Generation Of Human CD8 Research Articles

Adoptive Transfer of Interleukin-21-stimulated Human CD8+ T Memory Stem Cells Efficiently Inhibits Tumor Growth.

Memory stem T (TSCM) cells, a new subset of memory T cells with self-renewal and multipotent capacities, are considered as a promising candidates for adoptive cellular therapy. However, the low proportion of human TSCM cells in total CD8+ T cells limits their utility. Here, we aimed to induce human CD8+ TSCM cells by stimulating naive precursors with interleukin-21 (IL-21). We found that IL-21 promoted the generation of TSCM cells, described as CD45RA+CD45RO−CD62L+CCR7+CD122+CD95+ cells, with a higher efficiency than that observed with other common γ-chain cytokines. Upon adoptive transfer into an A375 melanoma mouse model, these lymphocytes mediated much stronger antitumor responses. Further mechanistic analysis revealed that IL-21 activated the Janus kinase signal transducer and activator of transcription 3 pathway by upregulating signal transducer and activator of transcription 3 phosphorylation and consequently promoting the expression of T-bet and suppressor of cytokine signaling 1, but decreasing the expression of eomesodermin and GATA binding protein 3. Our findings provide novel insights into the generation of human CD8+ TSCM cells and reveal a novel potential clinical application of IL-21.

Role of bone marrow stromal cells in the generation of human CD8 + regulatory T cells

Fibroblast-like stromal cells exert a strong inhibitory effect on lymphocyte proliferation, both directly by interacting with responding lymphocytes and indirectly by inducing the generation of regulatory T cells. Indeed, upon triggering via the CD3/TCR complex, highly effective CD8 +regulatory cells (CD8 +Reg c) are generated from cocultures of peripheral blood CD8 +T cells and bone-marrow-derived stromal cells. When cell-to-cell interactions occur, CD8 +Reg c strongly inhibit lymphocyte proliferation at a ratio of 1:1 to 1:100 between CD8 +Reg c and responding lymphocytes. Phenotypic analysis indicated that CD8 +Reg c are CD25 +CD28 + and express low levels of mRNA for Foxp3 but they do not bear CTLA4 and glucocorticoid-induced tumor necrosis factor receptor antigens. Soluble mediators such as interleukin-10, transforming growth factor-β, and prostaglandin E 2 are not involved in the generation of CD8 +Reg c from CD8 + precursors or in the immunosuppressive mechanism mediated by CD8 +Reg c on lymphocyte proliferation. Cyclosporin A (CSA) slightly downregulated generation of CD8 +Reg c indicating that only a small fraction of precursors of CD8 +Reg c are sensitive to this immune-suppressive drug. Along this line, treatment of effector CD8 +Reg cwith CSA does not affect their immunosuppressive effect, indicating that the molecular mechanism of CD8 +Reg c-mediated regulation is independent of the function of CSA biochemical target molecules.

PREVENTING HUMAN CD8+ CYTOTOXIC T LYMPHOCYTES (CTL)-MEDIATED CYTOTOXICITY AGAINST SWINE ENDOTHELIAL CELLS BY OVEREXPRESSION OF HUMAN DECOY FAS ANTIGEN

P1060 Aims: Human CD8+ cytotoxic T lymphocytes (CTL)-mediated killing in xenograft recipients may be a new immunological obstacle for long-term graft survival. The objectives in this study are to assess the mechanism of human CTL-mediated xenocytotoxicity and to explore the method to prevent this killing with overexpression of human decoy Fas antigen by binding competition with endogenous Fas antigen on swine cells for common ligand, human FasL. Furthermore, we generate a membrane-bound form of human FasL that can not be cleaved with metalloproteinase and assess the usefulness of this molecule in inhibition of CTL cytotoxicity. Methods: Generation of human CD8+ CTL: PBMC freshly obtained from healthy volunteers were separated by centrifugation onto a cushion of Histopaque-1077. The fresh lymphocytes were co-cultured for 14 days with irradiated swine endothelial cells (SEC) and human IL-2 (100 U/ml). We employed cultured lymphocytes as effector cells in cytotoxicity assay. Cytotoxicity assay and blockade of lymphocytes killing in the cytotoxicity assay: The Cytotoxicity of human CTL incubated under various effector-to-target ratios was assessed by Terasaki plates. Parental SEC and SEC transfectants were plated as target cells in those plates. Furthermore, the blocking experiments were performed using Concanamycin A to inhibit perforin-dependent lysis and an anti-human FasL mAb to block Fas/FasL pathway. Gene: cDNAs of human decoy Fas which does not contain the death domain in its cytoplasmic tail and FasL carrying the deletion at the cleavage site with metalloproteinase, were subcloned into pEF-BOS expression vector (promoter of human elongation factor 1α), respectively. Expression of plasmids: These plasmids were transfected into SEC by lipofection, respectively. The expression of these molecules on SEC transfectants were assessed with FACS analysis using PE-conjugated mAbs. Results: Mechanism of human CTL cytotoxicity: The human CTL killing was dramatically inhibited by anti-FasL mAb, whereas the killing with them was partially suppressed by Concanamycin A. These results are averages of 3∼6 experiments in the table.FigureConclusions: The Fas/FasL pathway is a main contributor to human CTL killing against xenograft cells. SEC transfectants with either human decoy Fas antigen or membrane-bound FasL gene were highly resistant to human CTL-mediated lysis. Our data demonstrate that these novel molecules may represent a step forward toward preventing cellular xenograft rejection and that the combinatorial expression of both molecules may be more beneficial to protect xenograft cells.

Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells.

Although CD8 T cell–mediated immunosuppression has been a well-known phenomenon during the last three decades, the nature of primary CD8 T suppressor cells and the mechanism underlying their generation remain enigmatic. We demonstrated that naive CD8 T cells primed with allogeneic CD40 ligand–activated plasmacytoid dendritic cells (DC)2 differentiated into CD8 T cells that displayed poor secondary proliferative and cytolytic responses. By contrast, naive CD8 T cells primed with allogeneic CD40 ligand–activated monocyte-derived DCs (DC1) differentiated into CD8 T cells, which proliferated to secondary stimulation and killed allogeneic target cells. Unlike DC1-primed CD8 T cells that produced large amounts of interferon (IFN)-γ upon restimulation, DC2-primed CD8 T cells produced significant amounts of interleukin (IL)-10, low IFN-γ, and no IL-4, IL-5, nor transforming growth factor (TGF)-β. The addition of anti–IL-10–neutralizing monoclonal antibodies during DC2 and CD8 T cell coculture, completely blocked the generation of IL-10–producing anergic CD8 T cells. IL-10–producing CD8 T cells strongly inhibit the allospecific proliferation of naive CD8 T cells to monocytes, and mature and immature DCs. This inhibition was mediated by IL-10, but not by TGF-β. IL-10–producing CD8 T cells could inhibit the bystander proliferation of naive CD8 T cells, provided that they were restimulated nearby to produce IL-10. IL-10–producing CD8 T cells could not inhibit the proliferation of DC1-preactivated effector T cells. This study demonstrates that IL-10–producing CD8 T cells are regulatory T cells, which provides a cellular basis for the phenomenon of CD8 T cell–mediated immunosuppression and suggests a role for plasmacytoid DC2 in immunological tolerance.

Cationic liposome-mediated enhanced generation of human HLA-restricted RSV-specific CD8+ CTL+.

Generation of human CD8+ cytotoxic T-lymphocyte (CTL) activity against respiratory syncytial virus (RSV) using peripheral blood leukocytes (PBL) in vitro is inefficient. Lipofectamine, a polycationic liposome, previously shown to enhance the transfection efficiency of DNA in cells, was evaluated for enhancing RSV CTL activity. Stimulator cells were prepared by infecting human PBL with RSV with or without Lipofectamine for 3 hr and then transferred to responder cells. After 8 days of incubation, CTL lysis of autologous target cells infected with RSV (also treated with Lipofectamine) was determined in a 4-hr 5'chromium release assay. Lipofectamine treatment significantly enhanced HLA-restricted RSV-specific CD8+ CTL activity (up to sevenfold, P < 0.05-0.001). Lipofectamine treatment also enhanced cell surface RSV antigen expression and increased the frequencies of HLA-A,B,C+/RSV+ and HLA-DR+/RSV+ leukocytes as demonstrated by flow cytometry. These results demonstrate the usefulness of cationic liposomes in augmenting cell surface antigen expression and increasing the efficiency of generation of human RSV-specific CD8+ CTL activity.