Blockade of lymphocyte chemotaxis in visceral graft-versus-host disease

Abstract

Background: Graft-versus-host disease (GVHD) is a major barrier to successful allogeneic hematopoietic stem-cell transplantation (HSCT). The chemokine receptor CCR5 appears to play a role in alloreactivity. We tested whether CCR5 blockade would be safe and limit GVHD in humans.

Methods: We tested the in vitro effect of the CCR5 antagonist maraviroc on lymphocyte function and chemotaxis. We then enrolled 38 high-risk patients in a single-group phase 1 and 2 study of reduced-intensity allogeneic HSCT that combined maraviroc with standard GVHD prophylaxis.

Results: Maraviroc inhibited CCR5 internalization and lymphocyte chemotaxis in vitro without impairing T-cell function or formation of hematopoietic-cell colonies. In 35 patients who could be evaluated, the cumulative incidence rate (±SE) of grade II to IV acute GVHD was low at 14.7±6.2% on day 100 and 23.6±7.4% on day 180. Acute liver and gut GVHD were not observed before day 100 and remained uncommon before day 180, resulting in a low cumulative incidence of grade III or IV GVHD on day 180 (5.9±4.1%). The 1-year rate of death that was not preceded by disease relapse was 11.7±5.6% without excessive rates of relapse or infection. Serum from patients receiving maraviroc prevented CCR5 internalization by CCL5 and blocked T-cell chemotaxis in vitro, providing evidence of antichemotactic activity.

Conclusions: In this study, inhibition of lymphocyte trafficking was a specific and potentially effective new strategy to prevent visceral acute GVHD. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00948753.).

Figure 1 (facing page). Specific Inhibition of CCL5- and CCL3-Induced Internalization of CCR5 and Inhibition of T-Cell Chemotaxis Associated with Maraviroc

In Panel A, a representative flow-cytometric analysis shows a reduction in surface expression of CCR5 on normal donor CD8+ T cells after incubation with 100 nM of CCL5 for 30 minutes. Preincubation with 1 μM of maraviroc (MVC) for 30 minutes fully abrogated internalization of the receptor. Panel B shows the effect of various concentrations of maraviroc on CCL5-induced internalization of T cells. The mean fluorescence intensity (MFI) of CCR5 was measured by means of flow cytometry after incubation with escalating concentrations of maraviroc and then incubation with 100 nM of CCL5 or CCL3. The surface expression of CD4, CD8, CD45RA, and CCR7 was used to define T-cell subsets. Plots represent means of three different experiments. All T-cell subsets other than naive CD4+ and CD8+ cells showed a significant response to CCL5 and efficient inhibition by maraviroc (P<0.01 by analysis of variance for central memory, effector memory, and effector cells; P>0.05 for naive cells). Similar results were observed with CCL3 (not shown). Panel C shows the lack of effect of maraviroc on CCL5-induced internalization of CCR1 on T cells and CCL2-induced internalization of CCR2 on monocytes (P>0.05 by analysis of variance). Panel D shows the results of chemotaxis assays in the presence of escalating concentrations of maraviroc. Peripheral-blood mononuclear cells from five normal donors and from three donors who were homozygous for the CCR5 Δ32 allele (CCR5-Δ32) were preincubated with maraviroc and then allowed to migrate in a Boyden chamber in response to 100 nM of CCL5 (left panel) or 0.5 nM of CCL3 (right panel) for 3 hours. Migrating cells were stained with surface markers and counted by means of flow cytometry. Chemotaxis ratios represent the number of migrating cells in each experimental condition divided by the number of cells in the positive control sample (chemokine without maraviroc). Means and standard errors (represented by T bars) are shown. P values represent a two-sided t-test between normal donor cells and Δ32 cells for each condition. Asterisks indicate P<0.01.

Figure 2. Clinical Trial Outcomes of Acute GVHD, Moderate-to-Severe Chronic GVHD, and Organ-Specific Acute GVHD

Shown are cumulative incidence plots of grade II to IV and grade III or IV acute GVHD (Panel A), moderate-to-severe chronic GVHD (Panel B), and organ-specific acute GVHD (in the skin, liver, and gut) (Panel C) in 35 patients undergoing reduced-intensity conditioned hematopoietic stem-cell transplantation with maraviroc added to standard GVHD prophylaxis.

Figure 3 (facing page). Inhibition of CCL5-Induced CCR5 Internalization and CCL5-Induced T-Cell Chemotaxis by Serum from Patients Receiving Maraviroc

Panels A and C show the loss of responsiveness of CD8+ T cells to CCL5 after incubation with serum obtained from patients receiving maraviroc (MVC) at a dose of 300 mg twice daily on day 12 after transplantation; serum obtained on day 60 (30 days after the last dose of maraviroc) still allowed the cells to respond to CCL5. Panel A includes representative results on flow cytometry that show the expression of CCR5 on normal donor CD8+ T cells after incubation with serum for 1 hour and stimulation by 100 nM of CCL5, 100 nM of CCL5 plus excess maraviroc (1 mM), or control media. CCR5 internalization was reversed by adding excess maraviroc to the day 60 sample but not to the day 12 sample. Panel C shows the ratios of mean fluorescent intensities (MFI) of CCR5 on normal donor CD8+ T cells, expressed as the MFI ratio between each experimental condition and its media control. Serum obtained from six patients on day 12 at trough levels (before drug administration) or peak levels (3 or 4 hours after administration) abrogated the internalization of CCR5 by CCL5. Serum obtained on day 60 and control media did not have an inhibitory effect on the internalization of CCR5 by CCL5, which was efficiently blocked by adding excess maraviroc (1 mM). The asterisks indicate P<0.01 in a paired two-sided t-test. Similar results were seen with CD4+ cells. Panels B and D show the results of chemotaxis experiments in which normal donor peripheral-blood mononuclear cells were suspended for 1 hour in serum from patients obtained on day 0 (maraviroc trough or peak levels) and on day 60 (off maraviroc). The cells were then allowed to migrate in response to 100 nM of CCL5 or control media. Chemotaxis ratios represent the number of migrated CD3+ cells with CCL5 stimulation divided by the number of cells in the control sample. Panel B shows a heat map of chemotaxis ratios on day 0 and day 60, showing consistent inhibition of CCL5-induced chemotaxis in patients receiving maraviroc (at a dose of 300 mg twice daily). Panel D shows the chemotaxis ratios for the first six patients who received maraviroc, showing inhibition of CCL5-induced chemo-taxis at maraviroc peak and trough levels, as compared with those on day 60. The asterisk indicates P<0.01 in a paired two-sided t-test. The addition of 1 mM of maraviroc decreased the chemotaxis ratio only on day 60. The I bars indicate standard errors.