RESUMO
CD28/B7 co-stimulation blockade with belatacept prevents alloreactivity in kidney transplant patients. However, cells lacking CD28 are not susceptible to belatacept treatment. As CD8(+) CD28(-) T-cells have cytotoxic and pathogenic properties, we investigated whether mesenchymal stem cells (MSC) are effective in controlling these cells. In mixed lymphocyte reactions (MLR), MSC and belatacept inhibited peripheral blood mononuclear cell (PBMC) proliferation in a dose-dependent manner. MSC at MSC/effector cell ratios of 1:160 and 1:2·5 reduced proliferation by 38·8 and 92·2%, respectively. Belatacept concentrations of 0·1 µg/ml and 10 µg/ml suppressed proliferation by 20·7 and 80·6%, respectively. Both treatments in combination did not inhibit each other's function. Allostimulated CD8(+) CD28(-) T cells were able to proliferate and expressed the cytolytic and cytotoxic effector molecules granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. While belatacept did not affect the proliferation of CD8(+) CD28(-) T cells, MSC reduced the percentage of CD28(-) T cells in the proliferating CD8(+) T cell fraction by 45·9% (P = 0·009). CD8(+) CD28(-) T cells as effector cells in MLR in the presence of CD4(+) T cell help gained CD28 expression, an effect independent of MSC. In contrast, allostimulated CD28(+) T cells did not lose CD28 expression in MLR-MSC co-culture, suggesting that MSC control pre-existing CD28(-) T cells and not newly induced CD28(-) T cells. In conclusion, alloreactive CD8(+) CD28(-) T cells that remain unaffected by belatacept treatment are inhibited by MSC. This study indicates the potential of an MSC-belatacept combination therapy to control alloreactivity.
Assuntos
Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos/imunologia , Imunoconjugados/farmacologia , Leucócitos Mononucleares/imunologia , Células-Tronco Mesenquimais/imunologia , Abatacepte , Apoptose , Proliferação de Células , Células Cultivadas , Granzimas/biossíntese , Humanos , Interferon gama/biossíntese , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Due to their immunomodulatory properties, mesenchymal stem cells (MSC) are interesting candidates for cellular therapy for autoimmune disorders, graft-versus-host disease and allograft rejection. MSC inhibit the proliferation of effector T cells and induce T cells with a regulatory phenotype. So far it is unknown whether human MSC-induced CD4(+) CD25(+) CD127(-) forkhead box P3 (FoxP3)(+) T cells are functional and whether they originate from effector T cells or represent expanded natural regulatory T cells (nT(reg)). Perirenal adipose-tissue derived MSC (ASC) obtained from kidney donors induced a 2·1-fold increase in the percentage of CD25(+) CD127(-) FoxP3(+) cells within the CD4(+) T cell population from allostimulated CD25(-/dim) cells. Interleukin (IL)-2 receptor blocking prevented this induction. The ASC-induced T cells (iT(reg)) inhibited effector cell proliferation as effectively as nT(reg). The vast majority of cells within the iT(reg) fraction had a methylated FOXP3 gene T(reg)-specific demethylated region (TSDR) indicating that they were not of nT(reg) origin. In conclusion, ASC induce T(reg) from effector T cells. These iT(reg) have immunosuppressive capacities comparable to those of nT(reg). Their induction is IL-2 pathway-dependent. The dual effect of MSC of inhibiting immune cell proliferation while generating de-novo immunosuppressive cells emphasizes their potential as cellular immunotherapeutic agent.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Fatores de Transcrição Forkhead/metabolismo , Células-Tronco Mesenquimais/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Tecido Adiposo/citologia , Anticorpos Monoclonais/farmacologia , Basiliximab , Antígenos CD4/metabolismo , Separação Celular , Células Cultivadas , Metilação de DNA , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Humanos , Tolerância Imunológica , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Receptores de Interleucina-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Mesenchymal stem cells (MSCs) have potential for therapeutic application as an immunomodulatory and regenerative agent. The immunogenicity and survival of MSCs after infusion are, however, not clear and evidence suggests that allogeneic but also autologous MSCs disappear rapidly after infusion. This may be associated with the susceptibility of MSCs to lysis by natural killer (NK) cells, possibly a result of culture-induced stress. In the present study we examined whether NK cell-mediated lysis of MSCs could be inhibited by immunosuppressive drugs. Human MSCs were isolated from adipose tissue and expanded in culture. Peripheral blood mononuclear cells were activated with interleukin (IL)-2 (200 U/ml) and IL-15 (10 ng/ml) for 7 days. CD3(-)CD16(+)CD56(+) NK cells were then isolated by fluorescence-activated cell sorting and added to europium-labeled MSCs for 4 hr in the presence or absence of immunosuppressive drugs. Lysis of MSCs was determined by spectrophotometric measurement of europium release. Nonactivated NK cells were not capable of lysing MSCs. Cytokine-activated NK cells showed upregulated levels of granzyme B and perforin and efficiently lysed allogeneic and autologous MSCs. Addition of tacrolimus, rapamycin or sotrastaurin to the lysis assay did not inhibit MSC killing. Furthermore, preincubation of activated NK cells with the immunosuppressive drugs for 24 hr before exposure to MSCs had no effect on MSC lysis. Last, addition of the immunosuppressants before and during the activation of NK cells, reduced NK cell numbers but did not affect their capacity to lyse MSCs. We conclude that the immunosuppressive drugs tacrolimus, rapamycin, and sotrastaurin are not capable of inhibiting the lysis of allogeneic and autologous MSCs by activated NK cells. Other approaches to controlling lysis of MSCs should be investigated, as controlling lysis may determine the efficacy of MSC therapy.