Phenotypic and functional characterization of porcine T follicular helper and regulatory cells

T follicular helper (Tfh) cells provide help to B cells for antibody class switching and affinity maturation and are critical for germinal center (GC) formation. The frequency of Tfh cells has proven to be a valuable marker for predicting durable neutralizing antibody responses; conversely, the expansion of dysfunctional Tfh cells and increased T follicular regulatory (Tfr) cells are associated with suboptimal antibody responses. Given that the pig is becoming an appealing preclinical model for vaccine evaluation, a better understanding of porcine Tfh and Tfr cell populations is required. Novel reagents to detect CXCR5 and PD-1 were generated and deployed in a high-dimensional flow cytometry panel to identify putative Tfh and Tfr cells in porcine blood and lymphoid tissues. Higher frequencies of CXCR5+PD-1-BCL6-CD4+ and CXCR5hiPD-1+BCL6+CD4+ T cells, putative Tfh and GC Tfh cells, respectively, were detected in tonsils compared with other tissues, while higher frequencies of FoxP3+CXCR5+BCL6-CD4+ (putative Tfr) and FoxP3+CXCR5hiBCL6+CD4+ (putative GC Tfr) cells were present in the tracheobronchial lymph nodes. In an in vitro coculture model, both CXCR5hiPD-1+CD4+ and CXCR5+PD-1-CD4+ Tfh cells supported greater B cell proliferation, plasma cell formation, and antibody production than CXCR5-CD4+ T cells. A staining panel developed to profile cytokine and activation-induced marker expression revealed that following T cell receptor stimulation, CXCR5hiBCL6+CD4+ and CXCR5+BCL6-CD4+ cells expressed greater levels of CD40L, ICOS, and IL-4 than non-Tfh cells, resembling findings in human Tfh cells. These data lay the foundation for further studies on porcine Tfh and Tfr cells, and the exploration of strategies to harness these cells for improved vaccine-induced immunity.