported to mediate FOXP3-dependent gene silencing, is expressed in Helios+ Tregs, Aiolos, which is involved in the differentiation of TH17 and induced Tregs, is instead expressed in Helios− Tregs. In addition, whereas both subsets are suppressive ex vivo, Helios− Tregs display increased suppressive capacity in comparison to Helios+ Tregs, but respond to IL-1β by downregulating their suppressive activity. Together, these data support the concept that human Helios− memory Tregs encompass induced Tregs that can readily respond to changes in the environment by modulating their suppressive capacity.

Decreased binding of peptides-MHC class I (pMHC) multimeric complexes to CD8 affects their binding avidity for the TCR but does not significantly impact on pMHC/TCR dissociation rate.

The CD8 coreceptor plays a crucial role in both T cell development in the thymus and in the activation of mature T cells in response to Ag-specific stimulation. In this study we used soluble peptides-MHC class I (pMHC) multimeric complexes bearing mutations in the CD8 binding site that impair their binding to the MHC, together with altered peptide ligands, to assess the impact of CD8 on pMHC binding to the TCR. Our data support a model in which CD8 promotes the binding of TCR to pMHC. However, once the pMHC/TCR complex is formed, the TCR dominates the pMHC/TCR dissociation rates. As a consequence of these molecular interactions, under physiologic conditions CD8 plays a key role in complex formation, resulting in the enhancement of CD8 T cell functions whose specificity, however, is determined by the TCR.

Regulation of CD4+NKG2D+ Th1 Cells in Patients with Metastatic Melanoma Treated with Sorafenib: Role of IL-15Rα and NKG2D Triggering

Beyond cancer-cell intrinsic factors, the immune status of the host has a prognostic impact on patients with cancer and influences the effects of conventional chemotherapies. Metastatic melanoma is intrinsically immunogenic, thereby facilitating the search for immune biomarkers of clinical responses to cytotoxic agents. Here, we show that a multi-tyrosine kinase inhibitor, sorafenib, upregulates interleukin (IL)-15Rα in vitro and in vivo in patients with melanoma, and in conjunction with natural killer (NK) group 2D (NKG2D) ligands, contributes to the Th1 polarization and accumulation of peripheral CD4(+)NKG2D(+) T cells. Hence, the increase of blood CD4(+)NKG2D(+) T cells after two cycles of sorafenib (combined with temozolomide) was associated with prolonged survival in a prospective phase I/II trial enrolling 63 patients with metastatic melanoma who did not receive vemurafenib nor immune checkpoint-blocking antibodies. In contrast, in metastatic melanoma patients treated with classical treatment modalities, this CD4(+)NKG2D(+) subset failed to correlate with prognosis. These findings indicate that sorafenib may be used as an adjuvant molecule capable of inducing or restoring IL-15Rα/IL-15 in tumors expressing MHC class I-related chain A/B (MICA/B) and on circulating monocytes of responding patients, hereby contributing to the bioactivity of NKG2D(+) Th1 cells.

Lentivector immunization induces tumor antigen‐specific B and T cell responses in vivo

Expression of the cancer/germ‐line antigen NY‐ESO‐1 by tumors elicits spontaneous humoral and cellular immune responses in some cancer patients. Development of vaccines capable of stimulating such comprehensive immune responses is desirable. We have produced recombinant lentivectors directing the intracellular synthesis of NY‐ESO‐1 (rLV/ESO) and have analyzed the in vivo immune response elicited by this vector. Single injection of rLV/ESO into HLA‐A2‐transgenic mice elicited long‐lasting Bu2004and T cell responses against NY‐ESO‐1. CD8+ T cells against the HLA‐A2‐restricted peptide NY‐ESO‐1157–165 were readily detectable ex vivo and showed restricted TCR Vβ usage. Moreover, rLV/ESO elicited a far greater anti‐NY‐ESO‐1157–165 CD8+ T cell response than peptide‐ or protein‐based vaccines. Anti‐NY‐ESO‐1 antibodies were rapidly induced after immunization and their detection preceded that of the antigen‐specific CD8+ T cells. The rLV/ESO also induced CD4+ T cells. These cells played an essential role as their depletion completely abrogated B cell and CD8+ T cell responses against NY‐ESO‐1. The induced CD4+ T cells were primarily directed against a single NY‐ESO‐1 epitope spanning amino acidsu200481–100. Altogether, our study shows that rLV/ESO induces potent and comprehensive immune responses in vivo.