Recognition of different subsets of alloreactive T cells by activation-induced markers

Alloreactive T-cells can be visualized using activation-induced markers (AIMs) including CD69, CD134, CD137 and CD154. Whether these AIMs recognize similar subsets of alloreactive T-cells is largely unknown. AIM-expressing alloreactive CD4+ T cells were analyzed in detail for phenotype by dissecting different T-cell subsets using antibodies directed to CCR7 and CD45RA. Moreover, detailed functional analysis was performed by determining proportions of cytokine producing cells within AIM-expressing CD4+ T cells using multiparameter flowcytometry. CD154 was predominantly expressed by naïve and central-memory alloreactive CD4+ T cells, CD134 by central-memory alloreactive CD4+ T cells and CD137 by CD4+ alloreactive memory T cells. Alloreactive CD8+ T cells could only be recognized by CD137 expression. The majority of alloreactive CD4+ T cells were single AIM-positive (72 %) and co-expression of all AIMs was infrequent. Polyclonal stimulation with anti-CD3/anti-CD28 resulted in a high frequency of CD4+ T cells co-expressing AIMs which was a dose-dependent phenomenon. Alloreactive memory CD4+ T cells expressing >1 AIM showed the highest proportion of polyfunctional cells. Allogeneic stimulation of sorted naïve CD4+ T cells yielded a population of proliferating T cells, progressing to effector-memory T cells expressing >1 AIM. In conclusion, different AIMs are preferentially expressed by different subsets of circulating alloreactive CD4+ T cells and expression of AIMs is determined by proliferation/differentiation and strength of the T cell receptor (TCR)-stimulation.