Figure 1. Nr2f6 Loss Leads to Tfh Cell Accumulation and Increased GC Responses following OVA-Alum Immunization.

(A) Experimental setup used for OVA-alum immunization of Nr2f6^+/+ or Nr2f6^−/− mice.

(B and C) Total counts of Nr2f6^+/+ or Nr2f6^−/− splenocytes, B220⁺ cells, and CD4⁺ cells on (B) day 4 or (C) day 10 after immunization.

(D and E) Splenic Tfh cell numbers on (D) day 4 or (E) day 10 after immunization; representative flow cytometry plots for Nr2f6^+/+ and Nr2f6^−/− Tfh cells, total Tfh cell counts, and Tfh cell frequency within CD4 cells. Total Tfh cell counts are shown as total live non-Tfr cells (annexin V⁻, FoxP3⁻, and 7-AAD⁻).

(F) Total germinal center B cell counts on day 4.

(G) Day 10 representative flow cytometry plots, with total cell counts.

(H) GC LZ and DZ distribution in Nr2f6^+/+ or Nr2f6^−/− GCs 10 days after immunization.

(I and J) PC counts on day 4 (I), and representative flow cytometry plots and cell counts on day 10 (J).

(K) Immunofluorescence staining for CD3 (white), B220 (red), and peanut agglutinin (PNA) (green) on 5 μM sections of spleens harvested day 10 after OVA immunization.

(L and M) Frequency of live Tfh cells, GC B cells, and PCs (annexin V⁻ 7-AAD⁻) on (L) day 4 or (M) day 10 after OVA-alum immunization.

(N) Tfr (CD4⁺, CXCR5⁺, PD-1⁺, and FoxP3⁺) total cell counts on day 4 (left panel), and the Tfh cell to Tfr cell ratio (right panel).

(O) Representative FoxP3 staining of Nr2f6^+/+ or Nr2f6^−/− CD4⁺ CXCR5⁺ PD-1⁺ T cells, total Tfr cell counts, total Tfr frequency of all CD4 cells, and the Tfh cell to Tfr cell ratio on day 10 post-immunization.

Data shown are from at least two separate experiments with n ≥ 5. The middle bar represents the average for the dataset. Error bars represent SD, and an asterisk indicates statistically significant differences calculated using two-tailed Student’s t test or Mann-Whitney U test. A p value of <0.05 was considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001.