Clémence Granier

Induction of resident memory T cells enhances the efficacy of cancer vaccine

Tissue-resident memory T cells (Trm) represent a new subset of long-lived memory T cells that remain in tissue and do not recirculate. Although they are considered as early immune effectors in infectious diseases, their role in cancer immunosurveillance remains unknown. In a preclinical model of head and neck cancer, we show that intranasal vaccination with a mucosal vector, the B subunit of Shiga toxin, induces local Trm and inhibits tumour growth. As Trm do not recirculate, we demonstrate their crucial role in the efficacy of cancer vaccine with parabiosis experiments. Blockade of TFGβ decreases the induction of Trm after mucosal vaccine immunization, resulting in the lower efficacy of cancer vaccine. In order to extrapolate this role of Trm in humans, we show that the number of Trm correlates with a better overall survival in lung cancer in multivariate analysis. The induction of Trm may represent a new surrogate biomarker for the efficacy of cancer vaccine. This study also argues for the development of vaccine strategies designed to elicit them.

Long-term immune reconstitution and infection burden after mismatched hematopoietic stem cell transplantation.

Mismatched unrelated donor (MMUD) or umbilical cord blood (UCB) can be chosen as alternative donors for allogeneic stem cell transplantation but might be associated with long-lasting immune deficiency. Sixty-six patients who underwent a first transplantation from either UCB (n = 30) or 9/10 MMUD (n = 36) and who survived beyond 3 months were evaluated. Immune reconstitution was prospectively assessed at sequential time points after transplantation. NK, B, CD4(+), and CD8(+) T cells and their naïve and memory subsets, as well as regulatory T cells (Treg), were studied. Detailed analyses on infections occurring after 3 months were also assessed. The 18-month cumulative incidences of infection-related death were 8% and 3%, and of infections were 72% and 57% after MMUD and UCB transplantation, respectively. Rates of infection per 12 patient-month were roughly 2 overall (1 for bacterial, .9 for viral, and .3 for fungal infections). Memory, naïve CD4(+) and CD8(+)T cells, naïve B cells, and Treg cells reconstitution between the 2 sources were roughly similar. Absolute CD4(+)T cells hardly reached 500 per μL by 1 year after transplantation and most B cells were of naïve phenotype. Correlations between immune reconstitution and infection were then performed by multivariate analyses. Low CD4(+) and high CD8(+)T cells absolute counts at 3 months were linked to increased risks of overall and viral (but not bacterial) infections. When assessing for the naïve/memory phenotypes at 3 months among the CD4(+) T cell compartment, higher percentages of memory subsets were protective against late infections. Central memory CD4(+)T cells protected against overall and bacterial infections; late effector memory CD4(+)T cells protected against overall, bacterial, and viral infections. To the contrary, high percentage of effector- and late effector-memory subsets at 3 months among the CD8(+) T cell compartment predicted higher risks for viral infections. Patients who underwent transplantation from alternative donors represent a population with very high risk of infection. Detailed phenotypic analysis of immune reconstitution may help to evaluate infection risk and to adjust infection prophylaxis.

Immunotherapy of HPV-associated head and neck cancer: Critical parameters

Various arguments support the development of a vaccine targeting human papillomavirus (HPV) for the treatment of HPV-associated head and neck cancer. However, the mucosal localization of this tumor, the HPV-driven downregulation of MHC Class I molecules and various other immunosuppressive mechanisms must be carefully considered to improve the clinical efficacy of such an immunotherapeutic strategy.

Pressure from TRIM5α Contributes to Control of HIV-1 Replication by Individuals Expressing Protective HLA-B Alleles

ABSTRACT The expression of certain HLA class I alleles, including HLA-B*27 and HLA-B*57, is associated with better control of human immunodeficiency virus type 1 (HIV-1) infection, but the mechanisms responsible are not fully understood. We sought evidence that pressure from the human restriction factor TRIM5α (hTRIM5α) could contribute to viral control. The hTRIM5α sensitivity of viruses from both HLA-B*57-positive (HLA-B*57+) and HLA-B*27+ patients who spontaneously controlled viral replication, but not viruses from viremic patients expressing these alleles, was significantly greater than that of viruses from patients not expressing these protective HLA-B alleles. Overall, a significant negative correlation between hTRIM5α sensitivity and viral load was observed. In HLA-B*57+ patients, the T242N mutation in the HLA-B*57-restricted TW10 CD8+ T lymphocyte (CTL) epitope was strongly associated with hTRIM5α sensitivity. In HLA-B*27+ controllers, hTRIM5α sensitivity was associated with a significant reduction in emergence of key CTL mutations. In several patients, viral evolution to avoid hTRIM5α sensitivity was observed but could be associated with reduced viral replicative capacity. Thus, in individuals expressing protective HLA-B alleles, the combined pressures exerted by CTL, hTRIM5α, and capsid structural constraints can prevent viral escape both by impeding the selection of necessary resistance/compensatory mutations and forcing the selection of escape mutations that increase hTRIM5α sensitivity or impair viral replicative capacity.

Following up tumor-specific regulatory T cells in cancer patients.

Regulatory T cells (Treg) represent a subpopulation of immunosuppressive cells that preferentially expand during tumor progression. The primary role of Tregs is to dampen antitumor effector T-cell responses, but they also modulate inflammatory reactions and promote angiogenesis. Until now, the great majority of studies analyzed the total number of Tregs without focusing on their antigen specificity, due to the lack of available analytic tools. Philipp Beckhove’s group has now reported a high frequency of endogenous Tregs directed against the self antigen mammaglobin (mam) in primary breast carcinoma patients.1 The authors detected the presence of these cells by a functional assay based on the amplification of anti-mam effector T cells after Treg depletion. In addition, they confirmed their results by manufacturing specific tetramers loaded with MHC Class II-restricted peptides derived from mammaglobin (mam34–48). A mean frequency of 0.21% anti-mam Tregs was found in the peripheral blood of breast carcinoma patients. The use of HLA Class II tetramers is still in an early stage due to paucity of reagents and tools to validate them. Beckhove and colleagues derived specific anti-mam CD4+ T-cell clones to control the specificity of their mam-targeted HLA Class II tetramers, significantly reinforcing the strength of the study. Wang et al. pioneered the detection of tumor-specific Treg directed against the cancer/testis antigen 2 CTAG2 (an homolog of NY-ESO-1 best known as LAGE-1) and against peptides derived from BBX (best known as ARTC1) in tumor-bearing mice.2 Next, pre-existing Tregs specific for a variety of antigens including differentiation (e.g., gp100, TRP2), cancer-testis (e.g., NY-ESO-1), overexpressed (e.g., CEA, EGFR, MUC1), universal (e.g., telomerase, surviving), and viral (e.g., HPV16-derived) antigens were detected in cancer patients. One striking feature of the Beckhove study is that mam-specific Tregs were detected in the peripheral blood of patients directly ex vivo. Indeed, in most studies, the identification of antitumor Tregs required an in vitro amplification step or the generation of T-cell clones.3 Usually, specific Tregs are identified within tumor-infiltrating lymphocytes, as they are highly enriched in this compartment as compared with the peripheral blood. Indeed, although an increased frequency of circulating Tregs has been observed in cancer patients, reaching 5–10% of blood CD4+ T cells, Tregs can account for 40–50% of CD4+ T cells infiltrating some human and murine tumors.3 This said, as specific Tregs have been observed both in peripheral blood and within neoplastic lesions, these cells may interfere with antitumor immune responses at both the induction and effector levels.4 The Beckhove study clearly demonstrated that the levels of mam-specific Tregs are higher in the blood of breast carcinoma patients than in healthy individuals, as previously observed for Tregs targeting other tumor-associated antigens. It would have been of interest to complete this study by assessing the levels of mam-specific Tregs in the tumor microenvironment of these patients. Various mechanisms may account for Treg deregulation in cancer patients and their accumulation within neoplastic lesions. The tumor microenvironment favors indeed the conversion of conventional T cells into Tregs, since the presentation of self tumor antigens prevails in the presence of transforming growth factor β (TGFβ), interleukin-10 (IL-10), and vascular endothelial growth factor (VEGF), most likely owing to immature dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs).5,6 In addition, specific chemokines produced in the tumor microenvironment such as CCL17 and CCL22 preferentially recruit Tregs.7,8 There is a debate as to whether intratumoral Tregs reflect the local amplification of natural Tregs or the in situ conversion of conventional T cells (Tconvs).9,10 Both mechanisms have been reported to occur, and Beckhove et al. showed that the same tumor-associated antigen could be recognized by both Tconvs and Tregs. The study of the TCR repertoire of mam-specific T cells may have helped in distinguishing their precise origin. It is clear that various subpopulations of Tregs endowed with various clinical significance co-exist in cancer patients.11 For example, Tregs expressing activation markers such as CCR4 may exert more robust immunosuppressive functions and hence be more closely associated with prognosis than the general Treg population.8 Although not performed in the study by Beckhove and colleagues, HLA Class II tetramers will also allow for an extensive phenotyping of tumor-specific Tregs, hence informing strategies to inhibit their function.12 Various arguments support the need for rigorously monitoring specific Treg subsets (instead of the whole pool of Tregs) in cancer. First, Beckhove et al. have previously shown that the depletion of Treg efficiently enhanced tumor-associated antigen-specific Tconvs only when Tregs recognizing the same antigen pre-existed, supporting the antigen specificity of optimal Treg-mediated Tconv inhibition.13 Second, in the course of anticancer vaccination, monitoring specific Treg subsets is highly recommended, as in both mice and humans these vaccines could increase specific Tregs and not only effector cells, an issue that may explain some recent clinical failures.3 Third, some anticancer vaccines do not modify the pool of Tregs, but decrease the ratio between antigen-specific Tregs and Tconvs, favoring TH1 immune responses.14 The importance of Tregs in the clinics has been shown for the first time in a randomized clinical trial testing an anticancer vaccine in renal cancer patients.15 In this study, a single dose of cyclophosphamide affected predominantly proliferative Tregs, possibly those specific for tumor-associated antigens, and only prolonged the survival of subjects exhibiting vaccine-elicited immune responses. It remains unclear whether therapeutic avenues to boosting Tconvs (e.g., the blockade of immunological checkpoints, immunogenic chemotherapies) may modulate tumor-specific Tregs. The emergence of new tools to directly access specific Tregs ex vivo will allow for a fine monitoring of these cells in cancer patients before and after therapeutic interventions, and will therefore help the design of future clinical trial.

Impact of the source of hematopoietic stem cell in unrelated transplants: Comparison between 10/10, 9/10-HLA matched donors and cord blood

In absence of available matched‐related or unrelated donor (MUD), mismatched unrelated donors (MMUD) and unrelated cord blood (UCB) are both considered to be suitable donors, with similar post‐transplant overall survival. In most of these retrospective comparisons, HLA typing of adult donors was performed at eight loci. The aim of this study was to compare the outcome of patients transplanted from UCB (N = 64) with those transplanted from 9/10‐HLA MMUD (N = 84) or 10/10‐HLA MUD (N = 196). In multivariate analysis, UCB was associated with less Grade II–IV acute GVHD in comparison with MUD (aHR 1.97, 95% CI 1.19–3.27, P = 0.009) and MMUD transplants (aHR 1.79, 95% CI 1.02–3.15, P = 0.042), while the cumulative incidence of chronic GVHD was not significantly different between the three groups. Overall survival (OS), non‐relapse mortality, and relapse were not different between MMUD and UCB transplantation, whereas OS was impaired after UCB in comparison with MUD (aHR 0.65, 95% CI 0.43–0.99, P = 0.043). Factors also impacting OS were the donor/recipient CMV serostatus (Donor−/Recipient+ aHR 1.76, 95% CI 1.23–2.52, P = 0.002 compared with D−/R−), the donor/recipient gender combination (Female/Male versus other combinations aHR 1.57, 95% CI 1.11–2.22, P = 0.012) and disease risk (aHR 1.58, 95% CI 1.05–2.38, P = 0.027 for high vs. low risk disease). Our data confirm that UCB and 9/10‐HLA MMUD are both relevant alternative options when no 10/10‐HLA donor is available. Donor/recipient gender combination and CMV serostatus had a significant impact on survival and may be taken into account, along with donor type, in the setting of MMUD and UCB transplants. Am. J. Hematol. 90:897–903, 2015.

Therapeutic cancer vaccine: building the future from lessons of the past

Anti-cancer vaccines have raised many hopes from the start of immunotherapy but have not yet been clinically successful. The few positive results of anti-cancer vaccines have been observed in clinical situations of low tumor burden or preneoplastic lesions. Several new concepts and new results reposition this therapeutic approach in the field of immunotherapy. Indeed, cancers that respond to anti-PD-1/PD-L1 (20–30%) are those that are infiltrated by anti-tumor T cells with an inflammatory infiltrate. However, 70% of cancers do not appear to have an anti-tumor immune reaction in the tumor microenvironment. To induce this anti-tumor immunity, therapeutic combinations between vaccines and anti-PD-1/PD-L1 are being evaluated. In addition, the identification of neoepitopes against which the immune system is less tolerated is giving rise to a new enthusiasm by the first clinical results of the vaccine including these neoepitopes in humans. The ability of anti-cancer vaccines to induce a population of anti-tumor T cells called memory resident T cells that play an important role in immunosurveillance is also a new criterion to consider in the design of therapeutic vaccines.

Les « immune checkpoints », comment ça marche

Costimulatory molecules allow the full lymphocyte activation, whereas co-inhibitory molecules are negative counterparts that act as immune regulators, avoiding excessive response. In some context of chronic inflammation such as cancer, co-inhibitory immune checkpoint as CTLA-4, PD-1, Lag-3, Tim-3 can accumulate at the membrane of T cells leading to a state of anergy and therefore the loss of tumor growth control. Consequently, these immune checkpoints are considered as potential target in the treatment of cancer. Immunotherapy by anti-CTLA-4 and anti-PD-1/PD-L1 early demonstrated very good proof of efficacy in the setting of several cancers types, supporting the role of these molecules in tumor immune escape. The aim of this review is to summarize the pathophysiology of immune checkpoints and their therapeutic applications in cancer.

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment. The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8+ T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.