Background: Human CD4⁺ T cell allo-immunity plays pivotal roles during cellular and humoral allograft rejection. Our previous analysis showed that allo-reactive precursors were present in both naïve and memory CD4⁺T cells. It is unclear their distribution within T follicular (T_FH:CD45RO⁺CXCR5⁺) and T conventional (T_CONV:CD45RO⁺CXCR5^–) subsets. To address this question, we measured their allo-specific polarization, proliferation, and TCR clonal repertoire.

Methods: FACS-sorted T_CONV or T_FH cells from 6 healthy control (HC) were used to set up CFSE-MLR assays with autologous monocytes pulsed with allogeneic PBMC lysates. Proliferation and cytokine production (IFN-γ, IL-10, IL-17a, IL-21) were assessed by multi-parameter flow cytometry. In addition, DNA from selected in vitro co-cultures was isolated and the TCR-CDR3 regions were profiled by PCR amplification followed by high throughput sequencing using the immunoSEQ platform (Adaptive Biotechnologies).

Results: Indirect allo-stimulation induced low proliferation, low effector cytokines (IFN-γ/IL-17a/IL-21) and significant high IL-10 responses within T_FH and T_CONV cells (considered naïve response) in all subjects but one, who displayed low IL-10 secretion paralleled by high IFN-γ/IL-21 in T_FH and high IFN-γ/IL-17a in T_CONV cells, indicating a memory allogeneic response. ImmunoSEQ was performed on one naïve and one who displayed functional memory. Although resting T_FH cells from both subjects displayed a more diverse (polyclonal) TCR repertoire compared to T_CONV, allo-stimulation triggered significant TCR clonotype expansion in both T_FH and T_CONV only in the HC who showed allo-reactive memory responses. Moreover, resting T_CONV cells contained most alloreactive TCR clonotypes that correlated positively with expanded allo-reactive TCR clonotypes after allo-stimulation. Two out of 38 TCR expanded clones were shared between T_FH and T_CONV after in vitro allo-specific stimulation, suggesting potential intraclonal diversity.

Conclusion: These comprehensive analyses of human circulating T_FH and T_CONV cells reflect the heterogeneity of allo-reactivity among humans. In addition, monitoring of circulating allo-reactive T_FH and T_CONV cells may be important for identifying patients with pre-formed donor-reactive memory responses (effector cytokines and high TCR clonotype expansion) that may be deleterious post-transplant.

CITATION INFORMATION: Macedo C., Yamada M., Gu X., Suchoroski M., Hamm D., Kaplan M., Metes D. Allogeneic T Follicular and T Conventional Responses Assessed by Proliferation, Cytokine Production, and TCR Clonality Am J Transplant. 2017;17 (suppl 3).

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