title: Regulatory T-cell expansion and function do not account for the impaired alloreactivity of ex vivo-expanded T cells.
abstract: CD3- and CD28-activated T cells expanded for 12 days ex vivo to produce suicide gene-modified T cells are hyporesponsive to alloantigens. To investigate whether this impaired alloreactivity is a result of preferential expansion of regulatory T (Treg) cells, we compared peripheral blood mononuclear cells (PBMC) activated with CD3 and CD28 antibodies co-immobilized on beads and expanded for 12 days with interleukin (IL)-2 (Co(CD3/CD28) cells) to the respective unactivated PBMC in terms of proliferation, cytokine production, and expression of Treg markers [cytotoxic T-lymphocyte antigen 4 (CTLA4), glucocorticoid-induced tumour necrosis factor receptor (GITR) and forkhead box P3 (FoxP3)] after allostimulation. Alloreactive cells were identified by carboxyfluoresceine succinimidyl ester staining dilution. Alloreactive cells in Co(CD3/CD28) cells had a lower proliferative response and a lower potential for IL-2 and interferon-gamma secretion than did those in PBMC, demonstrating a functional impairment of alloreactive cells during ex vivo expansion. Expression of Treg markers transiently increased during ex vivo expansion and was unaffected by depletion of CD25(+) cells (containing Treg cells) before ex vivo PBMC expansion. Such prior CD25(+) depletion did not restore the alloreactivity of Co(CD3/CD28) cells. After allostimulation, expression of Treg markers was restricted to proliferative (alloreactive) cells among PBMC or Co(CD3/CD28) cells. Lastly, CD4(+) CD25(+) cells purified from Co(CD3/CD28) cells lacked suppressive activity when used as a third party, in contrast to CD4(+) CD25(+) cells purified from PBMC. In conclusion, the impaired alloreactivity of T cells expanded ex vivo is not a result of preferential Treg cell expansion and/or enhanced suppressive Treg activity.
authors.affiliation: INSERM, Besançon, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Montcuquet
authors.forename: Patricia
authors.initials: P
authors.lastname: Mercier-Letondal
authors.forename: Sylvain
authors.initials: S
authors.lastname: Perruche
authors.forename: Anne
authors.initials: A
authors.lastname: Duperrier
authors.forename: Mélanie
authors.initials: M
authors.lastname: Couturier
authors.forename: Abdelghani
authors.initials: A
authors.lastname: Bouchekioua
authors.forename: Mark
authors.initials: M
authors.lastname: Bonyhadi
authors.forename: Christophe
authors.initials: C
authors.lastname: Ferrand
authors.forename: Pierre
authors.initials: P
authors.lastname: Tiberghien
authors.forename: Eric
authors.initials: E
authors.lastname: Robinet
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D060908:CTLA-4 Antigen; D049109:Cell Proliferation; D002478:Cells, Cultured; D051858:Forkhead Transcription Factors / Q000378:metabolism; D053450:Glucocorticoid-Induced TNFR-Related Protein; D006801:Humans; D007108:Immune Tolerance; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000096:biosynthesis; D007376:Interleukin-2 / Q000096:biosynthesis; D007959:Lymphocyte Culture Test, Mixed; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*
chemical_list: D015703:Antigens, CD; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D053450:Glucocorticoid-Induced TNFR-Related Protein; D007376:Interleukin-2; D017475:Receptors, Nerve Growth Factor; D018124:Receptors, Tumor Necrosis Factor; C491405:TNFRSF18 protein, human; D007371:Interferon-gamma