Immunologic Effects of CCR5 Blockade in Graft-Versus-Host Disease Prophylaxis

Background: Acute graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Experimental models and clinical studies suggest that blocking lymphocyte trafficking with the chemokine receptor CCR5 antagonist maraviroc (MVC) ameliorates visceral GVHD. However, the effects of CCR5 blockade on immune reconstitution and activation in allogeneic HSCT recipients remain unknown.

Methods: We previously reported a phase I/II study of 38 high-risk patients undergoing reduced-intensity allogeneic HSCT with standard GVHD prophylaxis (tacrolimus, methotrexate) combined with brief (33-day) MVC treatment. We compared the clinical and immunologic outcomes of these patients to a contemporary control cohort of 115 consecutive patients undergoing allogeneic HSCT with standard GVHD prophylaxis alone. Multivariate analysis was used to assess the effect of MVC treatment on the incidence of GVHD. Flow cytometry on day 30 and day 60 PBMC samples was used to monitor immune cell subsets, T cell activation markers and chemokine receptor expression. We also used a multiplex Luminex assay to quantify serum cytokine levels at day 30.

Results: Since our initial report, longer follow up (median 37 months) now shows that patients who received MVC have reduced incidence rates of acute grade II-IV GVHD (adjusted hazard ratio [aHR]=0.42, 95% CI 0.21-0.84, p=0.015) and grade III-IV GVHD (aHR=0.43, 95% CI 0.17-1.09, p=0.075) compared to our control cohort (Figure 1). There was no difference in chronic GVHD between the groups. Early lymphocyte recovery was similar between MVC and control cohorts, with no significant difference in absolute lymphocyte, CD4 T cell and CD8 T cell counts at day 30. We observed increased CCR5 expression on CD4 T cells, CD8 T cells and B cells at day 30 in response to MVC treatment (Figure 2A-B). However, CCR5 expression on these cell types equalized between MVC and control patients by day 60. Day 30 MVC samples also demonstrated decreased percentages of CD38⁺ CD4 T cells and HLA-DR⁺ CD8 T cells (Figure 2C-D), an effect that also faded by day 60. These data suggest that inhibition of lymphocyte trafficking with CCR5 blockade dampens peripheral T cell activation.

Notably, some patients developed acute GVHD despite MVC prophylaxis, and therefore we investigated differences between MVC responders and non-responders. Patients who developed GVHD had a higher percentage of activated CD38⁺ CD4 T cells in peripheral blood (Figure 3A) and an increased proportion of naive CD4 (36.5% vs 18.1% p=0.005) and CD8 (35.5% vs 11.9% p=0.0008) T cells, which has been associated with the development of GVHD. We also observed higher CCR5 expression on CD8 T cells from MVC non-responders (CCR5 MFI 8800 vs 6595, p=0.02). Intriguingly, while MVC responders and non-responders had similar serum concentrations of CCR5 ligands (MIP-1a, MIP-1b, Rantes), MVC non-responders had elevated levels of CXCR3 ligands (IP-10, MIG) (Figure 3B-C). Taken together, these data suggest that lymphocyte migration via CXCR3 may be a potential escape mechanism for GVHD initiation and T cell activation in the setting of CCR5 blockade.

Conclusion: Brief CCR5 blockade in high-risk patients inhibits T cell activation and reduces the incidence of GVHD. MVC does not inhibit early lymphocyte recovery, which was similar in treated and control patients. This supports the hypothesis that MVC treatment would not impair graft-versus-leukemia activity. Our analysis of immunologic outcomes of MVC reveals a distinct immunologic effect for CCR5 blockade in allogeneic HSCT recipients and suggests a novel resistance mechanism. These studies bolster CCR5 antagonism as an effective strategy for GVHD prevention and provide support for extended MVC treatment duration and investigation into CXCR3 as a therapeutic target.

Figure 1. Incidence of GVHD in control and MVC cohorts

 

Figure 2. MVC increases CCR5 expression and dampens T cell activation at day 30   *p<0.05; **p<0.01; ***p<0.001

 

Figure 3. Increased T cell activation and serum CXCR3 ligands in MVC non-responders   *p<0.05; ***p<0.001