Fig 2: Combined deficiency in Lamp2 and B2m specifically reduces CD4 T cell development, without redirecting MHC II-restricted cells into the CD8 lineage. (A-B) Flow cytometry analysis of T cell development in b2m KO (b2m KO-WT) and in lamp2 and b2m double-KO (lamp2 b2m dKO-WT) thymus grafted into WT recipients. (A) Representative dot plots of CD4 and CD8 expression on total thymocytes, with numbers indicating the frequencies of the different subsets. Graphs represent the cellularity of total thymocytes and the indicated subsets in b2m KO (gray) and lamp2 b2m dKO (blue). (B) Graph represents the absolute numbers of SP4 and SP8 within mature TCRB+ thymocytes. Data shown represent an average of 9 b2m KO and 10 lamp2 b2m dKO ectopic thymi from 3 independent experiments. (C) Dot plots show the flow cytometric profile for CD69 and TCRB on total thymocytes from b2m KO-WT and lamp2 b2m dKO-WT ectopic thymi. Stage I (TCRBneg/int CD69neg), II (TCRBint CD69int), III (TCRBhi CD69hi) and IV (TCRBhi CD69lo/neg) were defined as represented. Graphs represent the average percentages of cells in the indicated gates in b2m KO-WT (gray) and lamp2 b2m dKO-WT (blue) ectopic thymi. (D) Graphs represent the average frequency of IKZF2/HELIOS+ cells in DP and SP4 from b2m KO-WT (gray) and lamp2 b2m dKO-WT (blue) (defined as shown in Figure S3B). Data include an average of 2 independent experiments (n = 6 b2m KO and 8 lamp2 b2m dKO-WT). Results in A-D are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig 3: T cell development in WT and lamp2 KO thymic grafts. Representative dot plots of CD4 and CD8 expression within (A) total thymocytes and (B) TCRB+ thymocytes in thymic grafts of Nude recipients transplanted with WT (WT-Nu) and lamp2 KO (lamp2 KO-Nu) ectopic thymus, 8–10 weeks after transplantation. Numbers on the plots indicate the frequencies of the different subsets. Bar graphs in A and B depict the average absolute cellularity of the thymus and the indicated thymocyte subsets in WT-Nu (gray) and lamp2 KO-Nu (blue). In B, ratio of the frequency of SP4:SP8 is shown in WT-Nu and lamp2 KO-Nu, as well in the endogenous thymus. (C) Analysis of peripheral T cells in spleens from WT-Nu and lamp2 KO-Nu mice. Graphs (left) represent the average % and numbers of TCRB+ T cells in the spleen. Dot plots show CD44 and SELL/CD62L expression within CD4 and CD8 T cells. Graphs (right) show average cellularity of naïve (SELL/CD62L+ CD44lo) and effector/memory (SELL/CD62L± CD44hi) CD4 and CD8 T cells. Data represent an average of 3 independent experiments (n = 10 WT and lamp2 KO ectopic thymi). Results in A-C are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig 4: Analysis of SP4+ TCR repertoire in WT and lamp2 KO thymus. (A) T cell development in WT (WT-TCR:Vß6TG) and lamp2 KO (lamp2 KO-TCR:Vß6TG) ectopic thymus grafted into fixed TCRB/TCRßTG recipients. CD4 and CD8 expression within mature TCRB+ thymocytes. Graphs represent the absolute number of the indicated thymocyte subsets in WT-TCR:Vß6TG (gray) and lamp2 KO-TCR:Vß6TG (blue) thymi. Data shown represent an average of 2 independent experiments (n = 6 WT and 6 lamp2 KO). Graphs indicated (B) the number and (C) frequency of unique sequences of Tcr clones found in SP4 thymocytes developing within WT and lamp2 KO ectopic thymus. Pie graphs in C depict a colorimetric representation of the most abundant clonotypes found in each thymi. Results in A-B are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.