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Requirement of cognate CD4+ T-cell recognition for the regulation of allospecific CTL by human CD4+ CD127- CD25+ FOXP3+ cells generated in MLR.

Yu Y, Miller J, Leventhal JR, Tambur AR, Chandrasekaran D, Levitsky J, Luo X, Mathew JM - PLoS ONE (2011)

Bottom Line: Therefore, it was concluded that human CD4(+)CD127(-)CD25(+)FOXP3(+) MLR-Tregs down-regulate alloreactive cytotoxic responses.Regulatory allospecificity, however, requires the presence of cognate responding CD4(+) T cells.CD8(+) CTL regulatory mechanisms include impaired proliferation, reduced expression of cytolytic molecules and CD25(+) activation epitopes.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT
Although immunoregulation of alloreactive human CTLs has been described, the direct influence of CD4(+) Tregs on CD8(+) cytotoxicity and the interactive mechanisms have not been well clarified. Therefore, human CD4(+)CD127(-)CD25(+)FOXP3(+) Tregs were generated in MLR, immunoselected and their allospecific regulatory functions and associated mechanisms were then tested using modified (51)Chromium release assays (Micro-CML), MLRs and CFSE-based multi-fluorochrome flow cytometry proliferation assays. It was observed that increased numbers of CD4(+)CD127(-)CD25(+)FOXP3(+) cells were generated after a 7 day MLR. After immunoselection for CD4(+)CD127(-)CD25(+) cells, they were designated as MLR-Tregs. When added as third component modulators, MLR-Tregs inhibited the alloreactive proliferation of autologous PBMC in a concentration dependent manner. The inhibition was quasi-antigen specific, in that the inhibition was non-specific at higher MLR-Treg modulator doses, but non-specificity disappeared with lower numbers at which specific inhibition was still significant. When tested in micro-CML assays CTL inhibition occurred with PBMC and purified CD8(+) responders. However, antigen specificity of CTL inhibition was observed only with unpurified PBMC responders and not with purified CD8(+) responders or even with CD8(+) responders plus Non-T "APC". However, allospecificity of CTL regulation was restored when autologous purified CD4(+) T cells were added to the CD8(+) responders. Proliferation of CD8(+) cells was suppressed by MLR-Tregs in the presence or absence of IL-2. Inhibition by MLR-Tregs was mediated through down-regulation of intracellular perforin, granzyme B and membrane-bound CD25 molecules on the responding CD8(+) cells. Therefore, it was concluded that human CD4(+)CD127(-)CD25(+)FOXP3(+) MLR-Tregs down-regulate alloreactive cytotoxic responses. Regulatory allospecificity, however, requires the presence of cognate responding CD4(+) T cells. CD8(+) CTL regulatory mechanisms include impaired proliferation, reduced expression of cytolytic molecules and CD25(+) activation epitopes.

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Flow diagram depicting the culture system for the generation of MLR-Tregs (step #1) and their utilization in various MLR, micro- CML and flow cytometric assays (step #2).
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pone-0022450-g001: Flow diagram depicting the culture system for the generation of MLR-Tregs (step #1) and their utilization in various MLR, micro- CML and flow cytometric assays (step #2).

Mentions: MLR-Tregs were generated as we previously reported [17] and as shown in the top portion of Figure 1. Briefly, PBMC were isolated by Ficoll-Hypaque density gradient centrifugation and 40×106 responder cells were stimulated with 40×106 irradiated (3000 R) stimulator cells in culture medium [NAB-CM; RPMI-1640 supplemented with 2 mM L-glutamine, 10 mM HEPES, 100 U/ml Penicillin-Streptomycin (all from Mediatech, Manassas, VA) and 15% normal human AB serum (Gemini Bio-Products, W. Sacramento, CA)] at 1×106 cells/ml at 37°C in 5% CO2 in multiple T-75 flasks. After 7 days, the CD4+CD127−CD25+ cells were purified using the Treg isolation kit and the AutoMACS (Miltenyi Biotech, Auburn, CA) as previously described [17].


Requirement of cognate CD4+ T-cell recognition for the regulation of allospecific CTL by human CD4+ CD127- CD25+ FOXP3+ cells generated in MLR.

Yu Y, Miller J, Leventhal JR, Tambur AR, Chandrasekaran D, Levitsky J, Luo X, Mathew JM - PLoS ONE (2011)

Flow diagram depicting the culture system for the generation of MLR-Tregs (step #1) and their utilization in various MLR, micro- CML and flow cytometric assays (step #2).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3142165&req=5

pone-0022450-g001: Flow diagram depicting the culture system for the generation of MLR-Tregs (step #1) and their utilization in various MLR, micro- CML and flow cytometric assays (step #2).
Mentions: MLR-Tregs were generated as we previously reported [17] and as shown in the top portion of Figure 1. Briefly, PBMC were isolated by Ficoll-Hypaque density gradient centrifugation and 40×106 responder cells were stimulated with 40×106 irradiated (3000 R) stimulator cells in culture medium [NAB-CM; RPMI-1640 supplemented with 2 mM L-glutamine, 10 mM HEPES, 100 U/ml Penicillin-Streptomycin (all from Mediatech, Manassas, VA) and 15% normal human AB serum (Gemini Bio-Products, W. Sacramento, CA)] at 1×106 cells/ml at 37°C in 5% CO2 in multiple T-75 flasks. After 7 days, the CD4+CD127−CD25+ cells were purified using the Treg isolation kit and the AutoMACS (Miltenyi Biotech, Auburn, CA) as previously described [17].

Bottom Line: Therefore, it was concluded that human CD4(+)CD127(-)CD25(+)FOXP3(+) MLR-Tregs down-regulate alloreactive cytotoxic responses.Regulatory allospecificity, however, requires the presence of cognate responding CD4(+) T cells.CD8(+) CTL regulatory mechanisms include impaired proliferation, reduced expression of cytolytic molecules and CD25(+) activation epitopes.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT
Although immunoregulation of alloreactive human CTLs has been described, the direct influence of CD4(+) Tregs on CD8(+) cytotoxicity and the interactive mechanisms have not been well clarified. Therefore, human CD4(+)CD127(-)CD25(+)FOXP3(+) Tregs were generated in MLR, immunoselected and their allospecific regulatory functions and associated mechanisms were then tested using modified (51)Chromium release assays (Micro-CML), MLRs and CFSE-based multi-fluorochrome flow cytometry proliferation assays. It was observed that increased numbers of CD4(+)CD127(-)CD25(+)FOXP3(+) cells were generated after a 7 day MLR. After immunoselection for CD4(+)CD127(-)CD25(+) cells, they were designated as MLR-Tregs. When added as third component modulators, MLR-Tregs inhibited the alloreactive proliferation of autologous PBMC in a concentration dependent manner. The inhibition was quasi-antigen specific, in that the inhibition was non-specific at higher MLR-Treg modulator doses, but non-specificity disappeared with lower numbers at which specific inhibition was still significant. When tested in micro-CML assays CTL inhibition occurred with PBMC and purified CD8(+) responders. However, antigen specificity of CTL inhibition was observed only with unpurified PBMC responders and not with purified CD8(+) responders or even with CD8(+) responders plus Non-T "APC". However, allospecificity of CTL regulation was restored when autologous purified CD4(+) T cells were added to the CD8(+) responders. Proliferation of CD8(+) cells was suppressed by MLR-Tregs in the presence or absence of IL-2. Inhibition by MLR-Tregs was mediated through down-regulation of intracellular perforin, granzyme B and membrane-bound CD25 molecules on the responding CD8(+) cells. Therefore, it was concluded that human CD4(+)CD127(-)CD25(+)FOXP3(+) MLR-Tregs down-regulate alloreactive cytotoxic responses. Regulatory allospecificity, however, requires the presence of cognate responding CD4(+) T cells. CD8(+) CTL regulatory mechanisms include impaired proliferation, reduced expression of cytolytic molecules and CD25(+) activation epitopes.

Show MeSH
Related in: MedlinePlus