Background: It is well accepted that human CD4⁺ T cell allo-immunity plays pivotal roles during cellular and humoral allograft rejection. While our previous analysis showed that allo-reactive precursors were equally present in both naïve and memory CD4⁺ T cells, it is yet unclear what is the contribution of T_FH vs T_CONV CD4⁺ T to the allo-immune response. One approach to address this question was to assess the direct and the indirect allo-reactivity of human blood T_FH and T_CONV cells by measuring their functional polarization and ability to proliferate to allo-Ag in CFSE-MLR assays.

Methods: Bulk PBMC and FACS sorted blood memory helper T_FH (CD45RO⁺CXCR5⁺) or T_CONV (CD45RO⁺CXCR5^–) CD4⁺ T cells from 3 healthy subjects were CFSE stained and used to set up ex vivo CFSE-MLR assays all at 1:1 ratio for 5 days. To analyze the direct pathway of allo-presentation, bulk PBMC or sorted CD4⁺ T cells were incubated with PKH16-stained allogeneic PBMC, while for indirect-pathway of allo-recognition, flow-sorted autologous PKH16-stained monocytes, were pulsed or not with donor-derived PBMC lysates. Specific functional responses such as proliferation by CFSE-dilution and cytokine production were all assessed by multi-parameter flow cytometry.

Results: Direct allo-stimulation triggered (i) proliferation, IFN-γ responses and negligible IL-10 release within T_FH and T_CONV cells in all subjects tested, (ii) IL-17a production by T_CONV in two subjects and IL-21 release by T_FH >T_CONV from one of the three subjects. In contrast, the indirect allo-stimulation induced (i) low proliferation, low effector cytokines (including IFN-γ) and significant high IL-10 responses within T_FH and T_CONV cells in all subjects but one, who displayed low IL-10 secretion paralleled by high IFN-γ and IL-21 in T_FH cells or by IFN-γ and IL-17a in the T_CONV cells.

Conclusion: These comprehensive analyses of human circulating allo-reactive T_FH and T_CONV cell responses reflect the heterogeneity of allo-reactivity among humans, and are critical for the understanding of how distinct pathways of allo-reactivity are regulated. In addition, monitoring circulating allo-Ag reactive T_FH cells using the indirect pathway of allo-stimulation may be important at identifying patients with defective IL-10 responses, that may be permissive for deleterious effector cytokine responses post-Tx.

CITATION INFORMATION: Macedo C, Paul F, Masaki Y, Kevin H, Diana M. Indirect Allo-Antigen Stimulation Identifies the Presence of Either Regulatory IL-10 or of Effector Cytokines IFN-g, IL-21, IL-17a Production by Human Blood Allo-Reactive Memory T_FH and T_CONV CD4⁺ T Cells. Am J Transplant. 2016;16 (suppl 3).

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