Subsets of transgenic T cells that recognize CD1 induce or prevent murine lupus: role of cytokines

Abstract

T cells with T cell receptor (TCR) transgenes that recognized CD1 on syngeneic B cells stimulated B cells to secrete immunoglobulins in vitro. The CD4+, CD8+, or CD4-CD8- T cells from the spleen of the TCR transgenic BALB/c donors induced lupus with anti-double stranded DNA antibodies, proteinuria, and immune complex glomerulonephritis in irradiated BALB/c nude mice reconstituted with nude bone marrow. Injection of purified CD4-CD8- T cells from the marrow of transgenic donors prevented the induction of lupus by the transgenic T cells. Transgenic T cells that induced lupus secreted large amounts of interferon (IFN)-gamma and little interleukin (IL)-4, and those that prevented lupus secreted large amounts of IL-4 and little IFN-gamma or IL-10.

Figure 1

Mutual activation of Transgenic T cells and nontransgenic B cells. (A), 5 × 10⁵ sorted B (B220⁺) cells from the nontransgenic BALB/c spleen were incubated with graded numbers of sorted T (Thy1.2⁺) cells from the SP transgenic (Tg) BALB/c spleen. Cells were cultured for 5 d, and concentrations of IgM and IgG in the culture supernatants were determined. In control experiments, nontransgenic, wild-type (WT), sorted T cells were substituted for transgenic T cells. (B and C) 5 × 10⁵ sorted nontransgenic B cells and 5 × 10⁵ sorted transgenic T cells were incubated for 5 d in the presence of anti-CD1 (1B1) antibody or isotype control (rat IgG2b), and the concentrations of IgM and IgG in culture supernatants were measured. (D) shows [³H]thymidine incorporation of 10⁴ V_β9, V_α4.4 cloned BALB/c T cells (parent line) after incubation with graded numbers of irradiated (4,000 cGy) CD1 transfected A20 cells or nontransfected A20 cells for 48 h. (E) [³H]thymidine incorporation of SP transgenic spleen cells (10⁵) incubated with graded numbers of irradiated (4,000 cGy) BCL₁ cells or BALB/c spleen cells activated with LPS (Blasts). (F and G), Transgenic spleen cells (10⁵) were incubated with irradiated BCL₁ cells (10⁵) or LPS-activated spleen cells (10⁵), respectively, in the presence of graded concentrations of anti-CD1 (3C11) antibody or isotype control. (H–J) The one-color flow cytometric analyses of nontransfected A20, CD1 transfected A20, and BCL₁ cells, respectively, after staining with anti-CD1 (3Cll) antibody. Solid lines show 3Cll and dotted lines show isotype control. (K) BALB/c spleen cells after staining for B220 versus CD1 receptors. Box encloses B220⁺CD1^hi cells.

Figure 2

Expression of the V_β9 transgene on T cells in the blood of irradiated BALB/c athymic or euthymic recipients given transgenic BM cells. (A and B) Two-color flow cytometric analysis of peripheral blood mononuclear cells obtained from irradiated athymic or euthymic recipients, respectively, 4 wk after the injection of 2.5 × 10⁶ SP BM cells. Cells were stained for CD4 and CD8 receptors versus V_β9 receptors. Boxes 1 and 2 enclose CD4⁺ and CD8⁺ cells that were V_β9⁻ or V_β9⁺, respectively. (C and D) Two-color analysis of blood cells from athymic and euthymic recipients, respectively, 4 wk after the injection of 2.5 × 10⁶ DN BM cells. Boxes 1, 2, and 3 enclose CD4⁺V_β9⁻ and CD8⁺V_β9⁻, CD4⁺V_β9⁺ and CD8⁺V_β9⁺, and CD4⁻CD8⁻V_β9⁺ cells, respectively. Panels are representative of groups of four to six recipient mice. Cells in A and B were derived from mice from two groups (athymic and euthymic) in one experiment, and cells in C and D were derived from mice from two groups in another.

Figure 3

Immunohistopathology of kidneys from BALB/c nu/nu recipients given transgenic BM cells. (A and B) Two-color flow cytometric analysis of enriched T cells from the BM of SP or DN transgenic mice, respectively, stained for CD4 and CD8 receptors versus V_β9 receptors. Upper boxes enclose CD4⁺ and CD8⁺ T cells and lower box encloses CD4⁻CD8⁻ T cells. T cells were enriched by depleting B220⁺, Gr-1⁺, and Mac-1⁺ cells (16). (C and D) The immunofluorescent staining of histological sections of kidneys from recipients given BM cells from SP or DN transgenic mice, respectively, with FITC-conjugated anti–mouse IgG antibodies. Arrows show staining of capillary loops. E and F show the appearance of recipients given BM cells from SP or DN transgenic mice, respectively. The mouse in E has ascites and generalized edema.

Figure 3

Immunohistopathology of kidneys from BALB/c nu/nu recipients given transgenic BM cells. (A and B) Two-color flow cytometric analysis of enriched T cells from the BM of SP or DN transgenic mice, respectively, stained for CD4 and CD8 receptors versus V_β9 receptors. Upper boxes enclose CD4⁺ and CD8⁺ T cells and lower box encloses CD4⁻CD8⁻ T cells. T cells were enriched by depleting B220⁺, Gr-1⁺, and Mac-1⁺ cells (16). (C and D) The immunofluorescent staining of histological sections of kidneys from recipients given BM cells from SP or DN transgenic mice, respectively, with FITC-conjugated anti–mouse IgG antibodies. Arrows show staining of capillary loops. E and F show the appearance of recipients given BM cells from SP or DN transgenic mice, respectively. The mouse in E has ascites and generalized edema.

Figure 4

Serum IgM and IgG concentrations in BALB/c nu/ nu recipients. Concentrations of serum IgM and IgG were measured at serial time points in experimental recipients given an injection of 2.5 × 10⁶ BM cells from SP transgenic mice or in control recipients given 2.5 × 10⁶ BM cells from nontransgenic BALB/c nu/nu mice. Each point represents the mean of four experimental mice that developed ascites, six experimental mice that did not develop ascites, or four control mice. Bars show standard errors of the means. A and B show IgG and IgM levels, respectively.

Figure 4

Serum IgM and IgG concentrations in BALB/c nu/ nu recipients. Concentrations of serum IgM and IgG were measured at serial time points in experimental recipients given an injection of 2.5 × 10⁶ BM cells from SP transgenic mice or in control recipients given 2.5 × 10⁶ BM cells from nontransgenic BALB/c nu/nu mice. Each point represents the mean of four experimental mice that developed ascites, six experimental mice that did not develop ascites, or four control mice. Bars show standard errors of the means. A and B show IgG and IgM levels, respectively.