
==== Front
Int Immunol
Int Immunol
intimm
International Immunology
0953-8178
1460-2377
Oxford University Press UK

38778574
10.1093/intimm/dxae033
dxae033
Original Research
AcademicSubjects/MED00730
Blockade of CCR5 and CXCR3 attenuates murine acute graft-versus-host disease through modulating donor-derived T-cell distribution and function
Tang Bo Department of Hematology, Peking University First Hospital, Beijing, China

Qin Chenchen Department of Hematology, Peking University First Hospital, Beijing, China

Liu Huihui Department of Hematology, Peking University First Hospital, Beijing, China

Miao Shengchao Department of Hematology, Peking University First Hospital, Beijing, China

Xue Chao Department of Hematology, Peking University First Hospital, Beijing, China

Wang Zhenhua Department of Hematology, Peking University First Hospital, Beijing, China

Zhang Yang Department of Hematology, Peking University First Hospital, Beijing, China

Dong Yujun Department of Hematology, Peking University First Hospital, Beijing, China

Liu Wei Department of Hematology, Peking University First Hospital, Beijing, China

Ren Hanyun Department of Hematology, Peking University First Hospital, Beijing, China

Bo Tang, Chenchen Qin and Huihui Liu contributed equally to this work.

Correspondence to: H. Ren; E-mail: renhy0813@163.com
Correspondence to: W. Liu; E-mail: liuwei_abc@sina.com
10 2024
23 5 2024
23 5 2024
36 10 541552
21 4 2023
22 5 2024
12 5 2024
03 7 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society for Immunology.
2024
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Abstract

Lymphocyte trafficking via chemokine receptors such as C-C chemokine receptor 5 (CCR5) and CXCR3 plays a critical role in the pathogenesis of acute graft-versus-host disease (aGVHD). Our previous studies showed that the addition of CCR5 or CXCR3 antagonists could only slightly alleviate the development of aGVHD. Given the specificity of T lymphocytes bearing CXCR3 and CCR5, we investigated whether combined CCR5 and CXCR3 blockade could further attenuate murine aGVHD. A mouse model of aGVHD was established to assess the efficacy of CCR5 and/or CXCR3 blockade on the development of aGVHD. The distribution of lymphocytes was calculated by quantification of immunostaining cells. The immunomodulatory effect on T cells was assessed by evaluating T-cell proliferation, viability, and differentiation. Using the murine allogeneic hematopoietic stem cell transplantation model, we demonstrated that blockade of both CCR5 and CXCR3 could efficiently alleviate the development of aGVHD. Further investigation on the immune mechanisms for this prophylactic effect showed that more T cells were detained into secondary lymphoid organs (SLOs), which may lead to reduced infiltration of T cells into GVHD target organs. Our study also showed that T cells detained in SLOs dampened the activation, suppressed the polarization toward T helper type 1 (Th1) and T cytotoxic type 1 (Tc1) cells, and induced the production of Treg cells. These data suggest that concurrent blockade of CCR5 and CXCR3 attenuates murine aGVHD through modulating donor-derived T-cell distribution and function, and this might be applicable for aGVHD prophylaxis in clinical settings.

Combined CCR5 and CXCR3 blockade alleviates GVHD

Graphical Abstract

Graphical Abstract

acute GVHD
mouse model
National Natural Science Foundation of China 10.13039/501100001809 81970160 81800181 81570160 81100351
==== Body
pmcIntroduction

Acute graft-versus-host disease (aGVHD) remains a significant barrier to successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) because of high morbidity and mortality (1). aGVHD occurs when infused donor-derived T cells recognize recipient antigens as foreign and consequently mediate tissue destruction (2). The current major therapy for aGVHD is immunosuppressive therapy, which often antagonizes T-cell-mediated graft-versus tumor responses and delays immune reconstitution (3, 4). How to prevent aGVHD without intensive immune suppression remains a major challenge for HSCT recipients.

Recruitment of T lymphocytes into end-organs is critical to initiate aGVHD (5). Alloreactive T cells in the graft must first migrate to secondary lymphoid organs (SLOs) where T cells are activated after recognizing alloantigens presented by antigen-presenting cells and then to target tissues and cause tissue injury (6). During this process, the migration of T cells is carefully directed by adhesion molecules and chemokine receptors expressed on these lymphocytes (5).

C-C chemokine receptor 5 (CCR5) and its natural ligands CCL3–5 are critical in the pathogenesis of aGVHD. CCR5 is highly expressed in activated Th1/Tc1 (T helper type 1 / T cytotoxic type 1) cells, natural killer cells, dendritic cells, and macrophages (7). GVHD could be prevented by blocking alloreactive CCR5+ T-cell homing in a mouse model (8). In the clinical setting, Maraviroc (MVC), a CCR5 antagonist, was demonstrated to further protect against GVHD in allo-HSCT recipients when combined with tacrolimus and methotrexate (9). CXCR3 is another chemokine receptor involved in the pathogenesis of aGVHD (10). In animal models, the blockade of CXCR3 or CXCR3 deficiency inhibited the development of GVHD (11). However, previous studies showed that intervention with CCR5 or CXCR3 antagonist could only slightly alleviate the development of aGVHD (12, 13). In fact, CXCR3 blockade in mice receiving CCR5−/− donor cells greatly attenuated rejection and improved survival (14). A recent clinical study reported that a subgroup of patients developed GVHD despite MVC prophylaxis (15). Further analysis revealed that increased expression of CXCR3 ligands is related to noneffective prophylaxis for the development of GVHD, suggesting that CXCR3 signaling may be a resistance mechanism to bypass the CCR5 blockade in GVHD (16).

Given the complexity of the chemokine system with numerous ligands/receptors and redundant functions, the blockade of one chemokine receptor might not be sufficient to prevent the recruitment of effector cells to GVHD target organs during the GVHD process. Therefore, we hypothesize that combined blockade of two or more chemokine receptors may be an efficient way to prevent GVHD development. Given that both CCR5 and CXCR3 are expressed on activated T cells and both are involved in the development of aGVHD, we aimed to investigate whether combined blockade of CCR5 and CXCR3 could have a synergistic effect on the prophylaxis of acute GVHD in this study. The results showed that blockade of both CCR5 and CXCR3 synergistically attenuates murine aGVHD by modulating donor-derived T-cell distribution and function.

Methods

Animals and reagents

Female C57BL/6J (H2b) and male BALB/c (H2d) mice were purchased from Vital River, Charles River China. All mice were bred and housed in a pathogen-free environment at the center animal laboratory of Peking University First Hospital. Mice were used in the experiments at 8–10 weeks of age. MVC (Sigma-Aldrich) is the first CCR5 antagonist, with specific high-affinity binding to CCR5. The IC50 value of MVC was reported in the range of 3.3–7.2 nM by inhibition of CCR5 binding to all three ligands (14). Calcium flux and cAMP levels were inhibited with IC50 at 4–30 nM. Thus, the MVC was administered by intraperitoneal injection (i.p.) once daily at a dose of 10 mg/kg/day, and the dose was enough to block primate CCR5. AMG487 (R&D) is the antagonist of CXCR3, which can inhibit CXCR3-mediated cell migration by the chemokines IP-10, ITAC, and MiG (13). The in vivo formulation of AMG487 was prepared in 20% hydroxypropyl-β-cyclodextrin (Sigma, St. Louis, MO) as described previously and was used to i.p. treat mice once daily at 5 mg/kg.

The drugs, MVC and AMG487 (see reagents above), were administered daily after transplantation from day 0 to 7.

CD3+ T-cell activation in vitro

Splenocytes derived from BALB/c mice were acquired. CD3+ T cells from the splenocytes were used as responder cells using a Mouse CD3+ T-cell isolation kit (BioLegend, San Diego, USA) according to the manufacturer’s instructions. The purity of CD3+ T cell was >90% assessed by flow cytometry. The CD3+ T cells (3 × 105 in 300 ml complete culture medium) were stimulated by the addition of anti-mouse CD3 antibody (2 mg/ml) and CD28 antibody (1 mg/ml). Meanwhile, vehicle [0.01% dimethyl sulfoxide (DMSO)], 0.1 µM AMG487, 1 µM AMG487, 10 µM AMG487, 1 µM MVC, 10 µM MVC, and 100 µM MVC were added into the culture medium. We found AMG487 at 10 µM showed cytotoxic effects. Here, we examined AMG487 at 0.1 and 1 µM with different concentrations of MVC at 1, 10, and 100 µM. After another 48-h treatment, T-cell differentiation was examined by flow cytometry.

Establishment of the murine GVHD model and intervention with chemokine receptor blockers

Experiments were carried out under protocols approved by the Institutional Animal Ethics Committee. Bone marrow cells (BMCs) were collected from C57BL/6J or BALB/c mice followed by red blood cell lysis. Splenic mononuclear cells (SPMNCs) were isolated by Ficoll gradient centrifugation. BALB/c recipients received 750 cGy of total body irradiation (TBI; 60Coγ source) on day 0 followed by infusion of 107 BMCs and 107 SPMNCs from either syngeneic (BALB/c) or allogeneic (C57BL/6J) wild-type donors. Controls were given BMCs alone. The drugs, MVC and AMG487 (see reagents above), were administered daily after transplantation from day 0 to 7. Groups in this experiment are shown in Table 1. There were 10 mice in each group. Survival was monitored daily. The recipient’s body weight was recorded every 3 days and the GVHD clinical score was measured weekly. The degree of aGVHD was assessed by a scoring system incorporating five clinical parameters: weight loss, activity, skin integrity, fur ruffling, and posture as published previously (17).

Table 1. Transplantation groups in this experiment.

Groups	Graft/mouse	Drug treatment	
BM group	107 BMC	-	
GVHD group	107 BMC + 107 SPa	-	
MVC group	107 BMC + 107 SP	MVC (10 mg/kg/day)	
AMG487 group	107 BMC + 107 SP	AMG487 (5 mg/kg/day)	
MVC + AMG487 group	107 BMC + 107 SP	MVC + AMG487	
aSP, SPMNCs.

Histologic analysis

Representative samples of liver, colon, lung, and SLOs were taken from recipients for pathologic analysis 30 days after transplantation. Tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, cut into 5 µm thick sections, and stained with H&E. Histopathology of GVHD was graded using a semiquantitative scoring system according to Kaplan et al. (17). All slides for GVHD analysis were coded and read in a blinded fashion by one experienced staff member.

Immunohistochemistry

Infiltrating CD4+ and CD8+ T cells were examined in the tissues of recipient mice at 7 days post-transplantation. Briefly, the tissues were embedded in paraffin, and were dewaxed, rehydrated, and treated with 0.5% hydrogen peroxide solution in methanol. 1% BSA was used to block after antigen retrieval. Then, antibodies of CD4 (Novus) and CD8 (Abcam) were incubated overnight followed by incubation with the appropriate secondary antibody.

Donor SPMNCs labeled with carboxyfluorescein diacetate succinimidyl ester in vitro

In carboxyfluorescein diacetate succinimidyl ester (CSFE) studies, donor SPMNC were stained with CFSE using the Vybrant Cell Tracker Kit (Invitrogen) prior to injection into recipient mice. CFSE was reconstituted with DMSO to a final concentration of 5 µM. Cells were resuspended in phosphate-buffered saline (PBS) + 5% foetal calf serum (FCS) (Gibco) at 10 × 106/ml and were incubated with CFSE for 5 min at room temperature and protected from light. Five times the original staining volume of culture medium (containing at least 5% FCS) was then added to the cells. Cells were resuspended in a medium with a final concentration of 1 × 108 cells/ml prior to injection into irradiated bone marrow transplantation (BMT) recipients.

In vivo distribution of transplanted donor-derived T cells

To detect the in vivo distribution of donor T cells, SPMNCs were labeled with CFSE as described above and intravenously injected into irradiated BMT recipients. Seven days later, samples of SLOs, including mesenteric lymph nodes, and spleen, from the recipients were collected for cryosections. The samples were cut into 5 µm thick sections and fixed in cold acetone for 10 min. Samples were then washed for 10 min in PBS three times. Slides examined by fluorescence microscopy were counterstained with 4ʹ,6ʹ-diamidino-2-phenylindole (DAPI).

Flow cytometry

Single-cell suspensions were collected from the spleens of each group for fluorescence-activated cell sorting (FACS) analysis. All antibodies used for FACS were obtained from BioLegend, including anti-mouse H2Kb-APC, CD3-APC/Cy7, CD4-FITC, CD8-PerCP/Cy5.5, IFN-γ-PE, IL4-PE, IL17A-PE/Cy7, CD4-PE, CD25-FITC, Foxp3-APC, and CD69-PE. For surface receptor staining, approximately one million cells were incubated with the appropriate antibody mixture for 30 min at 4°C. For intracellular cytokine analysis, leukocytes isolated from spleen cells and livers were stimulated with Cell Stimulation Cocktail plus protein transport inhibitors (500×, eBioscience) which was directly added to the culture medium at 1 × (2 µl/ml) for 4 h. After surface staining, cells were fixed and permeabilized using BD IntraSure Kit according to the instructions. Treg cells were determined using a FOXP3 Fix/Perm Buffer Set (BioLegend) according to the manufacturer’s instructions. All samples were acquired using a FACS Canto II equipped with Cell-Quest software (BD Pharmingen) and were analyzed using FlowJo software.

Cytokine enzyme-linked immunosorbent assay

Antibodies were purchased from eBioscience Systems and assays were performed according to the manufacturer’s protocol. The capture and detection mAbs were diluted at 1:1000 in the coating buffer. The sensitivity of the Interferon-gamma (IFN-γ) and Interleukin-10 (IL-10) enzyme-linked immunosorbent assay was <15 and <32 pg/ml, respectively. Plates were read at 450 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA). Samples and standards were duplicated.

Statistical analysis

The survival curve was analyzed using the Kaplan–Meier method. Differences between groups in survival studies were determined using log-rank statistics, while a Student's t-test was applied for the other comparisons. Results were considered significant if P < .05.

Results

The dynamic patterns of CCR5 and CXCR3 expression on T cells varied following allo-HSCT

Both CCR5 and CXCR3 are expressed on activated T-cell subsets and contribute to the recruitment of these cells to sites of inflammation in GVHD (5). However, the dynamic changes of CCR5 and CXCR3 expression are unclear during the early phase following allo-HSCT. As depicted in Fig. 1A, donor CD3+ T cells began to express CCR5 on day 1 after transplantation, reached the peak on day 4 (36.7 ± 5.6%), and declined to baseline on day 7 (3.1 ± 0.7%). In syngeneic transplantation, the expression of CCR5 in T cells was only slightly increased on day 4 and then declined gradually to the baseline by day 21. However, CXCR3 showed a different pattern of expression (Fig. 1B). The CXCR3+ T cells were about 15% before transplantation. The expression of CXCR3 on T cells increased and the proportion of CXCR3+ T cells was higher than CCR5+ T cells after both syngeneic and allotransplantation. The dynamic analysis showed that CXCR3 expression reached the peak on day 7 (53.3 ± 5.6%) and sustained the high expression level for at least 21 days after allo-HSCT. The cell subset analysis showed that CCR5 was upregulated on both CD4+ and CD8+ T cells, while CXCR3 positivity after transplantation was slightly higher on CD8+ T cells compared with CD4+ T cells (as depicted in Fig. 1C–F). These results suggest that the expression of both CCR5 and CXCR3 is enhanced during the GVHD process, with CCR5 expression only in the early phase while the expression of CXCR3 lasts longer after transplantation.

Figure 1. Expression of CCR5 and CXCR3 on T cells increased with different patterns in the spleen after allogeneic HSCT. Lethally irradiated BALB/c mice received transplantation of spleen cells from either syngeneic BALB/c or allogeneic B6 donors as described in the Methods section. The results show CCR5 (A, C, and E) and CXCR3 (B, D, and F) expression on T cells and their subsets at different time points after HSCT and compared with syngeneic controls. The values are from three animals per group and are expressed as mean ± SD.

Blockade of CCR5 and CXCR3 further reduced the severity of aGVHD

Our previous studies demonstrated that blockade of either CCR5 or CXCR3 combined with CsA could slightly improve survival (12, 13). In this study, we tested whether concurrent blockade of CXCR3 and CCR5 could further alleviate GVHD. The murine GVHD model was established with lethally irradiated BALB/c recipients given 107 C57B6L BM cells plus 107 spleen cells infusion. GVHD scores were determined by analyzing weight, activity, skin, fur ruffling, and posture. The result showed that transplantation of allogeneic BM and SCs induced severe GVHD with continuously decreasing body weight (Fig. 2B), high GVHD scores (Fig. 2C), and a lethality of 90% (Fig. 2A, red line). Blockage of either CCR5 (brown, MVC) or CXCR3 (green, AMG487) caused no significant reduction in GVHD scores and survival (Fig. 2A, P = .366 and .790, respectively). However, combined CCR5 and CXCR3 blockade (magenta, MVC + AMG487) could improve the survival rate by 30% (P = .0384, Fig. 2A). Consistent with the severity of aGVHD, the histopathologic analysis showed that more severe GVHD target tissue damage was observed in the GVHD group. Intervention with either CCR5 or CXCR3 antagonist only slightly mitigates the damage to these tissues. However, the double blockade of CCR5 and CXCR3 remarkably alleviates the histological appearance of GVHD targets (Fig. 2D).

Figure 2. Blockage of both CXCR3 and CCR5 further alleviated GVHD in a murine transplant model. Lethally irradiated BALB/c recipient mice were reconstituted with bone marrow (BM) from B6 mice without or with B6-derived spleen cells and then intervened with CXCR3 and/or CCR5 antagonists i.p. to explore the protective effect on the development of aGVHD. Survival (A), body weight (B), and GVHD scores (C) were determined in each experiment group. Paraffin sections of the liver, lung, and intestine of 4 animals/treatment groups were analyzed for histologic signs of GVHD 30 days after transplantation (D). Bars represent means ± SD. *P < .05; **P < .01; ***P < .001.

Blockade of CXCR3 and CCR5 decreased T lymphocyte infiltration in GVHD target organs

Next, we examined the infiltration of T cells and their subsets in the intestine and liver, two important target organs of GVHD. On day 7 post grafting, the numbers of CD4+ and CD8+ cells were quantified by immunohistochemistry. Compared with the GVHD group, the numbers of CD4+ and CD8+ cells only slightly decreased in the CCR5 or CXCR3 blockade group. However, there was significantly decreased CD4+ and CD8+ lymphocyte infiltration in the hepatic portal area and in the lamina propria in the combined blockade group (Fig. 3, P < .05).

Figure 3. The infiltration of CD4+ or CD8+ T cells in GVHD target organs decreased after the blockade of both CCR5 and CXCR3. The samples of the liver and intestine were obtained on day 7 after transplantation and then were stained with antibodies against CD4 or CD8. The number of CD4+ or CD8+ T cells in the liver (A) and intestine (B) was quantified as described in the Methods section. The results are shown as one representative histological section and calculated for means ± SD from three samples in each group. Bar = 50 µm. *P < .05; **P < .01; ***P < .001.

Blockade of CXCR3 and CCR5 increased the retention of T lymphocytes in SLOs

Because donor T cells in grafts accumulate in SLOs within the first hours after intravenous infusion (5), we supposed that the reduction of donor T-cell infiltration in GVHD target organs might be secondary to increased accumulation of T cells in SLOs after CCR5 and CXCR3 blockade. Thus, we analyzed the absolute number of donor-derived T cells in the spleen where T cells are retained. As shown in Fig. 4A, the average weight of the spleens in the combined CCR5 and CXCR3 blockade group was significantly heavier than that in other groups (P < .01). The number of H2Kb+CD3+ T cells in the spleen also increased in the CCR5 and CXCR3 blockade group compared with the other groups (Fig. 4B). These results suggested that more donor-derived T cells were retained in the spleen. The fluorescent tracer technique showed that the localization of CFSE-labeled donor-derived SPMNCs was also increased in the spleen and inguinal lymphoid nodes (iLN) at day 7 in the combined blockade group compared with other groups (Fig. 4C). These observations were also confirmed by the immunohistochemistry assay for quantification of CD4+ and CD8+ T cells localized in the spleen and iLN (Fig. 4D and E). These results indicated that donor-derived T cells were retained in SLOs after CCR5 and CXCR3 blockade.

Figure 4. Blockade of CCR5 and CXCR3 increased the retention of donor-derived T cells in SLOs. The samples of spleen and iLN were obtained on day 7 after transplantation. The weight of the spleen (A) and the absolute number of H2kb+CD3+ T cells in the spleens (B) in each group were analyzed. Donor SPMNCs were labeled with CFSE and intravenously injected into recipient mice. The presence of CFSE+ cells in the spleen (up) and iLN (down) after 7 days was examined by cryosection examination (CFSE green; DAPI blue) (C, D). Immunohistochemistry assay for the quantitation of CD4+ and CD8+ T cells in the spleen and the iLN (E) was performed as described in the Methods section. Bar = 50 µm. Bars represent means ± SD, n = 3. *P < .05; **P < .01; ***P < .001.

Antagonists of CCR5 and CXCR3 do not show significant cytotoxic effects on donor-derived T cells

In order to rule out the possibility of T-cell toxicities that may be responsible for the prophylactic effect on the development of aGVHD, we further examined the effect of CCR5 and CXCR3 antagonists on T-cell apoptosis and proliferation. First, we analyzed annexin V+ donor-derived T cells on day 7 after transplantation. The results showed that, compared with the GVHD group, the apoptotic T cells were comparable among each group (Fig. 5A). This demonstrated that the prophylactic effect of CCR5 and/or CXCR3 antagonists on aGVHD is not related to cytotoxic effects on donor T cells. To evaluate the proliferation of donor-derived T-cells in vivo, the CFSE-labeled donor T cells were transplanted after TBI conditioning, and CCR5 and/or CXCR3 antagonists were given i.p. as described in the Methods section. The results showed that CCR5 and/or CXCR3 antagonists had no significant effect on the proliferation of donor-derived T cells (Fig. 5B).

Figure 5. Antagonists for CCR5 and CXCR3 do not show significant cytotoxic effects on donor T cells. Recipient mice were euthanized on day 7 after HSCT, and spleens were harvested. (A) Annexin V+ spleen-derived T cells on day 7 after HSCT were analyzed using flow cytometry. (B) The proliferative effect on transplanted T cells labeled with CFSE was analyzed using the CFSE diluted method. The flow cytometric graphs are one of the representatives of three mice. The values are from three animals per group and expressed as mean ± SD. *P < .05; **P < .01; ****P < .0001.

Blockade of CCR5 and CXCR3 affected T-cell functions in the spleen

To further explore potential mechanisms for combined blockade of CCR5 and CXCR3 on T cells in SLOs, we investigated the activation and differentiation of H2Kb+ donor T cells at 7 days after transplantation. Our results showed that the ratio of activated donor-derived T cells (H2Kb+CD3+CD69+) in the spleen did not change in the MVC-treated group, and only slightly decreased in the AMG487-treated group compared with that of the GVHD group. However, it was noted that the activation of donor-derived T cells was significantly inhibited in the MVC and AMG487 combination group (Fig. 6A). The donor-derived T cells can further differentiate into effector T cells after activation. Among the four groups of mice, the proportions of both Th1 and Tc1 cells were the highest in mice in the GVHD group while only slightly decreased in the groups receiving single-drug treatment of MVC or AMG487. However, the proportions of Th1 and Tc1 cells significantly decreased in the combined CCR5 and CXCR3 blockade group (Fig. 6B). T-cell differentiation toward Treg cells was also obvious in recipients for both CCR5 and CXCR3 blockade (Fig. 6C). Consistent with the inhibition of T-cell differentiation toward Th1/Tc1 and promotion of the production of Treg cells of donor-derived T cells after treatment of recipient mice with CCR5 and CXCR3 antagonists, IFN-γ levels in sera in recipient mice treated with CCR5 and CXCR3 antagonists decreased dramatically in comparison with recipients of other groups. Also, the concentration of IL-10, a cytokine with anti-inflammatory properties, was elevated in the sera of recipient mice treated with CCR5 and CXCR3 blockade (Fig. 6D). In addition, we examined the effect of MVC and AMG487 combination treatment on T-cell differentiation of Th1 and Tc1 cells in vitro. As shown in Supplementary Fig. 1, the combined treatment of MVC and AMG487 showed no obvious effects on T-cell differentiation of Th1 and Tc1 cells in vitro. These results demonstrate that blockade of both CCR5 and CXCR3 can alter the polarization of T cells and this may influence the immunological effect after allo-HSCT.

Figure 6. Blockade of CCR5 and CXCR3 modulated donor-derived T cell function in the spleen. The samples of spleens were obtained on day 7 after transplantation. The activation and differentiation of donor-derived T cells were detected using flow cytometry. Mice were euthanized, and the early activation marker CD69 expression was detected on the donor T cells in the spleens of the recipient mice (A). Intracellular cytokines of donor T cells were detected by FACS analysis at day 7 after transfusion. The frequencies of Tc1 and Th1 were shown, respectively (B). The frequencies of Treg in each group were shown (C). The sera were collected on day 7 after transplantation, and the concentrations of IFN-γ and IL-10 were detected and shown in each group (D). Bars represent means ± SD. *P < .05; **P < .01; ***P < .001.

Discussion

The chemokine system is very complex with numerous ligands/receptors and redundant functions. More data suggested that CCR5 is an inflammatory chemokine receptor and is involved in the pathogenesis of aGVHD (16, 18). In animal experiments, anti-CCR5 antibodies could prevent GVHD by blocking alloreactive CCR5+CD8+ T-cell homing to target organs (8, 19), and certain human CCR5 polymorphisms affected the risk of GVHD development, relapse, and overall survival following allo-HSCT (20–22). As for CXCR3, it is also involved in the pathogenesis of GVHD (10, 11). The infusion of donor-derived T cells from CXCR3−/− mice caused reduced liver damage (10) and in vivo administration of anti-CXCR3 antibody could prevent murine GVHD by inhibiting alloreactive T-cell function (23). These results suggest that the blockade of lymphocyte migration using antagonists is a feasible strategy to improve the outcomes. However, our previous studies demonstrated that the blockade of CCR5 or CXCR3 individually could only slightly improve survival in the allo-HSCT mouse model (12, 13). The major explanation might be that one chemokine receptor-mediated lymphocyte migration mechanism might be circumvented by the function of other chemokine receptors after the blockade, and eventually, GVHD is induced. In this preview study, we found that CCR5 and CXCR3 were co-expressed on T cells with different patterns early after allo-HSCT, supporting the hypothesis that single blockade of one chemokine receptor may not be sufficient to alleviate the development of GVHD, and combined blockade of CCR5 and CXCR3 may have a synergistic effect on GVHD prevention.

In this study, we found the CXCR3 expression on CD8+ T cells was obviously upregulated even after syngeneic transplantation. We suggested a potential explanation for this finding. Firstly, conditioning, even in the syngeneic group, could cause the upregulation of inflammatory mediators such as TNF-α and IFN-γ, which are well-known proinflammatory cytokines, and it had been demonstrated that IFN-γ signaling mediates increased surface expression of CXCR3 on Th1 cells and Tc1 cells (24). Additionally, it was reported the IFN-inducible CXCR3 ligands, CXCL10 and CXCL11 were increased under irradiated conditions compared with nonirradiated recipients during the first week post-transplant, while expression of CXCR3 ligands in the spleens of irradiated recipients declined rapidly during the first week post-transplant (25). The expression trend of CXCR3 ligands is consistent with the expression of CXCR3 in our study, which may suggest that CXCR3 ligands may participate in the regulation of CXCR3.

Studies showed that T cells infiltrate into lymphoid tissues within hours after transplantation (5). As shown in this study, expression of CXCR3 maintains a certain level in T cells, especially in CD4+ T cells. The expression of both CCR5 and CXCR3 increased with different patterns in the early period of transplantation. Meanwhile, the expression of CXCL9, CXCL10, and CXCL11 which are the main ligands for CXCR3 increase first and the expression of ligands for CCR5 including CCL3, CCL4, and CCL5 in lymphoid tissues later at early days after transplantation, suggesting that T cells in the graft may accumulate in lymphoid tissues in such an environment with a high concentration of chemokines by the effect of chemotaxis (4). After activation and amplification by alloantigens presented by recipient antigen-presenting cells in lymphoid tissues, alloreactive T cells may emigrate to GVHD target tissues where they may cause the development of GVHD. When the interaction of chemokine with its cognate receptor is blocked, the donor T cells may not respond to the proinflammatory chemokines in lymphoid tissues and decrease the ability of the emigration of these cells to other tissues after allo-HSCT. Therefore, it can be explained that the blockade of both CXCR3 and CCR5, two main GVHD-related chemokine receptors, can retain donor-derived T cells within SLOs, and decrease the trafficking of alloreactive T cells to the sites of GVHD initiation. This is consistent with the previous findings that the absence of CCR5 on donor T cells and combined blockade of CXCR3 reduced alloantigen-specific T lymphocyte proliferation and effector cytokine production in a cardiac allograft model (14).

Many studies showed that SLOs are also immune tolerance organs in many situations (26). Fibroblastic reticular cells (FRCs) which are the main stromal cells located in the cortical and medullar regions of lymphoid organs, may be responsible for this immune regulatory effect (27). FRC-produced cytokines including CCL19 and CCL21, and homeostatic cytokines such as IL-7 are important for T-cell priming (28, 29). FRCs induce immune suppression via the expression of self-antigens and suppressive factors, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), or indirectly restrict T-cell expansion by lowering the immunogenicity of DCs (30, 31). Additionally, we found that combined treatment of MVC and AMG487 showed no obviously direct effects on T-cell differentiation of Th1 and Tc1 cells in vitro, which helped to support our hypothesis that it was not the direct effect of drug inhibitors, but the SLO with its specialized structure and complex cellular interactions altering T-cell polarization after combined blockade of CCR5 and CXCR3. When T cells are detained in SLOs by blocking the interaction between chemokines and their cognate receptors, they may increase contact with the immune regulatory microenvironment in SLOs. This may inhibit the activation and proliferation of donor-derived T cells by FRCs and their related microenvironment. The results in this study also showed that the detained T cells in SLOs possess low potential for activation and proliferation, and low ability for the differentiation toward Th1/Tc1 cells.

The expression of specific chemokine receptors, such as CCR4, CCR8, or CXCR3 on Treg cells supported their accumulation within inflamed environments (32). In our text, we demonstrated that more Treg cells were detected in SLOs after the blockade of both CCR5 and CXCR3, compared with the single blockade. This may suggest that the dynamic expression of multiple chemokine receptors was involved in Treg cell accumulation, and thus the examination of specific chemokine receptors in isolation was incomplete. Because of the complex microarchitecture and heterogeneous populations of hematopoietic and non-hematopoietic cells in SLOs, the upregulation of Treg cells may be the overall impact. After blocking both CCR5 and CXCR3, T cells in the spleen tended to be polarized into Treg cells. Treg cells, which are specialized suppressive cells, often showed suppressive function via secretion of inhibitory cytokines (IL-10 and Transforming growth factor β (TGF-β)) (33). Other colleagues in our research group found that functional inhibition of splenic dendritic cells (DCs) was induced by blockade of CCR5 and CXCR3, and infusion of the DCs induced foxp3, IL-10, and TGF-β expression at the RNA level (unpublished data). It helped to support our hypothesis that it is the SLO with its specialized structure and complex cellular interactions that made Treg cell induction after the combined blockade of CCR5 and CXCR3. These results demonstrated that the blockade of CCR5 and CXCR3 not only affected the distribution of T cells but also affected the function of these cells at early stages after transplantation.

There were some limitations in the study. (i) The survival curves were not remarkable, we analyzed the potential explanation for this finding as follows. Firstly, it may be related to higher lethally irradiated doses and greater severity of GVHD. We planned to make some improvements to the establishment of a murine GVHD model in the follow-up experiments so that, although GVHD occurs, it is not so serious. Additionally, the reason may be that short-term AMG487 and MVC could not effectively inhibit the migration of T cells to target organs, when donor T cells continued to express CXCR3 and CCR5 following stimulation by antigens, therefore, we aimed to explore whether long-term AMG487 and MVC would ameliorate aGVHD more efficiently in future studies. (ii) We did not show direct evidence of CD4+CD25+ cells as a source of IL-10, which may be addressed in future studies.

In conclusion, our study showed that combined CXCR3 and CCR5 blockade was more effective than individual blockade in preventing the development of GVHD in a murine model. This may provide evidence for the prophylaxis of GVHD using the combination of different chemokine receptor antagonists. Further elucidation of the mechanisms for combined chemokine receptor antagonists may pave the way for clinical studies using this strategy.

Supplementary data

Supplementary data are available at International Immunology Online.

dxae033_suppl_Supplementary_Material

Author contributions

H.R. conceived and designed the study. W.L. participated in the research design and reviewed the manuscript. B.T. designed the study and performed the most experiments of this study and wrote the paper. C.Q. and H.L. helped to establish aGVHD mouse models and performed part of the flow cytometric analysis. S.M., Z.W., and Y.Z. performed part of the flow cytometric analysis. C.X. helped to perform most experiments during the major revision. Y.D. participated in the research design. All authors contributed to the article and approved the submitted version.

Conflict of interest statement. The authors claim that the study was performed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This study was supported by the National Natural Science Foundation of China (81970160, 81800181, 81570160, and 81100351).
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