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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: Biol Blood Marrow Transplant. 2010 Mar 23;16(9):1222–1230. doi: 10.1016/j.bbmt.2010.03.015

Recipient B Cells Are Not Required For GVHD Induction

Catherine Matte-Martone 1,2, Xiajian Wang 8, Britt Anderson 4, Dhanpat Jain 5, Anthony J Demetris 7, Jennifer McNiff 6, Mark J Shlomchik 3,4,*, Warren D Shlomchik 1,2,3,*
PMCID: PMC3135976  NIHMSID: NIHMS190904  PMID: 20338255

Abstract

Recipient antigen presenting cells (APCs) are required for CD8-mediated GVHD and have an important and nonredundant role in CD4-mediated GVHD in mouse MHC-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 due to prior Rituximab. We therefore studied the role of recipient B cells in MHC-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.

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 skin GVHD (2). In MHC-mismatched GVHD, recipient APCs have also been shown to be pivotal and their depletion by alloreactive NK cells diminishes GVHD (3, 4).

Recipient dendritic cells, 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 dendritic cells 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 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 (6). 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 recipent B cells.

B cells are a particularly intriguing target as Rituximab, a humanized monoclonal antibody against human CD20 used to treat CD20+ lymphomas, profoundly depletes non-malignant 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), though 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 chronic GVHD, 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 (15-18) as well as antigen taken up via the B cell receptor. B cells can directly activate CD4 cells, which would be a prerequisite for promoting CD4-mediated GVHD (19-22). B cells can also stimulate CD8 cells in vitro (23-25) and their absence can diminish CD8 responses in vivo (26). On the other hand, some CD8 responses are B cell-independent (26-28) and B cells can even tolerize CD8 cells (29-31).

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 histo-pathologic 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 (32-34). 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.

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 (greater than 7 generations backcrossed to BALB/c), homozygous for the absence of all four 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. Lymph nodes were crushed through metal screens and red cells were lysed using ACK (0.15M NH4CL, 1mM KHCO3 and 0.1mM 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, Auburn, CA USA) magnetic cell separator. CD8 cells were >90% pure with CD4 T cell contamination of less than 2%. Bone marrow (BM) was flushed from tibias and femurs, followed by 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.

Bone marrow transplantation

All transplants were performed according to protocols approved by the Yale University Institutional Animal Care and Use Committee. B6 and B6 muMT hosts received 1000cGy 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 850-900cGy and were reconstituted with 107 unfractionated spleen cells and 107 TCD BM cells from B10.D2 mice.

B cell depletion

B6 recipients were injected i.p. with 200ug of anti-mouse CD20 (an IgG2aκ derivative of clone 18B12 (37)) or a control IgG2aκ derivative of clone 2B8, the parent anti-human CD20 monoclonal 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 necro-inflammatory activity were semi-quantitatively 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 three 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 two-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). The significance of differences in histologic score were determined by the Mann-Whitney test (one-tailed for comparisons between recipients of BM alone and CD8 cells; two-tailed for comparisons between groups that received CD8 cells).

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 three independent experiments, muMT recipients of donor BM and CD8 cells developed less weight loss than did wt control recipients (Figure 1). Nonetheless, in two of three experiments, muMT CD8 recipients developed significantly more weight loss than did recipients of only donor BM, indicative of clinical GVHD.

Figure 1. muMT recipients of donor CD8 cells have less weight loss than do wild-type recipients of CD8 cells.

Figure 1

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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 purifed C3H.SW CD8+ T cells (13-15 mice/group). Data are shown from 3 independent experiments.† indicates days in which P<0.05, 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<0.05) than did the muMT CD8-recipient group.

Histologic GVHD was clearly evident in muMT CD8-recipients. Liver GVHD developed in all three repetitions and when histopathology scores from the three 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 3/3 and 2/3 experiments, respectively. When histopathology scores from the three 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=0.55). In contrast, muMT and wt CD8-recipients developed colon GVHD in 1/3 and 2/3 repetitions, respectively. When colon scores from the three repetitions were analyzed together, pathology was more severe in wt than in muMT CD8-recipients (P=0.03). Significant clinical or histologic GVHD of non-ear skin was not observed in any experiment (not shown).

Figure 2. muMT recipients of donor CD8 cells develop histologic GVHD.

Figure 2

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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<0.0001 comparing liver scores in wt or muMT CD8-recipients compared to the respective BM alone controls. P<0.05 comparing ear scores in wt or muMT CD8-recipients compared to the respective BM alone controls. P=0.02 and P=0.12 comparing colon scores in wt and muMT CD8-recipients (respectively) to the appropriate BM alone control. P=0.08, P=0.55 and P=0.34 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 due to developmental differences rather than only the absence of B cells at the time of transplantation (32-34).

To distinguish these possibilities, we used a monoclonal antibody 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 200ug of anti-CD20 or an isotype-matched control antibody that recognizes human and not mouse CD20. Two weeks post injection, 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 1000cGy. 24 hours post irradiation, B cells in spleen and LN were enumerated. The addition of anti-CD20 prior to irradiation induced a greater than 30-fold further reduction of splenic and LN B cells than did pre-treatment with the control antibody (Figure 3A). Of note, without anti-CD20 treatment the number of splenic B cells 24 hours post irradiation is greater than the number of dendritic cells (mean of 2.7×105 dendritic cells/spleen in irradiated recipients of isotype-control antibody).

Figure 3. Similar GVHD develops in anti-CD20 and control treated CD8-recipients.

Figure 3

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A. On day -14 mice received 200ug anti-mouse CD20 or control. Some of these mice were irradiated on day 0 (IR) and all were sacrificed on day +1 post irradiation (which was day +16 after antibody treatment) to quantitate spleen and lymph node (LN) B cells. 3 mice per group were analyzed; however sufficient LN cells for analysis could only be obtained in 2/3 ant-CD20 treated mice and 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<0.04 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 one of two experiments in which significant clinical skin disease developed. P<0.05 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 two experiments. P=0.41 comparing colon or liver pathology in isotype to that in anti-CD20 CD8-recipients. P<0.031 comparing colon GVHD in isotype or anti-CD20-treated CD8-recipients to the appropriate bone marrow only control. P<0.0007 comparing liver GVHD in isotype or anti-CD20-treated CD8-recipients versus the bone marrow only control. Skin pathology is shown for the one (of two total) experiments with clinical skin GVHD. P=0.22 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 due to the absence of host B cells during priming, but rather due to 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 chronic GVHD. 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, 4E), 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, though there were modest differences in the penetrance of skin disease and the severity of weight loss between the two 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.

Figure 4. B cell deficient JhD BALB/c recipients of donor B10.D2 spleen cells develop GVHD.

Figure 4

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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 <0.03); weight change, C (* P <0.03). Experiment 2 data are in D, E and F: incidence of clinical skin disease, D (*, P<0.05); clinical skin score, E (*, P <0.05 at day 26 only); weight change, F (* P <0.006 at all time points beginning on day +17).

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 two 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. While there was some inter-experiment 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 (as compared with recipients of only donor BM) was statistically significant in 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 two of three 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 time of transplant or a consequence of a developmental defect in secondary LNs due to the life-long absence of B cells (32-34), 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 monoclonal antibody (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 a consequence of a developmental difference rather than the absence of recipient B cells that 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, though B cell depletion can last 3-4 weeks. It is therefore possible that there was sufficient residual anti-CD20 to transiently deplete donor-derived B cells. We point out that donor marrow-derived B cells do not begin to appear until 10-14 days post transplantation (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 as 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 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 reported that muMT mice developed more severe GVHD in the fully MHC-mismatched BALB/c→B6 strain pairing (48). 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-/- bone marrow chimeric hosts in which most B cells were IL-10-/-, though 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 dependent, 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 (19-22). Therefore it would not have been surprising to observe less GVHD in B cell deficient hosts. A small retrospective study reported a trend towards reduced acute GVHD in alloSCT patients who received Rituximab as part of an ablative conditioning regimen for treatment of ALL, as 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 chronic GVHD (50). In human studies, Ritz and colleagues found a correlation of donor-derived antibodies against an antigen encoded by the Y chromosome gene DBY in male recipients of female grafts with chronic GVHD (11, 51, 52). Because these antibodies were IgH class-switched, the B cells which 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 chronic GVHD wherein they may further activate CD4 cells which 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.

Supplementary Material

01

Acknowledgments

We thank the Yale Animal Resources Center for expert animal care. This work was supported by NIH HL-083072, HL-066279-08 and P01 AI064343. W.D.S is a recipient of a Clinical Scholar award from the Leukemia and Lymphoma Society.

Footnotes

Authorship 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. Xiajian Wang performed experiments and analyzed data. C.M., X.W. and B.A. contributed equally.

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