title: Requirement of cognate CD4+ T-cell recognition for the regulation of allospecific CTL by human CD4+ CD127- CD25+ FOXP3+ cells generated in MLR.
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.
authors.affiliation: Department of Surgery, Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.
authors.forename: Yuming
authors.initials: Y
authors.lastname: Yu
authors.forename: Joshua
authors.initials: J
authors.lastname: Miller
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Leventhal
authors.forename: Anat R
authors.initials: AR
authors.lastname: Tambur
authors.forename: Dhivya
authors.initials: D
authors.lastname: Chandrasekaran
authors.forename: Josh
authors.initials: J
authors.lastname: Levitsky
authors.forename: Xunrong
authors.initials: X
authors.lastname: Luo
authors.forename: James M
authors.initials: JM
authors.lastname: Mathew
mesh_terms: D017209:Apoptosis / Q000276:immunology; D015704:CD4 Antigens / Q000378:metabolism*; D002454:Cell Differentiation / Q000276:immunology; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism*; D053699:Interleukin-7 Receptor alpha Subunit / Q000172:deficiency*; D007959:Lymphocyte Culture Test, Mixed; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000276:immunology* / Q000378:metabolism
chemical_list: D015704:CD4 Antigens; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit