Patricia Corthesy

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 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.

Mouse interleukin-2 receptor alpha gene expression. Interleukin-1 and interleukin-2 control transcription via distinct cis-acting elements

We have shown that interleukin-1 (IL-1) and IL-2 control IL-2 receptor α (IL-2Rα) gene transcription in CD4CD8 murine T lymphocyte precursors. Here we map the cis-acting elements that mediate interleukin responsiveness of the mouse IL-2Rα gene using a thymic lymphoma-derived hybridoma (PC60). The transcriptional response of the IL-2Rα gene to stimulation by IL-1 + IL-2 is biphasic. IL-1 induces a rapid, protein synthesis-independent appearance of IL-2Rα mRNA that is blocked by inhibitors of NF-κB activation. It also primes cells to become IL-2 responsive and thereby prepares the second phase, in which IL-2 induces a 100-fold further increase in IL-2Rα transcripts. Transient transfection experiments show that several elements in the promoter-proximal region of the IL-2Rα gene contribute to IL-1 responsiveness, most importantly an NF-κB site conserved in the human and mouse gene. IL-2 responsiveness, on the other hand, depends on a 78-nucleotide segment 1.3 kilobases upstream of the major transcription start site. This segment functions as an IL-2-inducible enhancer and lies within a region that becomes DNase I hypersensitive in normal T cells in which IL-2Rα expression has been induced. IL-2 responsiveness requires three distinct elements within the enhancer. Two of these are potential binding sites for STAT proteins.

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.

Fine Structural Variations of αβTCRs Selected by Vaccination with Natural versus Altered Self-Antigen in Melanoma Patients

Immunotherapy of cancer is often performed with altered “analog” peptide Ags optimized for HLA class I binding, resulting in enhanced immunogenicity, but the induced T cell responses require further evaluation. Recently, we demonstrated fine specificity differences and enhanced recognition of naturally presented Ag by T cells after vaccination with natural Melan-A/MART-1 peptide, as compared with analog peptide. In this study, we compared the TCR primary structures of 1489 HLA-A*0201/Melan-A26–35-specific CD8 T cells derived from both cohorts of patients. Although a strong preference for TRAV12-2 segment usage was present in nearly all patients, usage of particular TRAJ gene segments and CDR3α composition differed slightly after vaccination with natural vs analog peptide. Moreover, TCR β-chain repertoires were broader after natural than analog peptide vaccination. In all patients, we observed a marked conservation of the CDR3β amino acid composition with recurrent sequences centered on a glycyl-leucyl/valyl/alanyl-glycyl motif. In contrast to viral-specific TCR repertoires, such “public” motifs were primarily expressed by nondominant T cell clonotypes, which contrasted with “private” CDR3β signatures frequently found in T cell clonotypes that dominated repertoires of individual patients. Interestingly, no differences in functional avidity were observed between public and private T cell clonotypes. Collectively, our data indicate that T cell repertoires generated against natural or analog Melan-A peptide exhibited slightly distinct but otherwise overlapping and structurally conserved TCR features, suggesting that the differences in binding affinity/avidity of TCRs toward pMHC observed in the two cohorts of patients are caused by subtle structural TCR variations.

Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites

The α chain of the interleukin-2 receptor (IL-2Rα) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5′-flanking region (base pairs −2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2Rα gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2Rα gene expression. These results show that regulation of the endogenous IL-2Rα gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5′-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5′ end of the endogenous IL-2Rα gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at −0.05 and −5.3 kilobase pairs, are present in resting T cells. A third site appears at −1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

In Vivo Persistence of Codominant Human CD8+ T Cell Clonotypes Is Not Limited by Replicative Senescence or Functional Alteration

T cell responses to viral epitopes are often composed of a small number of codominant clonotypes. In this study, we show that tumor Ag-specific T cells can behave similarly. In a melanoma patient with a long lasting HLA-A2/NY-ESO-1-specific T cell response, reaching 10% of circulating CD8 T cells, we identified nine codominant clonotypes characterized by individual TCRs. These clonotypes made up almost the entire pool of highly differentiated effector cells, but only a fraction of the small pool of less differentiated “memory” cells, suggesting that the latter serve to maintain effector cells. The different clonotypes displayed full effector function and expressed TCRs with similar functional avidity. Nevertheless, some clonotypes increased, whereas others declined in numbers over the observation period of 6 years. One clonotype disappeared from circulating blood, but without preceding critical telomere shortening. In turn, clonotypes with increasing frequency had accelerated telomere shortening, correlating with strong in vivo proliferation. Interestingly, the final prevalence of the different T cell clonotypes in circulation was anticipated in a metastatic lymph node withdrawn 2 years earlier, suggesting in vivo clonotype selection driven by metastases. Together, these data provide novel insight in long term in vivo persistence of T cell clonotypes associated with continued cell turnover but not replicative senescence or functional alteration.

Constitutive Expression of a Chimeric Receptor That Delivers IL-2/IL-15 Signals Allows Antigen-Independent Proliferation of CD8+CD44high But Not Other T Cells

We have prepared transgenic mice whose T cells constitutively express a chimeric receptor combining extracellular human IL-4R and intracellular IL-2Rβ segments. This receptor can transmit IL-2/IL-15-like signals in response to human, but not mouse, IL-4. We used these animals to explore to what extent functional IL-2R/IL-15R expression controls the capacity of T cells to proliferate in response to IL-2/IL-15-like signals. After activation with Con A, naive transgenic CD8+ and CD4+ T cells respond to human IL-4 as well as to IL-2. Without prior activation, they failed to proliferate in response to human IL-4, although human IL-4 did prolong their survival. Thus, IL-2-induced proliferation of activated T cells requires at least one other Ag-induced change apart from the induction of a functional IL-2R. However, a fraction of CD8+CD44high T cells proliferate in human IL-4 without antigenic stimulation or syngeneic feeder cells. In contrast, CD4+CD44high T cells are not constitutively responsive to human IL-4. We conclude that although all transgenic T cells express a functional chimeric receptor, only some CD8+CD44high T cells contain all molecules required for entry into the cell cycle in response to human IL-4 or IL-15.

Dominant Human CD8 T Cell Clonotypes Persist Simultaneously as Memory and Effector Cells in Memory Phase

The adaptive immune system plays a critical role in protection at the time of secondary infection. It does so through the rapid and robust reactivation of memory T cells which are maintained long-term, in a phenotypically heterogeneous state, following their primary encounter with Ag. Although most HLA-A*0201/influenza matrix protein58–66-specific CD8 T cells from healthy donors display characteristics typical of memory T cells, through our extensive phenotypic analysis we have further shown that up to 20% of these cells express neither the IL-7 receptor CD127 nor the costimulatory molecule CD28. In contrast to the majority of CD28pos cells, granzyme B and perforin were frequently expressed by the CD28neg cells, suggesting that they are effector cells. Indeed, these cells were able to kill target cells, in an Ag-specific manner, directly ex vivo. Thus, our findings demonstrate the remarkable long-term persistence in healthy humans of not only influenza-specific memory cells, but also of effector T cells. We further observed that granzyme B expression in influenza-specific CD8 T cells paralleled levels in the total CD8 T cell population, suggestive of Ag-nonspecific bystander activation. Sequencing of TCR α- and β-chains showed that the TCR repertoire specific for this epitope was dominated by one, or a few, T cell clonotype per healthy donor. Moreover, our sequencing analysis revealed, for the first time in humans, that identical clonotypes can coexist as both memory and effector T cells, thereby supporting the principle of multipotent clonotypic differentiation.

A Conserved IL-2 Responsive Enhancer in the IL-2Rα Gene

Abstract IL-2 stimulates expression of the a subunit of the high affinity IL-2 receptor (IL-2Rα) in antigen-activated T lymphocytes, by increasing IL-2Rα gene transcription. This response is mediated by a 52 nt IL-2 responsive enhancer (IL-2rE) that is conserved between mouse and man. The mouse enhancer is 1.3 kb upstream of the transcription start site and co-localizes with an inducible DNasel hypersensitive site, whereas the human homologue maps to −4 kb. The human IL-2rE is functional in rodent cells. Both enhancers contain two potential STAT binding sites and an Ets consensus motif. One of the STAT motifs overlaps with a binding site for GATA factors. Functional analysis of the mouse and human enhancers indicates that IL-2-activated STAT5 and the constitutively active Ets protein Elf-1 play a predominant role in controlling IL-2rE activity.