Estelle Devevre

Exhaustion of tumor-specific CD8+ T cells in metastases from melanoma patients

In chronic viral infections, CD8⁺ T cells become functionally deficient and display multiple molecular alterations. In contrast, only little is known of self- and tumor-specific CD8⁺ T cells from mice and humans. Here we determined molecular profiles of tumor-specific CD8⁺ T cells from melanoma patients. In peripheral blood from patients vaccinated with CpG and the melanoma antigen Melan-A/MART-1 peptide, we found functional effector T cell populations, with only small but nevertheless significant differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1-specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion. The identified exhaustion profile revealed extended molecular alterations. Our data demonstrate a remarkable coexistence of effector cells in circulation and exhausted cells in the tumor environment. Functional T cell impairment is mediated by inhibitory receptors and further molecular pathways, which represent potential targets for cancer therapy.

Four Functionally Distinct Populations of Human Effector-Memory CD8+ T Lymphocytes

In humans, the pathways of memory and effector T cell differentiation remain poorly defined. We have dissected the functional properties of ex vivo effector-memory (EM) CD45RA−CCR7− T lymphocytes present within the circulating CD8+ T cell pool of healthy individuals. Our studies show that EM T cells are heterogeneous and are subdivided based on differential CD27 and CD28 expression into four subsets. EM1 (CD27+CD28+) and EM4 (CD27−CD28+) T cells express low levels of effector mediators such as granzyme B and perforin and high levels of CD127/IL-7Rα. EM1 cells also have a relatively short replicative history and display strong ex vivo telomerase activity. Therefore, these cells are closely related to central-memory (CD45RA−CCR7+) cells. In contrast, EM2 (CD27+CD28−) and EM3 (CD27−CD28−) cells express mediators characteristic of effector cells, whereby EM3 cells display stronger ex vivo cytolytic activity and have experienced larger numbers of cell divisions, thus resembling differentiated effector (CD45RA+CCR7−) cells. These data indicate that progressive up-regulation of cytolytic activity and stepwise loss of CCR7, CD28, and CD27 both characterize CD8+ T cell differentiation. Finally, memory CD8+ T cells not only include central-memory cells but also EM1 cells, which differ in CCR7 expression and may therefore confer memory functions in lymphoid and peripheral tissues, respectively.

A Role for cis Interaction between the Inhibitory Ly49A Receptor and MHC Class I for Natural Killer Cell Education

Natural killer (NK) cells show enhanced functional competence when they express inhibitory receptors specific for inherited major histocompatibility complex class I (MHC-I) molecules. Current models imply that NK cell education requires an interaction of inhibitory receptors with MHC-I expressed on other cells. However, the inhibitory Ly49A receptor can also bind MHC-I ligand on the NK cell itself (in cis). Here we describe a Ly49A variant, which can engage MHC-I expressed on other cells but not in cis. Even though this variant inhibited NK cell effector function, it failed to educate NK cells. The association with MHC-I in cis sequestered wild-type Ly49A, and this was found to relieve NK cells from a suppressive effect of unengaged Ly49A. These data explain how inhibitory MHC-I receptors can facilitate NK cell activation. They dissociate classical inhibitory from educating functions of Ly49A and suggest that cis interaction of Ly49A is necessary for NK cell education.

Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen.

Human cancer vaccines are often prepared with altered “analog” or “heteroclitic” antigens that have been optimized for HLA class I binding, resulting in enhanced immunogenicity. Here, we take advantage of CpG oligodeoxynucleotides as powerful vaccine adjuvants and demonstrate the induction of high T cell frequencies in melanoma patients, despite the use of natural (unmodified) tumor antigenic peptide. Compared with vaccination with analog peptide, natural peptide induced T cell frequencies that were approximately twofold lower. However, T cells showed superior tumor reactivity because of (i) increased functional avidity for natural antigen and (ii) enhancement of T cell activation and effector function. Thus, novel vaccine formulations comprising potent immune stimulators may allow to circumvent the need for modified antigens and can induce highly functional T cells with precise antigen specificity.

Human Effector CD8+ T Lymphocytes Express TLR3 as a Functional Coreceptor

TLR are evolutionarily conserved molecules that play a key role in the initiation of innate antimicrobial immune responses. Through their influence on dendritic cell maturation, these receptors are also thought to indirectly shape the adaptive immune response. However, no data are currently available regarding both TLR expression and function in human CD8+ T cell subsets. We report that a subpopulation of CD8+ T cells, i.e., effector, but neither naive nor central memory cells, constitutively expresses TLR3. Moreover, the ligation of the receptor by a specific agonist in TLR3-expressing CD8+ T cells increased IFN-γ secretion induced by TCR-dependent and -independent stimulation, without affecting proliferation or specific cytolytic activity. These results thereby suggest that TLR3 ligands can not only indirectly influence the adaptive immune response through modulation of dendritic cell activation, but also directly increase IFN-γ production by Ag-specific CD8+ T cells. Altogether, the present work might open new perspectives for the use of TLR ligands as adjuvants for immunotherapy.

Memory and effector CD8 T-cell responses after nanoparticle vaccination of melanoma patients

Induction of cytotoxic CD8 T-cell responses is enhanced by the exclusive presentation of antigen through dendritic cells, and by innate stimuli, such as toll-like receptor ligands. On the basis of these 2 principles, we designed a vaccine against melanoma. Specifically, we linked the melanoma-specific Melan-A/Mart-1 peptide to virus-like nanoparticles loaded with A-type CpG, a ligand for toll-like receptor 9. Melan-A/Mart-1 peptide was cross-presented, as shown in vitro with human dendritic cells and in HLA-A2 transgenic mice. A phase I/II study in stage II-IV melanoma patients showed that the vaccine was well tolerated, and that 14/22 patients generated ex vivo detectable T-cell responses, with in part multifunctional T cells capable to degranulate and produce IFN-γ, TNF-α, and IL-2. No significant influence of the route of immunization (subcutaneous versus intradermal) nor dosing regimen (weekly versus daily clusters) could be observed. It is interesting to note that, relatively large fractions of responding specific T cells exhibited a central memory phenotype, more than what is achieved by other nonlive vaccines. We conclude that vaccination with CpG loaded virus-like nanoparticles is associated with a human CD8 T-cell response with properties of a potential long-term immune protection from the disease.

A Novel Approach to Characterize Clonality and Differentiation of Human Melanoma-Specific T Cell Responses: Spontaneous Priming and Efficient Boosting by Vaccination

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8+ T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.

Enrichment of Human CD4+ Vα24/Vβ11 Invariant NKT Cells in Intrahepatic Malignant Tumors

Invariant NKT cells (iNKT cells) recognize glycolipid Ags via an invariant TCR α-chain and play a central role in various immune responses. Although human CD4+ and CD4− iNKT cell subsets both produce Th1 cytokines, the CD4+ subset displays an enhanced ability to secrete Th2 cytokines and shows regulatory activity. We performed an ex vivo analysis of blood, liver, and tumor iNKT cells from patients with hepatocellular carcinoma and metastases from uveal melanoma or colon carcinoma. Frequencies of Vα24/Vβ11 iNKT cells were increased in tumors, especially in patients with hepatocellular carcinoma. The proportions of CD4+, double negative, and CD8α+ iNKT cell subsets in the blood of patients were similar to those of healthy donors. However, we consistently found that the proportion of CD4+ iNKT cells increased gradually from blood to liver to tumor. Furthermore, CD4+ iNKT cell clones generated from healthy donors were functionally distinct from their CD4− counterparts, exhibiting higher Th2 cytokine production and lower cytolytic activity. Thus, in the tumor microenvironment the iNKT cell repertoire is modified by the enrichment of CD4+ iNKT cells, a subset able to generate Th2 cytokines that can inhibit the expansion of tumor Ag-specific CD8+ T cells. Because CD4+ iNKT cells appear inefficient in tumor defense and may even favor tumor growth and recurrence, novel iNKT-targeted therapies should restore CD4− iNKT cells at the tumor site and specifically induce Th1 cytokine production from all iNKT cell subsets.

Differentiation associated regulation of microRNA expression in vivo in human CD8+ T cell subsets.

BackgroundThe differentiation of CD8+ T lymphocytes following priming of naïve cells is central in the establishment of the adaptive immune response. Yet, the molecular events underlying this process are not fully understood. MicroRNAs have been recently shown to play a key role in the regulation of haematopoiesis in mouse, but their implication in peripheral lymphocyte differentiation in humans remains largely unknown.MethodsIn order to explore the potential implication of microRNAs in CD8+ T cell differentiation in humans, microRNA expression profiles were analysed using microarrays and quantitative PCR in several human CD8+ T cell subsets defining the major steps of the T cell differentiation pathway.ResultsWe found expression of a limited set of microRNAs, including the miR-17~92 cluster. Moreover, we reveal the existence of differentiation-associated regulation of specific microRNAs. When compared to naive cells, miR-21 and miR-155 were indeed found upregulated upon differentiation to effector cells, while expression of the miR-17~92 cluster tended to concomitantly decrease.ConclusionsThis study establishes for the first time in a large panel of individuals the existence of differentiation associated regulation of microRNA expression in human CD8+ T lymphocytes in vivo, which is likely to impact on specific cellular functions.

Clonotype Selection and Composition of Human CD8 T Cells Specific for Persistent Herpes Viruses Varies with Differentiation but Is Stable Over Time

Protection from reactivation of persistent herpes virus infection is mediated by Ag-specific CD8 T cell responses, which are highly regulated by still poorly understood mechanisms. In this study, we analyzed differentiation and clonotypic dynamics of EBV- and CMV-specific T cells from healthy adults. Although these T lymphocytes included all subsets, from early-differentiated (EM/CD28pos) to late-differentiated (EMRA/CD28neg) stages, they varied in the sizes/proportions of these subsets. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28neg subset were also found within the pool of less differentiated “memory” cells. However, striking differences in the patterns of dominance were observed among these subsets, because some clonotypes were selected with differentiation while others were not. Late-differentiated CMV-specific clonotypes were mostly characterized by TCR with lower dependency on CD8 coreceptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of 4 years. Remarkably, clonotypic distribution was stable not only in late-differentiated but also in less-differentiated T cell subsets. Thus, T cell clonotypes segregate with differentiation, but the clonal composition once established is kept constant for at least several years. These findings reveal novel features of the highly sophisticated control of steady state protective T cell activity in healthy adults.