Figure 1.
Decreased Foxp3+ Tregs and CD25+Foxp3- Treg precursors in NIK KO thymus.
Single-cell suspensions of nucleated thymocytes from intact NIK KO or WT mice were stained with the indicated fluorescently conjugated antibodies and analyzed by flow cytometry. A, Top plots are gated on lymphocytes, bottom plots are gated on SP4. Numbers indicate percent of gated cells. B, Quantitation of cell percentages among lymphocytes or SP4. C-E, Quantitation of absolute number of SP4 Foxp3+ thymocytes, total thymocytes, and SP4 thymocytes per mouse, respectively. F, Quantitation of Treg precursors as a percent of SP4. G, Overlay of CD25 expression gated on SP4 Foxp3+ cells; bold line, NIK KO; narrow line, WT. Data in bar graphs represent mean ± SEM of n=4 mice per group in one representative experiment of two.
Figure 2.
Normal Foxp3+ Tregs and CD25+Foxp3- Treg precursors in thymus of WT recipients of NIK KO BM.
Single-cell suspensions of nucleated thymocytes from NIK KO or WT BM chimeric mice were stained with the indicated fluorescently conjugated antibodies and analyzed by flow cytometry. A, Top plots are gated on donor CD45.1- lymphocytes (BM derived, NIK KO or WT littermate); bottom plots are gated on CD45.1- SP4. Numbers indicate percent of gated cells. B, Quantitation of cell percentages among CD45.1- lymphocytes or SP4. C-E, Quantitation of absolute number of SP4 Foxp3+ CD45.1- thymocytes, total CD45.1- thymocytes, and SP4 CD45.1- thymocytes per mouse, respectively. F, Quantitation of Treg precursors as a percent of CD45.1- SP4. G, Overlay of CD25 expression gated on CD45.1- SP4 Foxp3+; bold line, NIK KO; narrow line, WT. Data in bar graphs represent mean ± SEM of n=3-4 mice per group in one representative experiment of two.
Figure 3.
Decreased peripheral Foxp3+ Tregs in both intact NIK KO and NIK KO single BM chimeric mice.
Single cell suspensions of nucleated splenocytes from intact (A-F) or BM chimeric (G-L) mice were stained with the indicated fluorescently labeled antibodies. A, Plots are gated on CD4+; numbers indicate percent of gated cells. B and E, Quantitation of percent of Foxp3+ and CD4+ splenocytes, respectively. C, D, and F, Quantitation of absolute number of Foxp3+, total, and CD4+ splenocytes. G, Plots are gated on CD4+CD45.1- cells (BM-derived, NIK KO or WT littermate). Numbers indicate percent of gated cells. H and K, Quantitation of percent of Foxp3 + CD45.1- and CD4+CD45.1- cells, respectively. I, J, and L, Quantitation of absolute number of Foxp3 + CD45.1-, total CD45.1-, and CD4+CD45.1- splenocytes, respectively. Data in bar graphs represent mean ± SEM of n=3-4 mice per group in one representative experiment of two (B-F) or three (G-L).
Figure 4.
Decreased peripheral Foxp3+ Tregs in NIK KO BM chimeric mice is cell-intrinsic.
Single cell suspensions of nucleated splenocytes (A-F) or thymocytes (G) from NIK KO (CD45.2) + WT (CD45.1xCD45.2) F1 or WT littermate (CD45.2) + WT (CD45.1xCD45.2) F1 mixed BM chimeric mice were stained with the indicated fluorescently labeled antibodies. A, Gating scheme for identifying cells in mixed BM chimeras. CD45.1 + CD45.2- cells (ungated) are radioresistant host cells. Numbers indicate percent of gated cells. B and C, Quantitation of percent and absolute number of donor CD45.2 + CD45.1-Foxp3+ cells. D and E, Quantitation of percent and absolute number of donor CD45.2 + CD45.1-CD4+ cells. These data compare NIK KO cells with WT littermate control cells. Because WT (CD45.1xCD45.2) F1 cells appear to have a slight, but consistent advantage over CD45.2 + CD45.1- WT littermates in terms of both overall proportion and proportion of Foxp3+ cells, comparing NIK KO T cells to WT littermate T cells is the most conservative and appropriate comparison. When we compare NIK KO T cells to WT (CD45.1xCD45.2) F1 T cells, the differences are even greater. F, CD25 expression on NIK KO (bold line) versus WT (dashed line) Foxp3+ splenocytes. G, Quantitation of percent of NIK KO and WT SP4 and Foxp3+ thymocytes in mixed BM chimeras. Data in bar graphs represent mean ± SEM of n=3-4 mice per group in one representative experiment of two.
Figure 5.
Altered phenotype of NIK KO Tregs depends on NIK KO stromal cells.
Single cell suspensions of nucleated splenocytes from intact (A and B), single BM chimeric (C and D), or mixed BM chimeric (E and F) mice were stained with the indicated fluorescently labeled antibodies. A, C, and E, Quantitation of percent CD62Llo among Foxp3+ NIK KO or WT cells. B, D, and F, Quantitation of mean fluorescence intensity of CD44 and CTLA4 on Foxp3+ NIK KO or WT cells. Data represent mean ± SEM of n=3-4 mice per group in one representative experiment of two (A, B, E, F) or three (C, D).
Figure 6.
NIK is required cell-intrinsically for normal proportions of memory phenotype conventional T cells.
Single cell suspensions of nucleated splenocytes from intact (A-E), single BM chimeric (F-J), or mixed BM chimeric (K-O) mice were stained with the indicated fluorescently labeled antibodies. Dot plots are gated on CD4+ (A), CD4+CD45.1- (F), or CD4+CD45.1-CD45.2+ (K) cells. Numbers on plots indicate percent of gated cells. B, G, and L, Quantitation of percent Foxp3-CD44hi within the gated populations described in A, F, and K, respectively. C, H, and M, Quantitation of absolute number of CD4+ memory T cells of the indicated genotype per spleen. D, I, and N, Quantitation of absolute number of CD4+ naive T cells of the indicated genotype per spleen. E, J, and O, Quantitation of naïve: memory CD4+ T cell ratio of the indicated genotype. Data in bar graphs represent mean ± SEM of n=3-4 mice per group in one representative experiment of two (A-E and K-O) or three (F-J).