Biology of Blood and Marrow Transplantation
Volume 16, Issue 9 , Pages 1222-1230, September 2010

Recipient B Cells Are Not Required for Graft-Versus-Host Disease Induction

  • Catherine Matte-Martone

      Affiliations

    • Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut
    • Department of Medicine, Yale University School of Medicine, New Haven, Connecticut
    • ,
  • Xiajian Wang

      Affiliations

    • Institute of Immunology, Zhejiang University, Hangzhou, People's Republic of China
    • ,
  • Britt Anderson

      Affiliations

    • Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut
    • ,
  • Dhanpat Jain

      Affiliations

    • Department of Pathology, Yale University School of Medicine, New Haven, Connecticut
    • ,
  • Anthony J. Demetris

      Affiliations

    • Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
    • ,
  • Jennifer McNiff

      Affiliations

    • Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut
    • ,
  • Mark J. Shlomchik

      Affiliations

    • Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut
    • Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut
    • ,
  • Warren D. Shlomchik

      Affiliations

    • Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut
    • Department of Medicine, Yale University School of Medicine, New Haven, Connecticut
    • Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut
    • Corresponding Author InformationCorrespondence and reprint requests: Warren D. Shlomchik, MD, Medical Oncology and Immunobiology, Yale University of Medicine, P.O. Box 208032, New Haven, CT 06620.

Received 18 January 2010; accepted 16 March 2010. published online 24 March 2010.

Article Outline

Recipient antigen presenting cells (APCs) are required for CD8-mediated graft-versus-host disease (GVHD), and have an important and nonredundant role in CD4-mediated GVHD in mouse major histocompatibility complex-matched allogeneic bone marrow transplantation (alloBMT). However, the precise roles of specific recipient APCs—dendritic cells, macrophages, and B cells—are not well defined. If recipient B cells are important APCs they could be depleted with rituximab, an anti-CD20 monoclonal antibody. On the other hand, B cells can downregulate T cell responses, and consequently, B cell depletion could exacerbate GVHD. Patients with B cell lymphomas undergo allogeneic hematopoietic stem cell transplantation (alloSCT) and many are B-cell-deficient because of prior rituximab. We therefore studied the role of recipient B cells in major histocompatibility complex-matched murine models of CD8- and CD4-mediated GVHD by using recipients genetically deficient in B cells and with antibody-mediated depletion of host B cells. In both CD4- and CD8-dependent models, B cell-deficient recipients developed clinical and pathologic GVHD. However, although CD8-mediated GVHD was clinically less severe in hosts genetically deficient in B cells, it was unaffected in anti-CD20-treated recipients. These data indicate that recipient B cells are not important initiators of GVHD, and that efforts to prevent GVHD by APC depletion should focus on other APC subsets.

Key Words: GVHD, B cells, Antigen presenting cells, Graft-versus-host disease

 

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Introduction 

Graft-versus-host disease (GVHD) remains a major toxicity that greatly limits the application and efficacy of allogeneic stem cell transplantation (alloSCT). Most patients who undergo alloSCTs receive stem cells from major histocompatibility complex (MHC)-identical or matched donors. In these patients, GVHD is initiated by donor T cells that recognize a subset of host peptides, called minor histocompatibility antigens (miHAs), which are derived from the expression of polymorphic genes that differ in host from donor. We have previously shown that intact recipient-type antigen presenting cells (APCs) are absolutely required for GVHD in an MHC-matched, multiple miHA-mismatched murine model of GVHD induced only by donor CD8+ T cells [1]. In contrast, either recipient or donor type APCs are sufficient for CD4-mediated GVHD across only miHAs, although host APCs are required for a high penetrance of skin GVHD [2]. In MHC-mismatched GVHD, recipient APCs have also been shown to be pivotal, and their depletion by alloreactive natural killer (NK) cells diminishes GVHD 3, 4.

Recipient dendritic cells (DCs), macrophages, and B cells could theoretically be important APCs for donor T cell priming in alloSCT, and ablation of the appropriate APC subsets could ameliorate GVHD. In MHC class I (MHCI) and MHC Class II (MHCII) disparate models of GVHD, add-back of host type B cells to otherwise GVHD-resistant MHCII− or donor→host chimeras did not restore GVHD, whereas splenic DC partially did so [5]. These experiments addressed whether host-type B cells are sufficient to promote GVHD, but did not address their role in a situation where all other APCs are intact. Add-back experiments also rely on the correct trafficking of infused cells, which cannot be assured. Moreover, these experiments did not address the role of B cells in an MHC-matched, multiple miHA disparate GVHD model, akin to the majority of human alloSCTs.

Schulz and colleagues [6] examined the role of recipient and donor B cells in GVHD mediated by a mix of CD4 and CD8 cells by depleting B cells in neonatal mice with anti-mu antibodies. Initial T cell priming was reduced in B cell-depleted recipients; however, GVHD was not significantly different in B cell-replete and B cell-depleted hosts. In these experiments, donor cells were also B cell depleted, and thus potential differences could not specifically be ascribed to recipient B cells.

B cells are a particularly intriguing target as rituximab, a humanized monoclonal antibody (mAb) against human CD20 used to treat CD20+ lymphomas, profoundly depletes nonmalignant B cells [7] and has been efficacious in treating patients with autoimmune diseases [8]. Recent clinical data also suggests that rituximab may be efficacious in treating a subset of patients with chronic GVHD (cGVHD) 9, 10, although in this case rituximab likely targets donor B cells. The presence of class-switched donor-derived antibodies against miHAs has also correlated with the presence of cGVHD, suggesting that alloreactive CD4 cells interact with donor B cells, and it is therefore reasonable that donor T cells also interact with recipient B cells [11]. Also, a growing number of patients with B cell lymphomas now undergo alloSCT, and most of these will have received rituximab during primary therapy, as part of the transplant preparative regimen, or both 12, 13. Therefore, it is a clinically important question to understand the role of recipient B cells in GVHD.

Recipient B cells should be capable of presenting self antigen acquired by pinocytosis [14] or by endogenous presentation of peptides derived from intracellular proteins 15, 16, 17, 18 as well as antigens taken up via the B cell receptor. B cells can directly activate CD4 cells, which would be a prerequisite for promoting CD4-mediated GVHD 19, 20, 21, 22. B cells can also stimulate CD8 cells in vitro 23, 24, 25 and their absence can diminish CD8 responses in vivo [26]. On the other hand, some CD8 responses are B cell-independent 26, 27, 28 and B cells can even tolerize CD8 cells 29, 30, 31.

To determine if recipient B cells either augment or suppress GVHD reactions, we compared GVHD in B cell-replete and B cell-deficient hosts in MHC-matched, multiple miHA-mismatched models of CD8- and CD4-dependent GVHD in which host APCs have essential or nonredundant roles 1, 2. Clinical and pathologic GVHD clearly occurred in hosts that were genetically deficient in mature B cells in both models of GVHD. However, CD8-mediated clinical and histopathologic colon GVHD were less severe in B cell-deficient hosts, suggesting a potential role for host B cells either as APCs or as cells important for establishing an optimal lymphoid environment for donor CD8+ T cell activation. Alternatively, the genetic deficiency of B cells could have adversely affected the development of secondary lymphoid tissues, thereby decreasing the efficiency of donor CD8+ T cell activation 32, 33, 34. To distinguish these, we depleted B cells in wild-type hosts by treatment with an antibody against mouse CD20 prior to transplantation. In these B cell-depleted recipients, both clinical and pathologic GVHD were indistinguishable from that in control antibody-treated recipients.

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Materials and Methods 

Mice 

C3H.SW (H-2b), B10.D2 (H-2d), and B6 muMT mice (H-2b) [35], which have a targeted disruption of the membrane exon of the immunoglobulin mu chain gene and do not have mature B cells, were obtained from Jackson Labs (Bar Harbor, MA). B6 mice (H-2b) were obtained from either the Jackson Labs or from the NCI (Frederick, MD). BALB/c (H-2d) mice were obtained from the NCI. B cell-deficient BALB/c JhD mice (>7 generations backcrossed to BALB/c), homozygous for the absence of all 4 JH gene segments [36], and thus lacking B cells, were bred at Yale University. All mice were between 8 and 10 weeks of age.

Cell Purifications 

CD8 cells were purified via depletion from lymph node (LN) cells. LNs were crushed through metal screens and red cells were lysed using ACK (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA). Cells were washed, and LN suspensions were stained with biotin conjugated antibodies against CD4 (clone GK1.5, lab grown and conjugated), B220 (clone 6B2; lab grown and conjugated), and CD11b (clone M1/70; BD Pharmingen, San Diego, CA). Cells were washed and then stained with streptavidin-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA) and separated on an AutoMACS (Miltenyi Biotec) magnetic cell separator. CD8 cells were >90% pure with CD4 T cell contamination of <2%. Bone marrow (BM) was flushed from tibias and femurs, followed by red blood cell (RBC) lysis with ACK. BM was depleted of T cells with anti-Thy1.2 conjugated magnetic microbeads (Miltenyi Biotec) followed by separation on the AutoMACs or with biotin-conjugated anti-Thy1.2 (clone 30H12), SA beads and the AutoMACs.

BM Transplantation (BMT) 

All transplants were performed according to protocols approved by the Yale University Institutional Animal Care and Use Committee. B6 and B6 muMT hosts received 1000 cGy, and were reconstituted with 7 × 106 C3H.SW T cell-depleted (TCD) BM with or without 2-3 × 106 C3H.SW CD8+ T cells. BALB/c and JhD BALB/c hosts received 850-900 cGy, and were reconstituted with 107 unfractionated spleen cells and 107 TCD BM cells from B10.D2 mice.

B Cell Depletion 

B6 recipients were injected intraperitoneally (i.p.) with 200 μg of antimouse CD20 (an IgG2aκ derivative of clone 18B12 [37]) or a control IgG2aκ derivative of clone 2B8, the parent antihuman CD20 mAb for rituximab (all provided by Marilyn Kehry, Biogen/IDEC, San Diego, CA). In alloBMT experiments, mice were treated 2 weeks prior to irradiation.

Histologic Analysis 

Tissues were fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Slides were numbered and read by pathologists expert in skin and gastrointestinal disease without knowledge as to experimental group. Skin scoring was as described previously 1, 38. Bowel and liver GVHD scoring in experiments performed with muMT and JhD recipients were by D. Jain as previously described 2, 39. Colon and liver GVHD in experiments with anti-CD20 treatment (by A. Demetris) were assessed as follows. In the liver, portal inflammation, inflammatory bile duct damage, central perivenulitis, and lobular necroinflammatory activity were semiquantitatively evaluated on a scale of 0 to 3. Weighted scores were calculated by multiplying the portal inflammation and central perivenulitis scores by a factor of 1. Bile duct injury and lobular necro-inflammatory activity were multiplied by a factor of 0.5. Total scores for each animal were obtained by adding the weighted scores together. Features evaluated for colon GVHD included overall inflammation/cryptitis, average number of apoptotic bodies per 10 crypts in the most severely affected areas, crypt abscesses, crypt loss, and ulceration. Each parameter was scored on a scale of 0 to 3. Weighted scores for each parameter were calculated as follows: inflammation/cryptitis × 0.9; apoptotic bodies per 10 crypts × 0.1; neutrophilic cryptitis/crypt abscess × 0.4; crypt loss × 1 and ulceration × 2. The total score for each animal was derived by adding together each of the weighted scores.

Monitoring of Clinical GVHD 

Mice were weighed approximately every 3 days following BMT. Skin disease was scored in the C3H.SW→B6 [39] and B10.D2→BALB/c [2] models as previously described.

Statistical Analysis 

The significance of differences in weight and clinical disease score were calculated by an unpaired t-test (one-tailed when comparing BM alone versus T cell recipients and 2-tailed when comparing wild type and B cell-deficient T cell recipients). The significance of differences in GVHD incidence were calculated by Log Rank using Prism (GraphPad Software, Inc., San Diego, CA). The significance of differences in histologic score were determined by the Mann-Whitney test (1-tailed for comparisons between recipients of BM alone and CD8 cells; 2-tailed for comparisons between groups that received CD8 cells).

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Results 

Role of B Cells in CD8-Mediated GVHD 

To investigate whether recipient B cells are important APCs we compared GVHD in wild-type (wt) B6 mice to that in B cell-deficient B6 muMT mice [35]. We utilized the same MHC-matched strain pairing, C3H.SW (H-2b)→B6 (H-2b), in which we previously found an essential role for recipient APCs [1]. In each of 3 independent experiments, muMT recipients of donor BM and CD8 cells developed less weight loss than did wt control recipients (Figure 1). Nonetheless, in 2 of 3 experiments, muMT CD8 recipients developed significantly more weight loss than did recipients of only donor BM, indicative of clinical GVHD.

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  • Figure 1 

    muMT recipients of donor CD8 cells have less weight loss than do wild-type recipients of CD8 cells. Wild-type B6 and B6 muMT mice were irradiated and reconstituted with TCD C3H.SW BM (5 mice per group) with or without 2-3 × 106 purified C3H.SW CD8+ T cells (13-15 mice/group). Data are shown from 3 independent experiments. †Indicates days in which P < .05, comparing either B6 or B6 muMT CD8 recipients with the corresponding BM alone group. ∗ Indicates days in which the B6 CD8-recipient group had significantly more weight loss (P < .05) than did the muMT CD8-recipient group.

Histologic GVHD was clearly evident in muMT CD8 recipients. Liver GVHD developed in all 3 repetitions and when histopathology scores from the 3 repetitions were analyzed together, relative to BM alone controls, muMT recipients of CD8 cells had similar liver involvement as did wt CD8 recipients (P = 0.08; histopathology scores, Figure 2). Ear GVHD developed in muMT and wt CD8 recipients in 3 of 3 and 2 of 3 experiments, respectively. When histopathology scores from the 3 experiments were combined, ear GVHD was significant in both muMT and wt CD8 recipients versus BM only controls, yet there was no difference between the muMT and wt recipients of CD8+ T cells (P = .55). In contrast, muMT and wt CD8 recipients developed colon GVHD in 1 of 3 and 2 of 3 repetitions, respectively. When colon scores from the 3 repetitions were analyzed together, pathology was more severe in wt than in muMT CD8-recipients (P = 0.03). Significant clinical or histologic GVHD of nonear skin was not observed in any experiment (not shown).

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  • Figure 2 

    muMT recipients of donor CD8 cells develop histologic GVHD. Shown are the combined pathology scores from 3 independent experiments. Each circle is the score of an individual mouse; horizontal bars are mean values. P < .0001 comparing liver scores in wt or muMT CD8 recipients compared to the respective BM alone controls. P < .05 comparing ear scores in wt or muMT CD8 recipients compared to the respective BM alone controls. P = .02 and P = .12 comparing colon scores in wt and muMT CD8 recipients (respectively) to the appropriate BM alone control. P = .08, P = .55, and P = .34 comparing liver, ear, and colon scores (respectively) in wt CD8 recipients to those in muMT CD8 recipients.

In sum, these data unequivocally demonstrate that recipient B cells are not required for clinical or histologic GVHD in this model. However, clinical GVHD as measured by weight loss and pathologic colon GVHD was less severe in muMT CD8 recipients, which suggested that recipient B cells could play a role in promoting some aspects of CD8-mediated GVHD. B cells could directly prime donor CD8 cells or function as accessory cells that promote the activation and/or expansion of alloreactive CD8 cells. B cells also provide immunoglobulin, which has a variety of immunomodulatory functions. Alternatively, because B cells contribute to the ontogeny of secondary lymphoid tissues, reduced GVHD in muMT hosts could have been because of developmental differences rather than only the absence of B cells at the time of transplantation 32, 33, 34.

To distinguish these possibilities, we used an mAb against mouse CD20 to deplete B cells from wt B6 recipients prior to using these mice in GVHD experiments. We first analyzed the efficacy of anti-CD20 in depleting splenic and LN B cells, as these are the primary sites for T cell priming in GVHD 40, 41. Mice were injected with 200 μg of anti-CD20 or an isotype-matched control antibody that recognizes human and not mouse CD20. Two weeks postinjection, B cell content in spleen and LN were determined. Anti-CD20 resulted in 240- and 50-fold reductions in splenic and LN B cells, respectively (Figure 3A). We next confirmed that prior treatment with anti-CD20 depleted B cells to a greater degree than did irradiation alone. Mice were treated with anti-CD20 or control antibody 2 weeks prior to receiving 1000 cGy. Twenty-four hours postirradiation, B cells in spleen and LN were enumerated. The addition of anti-CD20 prior to irradiation induced a >30-fold further reduction of splenic and LN B cells than did pretreatment with the control antibody (Figure 3A). Of note, without anti-CD20 treatment the number of splenic B cells 24 hours postirradiation is greater than the number of DCs (mean of 2.7 × 105 DCs/spleen in irradiated recipients of isotype-control antibody).

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  • Figure 3 

    Similar GVHD develops in anti-CD20 and control-treated CD8 recipients. (A) On day −14, mice received 200 μg anti-mouse CD20 or control. Some of these mice were irradiated on day 0 (IR) and all were sacrificed on day +1 postirradiation (which was day +16 after antibody treatment) to quantitate spleen and lymph node (LN) B cells. Three mice per group were analyzed; however, sufficient LN cells for analysis could only be obtained in 2/3 anti-CD20-treated mice and in 1/3 anti-CD20 and irradiated mice. Each symbol is data from an individual mouse. Horizontal lines are mean values. (B) Percent weight loss. Data combined from 2 experiments with similar results. P < .04 comparing weight change of anti-CD20 or isotype CD8 recipients with BM alone controls from days +6 and +11 onward, respectively. Weight change of anti-CD20 and control CD8 recipients did not differ at any measurement. (C) Skin disease incidence. Data from the 1 of 2 experiments in which significant clinical skin disease developed. P < .05 comparing anti-CD20 or control-treated CD8 recipients with the respective BM alone control. Incidences of skin disease in anti-CD20 and control IgG-treated CD8-recipients did not significantly differ. (D) Liver and colon pathology scores are shown for data combined from 2 experiments. P = .41 comparing colon or liver pathology in isotype to that in anti-CD20 CD8 recipients. P < .031 comparing colon GVHD in isotype or anti-CD20-treated CD8 recipients to the appropriate bone marrow only control. P < .0007 comparing liver GVHD in isotype or anti-CD20-treated CD8 recipients versus the bone marrow only control. Skin pathology is shown for the 1 (of 2 total) experiments with clinical skin GVHD. P = .22 comparing skin pathology in anti-CD20 versus isotype-treated CD8 recipients.

To determine whether reagent-based B cell depletion affects GVHD, anti-CD20- or isotype control-treated mice were irradiated and reconstituted with T cell-depleted C3H.SW BM, with or without 2 × 106 C3H.SW CD8 cells, and mice were followed for GVHD development. GVHD in anti-CD20 and isotype control-treated CD8 recipients was similar as measured by weight loss (Figure 3B; data combined from 2 experiments) incidence of skin disease (Figure 3C; data from the 1 of 2 experiments in which there was clinical skin disease), and blinded scoring of histopathology of liver, colon, skin, and ear (Figure 3D). As anti-CD20 and control-treated CD8 recipients developed similar GVHD, it is highly likely that the diminished GVHD seen in muMT mice was not because of the absence of host B cells during priming, but rather because of a developmental difference, perhaps abnormalities in secondary lymphoid organogenesis, or as a consequence of differences in gut flora secondary to life-long B cell depletion.

Role of Recipient B Cells in CD4 GVHD 

To analyze the role of recipient B cells in CD4-mediated GVHD we utilized the B10.D2 (H-2d)→BALB/c (H-2d) CD4-dependent model of cGVHD. GVHD in this system is manifested by alopecia, skin fibrosis, salivary, and lacrimal gland involvement, and mild portal inflammation 38, 42. Disease caused by unfractionated spleen cells is equivalent to that with purified CD4 cells, and in our hands CD8 cells do not induce clinical or histologic GVHD [43]. JhD or wild-type BALB/c mice were irradiated and reconstituted with TCD B10.D2 BM with or without 107 B10.D2 splenocytes. In Experiment 1, JhD and wt spleen cell recipients developed a similar incidence of clinical cGVHD (Figure 4A). However, the severity of skin disease in affected JhD recipients was greater than that in wt mice (Figure 4B), as was weight loss (Figure 4C). In Experiment 2, both incidence and skin scores were similar in JhD and wt recipients of spleen cells (Figure 4D and E), although again JhD mice had more weight loss (Figure 4F). Both JhD and wild type recipients of spleen cells developed skin fibrosis and interface dermatitis typical for this model (Supplemental Figure 1). Thus, although there were modest differences in the penetrance of skin disease and the severity of weight loss between the 2 experiments, they both showed that recipient B cells are not required for GVHD in this CD4 T cell-mediated model. If anything, their absence may increase the severity of GVHD.

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  • Figure 4 

    B cell-deficient JhD BALB/c recipients of donor B10.D2 spleen cells develop GVHD. JhD BALB/c or wild-type BALB/c recipients were irradiated and reconstituted with T cell-depleted B10.D2 BM (4 mice/group) without or with 107 B10.D2 spleen cells (10-11 mice per group). Shown are results from 2 independent experiments. All statistical comparisons are between JhD and wild-type recipients of donor spleen cells. Experiment 1 data are in A, B, and C: incidence of clinical skin disease, A; clinical skin score, B (∗P < .03); weight change, C (∗P < .03). Experiment 2 data are in D, E, and F: incidence of clinical skin disease, D (∗P < .05); clinical skin score, E (∗P < .05 at day 26 only); weight change, F (∗P < .006 at all time points beginning on day +17).

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Discussion 

These studies clearly demonstrate that recipient B cells are neither required for nor have a major impact on GVHD induced by CD4+ or CD8+ T cells in 2 models for which we have previously established essential roles for host APCs 1, 2. Nonetheless, depending on the model, the presence of B cells did affect the quality and/or severity of GVHD, albeit to a minor and somewhat variable extent. We observed unequivocal histologic GVHD in genetically B cell-deficient recipients of donor CD8 cells. Although there was some interexperiment variability in the severity of GVHD and pattern of organ involvement, which is characteristic of GVHD in this model (our unpublished observations over many experiments), in individual experiments, histologic GVHD (compared with recipients of only donor BM) was statistically significant in the ear, small bowel, and colon in muMT CD8 recipients, and significant in the liver in all repetitions. When the combined histology scores were analyzed, GVHD in both the ear and liver were similar in both wt and muMT CD8 recipients. Nevertheless, weight loss in muMT CD8 recipients was significantly less than that in wt CD8 recipients in 2 of 3 repetitions as was overall colon GVHD. To determine whether the reduction in clinical and pathologic GVHD was a consequence of the absence of B cells at the time of transplant or a consequence of a developmental defect in secondary LNs because of the life-long absence of B cells 32, 33, 34, or to other consequences of life-long B cell deficiency, for example on commensual flora, we compared GVHD in mice acutely depleted of B cells using an anti-CD20 mAb [37]. In this case, histologic and clinical GVHD was similar in anti-CD20 and isotype control-treated CD8 recipients. Thus, it is likely that the reduction in clinical and histopathologic colon GVHD observed in muMT mice was due to a developmental difference rather than the absence of recipient B cells, which directly promote alloreactive T cell generation. We do not know how a life-long absence of B cells specifically diminished colon GVHD. Perhaps this is a consequence of altered gut flora; alternatively, priming in mesenteric lymphoid tissues could be more affected by an absence of B cells.

Our goal was to assure B cell depletion by anti-CD20. We therefore transplanted mice 2 weeks after antibody injection, although B cell depletion can last 3 to 4 weeks. It is therefore possible that there was sufficient residual anti-CD20 to transiently deplete donor-derived B cells. We point out that donor BM-derived B cells do not begin to appear until 10 to 14 days posttransplantation 44, 45, making it likely that there were few donor B cells exposed to anti-CD20. If anti-CD20 treatment did suppress donor B cells, their additional depletion did not affect GVHD.

We also found that CD4-mediated GVHD was intact in B cell-deficient recipients. Indeed, host B cells may regulate rather than promote GVHD in this model, as JhD recipients of splenocytes had more severe cutaneous GVHD in Experiment 1 and greater weight loss in both experiments compared to wt recipients of spleen cells.

A regulatory role for B cells has been demonstrated in murine models of EAE, inflammatory bowel disease and diabetes. Regulatory B cells have been suggested to suppress inflammation via a number of mechanisms, including the elaboration of IL-10 and TGF-β and by acting as a suppressive second-line APC (reviewed in [46]). More recently, we have found a role for regulatory B cells in the nonobese diabetic (NOD) model of diabetes [47]. Thus, it is possible that the absence of such cells in JhD recipients augmented GVHD. In any case, our data definitively exclude a requirement for recipient B cells in CD4-mediated GVHD pathogenesis.

Hill and colleagues [48] reported that muMT mice developed more severe GVHD in the fully MHC-mismatched BALB/c→B6 strain pairing. They noted that irradiation increased IL-10 mRNA in residual host B cells, and that GVHD was more severe in IL-10−/− hosts and in mixed muMT+IL-10−/− BM chimeric hosts in which most B cells were IL-10−/−, although a fraction of other IL-10-producing cells, including regulatory T cells, may also have been Il-10−/−. It is possible that we observed more GVHD in JhD recipients because of a similar mechanism. However, that the absence of host B cells, by either genetic or reagent-based approaches, did not increase CD8-mediated GVHD in the C3H.SW→B6 strain pairing suggests that host B cell mitigation of GVHD is model-specific, and perhaps depends on whether CD4 cells are pathogenic.

B cells have a well established role in stimulating memory, and under certain circumstances, naïve CD4 cells 19, 20, 21, 22. Therefore, it would not have been surprising to observe less GVHD in B cell-deficient hosts. A small retrospective study reported a trend toward reduced acute GVHD in alloSCT patients who received rituximab as part of an ablative conditioning regimen for treatment of acute lymphoblastic leukemia, compared to similarly transplanted patients who did not receive rituximab [49]. A second retrospective study suggested that treatment with rituximab in the 6 months prior to nonmyeloablative alloSCT reduced the incidence of extensive cGVHD [50]. In human studies, Ritz and colleagues 11, 51, 52 found a correlation of donor-derived antibodies against an antigen encoded by the Y chromosome gene DBY in male recipients of female grafts with cGVHD. Because these antibodies were IgH class-switched, the B cells that expressed these B cell receptors must have had productive encounters with a CD40L+ donor CD4 cell. Consistent with this, a CD4 response against DBY has been found in a patient with antibody against DBY [52]. These studies raise the intriguing possibility that donor B cells might be important APCs for alloreactive donor CD4 cells. If so, this would be an elegant explanation for responses of cGVHD patients to rituximab, despite the minimal role for host B cells in GVHD induction 9, 10. One way of reconciling this result with our studies is that we assessed the importance of recipient B cells in priming naïve CD4 cells at the time of transplant whereas Ritz and colleagues may have revealed a role for B cells in established cGVHD, wherein they may further activate CD4 cells that may have been initially primed by an APC other than a B cell. Also consistent with this, in a mouse model of cGVHD and nephritis induced by transferring DBA/2 splenocytes into sublethally irradiated BALB/c mice, autoantibodies were donor-derived and skin thickening depended on donor B cells [53].

Taken together, our data indicate that recipient B cells are not required for the initiation of either CD8 or CD4-mediated GVHD across only minor H antigens. APC-targeted therapies to prevent GVHD should target other APC subsets.

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Acknowledgments 

The authors thank the Yale Animal Resources Center for expert animal care.

Financial disclosure: This work was supported by NIH HL-083072, HL-066279-08, and P01 AI064343. W.D.S was supported by Clinical Scholar award from the Leukemia and Lymphoma Society.

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Authorship Statement 

W.D.S. designed experiments, analyzed data, and wrote the manuscript. M.J.S. designed experiments, analyzed data, and helped to write the manuscript. B.E.A., C.M., and X.W. designed and performed experiments and analyzed data. D.J. and A.J.D. scored slides for evidence of GVHD involving the bowel and liver. J.M. scored slides for evidence of GVHD involving the skin.

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Supplementary Data 

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Supplemental Figure 1.

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References 

  1. Shlomchik WD, Couzens MS, Tang CB, et al. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science. 1999;285:412–415
  2. Anderson BE, McNiff JM, Jain D, Blazar BR, Shlomchik WD, Shlomchik MJ. Distinct roles for donor- and host-derived antigen-presenting cells and costimulatory molecules in murine chronic graft-versus-host disease: requirements depend on target organ. Blood. 2005;105:2227–2234
  3. Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science. 2002;295:2097–2100
  4. Teshima T, Ordemann R, Reddy P, et al. Acute graft-versus-host disease does not require alloantigen expression on host epithelium. Nat Med. 2002;8:575–581
  5. Duffner UA, Maeda Y, Cooke KR, et al. Host dendritic cells alone are sufficient to initiate acute graft-versus-host disease. J Immunol. 2004;172:7393–7398
  6. Schultz KR, Paquet J, Bader S, HayGlass KT. Requirement for B cells in T cell priming to minor histocompatibility antigens and development of graft-versus-host disease. Bone Marrow Transplant. 1995;16:289–295
  7. Maloney DG. Preclinical and phase I and II trials of rituximab. Semin Oncol. 1999;26:74–78
  8. Edwards JC, Cambridge G. B-cell targeting in rheumatoid arthritis and other autoimmune diseases. Nat Rev Immunol. 2006;6:394–403
  9. Ratanatharathorn V, Ayash L, Reynolds C, et al. Treatment of chronic graft-versus-host disease with anti-CD20 chimeric monoclonal antibody. Biol Blood Marrow Transplant. 2003;9:505–511
  10. Cutler C, Miklos D, Kim HT, et al. Rituximab for steroid-refractory chronic graft-versus-host disease. Blood. 2006;108:756–762
  11. Miklos DB, Kim HT, Miller KH, et al. Antibody responses to H-Y minor histocompatibility antigens correlate with chronic graft-versus-host disease and disease remission. Blood. 2005;105:2973–2978
  12. Khouri IF, Keating M, Korbling M, et al. Transplant-lite: induction of graft-versus-malignancy using fludarabine-based nonablative chemotherapy and allogeneic blood progenitor-cell transplantation as treatment for lymphoid malignancies. J Clin Oncol. 1998;16:2817–2824
  13. Khouri IF, Saliba RM, Giralt SA, et al. Nonablative allogeneic hematopoietic transplantation as adoptive immunotherapy for indolent lymphoma: low incidence of toxicity, acute graft-versus-host disease, and treatment-related mortality. Blood. 2001;98:3595–3599
  14. Zhong G, Reis e Sousa C, Germain RN. Antigen-unspecific B cells and lymphoid dendritic cells both show extensive surface expression of processed antigen-major histocompatibility complex class II complexes after soluble protein exposure in vivo or in vitro. J Exp Med. 1997;186:673–682
  15. Bogen B, Weiss S. Processing and presentation of idiotypes to MHC-restricted T cells. Int Rev Immunol. 1993;10:337–355
  16. Sant AJ. Endogenous antigen presentation by MHC class II molecules. Immunol Res. 1994;13:253–267
  17. Brooks AG, McCluskey J. Class II-restricted presentation of a hen egg lysozyme determinant derived from endogenous antigen sequestered in the cytoplasm or endoplasmic reticulum of the antigen presenting cells. J Immunol. 1993;150:3690–3697
  18. Munthe LA, Kyte JA, Bogen B. Resting small B cells present endogenous immunoglobulin variable-region determinants to idiotope-specific CD4(+) T cells in vivo. Eur J Immunol. 1999;29:4043–4052
  19. Constant S, Schweitzer N, West J, Ranney P, Bottomly K. B lymphocytes can be competent antigen-presenting cells for priming CD4+ T cells to protein antigens in vivo. J Immunol. 1995;155:3734–3741
  20. Croft M, Duncan DD, Swain SL. Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements. J Exp Med. 1992;176:1431–1437
  21. Croft M, Bradley LM, Swain SL. Naive versus memory CD4 T cell response to antigen. Memory cells are less dependent on accessory cell costimulation and can respond to many antigen-presenting cell types including resting B cells. J Immunol. 1994;152:2675–2685
  22. Ron Y, Sprent J. T cell priming in vivo: a major role for B cells in presenting antigen to T cells in lymph nodes. J Immunol. 1987;138:2848–2856
  23. Ke Y, Kapp JA. Exogenous antigens gain access to the major histocompatibility complex class I processing pathway in B cells by receptor-mediated uptake. J Exp Med. 1996;184:1179–1184
  24. Yefenof E, Zehavi-Feferman R, Guy R. Control of primary and secondary antibody responses by cytotoxic T lymphocytes specific for a soluble antigen. Eur J Immunol. 1990;20:1849–1853
  25. Barnaba V, Franco A, Alberti A, Benvenuto R, Balsano F. Selective killing of hepatitis B envelope antigen-specific B cells by class I-restricted, exogenous antigen-specific T lymphocytes. Nature. 1990;345:258–260
  26. Shen H, Whitmire JK, Fan X, Shedlock DJ, Kaech SM, Ahmed R. A specific role for B cells in the generation of CD8 T cell memory by recombinant Listeria monocytogenes. J Immunol. 2003;170:1443–1451
  27. Epstein MM, Di Rosa F, Jankovic D, Sher A, Matzinger P. Successful T cell priming in B cell-deficient mice. J Exp Med. 1995;182:915–922
  28. Asano MS, Ahmed R. CD8 T cell memory in B cell-deficient mice. J Exp Med. 1996;183:2165–2174
  29. Bennett SR, Carbone FR, Toy T, Miller JF, Heath WR. B cells directly tolerize CD8(+) T cells. J Exp Med. 1998;188:1977–1983
  30. Fuchs EJ, Matzinger P. B cells turn off virgin but not memory T cells. Science. 1992;258:1156–1159
  31. Hollsberg P, Batra V, Dressel A, Hafler DA. Induction of anergy in CD8 T cells by B cell presentation of antigen. J Immunol. 1996;157:5269–5276
  32. Fu YX, Huang G, Wang Y, Chaplin DD. B lymphocytes induce the formation of follicular dendritic cell clusters in a lymphotoxin alpha-dependent fashion. J Exp Med. 1998;187:1009–1018
  33. Ngo VN, Korner H, Gunn MD, et al. Lymphotoxin alpha/beta and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen. J Exp Med. 1999;189:403–412
  34. Kabashima K, Banks TA, Ansel KM, Lu TT, Ware CF, Cyster JG. Intrinsic lymphotoxin-beta receptor requirement for homeostasis of lymphoid tissue dendritic cells. Immunity. 2005;22:439–450
  35. Kitamura D, Rajewsky K. Targeted disruption of mu chain membrane exon causes loss of heavy-chain allelic exclusion. Nature. 1992;356:154–156
  36. Chen J, Trounstine M, Alt FW, et al. Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus. Int Immunol. 1993;5:647–656
  37. Ahuja A, Shupe J, Dunn R, Kashgarian M, Kehry MR, Shlomchik MJ. Depletion of B cells in murine lupus: efficacy and resistance. J Immunol. 2007;179:3351–3361
  38. Anderson BE, McNiff J, Yan J, et al. Memory CD4+ T cells do not induce graft-versus-host disease. J Clin Invest. 2003;112:101–108
  39. Matte-Martone C, Liu J, Jain D, McNiff J, Shlomchik WD. CD8+ but not CD4+ T cells require cognate interactions with target tissues to mediate GVHD across only minor H antigens, whereas both CD4+ and CD8+ T cells require direct leukemic contact to mediate GVL. Blood. 2008;111:3884–3892
  40. Beilhack A, Schulz S, Baker J, et al. Prevention of acute graft-versus-host disease by blocking T-cell entry to secondary lymphoid organs. Blood. 2008;111:2919–2928
  41. Anderson BE, Taylor PA, McNiff JM, et al. Effects of donor T-cell trafficking and priming site on graft-versus-host disease induction by naive and memory phenotype CD4 T cells. Blood. 2008;111:5242–5251
  42. Hamilton BL, Bevan MJ, Parkman R. Anti-recipient cytotoxic T lymphocyte precursors are present in the spleens of mice with acute graft versus host disease due to minor histocompatibility antigens. J Immunol. 1981;126:621–625
  43. Kaplan DH, Anderson BE, McNiff JM, Jain D, Shlomchik MJ, Shlomchik WD. Target antigens determine graft-versus-host disease phenotype. J Immunol. 2004;173:5467–5475
  44. Allman DM, Ferguson SE, Cancro MP. Peripheral B cell maturation. I. Immature peripheral B cells in adults are heat-stable antigenhi and exhibit unique signaling characteristics. J Immunol. 1992;149:2533–2540
  45. Allman DM, Ferguson SE, Lentz VM, Cancro MP. Peripheral B cell maturation. II. Heat-stable antigen(hi) splenic B cells are an immature developmental intermediate in the production of long-lived marrow-derived B cells. J Immunol. 1993;151:4431–4444
  46. Mizoguchi A, Bhan AK. A case for regulatory B cells. J Immunol. 2006;176:705–710
  47. Hu CY, Rodriguez-Pinto D, Du W, et al. Treatment with CD20-specific antibody prevents and reverses autoimmune diabetes in mice. J Clin Invest. 2007;117:3857–3867
  48. Rowe V, Banovic T, MacDonald KP, et al. Host B cells produce IL-10 following TBI and attenuate acute GVHD after allogeneic bone marrow transplantation. Blood. 2006;108:2485–2492
  49. Kebriaei P, Saliba RM, Ma C, et al. Allogeneic hematopoietic stem cell transplantation after rituximab-containing myeloablative preparative regimen for acute lymphoblastic leukemia. Bone Marrow Transplant. 2006;38:203–209
  50. van Dorp S, Pietersma F, Wölfl M, et al. Rituximab treatment before reduced-intensity conditioning transplantation associates with a decreased incidence of extensive chronic GVHD. Biol Blood Marrow Transplant. 2009;15:671–678
  51. Bellucci R, Alyea EP, Chiaretti S, et al. Graft-versus-tumor response in patients with multiple myeloma is associated with antibody response to BCMA, a plasma-cell membrane receptor. Blood. 2005;105:3945–3950
  52. Zorn E, Miklos DB, Floyd BH, et al. Minor histocompatibility antigen DBY elicits a coordinated B and T cell response after allogeneic stem cell transplantation. J Exp Med. 2004;199:1133–1142
  53. Zhang C, Todorov I, Zhang Z, et al. Donor CD4+ T and B cells in transplants induce chronic graft-versus-host disease with autoimmune manifestations. Blood. 2006;107:2993–3001

 C.M., X.W., and B.A. contributed equally to this article.

 Financial disclosure: See Acknowledgments on page 1229.

PII: S1083-8791(10)00121-7

doi:10.1016/j.bbmt.2010.03.015

Biology of Blood and Marrow Transplantation
Volume 16, Issue 9 , Pages 1222-1230, September 2010

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