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. Author manuscript; available in PMC: 2019 Mar 1.
Published in final edited form as: Biol Blood Marrow Transplant. 2017 Oct 20;24(3):594–599. doi: 10.1016/j.bbmt.2017.10.028

Pharmacodynamic monitoring predicts outcomes of CCR5 blockade as GVHD prophylaxis

Austin P Huffman 1,2, Lee P Richman 1,3, Lisa Crisalli 1, Alex Ganetsky 1, David L Porter 1, Robert H Vonderheide 1,3, Ran Reshef 1,2,*
PMCID: PMC5826857  NIHMSID: NIHMS928953  PMID: 29061535

Abstract

Blocking lymphocyte trafficking after allogeneic hematopoietic stem cell transplantation is a promising strategy to prevent graft-versus-host disease (GVHD) while preserving the graft-versus-tumor response. Maraviroc, a CCR5 antagonist, has shown promise in clinical trials, presumably by disrupting the migration of effector cells to GVHD target organs. We describe a phosphoflow assay to quantify CCR5 blockade during treatment with maraviroc and used it to evaluate 28 patients in a phase II study. We found that insufficient blockade of CCR5 was associated with significantly worse overall survival (HR=10.6, 95% CI 2.2–52.0, p=0.004) and higher rates of non-relapse mortality (HR=146, 95% CI 1.0–20,600, p=0.04), and severe acute GVHD (HR=12, 95% CI 1.9–76.6, p=0.009). In addition, we found that pre-transplant high surface expression of CCR5 on recipient T-cells predicted higher non-relapse mortality and worse GVHD- and relapse-free survival. Our results demonstrate that pharmacodynamic monitoring of CCR5 blockade unravels inter-patient variability in the response to therapy and may serve as a clinically informative biomarker.

Introduction

Graft-versus-host disease (GVHD) is a significant obstacle to successful allogeneic hematopoietic stem cell transplantation (allo-HSCT), a curative treatment for many hematologic malignancies. Acute GVHD occurs when alloreactive donor T-cells recognize recipient antigens as foreign and attack host tissues, especially in the liver, gut, and skin. Despite standard prophylaxis, acute GVHD incidence rates are approximately 30–50% in related donor HLA-matched transplants and as high as 50–70% in unrelated donor transplants (1).

Alloreactive donor T-cells home first to secondary lymphoid organs and then to target tissues, where they cause an inflammatory response (2). Cell migration is carefully directed by adhesion molecules and chemokine receptors on the cell surface and disruption of this process offers an opportunity for therapeutic intervention. In particular, the chemokine receptor CCR5 and its ligands CCL3, CCL4, and CCL5 appear to be critical to the development of GVHD. An anti-CCR5 antibody prevented murine GVHD by inhibiting CD8 T-cell homing to the gut and liver (3,4), and in humans certain CCR5 polymorphisms are associated with GVHD after allo-HSCT (5–7). Therefore, CCR5 blockade as GVHD prophylaxis may confer a clinical benefit.

We previously published the results of a phase I/II study of high-risk patients undergoing reduced-intensity allo-HSCT with standard GVHD prophylaxis (tacrolimus + methotrexate) combined with maraviroc (MVC), a non-allosteric CCR5 antagonist, given up to day 30 (8, 10). MVC treatment resulted in lower rates of acute GVHD compared to contemporary controls. We then conducted a follow-up phase II trial [NCT02208037] to investigate a longer treatment course (up to day 90) and to explore the pharmacodynamic properties of MVC (11).

In order to monitor the degree of CCR5 blockade in vivo in our phase II trial, we used a flow-cytometry-based assay on fresh whole blood at several peri-transplant time-points. The activated form of CCR5 is phosphorylated and internalized, and intracellular staining with a phosphospecific antibody allows quantification of the activated form of the receptor. It has been previously shown that pCCR5 levels increase in response to binding of the ligand CCL4 in T-lymphocytes (9). Here we demonstrate that treatment with MVC reduces levels of phosphorylated CCR5 in CD4 and CD8 T-cells and can be used to quantify the response. To our knowledge, phosphoflow has not yet been used in the context of a prospective clinical trial of chemokine receptor antagonists for any indication.

Materials and Methods

Study treatment

The trial methodology was similar to our previous phase I/II study that has been described in detail (8). Briefly, 37 patients receiving allo-HSCT with a reduced-intensity conditioning regimen at the University of Pennsylvania were enrolled in this study between January 2013 and June 2015. Patients received a uniform conditioning regimen of fludarabine and busulfan as well as standard GVHD prophylaxis of tacrolimus p.o. (0.06mg/kg/day) in two divided doses beginning two days before HSCT and intravenous methotrexate (15mg/m2 on day 1 and 10mg/m2 on days 3, 6, and 11). They also received MVC (300mg twice daily) beginning three days prior to transplantation until day 90. Patients received peripheral blood stem-cell grafts from unrelated donors. The study was approved by the Institutional Review Board of the University of Pennsylvania, and the patients provided written informed consent.

Phosphoflow

Whole blood was collected from patients on transplant days −6, 0, and 14 at drug trough into heparinized vacuum tubes and brought to the lab immediately after blood draw. Donor blood samples were obtained through the National Marrow Donor Program on the day of transplant. Whole blood aliquots of 200uL were dispensed directly into FACS tubes for each of four conditions: ligand-stimulated, antagonist-blocked, both, and neither (unstimulated control). Each condition was incubated for 30 minutes at 37C first with either 200uM MVC or an equivalent volume of DMSO control, followed by either 100nM CCL4 or an equivalent volume PBS control. Samples were then treated with Lyse/Fix Buffer (BD 558049) according to manufacturer’s instructions and permeabilized with ice-cold methanol at 4C for 20 minutes. Cells were then stained with APC-CD8 (IOTest IM0451), V450-CD3 (BD 560365), FITC-CD4 (BD 340133), and PE-pCCR5(Ser349) (Biolegend 321606), acquired on a BD FACS Canto flow cytometer, and analyzed with FlowJo single cell analysis software (TreeStar). pCCR5 MFI was recorded for CD3+CD4+ and CD3+CD8+ populations for each condition. Blockade insufficiency was calculated as a 20%+ drop in CD3+CD8+ pCCR5 MFI on day 0 when cells were treated with exogenous MVC, relative to the untreated condition.

Immune phenotyping

Fresh whole blood from donors and recipients was treated with ACK lysis buffer (Gibco A10492-01) according to manufacturer’s instructions and stained with PE-CCR5 (BD 550632), APC-H7-CD8 (BD 560179), V450-CD3 (BD 560365), and V500-CD4 (BD 560768).

Statistical analysis

Paired student t-tests were used to compare receptor expression levels over time. Time-to-event analyses were conducted using the Kaplan-Meier method or cumulative incidence function and comparisons between groups were done using the log-rank test or Gray test. Death was considered a competing risk for GVHD, relapse and non-relapse mortality outcomes. GVHD and relapse-free survival (GRFS) considered death, disease relapse, acute grade 3–4 GVHD and moderate-severe chronic GVHD as events. The impact of CCR5 blockade insufficiency and CCR5 expression levels was examined in a multivariable regression analysis, adjusting for variables that were found to be significant in univariable analysis for each outcome. Due to the small sample size, we limited the number of covariates to 2 in each model. Adjusted hazard ratios (HR) are presented throughout. Unadjusted univariate values are presented in Supplemental Table 1.

Results

Patient characteristics

Twenty-eight patients who had evaluable data at all 3 time points are included in this analysis. Patient characteristics are presented in Table 1. Median follow up was 29.9 months. No patients had active disease or active infections at the time of initiation of conditioning.

Table 1.

Patient Characteristics

N=28
Recipient age, median (range) 62 (49–72)
Recipient gender: M/F, % 61 / 39
Comorbidity Index, n (%):
 Low 5 (18)
 Intermediate 8 (29)
 High 15 (54)
Diagnosis, n:
 Acute myeloid leukemia 19
 Myelodysplastic syndrome 6
 Non-Hodgkin lymphoma 1
 Acute lymphoblastic leukemia 2
Donor, n(%):
 Matched unrelated (10/10) 23 (82)
 Mismatched unrelated (9/10) 5 (18)
Donor age, median (range) 32 (19–53)
Day 30 T-Cell Chimerism, median (range) 70 (0–100%)
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CCR5 blockade can be monitored and quantified by phosphoflow

As expected, pCCR5 levels in CD8 T-cells in pre-transplant samples prior to initiation of MVC (day −6) increased in response to stimulation with CCL4 and decreased in response to the addition of MVC, as measured by MFI (Fig. 1a, b). When cells were treated ex vivo with both CCL4 and MVC, we observed complete blockade in all patients and the MFI remained at similar levels of the MVC-only condition (Fig. 1a). On the day of transplant, which was the 4th day of MVC administration, CD8 T-cell pCCR5 levels were unresponsive to stimulation with CCL4 and did not decrease when treated with additional, exogenous MVC (Fig. 1b). However, we observed heterogeneity in the pCCR5 response on day 0, with a subset of patients who still demonstrated a decrease in pCCR5 levels on CD8 T-cells MFI when treated with exogenous MVC (Fig. 1c, d). We also examined the level of CCR5 blockade on CD4 T-cells and observed a similar trend, however the level of CCR5 expression on CD4 cells is consistently lower compared to CD8 cells and therefore it was more challenging to assess adequate blockade of the receptor.

Fig. 1. CCR5 blockade sufficiency can be determined by phosphoflow.

Fig. 1

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(A) Representative flow plot from patient 04712-015 on day −6 pre-transplant, prior to initiation of maraviroc (MVC). CD8 T-cell pCCR5 mean fluorescence intensity (MFI) increases in response to CCL4 stimulation and decreases in response to exogenous MVC. (B) Average fold-change in pCCR5 MFI on day −6 (patient not yet on MVC) and day 0 (patient on MVC) CD8 T-cells. Stimulation has an effect on day −6 but not day 0; exogenous MVC has an effect on day −6 that is ameliorated by day 0. (C) Representative flow plots and MFIs of sufficiently blocked patient 04712-018 (top) and insufficiently blocked patient 04712-005 (bottom). (D) Heterogeneity in %change in pCCR5 MFI on day 0 in response to exogenous MVC. Those patients whose MFI dropped by more than 20% compared to the unstimulated control were considered to have insufficient blockade, as additional MVC had an additional effect. *** = p<0.001.

Insufficient CCR5 blockade predicts worse outcomes

We then examined the associations between insufficient blockade of CCR5 on day 0 and clinical outcomes. On average, CD8 T-cell pCCR5 MFI decreased by approximately 20% when treated with MVC pre-transplant (Fig. 1b). Therefore, a drop greater than 20% in pCCR5 levels in response to exogenous MVC on day 0 was considered insufficient blockade of the receptor by endogenous MVC (Fig. 1c, d). Eight out of twenty-eight patients (29%) had insufficient blockade of CCR5 on day 0 based on this definition. We then compared the clinical outcomes for patients with insufficient CCR5 blockade on day 0 vs. patients with sufficient blockade. Multivariable regression analysis revealed that insufficient blockade was associated with a significantly worse overall survival (HR= 10.6, 95% CI [2.2,52.0], p=0.004; Fig. 2a), worse GRFS (HR= 3.4, 95% CI [1.1,10.6], p=0.04; Fig. 2b), increased risk of grade 3–4 acute GVHD (HR= 12, 95% CI [1.9, 76.6], p=0.009; Fig. 2c), and higher non-relapse mortality (HR= 146, 95% CI [1.0, 20,600], p=0.04; Fig. 2d). Blockade insufficiency was not associated with differences in incidence rates of relapse or grade 2–4 GVHD (Table 2).

Fig. 2. Blockade insufficiency is predictive of worse outcomes.

Fig. 2

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(A–D) Insufficient blockade on day 0 is associated with increased risk of death (A. HR=10.6, p=0.004), GRFS (B. HR=3.4, p=0.04), aGVHD3–4 incidence (C. HR=12, p=0.009), and non-relapse mortality (D. HR=146, p=0.04).

Table 2.

Multivariate associations between blockade sufficiency, patient pre-transplant CD8 CCR5 levels, and clinical outcomes

Variable Insufficient CCR5 Blockade Patient Pre-Transplant CD8 CCR5
OS HR 10.6 3.9
95% CI 2.2–52.0 1.0–16.4
P 0.004 0.06
GRFS HR 3.4 3.2
95% CI 1.1–10.6 1.1–9.2
P 0.04 0.03
NRM HR 146 6.20e08
95% CI 1.0–20,600 6.4e07–5.9e09
P 0.04 <0.001
aGVHD 2–4 HR 0.4 0.4
95% CI 0.09–2.2 0.10–1.5
P 0.30 0.20
aGVHD 3–4 HR 12 2.5
95% CI 1.9–76.6 0.4–16.2
P 0.009 0.30
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OS = Overall Survival, GRFS = GVHD- and Relapse-Free Survival, NRM = Non-Relapse Mortality, aGVHD 2–4 = acute Graft Versus Host Disease grades 2–4, aGVHD 3–4 = acute Graft Versus Host Disease grades 3–4, HR = adjusted Hazard Ratio, CI = Confidence Interval, P = P-value

T-cell CCR5 surface expression is dynamic peri-transplant and heterogeneous between individuals

Prior to transplant, recipient CD8 T-cells had elevated surface CCR5 expression compared to their healthy donor counterparts (Fig. 3a), supporting the role of this receptor as a marker for inflammation in cancer patients. CCR5 expression was further elevated by the day of transplant, but reduced to pre-transplant levels by day 14 post-transplant (Fig. 3a). The same pattern was observed in CD4 T-cells, which had lower CCR5 expression compared to CD8 T-cells (Fig. 3b). There was heterogeneity in both recipient (0.1–49.4%) and donor (0.3–17.3%) CD8 T-cell CCR5 surface expression. Day −6 and day 0 recipient CCR5 expression correlated with each other (R = 0.73, p = 0.004), but day 14 CCR5 expression did not correlate with day −6, day 0, or donor CCR5 expression, likely because day 14 T-cells represented a mix of donor and recipient cells, as donor chimerism has been shown to evolve over time after reduced-intensity transplant (12).

Fig. 3. Peri-transplant increase in T-cell CCR5 surface expression.

Fig. 3

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CCR5 expression on CD4 (A) and CD8 (B) T-cells. On both CD4 and CD8 T-cells, recipient pre-transplant CCR5 expression was elevated compared to healthy donor control, increased by day of transplant in response to maraviroc and chemotherapy, and dropped to an intermediate level. (C) Representative CCR5 expression flow plots of pre-transplant “CCR5lo” and “CCR5hi” patients’ CD8 T-cells. * = p<0.05, ** = p<0.01, *** = p<0.001.

Pre-transplant recipient CD8 T-cell CCR5 surface expression predicts survival

We then examined the predictive value of CCR5 expression in both donors and recipients on clinical outcomes. We grouped patients based on median CD8 CCR5 surface expression into CCR5hi and CCR5lo groups (Fig. 3c). We found that patients with higher pre-transplant CD8 T-cell CCR5 expression had worse overall survival, which approached statistical significance (HR= 3.9, 95% CI [1.0, 16.4], p=0.06; Fig. 4a), as well as significantly worse GRFS (HR= 3.2, 95% CI [1.1, 9.2], p=0.03; Fig. 4b), and non-relapse mortality (HR= 6.2e08, 95% CI [6.4e07, 5.9e09], p<0.001; Fig. 4c). Donor blood had a lower degree of heterogeneity in CCR5 expression and we did not identify associations between donor CCR5 expression and transplant outcomes.

Fig. 4. Recipient pre-transplant CD8 T-cell CCR5 expression is predictive of outcomes.

Fig. 4

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(A–C) High CD8 T-cell CCR5 surface expression is associated with increased risk of death (A. HR=3.9, p=0.06), GRFS (B. HR=3.2, p=0.03), and non-relapse mortality (C. HR=6.2e08, p<0.001).

Discussion

CCR5 blockade is a promising GVHD prophylactic therapy in allo-HSCT, but not all patients seem to have a similar degree of benefit. We developed an assay to evaluate the degree of blockade and discovered that in certain patients, treatment with MVC failed to block receptor activation completely, as evident by the addition of exogenous MVC into the assay leading to further deactivation of the receptor. These patients with insufficient receptor blockade were at higher risk for death, driven by higher rates of severe GVHD and non-relapse mortality. To our knowledge this is the first study in which the effectiveness of chemokine receptor blockade has been quantified and associated with clinical outcomes. Our real-time assay provides an important clinical measure that, if confirmed in a validation cohort, could inform the risk for complications and help guide dosing of MVC and potentially future drugs within the same class. Importantly, this assay can also be used ahead of the transplant to examine each patient’s individual response and allowing a dose increase in an attempt to achieve total blockade before proceeding to transplant. Our findings also imply that clinical benefit from MVC is not universal, and therefore studies like BMTCTN 1203 should examine differences between patient subsets with disparate clinical outcomes for blockade insufficiency and predictive pre-transplant biomarkers like CCR5 expression.

We also evaluated the value of pre-transplant CCR5 expression as a prognostic biomarker. Elevated CCR5 expression in the recipient correlated with worse outcomes. We think that increased CCR5 expression is indicative of pre-transplant systemic inflammation, which may increase the risk for various transplant complications. This finding is in accordance with previous preclinical and clinical studies that correlated increased inflammation with subsequent risk for transplant complications. For example, high pretransplant C-reactive protein levels predicted poor outcomes following reduced intensity conditioning (13). Additionally, chronic inflammatory IL-1 signaling has been shown to severely erode hematopoietic stem cell self-renewal capacity (14). Specifically for CCR5, a population of CCR5-expressing IL-17 producing T-cells has been shown to be involved in both acute and chronic GVHD (15, 16). As a surrogate for the effect of MVC against increased gut inflammation, we have previously shown that MVC decreases serum reg3a levels, implicating relief of intestinal inflammation as one potential mechanism for the drug’s anti-GVHD efficacy (10).

Limitations of our study include the small sample size, which may not be representative of certain patient types and precludes the use of more comprehensive multivariable models for prediction of outcomes. In addition, early measurement of CCR5 blockade on day 14 represents a mix of donor and recipient T-cells because mixed chimerism is common in early time points after Flu/Bu2 conditioning (12). Future studies might overcome these limitations and help confirm our findings.

Understanding the underlying mechanism of CCR5 blockade heterogeneity is an area of ongoing inquiry. Given that CCR5 polymorphisms have been previously shown to be clinically relevant in other settings (5–7) we believe there may be a genetic component in determining the response to CCR5 blockade. Whether incomplete blockade can be overcome by increased MVC dosage, leading to improvement in clinical outcome, should be examined prospectively.

Supplementary Material

Supp. Data

Acknowledgments

Funding sources: Amy Strelzer Manasevit Award from the National Marrow Donor Program; Career Development Award from the Conquer Cancer Foundation; National Institutes of Health grants P30-CA16520, K23-CA178202 & U01-HL069286. Pfizer supplied maraviroc for the clinical trial described in this manuscript.

The authors would like to thank the National Marrow Donor Program for providing donor samples. We acknowledge funding from the Amy Strelzer Manasevit Award from the National Marrow Donor Program; Career Development Award from the Conquer Cancer Foundation; National Institutes of Health grants P30-CA16520, K23-CA178202 & U01-HL069286.

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