Mouse Anti-Human CD40LG Hybridoma [N01]

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.

Objectives To explore the role of regulatory T (T-reg) cells in the establishment of immune tolerance induced by donor-specific transfusion (DST) in mice with skin-heart transplantation. Methods C57BL/6 mice received DST of splenocytes from CD47(+/+) or CD47(-/-) H-2(bm1) mice or no DST 7 days before skin-heart transplantation from major histocompatibility complex class I-mismatched H-2(bm1) donors. The number and proportion of T-reg cells in graft and lymphoid organs were measured by flow cytometry (FACS) and immunohistochemistry (IHC). The inhibitory function of T-reg cells and anti-donor T-cell responses were assessed by mixed lymphocyte reaction. Results We observed that mean survival time (MST) of skin or heart graft was significantly longer in C57BL/6 mice which received DST from CD47(+/+) H-2(bm1) mice than from CD47(-/-) H-2(bm1) mice. By FACS, we found that the number of T-reg cells in spleen was increased significantly in mice which received CD47(-/-) DST compared to mice which received CD47(+/+) DST. However, the percentages of T-reg cells in total splenocytes and lymph node cells were significantly higher in mice that received CD47(+/+) DST than mice which received CD47(-/-) DST. Immunohistochemistry showed an increased heart grafts infiltration of T-reg cells in the recipients with CD47(-/-) DST, but not CD47(+/+) DST. Supporting this, we found that donor T-cell proliferation was significantly suppressed in mice which received CD47(+/+) DST compared to mice which received CD47(-/-) DST. There was no difference of inhibitory function of T-reg cells between these two groups. Conclusion Our results indicated that CD47 expression on DST cells plays an important role in the induction of immune tolerance in mice with skin-heart transplantation. Increased percentage of T-reg cells may contribute to immune tolerance induced by CD47(+/+) DST.