Increased T follicular helper cells and germinal center B cells are required for cGVHD and bronchiolitis obliterans

Abstract

Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Having shown that germinal center (GC) formation and immunoglobulin deposition are required for multiorgan system cGVHD and associated bronchiolitis obliterans syndrome (BOS) in a murine model, we hypothesized that T follicular helper (Tfh) cells are necessary for cGVHD by supporting GC formation and maintenance. We show that increased frequency of Tfh cells correlated with increased GC B cells, cGVHD, and BOS. Although administering a highly depletionary anti-CD20 monoclonal antibody (mAb) to mice with established cGVHD resulted in peripheral B-cell depletion, B cells remained in the lung, and BOS was not reversed. BOS could be treated by eliminating production of interleukin-21 (IL-21) by donor T cells or IL-21 receptor (IL-21R) signaling of donor B cells. Development of BOS was dependent upon T cells expressing the chemokine receptor CXCR5 to facilitate T-cell trafficking to secondary lymphoid organ follicles. Blocking mAbs for IL-21/IL-21R, inducible T-cell costimulator (ICOS)/ICOS ligand, and CD40L/CD40 hindered GC formation and cGVHD. These data provide novel insights into cGVHD pathogenesis, indicate a role for Tfh cells in these processes, and suggest a new line of therapy using mAbs targeting Tfh cells to reverse cGVHD.

Figure 1

Increased Tfh cells in cGVHD. B10.BR mice were transplanted with BM only or BM and splenic T cells from B6 donor mice and spleens were harvested on day 60. (A) Representative spleens harvested from BM only and BM plus spleen mice on day 60 showing CD4 (blue), PD-1 (green), and peanut agglutinin (PNA) (red) or (B) CD19 (blue), GL7 (green), or PNA (red). Arrows indicate GC areas. (C) Composition of B cells in the spleen and peritoneal cavity. (D) Frequency of Tfh cells and (E) GC B cells. *P < .05; ***P < .001.

Figure 2

B-cell depletion by anti-CD20 therapy is not sufficient to prevent cGVHD and associated BOS. cGVHD was established in B10.BR mice transplanted with WT B6 BM and WT B6 spleen cells, and mice were treated with anti-CD20 or irrelevant mouse IgG1 antibody from day 28 to day 56. (A) PFTs for mice treated with irrelevant or anti-CD20 antibody. (B) Number of B cells purified from the spleens of mice on day 56. (C) Trichrome deposition and (D) Ig deposition in the lungs of mice harvested on day 56. (E) Number of CD138⁺ CD19 low plasma cells in the BM of D60 transplanted mice. (F) Representative images of frozen lung tissues stained with anti-CD19 FITC and 4,6 diamidino-2-phenylindole. Representative data from 2 experiments; n = 8 mice per group. *P < 0.5; **P < .01; ***P < .001.

Figure 3

Chemokine receptor CXCR5 on donor mature T cells is necessary for cGVHD. B10.BR mice were transplanted with WT B6 BM and WT B6 T cells or B6 CXCR5 KO T cells. (A) Mice were evaluated for pulmonary function. (B) Lung sections were stained with FITC-conjugated anti-mouse Ig, and the area of Ig deposition was measured as shown in (C) representative images. (D) Lungs of mice were stained with trichrome, and the ratio of collagen stain to total stain was measured surrounding the bronchioles as shown in (E) representative images. Representative data from 3 experiments; n = 8 mice per group. *P < .05; **P < .01; ***P < .001.

Figure 4

IL-21 production from donor T cells is required for cGVHD pathology. B10.BR mice were transplanted with WT B6 BM and splenocytes from either WT B6 or B6 IL-21 KO mice. (A) Mice were evaluated for pulmonary function. (B) Lungs of mice were stained with trichrome, and the ratio of collagen stain to total stain was measured surrounding the bronchioles. (C) Lung sections were stained with FITC-conjugated anti-mouse Ig, and the area of Ig deposition was measured. (D) Whole spleen sections were stained with rhodamine-conjugated PNA, and the size of the PNA-positive sections was measured. Frequency of (E) Tfh and (F) GC B cells in the spleen of transplanted mice on day 60. Representative data from 3 experiments; n = 8 mice per group. *P < .05; **P < .01; ***P < .001.

Figure 5

B cells need IL-21R for full maturation and progression of cGVHD. B10.BR recipients were transplanted with either WT BM or IL-21R KO BM and WT splenic (S) T cells. (A) Mice were evaluated for pulmonary function. (B) Whole spleen sections were stained with rhodamine-conjugated PNA, and the size of the PNA-positive sections was measured. Frequency of (C) Tfh and (D) GC B cells in the spleens of transplanted mice on D60. (E) Lung sections were stained with FITC-conjugated anti-mouse Ig, and the area of Ig deposition was measured, and (F) representative images. (G) Lungs of mice were stained with trichrome, and the ratio of collagen stain to total stain surrounding the bronchioles was measured with (H) representative images. Representative data from 3 experiments with n = 8 mice per group. *P < .05; **P < .01; ***P < .001.

Figure 6

ICOS or CD40L costimulatory pathway blockade is sufficient for reducing GCs and preventing BOS. cGVHD was established in B10.BR mice transplanted with WT B6 BM and WT B6 T cells, and mice were treated with either anti-ICOS, anti-CD40L, or irrelevant rat antibody from day 28 to day 56. (A) PFT. (B) GC size was analyzed in situ by measuring the PNA-positive sections in transplanted spleens from day 56. Splenocytes were harvested from transplanted mice on day 56, made into a single-cell suspension, and analyzed for (C) Tfh cells or (D) GC B cells. (E) Amount of Ig deposited in the lungs of transplanted mice on day 56 with (F) representative images. (G) Quantified trichrome present in the lungs of mice on day 56 with (H) representative images. Representative data from 2 experiments; n = 8. *P < .05; **P < .01; ***P < .001.

Figure 7

Anti-IL-21 mAb reverses BOS. cGVHD was established in B10.BR mice transplanted with WT B6 BM and WT B6 T cells, and mice were treated with either irrelevant mouse IgG1 or anti-IL-21. (A) PFTs from mice treated with either neutralizing anti-IL-21 mAb or irrelevant mAb. Splenocytes were isolated and analyzed for (B) GC B cells or (C) Tfh cells. (D) Ig and (E) ratio of trichrome-positive to total area surrounding bronchioles in trichrome stain. Representative data from 3 experiments; n = 8. *P < .05; **P < .01; ***P < .001.