IL-21-mediated potentiation of antitumor cytolytic and proinflammatory responses of human V gamma 9V delta 2 T cells for adoptive immunotherapy.

Vgamma9Vdelta2 T lymphocytes are a major human gammadelta T cell subset that react against a wide array of tumor cells, through recognition of phosphorylated isoprenoid pathway metabolites called phosphoantigens. Immunotherapeutic protocols targeting Vgamma9Vdelta2 T cells have yielded promising, yet limited, signs of antitumor efficacy. To improve these approaches, we analyzed the effects on gammadelta T cells of IL-21, a cytokine known to enhance proliferation and effector functions of CD8(+) T cells and NK cells. IL-21 induced limited division of phosphoantigen-stimulated Vgamma9Vdelta2 T cells, but did not modulate their sustained expansion induced by exogenous IL-2. Vgamma9Vdelta2 T cells expanded in the presence of IL-21 and IL-2 showed enhanced antitumor cytolytic responses, associated with increased expression of CD56 and several lytic molecules, and increased tumor-induced degranulation capacity. IL-21 plus IL-2-expanded Vgamma9Vdelta2 T cells expressed higher levels of inhibitory receptors (e.g., ILT2 and NKG2A) and lower levels of the costimulatory molecule NKG2D. Importantly, these changes were rapidly and reversibly induced after short-term culture with IL-21. Finally, IL-21 irreversibly enhanced the proinflammatory Th1 polarization of expanded Vgamma9Vdelta2 T cells when added at the beginning of the culture. These data suggest a new role played by IL-21 in the cytotoxic and Th1 programming of precommitted Ag-stimulated gammadelta T cells. On a more applied standpoint, IL-21 could be combined to IL-2 to enhance gammadelta T cell-mediated antitumor responses, and thus represents a promising way to optimize immunotherapies targeting this cell subset.

HLA-E-restricted cross-recognition of allogeneic endothelial cells by CMV-associated CD8 T cells: a potential risk factor following transplantation.

Although association between CMV infection and allograft rejection is well admitted, the precise mechanisms involved remain uncertain. Here, we report the characterization of an alloreactive HLA-E-restricted CD8 T cell population that was detected in the PBL of a kidney transplant patient after its CMV conversion. This monoclonal CD8 T cell population represents a sizable fraction in the blood (3% of PBL) and is characterized by an effector-memory phenotype and the expression of multiple NK receptors. Interestingly, these unconventional T cells display HLA-E-dependent reactivity against peptides derived from the leader sequences of both various HCMV-UL40 and allogeneic classical HLA-I molecules. Consequently, while HLA-E-restricted CD8 T cells have potential to contribute to the control of CMV infection in vivo, they may also directly mediate graft rejection through recognition of peptides derived from allogeneic HLA-I molecules on graft cells. Therefore, as HLA-E expression in nonlymphoid organs is mainly restricted to endothelial cells, we investigated the reactivity of this HLA-E-restricted T cell population towards allogeneic endothelial cells. We clearly demonstrated that CMV-associated HLA-E-restricted T cells efficiently recognized and killed allogeneic endothelial cells in vitro. Moreover, our data indicate that this alloreactivity is tightly regulated by NK receptors, especially by inhibitory KIR2DL2 that strongly prevents TCR-induced activation through recognition of HLA-C molecules. Hence, a better evaluation of the role of CMV-associated HLA-E-restricted T cells in transplantation and of the impact of HLA-genotype, especially HLA-C, on their alloreactivity may determine whether they indeed represent a risk factor following organ transplantation.

Cross-reactivity of herpesvirus-specific CD8 T cell lines toward allogeneic class I MHC molecules.

Although association between persistent viral infection and allograft rejection is well characterized, few examples of T-cell cross-reactivity between self-MHC/viral and allogeneic HLA molecules have been documented so far. We appraised in this study the alloreactivity of CD8 T cell lines specific for immunodominant epitopes from human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). CD8 T cell lines were generated after sorting with immunomagnetic beads coated with either pp65(495-503)/A*0201, BMLF1(259-267)/A*0201, or BZLF1(54-64)/B*3501 multimeric complexes. Alloreactivity of the CD8 T cell lines against allogeneic class I MHC alleles was assessed by screening of (i) TNF-alpha production against COS-7 cells transfected with as many as 39 individual HLA class I-encoding cDNA, and (ii) cytotoxicity activity toward a large panel of HLA-typed EBV-transformed B lymphoblastoid cell lines. We identified several cross-reactive pp65/A*0201-specific T cell lines toward allogeneic HLA-A*3001, A*3101, or A*3201. Moreover, we described here cross-recognition of HLA-Cw*0602 by BZLF1/B*3501-specific T cells. It is noteworthy that these alloreactive CD8 T cell lines showed efficient recognition of endothelial cells expressing the relevant HLA class I allele, with high level TNF-alpha production and cytotoxicity activity. Taken together, our data support the notion that herpes virus-specific T cells recognizing allo-HLA alleles may promote solid organ rejection.

Pattern of DAP12 expression in leukocytes from both healthy and systemic lupus erythematosus patients.

DAP12 is an ITAM-bearing transmembrane adaptor originally identified on the surface of Natural Killer cells. A broad expression among other immune cells was later found in myeloid and lymphoid cells. However, data on DAP12 expression pattern rely only on immunoblot and microarray analysis. Here, we describe the generation and the characterization of an anti-DAP12 monoclonal antibody. Using this novel reagent, we show that DAP12 expression is restricted to innate immune cells in basal condition. Since a decreased expression of DAP12 has been suggested in NK cells of systemic lupus erythematosus patients, we have further investigated the NK cell receptor repertoire and leukocyte expression of DAP12 in these patients and no major changes were detectable when compared to controls.

EBV-specific CD4+ T cell clones exhibit vigorous allogeneic responses.

Alloreactive T cells play a key role in mediating graft-vs-host disease and allograft rejection, and recent data suggest that most T cell alloreactivity resides within the CD4 T cell subset. Particularly, T cell responses to herpesvirus can shape the alloreactive repertoire and influence transplantation outcomes. In this study, we describe six distinct EBV-specific CD4(+) T cell clones that cross-reacted with EBV-transformed lymphoblastoid cell lines (LCLs), dendritic cells, and endothelial cells expressing MHC class II alleles commonly found in the population. Allorecognition showed exquisite MHC specificity. These CD4(+) T cell clones efficiently killed dendritic cells or LCLs expressing the cross-reactive allogeneic MHC class II molecules, whereas they did not kill autologous LCLs. Endothelial cells expressing the proper allogeneic MHC molecules were poorly killed, but they induced high-level TNF-alpha production by the EBV-specific CD4(+) T cell clones. As already proposed, the strong alloreactivity toward LCLs suggest that these cells could be used for selective depletion of alloreactive T cells.