Anti-CD4 monoclonal antibody prevents chronic graft-versus-host disease in mice by inducing immune tolerance of CD8+ T cells and alleviating thymus injury

Abstract

Background: Chronic graft-versus-host disease (cGVHD) manifests with characteristics of autoimmune disease with organs attacked by pathogenic helper T cells. Recent studies have highlighted the role of T cells in cGVHD pathogenesis. Due to limited understanding of underlying mechanisms, preventing cGVHD after allogenic hematopoietic cell transplantation (HCT) has become a major challenge.

Materials and methods: Here, we used a representative cGVHD model with the donor C57BL/6 to recipient BALB/c combination. Post-HCT, mice were treated with IgG or anti-CD4 monoclonal antibody. The severity of cGVHD was assessed by evaluating symptoms of cGVHD and histopathology examination (H&E) of target organs. Thymus gland damage and defects of the negative selection were assessed by analyzing the CD4⁺CD8⁺ double-positive thymocytes, cortical thymic epithelial cells and medullary thymic epithelial cells (mTECs). Immunotolerance of CD8⁺ T cells was assessed by detecting the expression of CD80, PD-1, GRAIL and IL-7Rα. Long-term cellular and humoral immunity associated with graft-versus-leukemia (GVL) effects were evaluated through detecting the percentage of CD4⁺ T cells, IgG, IgM and IgA concentrations, and performing tumor challenge experiment.

Results: Donor CD8⁺ T cells caused thymic epithelial cells damage and impaired negative selection in recipients, leading to generation of autoreactive T cells and causing cGVHD. Anti-CD4 mAb treatment promoted immune incompetence of thymus-infiltrating CD8⁺ T cells, facilitated recovery of CD4⁺CD8⁺ thymocytes and regeneration mTECs, and preserved negative-selection, but had no effects on the long-term cellular immunity and humoral immunity, resulting in preservation of GVL effect.

Conclusion: Our results indicate that anti-CD4 mAb therapy early post-HCT allows thymus recovery by inducing the immune tolerance of thymus infiltrated CD8⁺ T cells, thereby alleviating thymic epithelial cells damage, preserving negative selection, and preserving long-term GVL effect at the same time.

Keywords: CD4 + T cells; anti-CD4 monoclonal antibody; chronic graft-versus-host disease; immune tolerance; thymic recovery.

Figure 1

Anti-CD4 antibodies prevent the development of cGVHD. (A) Graphs showing percentage of body weight changes (left) and absence of diarrhea (right) in TCD-BM mice (TCD-BM), IgG treated mice (IgG) and Anti-CD4 treated mice (Anti-CD4) after HCT. n=8 per group. (B) Representative images of mice (top) at 50-60 days post-HCT and the cutaneous cGVHD score (bottom) from TCD-BM, IgG and Anti-CD4 groups. n=8 per group. (C) Graphs showing the survival curves of TCD-BM, IgG and Anti-CD4 groups. n=8 per group. (D) Representative H&E -stained sections of skin, salivary, liver, lung, small intestine and colon (top) from TCD-BM, IgG and Anti-CD4 group, with corresponding GVHD scores (mean ± SEM) (bottom). n=8 per group. ^†indicates death of all recipients in a group. The red arrows indicate representative changes in GVHD targeted tissues. The statistical significance was performed according to one-way ANOVA followed by Tukey’s post hoc. ***p < 0.001 versus TCM-BM. ^###p < 0.001 versus IgG. Scale bar: 100 μm.

Figure 2

Depletion of donor CD4+ T cells promotes recovery of CD4+CD8+ thymocytes and medullary thymic epithelial cells. (A) Representative FACS images (top) at day 60 post-HCT and mean ± SEM of percentage and yield (bottom) of CD8⁺CD4⁺ cells in the thymus of TCD-BM, IgG and Anti-CD4 groups. n=8 per group. (B) Representative immunostaining images of CK8 and UEA-I in the thymus of wild type mice (WT) TCD-BM, IgG and Anti-CD4 groups at day14 post-HCT. n=4 per group. Scale bar: 50 μm. The statistical significance was performed according to one-way ANOVA followed by Tukey’s post hoc. ***p < 0.001 versus TCM-BM. ^###p < 0.001 versus IgG.

Figure 3

Early depletion of donor CD4+ T cells post-transplantation enhances immune dysfunction in thymus infiltrating CD8+ T cells. (A) Yield of donor-derived CD8⁺ T cell in the thymus (left) and spleen (right) from IgG and Anti-CD4 groups at day 7 post-HCT. n=4 per group. (B) Representative FACS images (top) and percentage of CCR9 expression on CD8⁺ T cells in the spleen (mean ± SEM) (down) of IgG and Anti-CD4 groups collected at day 7 post-HCT. n=4 per group. (C) Representative FACS image and expression levels (mean ± SEM) of CD80, PD-1, GRAIL and IL-7Rα on thymus (THY) and spleen (SPL) infiltrated H2Kb⁺TCRβ⁺CD8⁺ T cells in IgG and anti-CD4 groups collected at day 7 post-HCT. n=4 per group. The statistical significance is performed according to Student’s t-tests (unpaired two-tailed). **p < 0.01 and ***p < 0.001 versus IgG.

Figure 4

Temporary depletion of CD4+ T cells in vivo does not impair long-term cellular and humoral immunity and the GVL effect. (A) Representative FACS images (left) and percentage (mean ± SEM) (right) of H2Kb⁺TCRβ⁺CD4⁺ T cells in the spleen from TCD-BM, IgG and Anti-CD4 groups at day 60 and day 100 after HCT. n=4 for each group. (B) ELISA results of the concentrations (mean ± SEM) of IgG (left), IgA (middle), and IgM (right) from serum of TCD-BM, IgG and Anti-CD4 groups on day 60 (n=4 per group) and day100 (n=4 per group) after HCT. (C) Representative image of in vivo bioluminescent imaging (BLI) and the statistic result from each time point after leukemia/lymphoma challenge. n=3 for each group. The statistical significance was performed according to one-way ANOVA followed by Tukey’s post hoc. **p < 0.01 and ***p < 0.001 versus TCM-BM. ^##p < 0.01 and ^###p < 0.001 versus IgG. NA, Not Applicable.

Figure 5

Model of role and mechanism of anti-CD4 in preventing cGVHD.