ICOS expression by effector T cells influences the ability of regulatory T cells to inhibit anti-chromatin B cell responses in recipient mice

Abstract

T regulatory cells are critical for the prevention of autoimmunity. Specifically, Treg cells can control anti-chromatin antibody production in vivo, and this correlates with decreased ICOS expression on CD4(+) T helper cells. Here we test the significance of high ICOS expression by T effector cells, firstly in terms of the anti-chromatin B cell response, and secondly on the ability of Treg cells to suppress T cell help. We bred CD4(+) T cell receptor transgenic mice with mice that carry the Roquin(san/san) mutation. The Roquin gene functions to limit ICOS mRNA such that CD4 T cells from mutant mice express elevated ICOS. Using an in vivo model, TS1.Roquin(san/san) Th cells were compared with wild-type TS1 Th cells with regard to their ability to help anti-chromatin B cells in the presence or absence of Treg cells. Both TS1 and TS1.Roquin(san/san) Th cells induced anti-chromatin IgM(a) antibodies, but the TS1.Roquin(san/san) Th cells resulted in the recovery of more class-switched and germinal center B cells. Neither source of Th cells were capable of inducing long-lived autoantibodies. Treg cells completely suppressed anti-chromatin IgM(a) antibody production and reduced anti-chromatin B cell recovery induced by TS1 Th cells. Importantly, this suppression was less effective when TS1.Roquin(san/san) Th cells were used. Thus, high ICOS levels on effector T cells results in autoimmunity by augmenting the autoreactive B cell response and by dampening the effect of Treg cell suppression.

Figure 1

Phenotype of BALB/c Roquin^san/san (san/san) mice A) This is representative immunohistochemical staining of the spleen showing B220 to mark B cells, and PNA to identify germinal centers (n=3). B) The graphs show the average percentage +/− standard deviation of Foxp3⁺ cells versus total CD4⁺ T cells (left) (n=5) and average total number of Foxp3 +/− the standard deviation in the spleen of the indicated mice (n=3). C) Graphs depict the ICOS expression levels on splenic CD4⁺ Foxp3⁻ and CD4⁺ Foxp3⁺ T cells. The numbers designate the geometric mean fluorescence intensity. Data from one of three independent experiments with similar results are shown.

Figure 2

TS1.Roquin^san/san Th cell proliferation is inhibited by Treg cells in vitro. These graphs are representative profiles of CFSE-labeled CD4⁺ CD25⁻ cells from the indicated mice after three days of culture stimulated with CD3. The numbers are the corresponding geometric mean fluorescence of the data from the shown experiment. Data from one of four independent experiments with similar results are shown.

Figure 3

Experimental model where CB17 (Ig^b) mice received CD4⁺ CD25⁻, TS1 or TS1.Roquin^san/san, and/or CD4⁺ CD25⁺ Treg cells from TS1 × HA28 mice and influenza virus at day 0 to prime the T cells. The following day, PR8 HA anti-chromatin B cells (Ig^a) were transferred from sd-VH3H9.HACII.Igκ^−/− donors. Spleen cells from recipient CB17 mice were analyzed on day 8.

Figure 4

TS1 and TS1.Roquin^san/san T cells induce anti-chromatin antibodies at day 8. Treg cells completely block this in the context of TS1 T cells, but only partially block with TS1 Roquin^san/san T cells. A) The graph shows the average optimal density +/− the standard deviation of the indicated serum titer of three independent experiments. (*) indicates that the serum titer of the group is significantly different compared to all other groups (n=3). B) The graphs show the average percent inhibition +/− the standard deviation of the 1/100 diluted serum anti-chromatin IgM^a from mice that received TS1 + Treg cells (white) or TS1.Roquin^san/san Th + Treg cells (black). The % inhibition of IgG_2a^a anti-chromatin by Treg cells is shown for the TS1 Roquin^san/san T cells only as the TS1 did not induce IgG_2a^a anti-chromatin antibodies. (*) indicates that the serum titer of the group is significantly different compared to all other groups (n=3). C) The graphs show the average IgM^a and IgG_2a^a B cell recovery +/− the standard deviation in the spleen. (*) denotes significant difference to all groups (IgM^a graph) or the specified groups (IgG_2a^a graph) (p < 0.05). The (^) marked groups are significantly different from all other groups but not from each other (n=3). No IgG₁^a B cells were detected in any of the groups tested (data not shown).

Figure 5

At day 8 mice that received TS1.Roquin^san/san Th cells have more anti-chromatin B cells with a germinal center phenotype. A) Representative dot plots of the splenic B220 ⁺ IgM^a+ cells that express the germinal center markers, PNA and Fas. The right box shows the cells that have a germinal center phenotype (PNA⁺ Fas⁺) and the left box indicates the anti-chromatin B cells that are activated without a germinal center phenotype, PNA⁻ Fas⁺ (n=3). B) The graph shows the average +/− standard deviation of the relative percentage of the B220⁺ IgM^a+ PNA⁻ Fas⁺ population in the spleen. The percentage of B220⁺ IgMa⁺ PNA⁻ Fas⁺ cells in the TS1 group was set at 100. The other groups were determined by the following formula: (% B220⁺ IgM^a+ PNA⁻ Fas⁺ in a non-TS1 group/% B220⁺ IgM^a+ PNA⁻ Fas⁺ in the TS1 group)*100. (*) indicates a significant difference (n=3). C) The graphs show the average IgM^a and IgG_2a^a germinal center (B220⁺ PNA⁺ Fas⁺) B cell recovery +/− the standard deviation in the spleen. (*) indicates significant difference compared to all groups (p < 0.05). The (^) marked groups are significantly different from all other groups but not from each other (n=3). D) Long term serum anti-chromatin antibody production. The graphs shows the O. D. +/− the standard deviation of the serum at the 1/100 titer from the indicated groups after immunization. (*) indicates significantly different compared to mice that did not receive Th cells or virus (n=3).

Figure 6

ICOS and CD40L expression on the transferred CD4⁺ Th cells. A) ICOS expression on CD4⁺ Foxp3⁻ cells pre-transfer (day 0) as well as ICOS and CD40L expression at day 8 post-transfer in the spleen on the indicated T cell populations. The numbers are the geometric mean fluorescence of the lines in the graph shown. For a reference point the black vertical line marks 1000 on the x axis of the histogram showing ICOS expression. The data are from one experiment that is representative of three independent experiments. B) The graph shows the average geometric mean for surface ICOS expression +/− the standard deviation on the CD4⁺ 6.5⁺ FoxP3⁻ cells at either day 0 or day 8 (n=3).

Figure 7

Intracellular cytokine production. A) The graph represents the average percent positive of the indicated cytokine +/− the standard deviation in the CD4⁺ 6.5⁺ T cell population (IL-2, n=3; IL-4, n=3; IL-10, n=2; IL-21, n=3; IFN-γ, n=3) at day 8 post-transfer. B) Representative histogram of IFN-γ and the graph shows the average geometric mean +/− the standard deviation of the IFN-γ⁺ cells from all experiments (n=3).