Protection of Mice from Acute Graft-versus-Host Disease Requires CD28 Co-stimulation on Donor CD4+ Foxp3+ Regulatory T Cells

Abstract

Acute graft-versus-host disease (aGvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell plus T cell transplantation (allo-HSCT). In this study, we investigated the requirement for CD28 co-stimulation of donor CD4⁺ conventional (CD4⁺CD25^-Foxp3^-, Tconv) and regulatory (CD4⁺CD25⁺Foxp3⁺, Treg) T cells in aGvHD using tamoxifen-inducible CD28 knockout (iCD28KO) or wild-type (wt) littermates as donors of CD4⁺ Tconv and Treg. In the highly inflammatory C57BL/6 into BALB/c allo-HSCT transplantation model, CD28 depletion on donor CD4⁺ Tconv reduced clinical signs of aGvHD, but did not significantly prolong survival of the recipient mice. Selective depletion of CD28 on donor Treg did not abrogate protection of recipient mice from aGvHD until about day 20 after allo-HSCT. Later, however, the pool of CD28-depleted Treg drastically declined as compared to wt Treg. Consequently, only wt, but not CD28-deficient, Treg were able to continuously suppress aGvHD and induce long-term survival of the recipient mice. To our knowledge, this is the first study that specifically evaluates the impact of CD28 expression on donor Treg in aGvHD. Moreover, the delayed kinetics of aGvHD lethality after transplantation of iCD28KO Treg provides a novel animal model for similar disease courses found in patients after allo-HSCT.

Keywords: CD28; acute graft-versus-host disease; co-stimulation; inducible deletion; regulatory T cells.

Figure 1

C57BL/6 donor Tconv proliferation and accumulation in BALB/c hosts during acute graft-versus-host disease is not impaired after induced CD28 deletion. (A) Lethally irradiated BALB/c mice were transplanted with 10⁷ C57BL/6 T cell-depleted bone marrow cells alone or together with 5 × 10⁵ unlabeled or 2.8 × 10⁶ CFSE labeled (day 3 data) Tconv from inducible CD28 knockout (iCD28KO) mice or wt littermates. CD28 deletion on iCD28KO donor Tconv was induced by tamoxifen treatment of the recipient mice from day 0 to day 3 after transplantation. (B) Donor Tconv were identified in the spleen of recipient mice by expression of Thy1.1 and stained for CD28 expression before transplantation (day 0) and on day 3 and day 7 after transplantation. Black histograms: specific staining. Gray histograms: istoype control staining. (C) CFSE dilution among splenic donor Tconv was analyzed on day 3 after transplantation. Proliferation index and percentage of divided cells are shown as mean ± SD of three independent experiments (n = 4 mice/group) and were tested with two-tailed Mann–Whitney test: p > 0.05. (D) Expression of Ki-67 and Foxp3 by donor iCD28KO or wt Tconv from spleen and mesenteric lymph node (mLN) of recipient mice on day 7 after transplantation. (E) Absolute numbers of donor Tconv per organ. Data of three independent experiments with a total of 8 mice per group are shown as mean percentages ± SD (D) or medians and range (E). White columns: wt Tconv; black columns: iCD28KO Tconv.

Figure 2

CD28 deletion on donor T cells before transplantation also does not affect accumulation of allogeneic T cells or phenotype of donor regulatory T cells (Treg) in recipient mice. (A) Transfer of 10⁷ C57BL/6 T cell-depleted bone marrow cells together with either 5 × 10⁵ wt or inducible CD28 knockout (iCD28KO) total CD4⁺ T cells into lethally irradiated BALB/c recipient mice. CD28 expression on donor T cells was deleted by tamoxifen treatment of the recipient mice after transplantation (see Figure 1A) or treatment of the donor mice from day −4 to day −1 before transplantation. (B) T cells from tamoxifen-treated donor mice were stained for CD28 expression before transfer (day 0) or 3 days after transplantation in the spleens of recipient mice [median fluorescence intensity (MFI)]. Black histograms: specific staining. Gray histograms: istoype control staining. (C–E) Tregs were identified by expression of CD25 and Foxp3 among freshly prepared donor T cells (day 0, before transplantation) or among splenic donor T cells (day 3 or 6 after transplantation). (C) Representative dot plots showing donor CD4⁺ T cells and Treg gating (D) Absolute donor T cell recovery from splenocytes of recipient mice and percentage of Treg among splenic donor T cells as shown in (C) (median + range). (E) MFI of CD25 and Foxp3 expressed by donor Treg after tamoxifen treatment of the donor or the recipient mice (mean + SD). (D,E) n = 3 recipient mice/group; white columns: wt T cells; black columns: iCD28KO T cells.

Figure 3

CD28 deficiency of donor Tconv reduces early clinical signs of acute graft-versus-host disease (aGvHD), but does not significantly mediate long-term protection from aGvHD-related mortality. Lethally irradiated recipients were reconstituted with 10⁷ C57BL/6 T cell-depleted bone marrow (TCD-BM) cells alone or together with 5 × 10⁵ inducible CD28 knockout (iCD28KO) or wt Tconv and treated with tamoxifen beginning on the day of transplantation. (A) Concentrations of TNF in the serum, (B) cumulative histological score of small and large bowel, and (C) clinical scores of recipient mice on day 7 after transplantation. Data were pooled from three independent experiments (n = 7–9 mice/group); two-tailed, unpaired Mann–Whitney test. (D) Median clinical score (E) survival of recipient mice and (F) post-mortem analysis of CD28 expression on splenic donor Tconv [rel. CD28 expression: ratio of median fluorescence intensity (MFI) of specific CD28 staining/MFI isotype control staining]. Mice that had to be killed prematurely for humane reasons are contained in the summary graph (D) until day 80 with the final clinical score assessed. Data of two independent experiments were pooled (n = 8 mice/group). (A–F) Triangles: TCD-BM only; circles: wt Tconv; squares: iCD28KO Tconv.

Figure 4

Regulatory T cells (Treg) do not require CD28 to suppress expansion of allogeneic Tconv in vivo. (A) Lethally irradiated BALB/c mice were reconstituted with T cell-depleted bone marrow cells alone or together with 2.5 × 10⁵ Thy1.1⁺ Tconv and 2.5 × 10⁵ Thy1.1⁺/Thy1.2⁺ Treg from inducible CD28 knockout (iCD28KO) donors or wt littermates and analyzed on day 6 after transplantation. (B) Gating strategy for identification of donor Treg in mesenteric lymph nodes (mLNs) of recipient mice and expression of Foxp3, CD25, and CD28 by donor Treg. Numbers indicate mean percentages ± SD for each quadrant. Black histograms: specific staining. Gray histograms: istoype control staining. Median fluorescence intensity of (C) Foxp3 and (D) CD25 of transferred Treg recovered from spleens and mLNs (mean + SD). (E) Absolute number of donor Treg and (F) donor Tconv per organ (median + range, one-tailed, unpaired Mann–Whitney test). (C–F) Data were pooled from three independent experiments, n = 7 mice/group. (C–F) white columns: wt Tconv; black columns: wt Tconv + wt Treg; gray columns: wt Tconv + iCD28KO Treg.

Figure 5

CD28-depleted regulatory T cells (Treg) can migrate to the gut and prevent tissue damage. T cell-depleted bone marrow (TCD-BM) cells were either transferred alone into BALB/c mice after lethal irradiation or together with 2.5 × 10⁵ Tconv and with or without 2.5 × 10⁵ Treg from inducible CD28 knockout (iCD28KO) donors or wt littermates. CD28 deletion was induced by treating recipient mice with tamoxifen from day 0 to day 3 after transplantation. On day 6 after transplantation (A) serum concentrations of TNF and (B) cumulative histological score of small and large bowel of recipient mice were assessed. Data were pooled from three independent experiments (n = 7 mice/group). (C,D) Paraffin sections of small and large bowel were immunohistochemically stained with Foxp3 eF660 antibody and Dapi. (C) Number of Foxp3 positive Treg as counted in 10 high power fields (200× magnification) of each small and large bowel. (D) Representative images of small bowel. Data were pooled from two independent experiments (n = 4 mice/group). p values refer to an unpaired Mann–Whitney test (comparisons between Treg recipients: two-tailed; all other comparisons: one-tailed). (A–C) Triangles: TCD-BM only; crosses: wt Tconv; circles: wt Tconv + wt Treg; squares: wt Tconv + iCD28KO Treg.

Figure 6

CD28-deficient donor regulatory T cells (Treg) fail to mediate long-term protection from aGvHD. Lethally irradiated recipients received either T cell-depleted bone marrow (TCD-BM) alone or together with 1.25 × 10⁵ Tconv and with or without 2.5 × 10⁵ Treg from inducible CD28 knockout (iCD28KO) donors or wt littermates. (A) Median clinical score (including final scores of animals that had to be killed for humane reasons) of all experimental groups (left) with individual clinical scores of Treg recipients (right). p values refer to a one-tailed Mann–Whitney test between Tconv only and Tconv + Treg recipients on day 7, 25, and 80. (B) Survival of all recipient mice and ratio of long-term survivors in the Treg recipient groups. p values refer to a Mantel–Cox test between the two groups receiving Treg until day 24 or from day 25 until the end of the experiment. (A,B) n = 8 mice/group; pool of two independent experiments.

Figure 7

Impaired survival of CD28-deficient donor regulatory T cells (Treg) during second wave of acute graft-versus-host disease. Preconditioned BALB/c recipients received T cell-depleted bone marrow (TCD-BM) alone or together with 4 × 10⁴ Tconv and 2.5 × 10⁵ Treg from inducible CD28 knockout (iCD28KO) donors or wt littermates before tamoxifen treatment. On day 19 after transplantation, mice were sacrificed and donor Treg were identified in spleen (SPL), mesenteric lymph nodes (mLN), and liver as Thy1.1⁺Thy1.2⁺ (see Figure 4B). (A) Percentage of Treg among donor T cells (median + range) and (B) donor Treg recovery. (C,D) Paraffin sections of small and large bowel were stained with Foxp3 eF660 antibody and Dapi and (C) the number of Foxp3⁺Dapi⁺ cells assessed in 10 high power fields (200× magnification) of small and large bowel each. Triangles: TCD-BM only; circles: wt Tconv + wt Treg; squares: wt Tconv + iCD28KO Treg. (D) Representative overlays of Dapi and Foxp3 staining in the small bowel. (E) Percentage of Ki-67⁺ Treg among transferred Treg in SPL and mLN (median + range). (F) Gating strategy to differentiate viable and dead donor Treg (left) and percentage of dead Treg (right) in SPL and mLN (median + range). (A,E,F) black columns: wt Tconv + wt Treg; gray columns: wt Tconv + iCD28KO Treg. (B,C) circles: wt Tconv + wt Treg; squares: wt Tconv + iCD28KO Treg. (A–F) n = 4–5 mice per group. p values refer to a two-tailed, unpaired Mann–Whitney test.