Background and Objectives Mesenchymal stem cells (MSC) have been proposed as a way to treat graft-versus-host disease based on their immunosuppressive effect. We analyzed whether regulatory T cells can be generated in co-cultures of peripheral blood mononuclear cells (PBMC) and MSC. Design and Methods MSC were obtained from the bone marrow of four healthy donors and nine patients with acute leukemia in complete remission following chemotherapy. Short-term (4 days) co-cultures of MSC and autologous or allogeneic PBMC were set up, the lymphocytes harvested and their regulatory activity assessed. Results Lymphocytes harvested from MSC-PBMC co-cultures strongly inhibit (up to 95%) mixed lymphocyte reaction (MLR), recall to alloantigen, and CD3- or phytohemagglutinin-induced lymphocyte proliferation. These lymphocytes, termed regulatory cells (Reg_c), were all CD45⁺CD2⁺ with variable proportions of CD25⁺ cells (range 40–75% n=10) and a minor fraction expressed CTLA4 (2–4%, n=10) or glucocorticoid-induced tumor necrosis factcor receptor-related gene (0.5–4% n=10). Both CD4⁺ and CD8⁺ Reg_c purified from MSC-PBMC co-cultures strongly inhibited lymphocyte proliferation at a 1:100 Reg_c:responder cell ratio. CD4⁺ Reg_c expressed high levels of forkhead box P3 (Foxp3) mRNA while CD8⁺ Reg_c did not. The effectiveness of Reg_c, whether CD4⁺ or CD8⁺, was 100-fold higher than that of CD4⁺CD25^+high regulatory T cells. Reg_c were also generated from highly purified CD25⁻ PBMC or CD4⁺ or CD8⁺ T cell subsets. Soluble factors, such as interleukin-10, transforming growth factor-β and prostaglandin E₂ did not appear to be involved in the generation of Reg_c or in the Reg_c-mediated immuno-suppressive effect. Furthermore, cyclosporine A did not affect Reg_c generation or the immunosuppression induced by Reg_c. Interpretation and Conclusions These findings indicate that powerful regulatory CD4⁺ or CD8⁺ lymphocytes are generated in co-cultures of PBMC with MSC. This strongly suggests that these regulatory cells may amplify the reported MSC-mediated immunosuppressive effect.

We analyzed 38 untreated patients with chronic lymphocytic leukemia of B-cell type (B-CLL): 24 low-, 8 intermediate-, and 6 high-risk stage. In 15 patients (13 low risk and 2 intermediate risk), circulating Vδ1 T lymphocytes were significantly increased (100 to 300 cells/μL) compared with most intermediate, all high-risk stage, and 15 healthy donors (50 to 100 cells/μL). We studied these Vδ1 T lymphocytes and observed that they proliferated in vitro and produced tumor necrosis factor α or IFN-γ in response to autologous leukemic B cells but not to normal lymphocytes. However, they were unable to kill resting autologous B cells, which lack the MHC-related MIC-A antigen and express low levels of the UL16-binding protein (ULBP) 3 and undetectable levels of ULBP1, ULBP2, and ULBP4. All these molecules are reported ligands for the NKG2D receptor, which is expressed by γδ T cells and activates their cytolytic function. The Vδ1 T lymphocytes studied were able to lyse the ULBP3⁺ C1R B-cell line upon transfection with MIC-A. More importantly, they also lysed autologous B-CLL cells when transcription and expression of MIC-A or up-regulation of ULBP3 were achieved either by activation or by exposure to trans-retinoic acid. The NKG2D receptor expressed on Vδ1 T cells was involved in the recognition of B-CLL. Finally, in six patients with low numbers of circulating Vδ1 T cells and undetectable ULBP3, the disease progressed over 1 year, whereas no progression occurred in patients with high Vδ1 T lymphocytes and detectable/inducible ULBP3. These data suggest that Vδ1 T lymphocytes may play a role in limiting the progression of B-CLL.

In early HIV-1 infection, Vδ1 T lymphocytes are increased in peripheral blood and this is related to chemokine receptor expression, chemokine response, and recirculation. Herein we show that, at variance with healthy donors, in HIV-1–infected patients ex vivo–isolated Vδ1 T cells display cytoplasmic interferon-γ (IFN-γ). Interestingly, these cells coexpress cytoplasmic interleukin-17 (IL-17), and bear the CD27 surface marker of the memory T-cell subset. Vδ1 T cells, isolated from either patients or healthy donors, can proliferate and produce IFN-γ and IL-17 in response to Candida albicans in vitro, whereas Vδ2 T cells respond with proliferation and IFN-γ/IL-17 production to mycobacterial or phosphate antigens. These IFN-γ/IL-17 double-producer γδ T cells express the Th17 RORC and the Th1 TXB21 transcription factors and bear the CCR7 homing receptor and the CD161 molecule that are involved in γδ T-cell transendothelial migration. Moreover, Vδ1 T cells responding to C albicans express the chemokine receptors CCR4 and CCR6. This specifically equipped circulating memory γδ T-cell population might play an important role in the control of HIV-1 spreading and in the defense against opportunistic infections, possibly contributing to compensate for the impairment of CD4+ T cells.

We show that HIV-1–infected patients have increased concentrations of circulating Vδ1 T cells (2.2%-9.0% of T lymphocytes; healthy donors, 1.0%-2%) and, in some instances, Vδ2 T cells (3.5%-4.8% vs 2.0%-3.3%). In these patients, both Vδ1 and Vδ2 T cells are CXCR3⁺CXCR4⁺, whereas in healthy donors CXCR4 was preferentially expressed on Vδ1 T lymphocytes. γδ T cells transmigrated across endothelial monolayers, in response to interferon-γ–inducing protein-10 (IP-10/CXCL10), stromal cell–derived factor-1 (SDF-1/CXCL12), or both, according to the expression of the specific receptors CXCR3 and CXCR4. Interestingly, 6Ckine/SLC/CCL21 was more effective than IP-10/CXCL10 on Vδ1 CXCR3⁺ cells, whereas Vδ2 CXCR3⁺ cells were driven more efficiently by IP-10/CXCL10. IP-10/CXCL10– and SDF-1/CXCL12–induced transmigration was dependent on phosphoinositide-3 kinase (PI-3K), as demonstrated by the use of the specific blockers wortmannin and LY294002 and by the activation of the downstream serine kinase Akt/PKB on ligation of CXCR3 and CXCR4. Occupancy of CXCR3, but not of CXCR4, led to CAMKII activation; accordingly, the CAMKII inhibitors KN62 and KN93 decreased IP-10/CXCL10– but not SDF-1/CXCL12–driven transmigration. Finally, HIV-1 Tat, which is present in the serum of HIV-1–infected patients, interferes with the chemotactic activity of these chemokines because of the cysteine-rich domain of the protein, which contains CXC and CC chemokine–like sequences.