The absence of MARCH 8 does not significantly alter the CD4⁺ T cell repertoire. (A and B) Frequency and number of CD4⁺ CD3⁺ thymocytes (A) or splenic CD4⁺ T cells (B) from wild-type or March8^−/− mice. The graphs display data pooled from a minimum of three independent experiments with symbols representing individual mice. Bars designate the mean. (C and D) Summary of the fold difference in geometric mean fluorescence intensity for T cell markers expressed by cells isolated from wild-type and March8^−/− mice: CD4⁺ SP thymocytes (C) or CD4⁺ splenic T cells (D). Graphs display mean + SD and represent data from three individual mice for each marker. (E) Percentage of CD25⁺ FoxP3⁺ T reg cells for CD4⁺ SP thymocytes and CD4⁺ splenic T cells for wild-type and March8^−/− mice. (F) Frequency of CD4⁺ OT-II (TCR Vα2/Vβ5⁺) T cells in blood lymphocytes for wild-type and March8^−/− mice. (G) Number of naive CD4⁺ tetramer⁺ progenitors in the spleen and lymph node of wild-type and March8^−/− mice. Data are pooled from two independent experiments. n.s., not significantly different relative to wild-type, Mann-Whitney U test. (E–G) Each symbol represents an individual mouse. Bars designate the mean. (H) Single I-Eα wild-type or March8^−/− CD4⁺ T cells were isolated, and the TCRβ usage was determined by multiplex single-cell reverse transcription and PCR amplification of TCR CDR3ε regions. Analysis was performed for two individual mice of each genotype.