Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class II-expressing dendritic cells

Abstract

Thymic T cell development is controlled by T cell receptor (TCR)-major histocompatibility complex (MHC) interactions, whereas a further dependence of peripheral mature T cells on TCR-MHC contact has not been described so far. To study this question, CD4 T cell survival was surveyed in mice lacking MHC class II expression and in mice expressing MHC class II exclusively on dendritic cells. Since neither of these mice positively select CD4 T cells in the thymus, they were grafted with MHC class II-positive embryonic thymic tissue, which had been depleted of bone marrow derived cells. Although the thymus grafts in both hosts were repopulated with host origin thymocytes of identical phenotype and numbers, an accumulation of CD4+ T cells in peripheral lymphoid organs could only be observed in mice expressing MHC class II on dendritic cells, but not in mice that were completely MHC class II deficient. As assessed by histology, the accumulating peripheral CD4 T cells were found to be in close contact with MHC class II+ dendritic cells, suggesting that CD4 T cells need peripheral MHC class II expression for survival and that class II+ dendritic cells might play an important role for the longevity of CD4 T cells.

Figure 1

(a) MHC class II I-E transgene expression correlates to CD11c expression in B6CD11c-E_α ^dI-A^−/− transgenic mice. Immunohistochemical analysis of normal adult lymph nodes (left) and spleen (right) from B6CD11c-E_α ^dI-A^−/− mice (13). Biotinylated antibodies specific for CD11b (Mac-1), CD11c, MHC class II I-E, or IgM were used on serial cryostat sections of frozen organs and developed with streptavidin– alkaline phosphatase. Transgenic I-E expression correlates to CD11c staining in lymph node and spleen, but spares IgM-positive B cells and most of the Mac-1–positive macrophages. All sections were originally photographed at ×200. (b) Transgenic I-E expression correlates with CD11c expression on DCs and can be found on less as well as more mature DC subpopulations. Three-color flow cytometric analysis of low density spleen cells from a low buoyant density gradient was performed. Only cells meeting high forward scatter and intermediate side scatter criteria are shown in this analysis. Cells falling within the gates shown in the top panels (CD11c, I-E) were further analyzed with the mAb NLDC-145 and 33D1 (lower histograms, solid lines). The controls for the second step reagent are shown as dotted lines (no first step mAb).

Figure 1

(a) MHC class II I-E transgene expression correlates to CD11c expression in B6CD11c-E_α ^dI-A^−/− transgenic mice. Immunohistochemical analysis of normal adult lymph nodes (left) and spleen (right) from B6CD11c-E_α ^dI-A^−/− mice (13). Biotinylated antibodies specific for CD11b (Mac-1), CD11c, MHC class II I-E, or IgM were used on serial cryostat sections of frozen organs and developed with streptavidin– alkaline phosphatase. Transgenic I-E expression correlates to CD11c staining in lymph node and spleen, but spares IgM-positive B cells and most of the Mac-1–positive macrophages. All sections were originally photographed at ×200. (b) Transgenic I-E expression correlates with CD11c expression on DCs and can be found on less as well as more mature DC subpopulations. Three-color flow cytometric analysis of low density spleen cells from a low buoyant density gradient was performed. Only cells meeting high forward scatter and intermediate side scatter criteria are shown in this analysis. Cells falling within the gates shown in the top panels (CD11c, I-E) were further analyzed with the mAb NLDC-145 and 33D1 (lower histograms, solid lines). The controls for the second step reagent are shown as dotted lines (no first step mAb).

Figure 2

Identical thymocyte repopulation of thymic grafts in B6CD11c-E_α ^dI-A^−/− and B6I-A^−/− mice. Three-color FACS^® analysis of fetal thymic grafts at wk 17 after transplantation. CD4 lineage thymocytes were divided into the indicated gates 1, 2, and 3 and further analyzed for expression of CD3, HSA, and CD69. In TG from both hosts, total thymocyte numbers were ∼3 × 10⁶ from wk 3–8 and ∼10⁶ in wk 17–21 after transplantation.

Figure 3

Accumulation of peripheral CD4⁺ T cells can be detected in thymus-grafted B6CD11c-E_α ^dI-A^−/− mice, but not in nontransgenic thymus-grafted B6I-A^−/− mice. Animals were bled, leukocytes purified by ficoll gradient centrifugation, stained with mAbs specific for CD4 and CD8, and analyzed by flow cytometry. The percentages of CD4⁺ cells of total blood leukocytes from 9 individual mice/ group are shown.

Figure 4

Analysis of lymph node cells from TG⁻ and TG⁺ B6I-A^−/−, TG⁺ B6CD11c-E_α ^dI-A^−/−, and B6-E_α ^dI-A^−/− mice (control mice expressing transgenic I-E under control of the MHC class II promoter; references 13, 14). Cell suspensions from lymph nodes of the indicated mice were analyzed by flow cytometry. Three-color analysis with mAbs specific for CD4, CD8, and α/β-TCR is shown in the two left sets of panels. The histograms for α/β-TCR staining and the Mel-14/CD69 staining was performed on gated CD4⁺ T cells. The percentages and total cell numbers falling within the indicated regions from the Mel-14/CD69 stainings are shown on the right side and are an average of 3 animals/group (percentage, total cell numbers).

Figure 5

Peripheral CD4⁺ T cells accumulate in T cell areas of lymph nodes in close proximity to I-E⁺ DCs. Double immunofluorescense analysis of a lymph node from a TG⁺ B6CD11c-E_α ^dI-A^−/− mouse at 8 wk after thymus transplantation. (A) CD4⁺ T cells (anti–CD4-FITC, green) are found in the paracortical area of the lymph node, whereas I-E–expressing DCs (anti–I-E-PE, red) are localized, but not in the primary B follicle (F) (original magnification: 200). (B) Higher magnification (1,000) of the same area.