Each T‑cell possesses a unique entity called T‑cell receptor (TCR). Protective immune responses rely on the specific recognition of antigen-derived peptides bound to self-MHC molecules provided by the TCRαβ. Numerous studies have attempted to correlate TCR usage by T‑cells with the recognition of specific viral-peptide-MHC complexes in humans and mice. Responses to a given T‑cell antigen often showed strong biases in TCR selection, resulting in the preferential usage of particular TCR gene segments. One of the best-documented examples is the human CD8⁺ T‑cell response specific for the influenza virus matrix peptide. The observation that unique TCRs are associated with anti-viral protection raises the questions whether such dominant T‑cell clones are generally required for protective immunity, and whether similar principles may also apply for anti-tumor immunity. One of our major goals is to deepen our knowledge on the functional and clonal diversity, frequencies, persistence and turnover of tumor-reactive T lymphocytes present in various biological compartments over the course of vaccination/immunotherapy.

Tracking tumor-reactive cytolytic T lymphocytes in melanoma patients

Spontaneous robust CD8⁺ T‑cell responses such as those directed against the Melan-A/MART‑1 antigen, a human tumor-associated self-antigen, have been reported to occur in a small fraction of studied melanoma patients. Studies devoted on such patients are of central importance, since they will likely reveal new key information about the generation of dominant and potentially protective T‑cell responses. To specifically address these points, we developed a new approach that combines flow cytometry-based cell sorting, gene expression profiling, TCR spectratyping and sequencing at the single-cell level for ex vivo molecular characterization of human tumor-specific CD8⁺ T‑cell responses. At present, we have identified dominant tumor-reactive T‑cell clones with high frequencies and long-term persistence in several melanoma patients. Complete in-depth studies in two patients showed that spontaneous priming led to the selection and expansion of dominant clones of high TCR avidity to cognate tumor antigen and strong tumor reactivity. Remarkably, peptide vaccination successfully boosted the frequencies of these pre-existing clones within the circulating CD8 pool, indicating that spontaneously primed T‑cell clones with high TCR avidity could be preferentially promoted through vaccination. Furthermore, the identified T‑cell clones expressed high levels of effector mediators such as IFN‑g, perforin and granzyme B ex vivo and were also dominantly found at metastatic tumor sites in those patients.

Overall, our molecular-based strategy allows for the first time to precisely assess T‑cell responses ex vivo by dissecting distinct subsets and anatomical compartments, over time, and during subsequent immunotherapy. The identification of those “super” anti-tumor selected T‑cell clones bearing high TCR avidity and their subsequent fine characterization is a key step to better understand the functional requirements of T‑cell receptors to efficiently recognize tumor cells. Expected results should allow streamlining the development of antigen specific immunotherapy.

Model for the generation of a protective immune response

Previous reports revealed large TCR repertoires among T‑cells responding to a given tumor antigen. Here, we were able to identify dominant T‑cell clonotypes because we focused our analysis on distinct T‑cell subsets (i.e. intermediate versus differentiated cells). In particular, we observed a highly restricted TCR repertoire in the most differentiated T‑cell compartment, supporting the notion that restricted but dominant antigen-specific T‑cell responses are composed of functionally differentiated effector cells with efficient cytolytic properties. Thus, high-resolution characterization of dominant tumor-reactive T‑cell responses provides the basis to identify biological parameters associated with protective T‑cell immunity. Moreover, the mechanisms involved in the selection of particular T‑cell clonotypes and the impact of vaccination can now be precisely assessed.

In line with these observations, we propose the following working model in which protective immune responses against tumor antigens may involve the selection and generation of differentiated T‑cells with potent effector functions, restricted clonal diversity and increased avidity of their T‑cell receptors (Fig. 1). This type of immune response is typically observed following viral infections with e.g. cytomegalovirus. If our model were to be confirmed, the development of new vaccines allowing the generation of such efficient tumor-specific immune T‑cell responses will be of central importance for cancer therapy.

Figure 1: Working model for the generation of a protective immune response upon CD8⁺ T‑cell differentiation. This involves the selection and generation of dominant differentiated cytolytic T lymphocytes with potent effector functions, restricted clonal diversity and increased avidity of their TCRs. CMV; cytomegalovirus.

Defining the functional diversity of tumor‑reactive T lymphocytes

This part of the project is devoted to the isolation and functional characterization of tumor-specific T lymphocytes that were identified from melanoma patients ex vivo (see previous sections) and is performed in collaboration with Dr. D. Speiser (LICR).
What remains intriguing, at present, is why oliclonal expansions of particular T‑cells are mostly observed in the differentiated subset (effector-memory CD28‑; EM28‑), but not in the less differentiated (effector‑memory CD28+; EM28+) one? One potential explanation may come from the « yet-to-be defined » function of both EM28+ and EM28‑ subsets during immune responses. Here, we further characterized and compared these subsets in the context of the melanoma cancer model. To address this point, we generated over 110 representative T‑cell clones in vitro by limiting-dilution from several patients with dominant tumor-reactive T‑cell responses. Whereas clones derived from differentiated EM28‑ T‑cells were able to kill Melan‑A peptide pulsed target cells and tumor cell lines in ⁵¹Cr release assays, at least half of the EM28+ derived T‑cell clones killed only poorly. TCR signaling was intact since EM28+ derived clones were able to secrete IFN‑g in a peptide specific manner in Elispot assays. In contrast, intracellular granzyme B staining showed variability between the two clonal populations. EM28‑ derived clones with the highest and EM28+ derived clones with very low granzyme B content. Together, these data reinforce the existence of two subsets of effector-memory T‑cells with distinct functions, whereby EM28‑ but not EM28+ differentiate into T‑cells with fully developed lytic properties (see our working model; Fig. 1).

Replicative senescence and immortalization of cytolytic T lymphocytes

Human T lymphocytes can be numerically expanded in vitro to a limited extend. Thus, although in vitro expansion of tumor-specific cytolytic T‑cells is widely used for clinical and research purposes, the finite life span of those cells has become a barrier for immunotherapy-based strategies. The factors that cause cultured T lymphocytes to cease responding to stimulation and enter a state of irreversible growth arrest have not yet been clearly identified. One of the better-understood causes of replicative senescence involves telomere attrition. Telomeres are specialized structures at the end of eukaryotic chromosomes, capped by cellular proteins, and important in maintaining the integrity of chromosomes. In most somatic cells, telomere shortening during cell division represents a molecular clock that triggers the entry of cells into senescence. An alternative mechanism that has been largely associated with irreversible growth arrest depends on the expression of the cyclin-dependent kinase inhibitor p16^Ink4a. We recently reported that p16^Ink4a expression could directly be induced as a consequence of T lymphocyte activation and was not related to activation-induced cell death. Importantly, the accumulation of p16^Ink4a was responsible for the exit of a significant fraction of T‑cells from the proliferative population, thus limiting their numerical expansion in vitro (Migliaccio et al., 2005, 2006).

We and others have previously found that the replicative life span of human T lymphocytes can be prolonged by induced expression of the human telomerase reverse transcriptase (hTERT) gene without inducing changes associated with transformation. However, it is yet unknown whether somatic cells that over-express telomerase are physiologically indistinguishable from normal cells. Recent data proposed that telomerase might mediate additional functions in DNA repair, cell survival and cell growth. Using CD8⁺ T lymphocyte clones over-expressing telomerase we investigated the molecular mechanisms that regulate T‑cell proliferation and senescence. We found striking differences in the control of the expansion potential between (i) early-passage CD8⁺ T‑cell clones (30 PDs) over-expressing or not hTERT, (ii) late-passage hTERT-transduced T‑cell clones with elongated telomeres and extended life span (>150 PDs), and (iii) normal aged T‑cell clones (70-80 PDs). Indeed, early-passage T‑cell clones transduced or not with hTERT displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in hTERT-transduced cells with extended life span. These cells, despite maintaining high expression level of genes involved in the cell cycle progression, also showed increased expression in several genes found in common with normal aging T lymphocytes. In particular, late-passage T‑cells over-expressing telomerase accumulated the cyclin-dependent kinase inhibitors p16^Ink4a and p21^Cip1, both associated with in vitro growth arrest. Whether tumor-reactive CD8⁺ T‑cells that ectopically express telomerase could now be used for adoptive transfer therapy in cancer patients remains unclear at this point. Nevertheless, our results regarding the safe and effective use of hTERT-transduced lymphocytes are encouraging, since they indicate that alternative growth arrest mechanisms such as p16^Ink4a and p21^Cip1 are still functional in these cells and regulate their growth potential.

Keywords

Melanoma patients, peptide vaccination, cytolytic T lymphocytes, T‑cell repertoire diversity