Inflammation causes resistance to anti-CD20 mediated B cell depletion

Abstract

B cells play a central role in antibody-mediated rejection and certain auto-immune diseases. However, B-cell-targeted therapy such as anti-CD20 B cell-depleting antibody(aCD20) has yielded mixed results in improving outcomes. In this study, we investigated whether an accelerated B-cell reconstitution leading to aCD20 depletion-resistance could account for these discrepancies. Using a transplantation model, we found that antigen-independent inflammation, likely through TLR signals, was sufficient to mitigate B cell depletion. Secondary lymphoid organs had a quicker recovery of B cells when compared to peripheral blood. Inflammation altered the pharmacokinetics(PK) and pharmacodynamics(PD) of aCD20 therapy by shortening drug half-life and accelerating the reconstitution of the peripheral B-cell pool by bone marrow-derived B cell precursors. IVIG co-administration also shortened aCD20 drug half-life and led to accelerated B cell recovery. Repeated aCD20 dosing restored B cell depletion and delayed allograft rejection, especially B cell-dependent, antibody-independent allograft rejection. These data demonstrate the importance of further clinical studies of the PK/PD of monoclonal antibody treatment in inflammatory conditions and highlight the disconnect between B cell depletion on peripheral blood and on secondary lymphoid organs, the deleterious effect of IVIG when given with aCD20, and the relevance of re-dosing of aCD20 for effective B cell depletion in alloimmunity.

Introduction

B cells are central to the development of antibody-mediated rejection and there is increasing evidence that they also play a major role in chronic allograft loss, both through antibody-dependent (1) and antibody-independent mechanisms (2). Thus it is surprising that a number of clinical trials using rituximab, a chimeric anti-CD20 monoclonal antibody that depletes B cells, have failed to show efficacy in the treatment of antibody-mediated rejection (AMR) (3) or as induction therapy, where one study was halted due to increase episodes of rejection (4). In contrast, other studies have suggested its efficacy in AMR (5), and in induction therapy for highly-sensitized recipients (6) and trends towards fewer and milder rejections and less de novo donor-specific antibody (DSA) formation (7).

The reasons for the failure to show benefit in some trials are not entirely clear, but are important to understand in considering whether to move forward with the use of B cell-targeting therapies. Amongst the hypotheses that have been entertained include the inability of rituximab to deplete memory B cells and plasma cells (which are CD20 negative) and the potential depletion of a regulatory B cell subset that protects against graft rejection (8, 9).

While these mechanisms likely contribute, we propose the additional possibility that current regimens may not be dosing rituximab optimally. Incomplete or non-sustained B cell depletion has been reported in aCD20-treated malignancies, autoimmunity and transplantation (4, 7, 10, 11) and has been associated with poor therapeutic outcome (4, 7, 10-13). Here, we demonstrate that inflammation mitigates B cell depletion by altering the pharmacokinetic and pharmacodynamics of anti-CD20 mAb therapy leading to accelerated reconstitution of the B cell pool. A single dose of anti-CD20 mAb at the time of transplant fails to maintain B cell depletion or prolong allograft survival, but repeated dosing restores B cell depletion in secondary lymphoid organs and delays graft rejection. Thus insufficient dosing of rituximab may contribute to the lack of efficacy seen in some clinical trials.

Materials and Methods

Mice

C57BL/6 (B6; H-2b) and BALB/c (H-2d) were purchased from the Jackson Laboratory. BALB/c.IgMi mice (IgMi; H-2d), which contain B cells but no secreted antibody, have been previously described (14). In experiments where differentiation between donor and recipient B cells was required, congenic B6 CD45.1 and BALB/c CD45.2 were used to easily identify their origin. All animals were bred and maintained under specific pathogen-free conditions. The Institutional Animal Care and Use Committee at Oregon Health & Science University approved animal care and usage.

In vivo treatments

aCD20 antibody clone 5D2 (murine IgG2a, Genentech), 200 mcg (10 mg/kg) in PBS, was given intravenously (IV). This dose is similar to human rituximab dosing. Isotype control murine IgG2a and IgG2b were purchased from BioXcell. Unless specifically indicated, aCD20 was administered the day prior to surgery or treatment with immune stimulus. LPS (List, #201, 5 mcg) and CpG (Invivogen, ODN1846, 40 mcg) were given intraperitoneally (IP). These doses are < 10% of the reported LD50 for these agents. To inhibit the alloreactive T cell response, we used cyclosporine in BALB/c transplant recipients. Cyclosporine was dosed to achieve blood levels similar to what is used in patients (cyclosporine: 200 - 300 ng/ml, 600 mcg/day, subcutaneously).

Surgeries

Heterotopic, abdominal cardiac transplants were performed with the donor ascending aorta and pulmonary vein anastomosed to the recipient infra-renal aorta and vena cava, respectively (15). Transplants were examined daily for rejection by palpation and at euthanasia by direct visualization. All syngeneic transplants, regardless of antibody treatment, were beating and had no evidence of rejection upon gross and microscopic examination. The ischemia-reperfusion injury (IRI) surgery mimicked the cardiac transplant procedure with 30 minutes of clamp time on the infra-renal aorta and vena cava to cause lower limb ischemia.

Flow Cytometry

Reagents were purchased from Biolegend, BD, eBioscience, or Invitrogen except for 5D2 and peptide MHC class I-monomers (pMHC class I-monomers, provided by the NIH Tetramer Core Facility, H-2K(b)/SSIEFARL and H-2K(d)/SYIGSINNI). pMHC-monomers and 5D2 were fluorochrome-conjugated using protein labeling kits per the manufacture's instructions (Invitrogen). For pMHC-monomer and intracellular-IgG⁺ staining, cell surface binding of these reagents was blocked with purified IgG1 and Fc-block (included with cell surface staining). Cells were then fixed with BD Fix/Perm solution. Intracellular staining for pMHC-monomers and anti-IgG subclass antibodies (FITC-conjugated IgG 1, A85-1, IgG2a, R19-15, IgG2b R12-3, IgG3 R40-82, all from BD) was performed in 0.25% saponin, 5% rat serum, in PBS at RT for 1 hr. Sample data were recorded on a LSRII (BD). Counts of BM cells were adjusted for total BM population by multiplication by 14.3 (16). Analyses were performed using FlowJo software (TreeStar). Gating for all flow cytometry plots was for lymphocytes off a forward/side scatter gate and doublet discrimination. Additional gating is indicated at the top of individual plots.

Measurement of serum protein and antibody concentrations

For measurement of BAFF (R&D Systems), and rat IgG2b concentrations (BD), we used commercially prepared kits per manufacture's instructions. For multiplex cytokine measurement, we used a kit from Life Technologies. For measurement of alloantibody and aCD20 concentrations, we developed cellular ELISAs modeled after the protocol developed by Fan (17). The major difference between the cellular ELISAs for alloantibody detection and measurement of aCD20 was the target cells. For the alloantibody protocol, donor splenocytes were the target, and for the aCD20 protocol, syngeneic CD19⁺ cells (purified by positive selection) were the target. Cells were incubated for one hour at 4°C in diluted sera, followed by one hour 4°C with monoclonal goat anti-mouse IgG-HRP for the alloantibody ELISA or anti-mouse IgG2a for the aCD20 ELISA. TMB was used as the substrate. Optical density was measured at 450nm. Known concentrations of purified monoclonal anti-H-2Kd/anti-H-2Ld (BD) antibodies and aCD20 were used for a standard curve for alloantibody and aCD20 concentrations, respectively.

Statistics

Prism5 (GraphPad) was used for all statistical studies. ANOVA with Bonferroni's multiple comparison post-test was used for multiple statistical comparisons on a single data set. An unpaired t test was used for comparison of two independent groups. Log-rank test was used to analyze statistical significance of differences in survival. Determination of statistical significant difference was based on 95% confidence intervals, and error bars indicate standard error of the mean throughout.

Results

Transplantation mitigates the efficacy of aCD20

Despite the ability of aCD20 to effectively deplete naïve B cells, which are the precursors to allospecific antibody-secreting cells (ASCs), prior studies have shown that B cell depletion may not correlate with suppression of alloantibody expression post-transplant even in unsensitized mouse recipients (18, 19). To investigate this further, we examined B cell depletion, reconstitution, differentiation, and antibody production after aCD20 treatment in transplantation. We first confirmed that administration of the aCD20 (clone 5D2) in naïve mice results in rapid and profound depletion of blood, splenic, and bone marrow (BM) B cell pools (Fig. S1). These B cell compartments recovered in parallel 8 – 10 weeks after a single dose of aCD20, similar to other murine aCD20 B cell depleting antibodies (18, 20).

To then test its efficacy in transplantation, we mimicked the design of the largest randomized trial of aCD20 induction therapy in transplantation by treating mice with aCD20 or control IgG one day prior to vascularized allogeneic transplantation (BALB/c hearts into C57BL/6, B6, mice) (7). B6 recipients of BALB/c allografts had an equivalent mean survival time (MST) regardless of treatment with aCD20 (control IgG-treated: 8.4 ± 0.7 days; aCD20-treated: 9.1 ± 1.1 days, p =0.7, n =10 in both groups). At various time points following transplantation, recipients were sacrificed and B cell populations in the blood, spleen, and bone marrow were counted. Two weeks post-transplant, aCD20-treated mice showed significant reductions in their circulating CD19⁺ B cell populations compared with IgG-treated recipients, and their numbers were similar in mice that received no transplant, allogeneic transplant, or syngeneic transplant (Fig. 1A, P > 0.05). In contrast, examination of the splenic compartment showed marked differences in the efficacy of aCD20-mediated depletion, depending on whether mice also received a transplant. In aCD20-treated mice that did not receive a transplant, near-complete depletion of splenic naïve B cells (CD19⁺IgD⁺) was seen for >4 weeks (Fig S1), while in transplant recipients aCD20 treatment led to near-complete depletion at 1 week, but full recovery to pre-depletion levels was seen by three weeks (Fig. 1B). Using CD45.2 (exclusively on donor BALB/c cells) and B cell markers, we observed that this accelerated reconstitution of the naïve B cell pool was not due to transfer of “passenger B cells” from the donor organ (data not shown). While alloantibody levels were initially suppressed by aCD20 treatment, these quickly rebounded to levels seen in recipients who did not receive aCD20 treatment (Fig. 1C), correlating with the recovery of the splenic B cell pool. The observation that B cell recovery is accelerated regardless of whether mice received an allogeneic or syngeneic graft suggests that antigen-independent immune activation from ischemia-reperfusion injury (IRI) is sufficient to cause aCD20 depletion-resistance. To test this, we evaluated the effect of B cell depletion under different inflammatory conditions, including administration of lipopolysaccharide (LPS, TLR2/4), unmethylated DNA (CpG, TLR9 ligand) or lower limb IRI. All these conditions led to accelerated B cell reconstitution (Fig. 1D), confirming that inflammation mitigates the efficacy of aCD20. These data also show that while aCD20-mediated depletion of lymphoid tissue B cells correlates well with circulating B cell numbers in naïve mice, this correlation is lost in transplant recipients. Since monitoring of peripheral B cells has been used to guide re-dosing of aCD20 in humans, the disconnection between peripheral blood and secondary lymphoid organ B cell depletion is clinically relevant.

Fig. 1. Inflammation mitigates the efficacy aCD20 therapy.

B6 (H-2b) mice received aCD20 or control IgG on day -1, and allogeneic heart transplants (hearts from BALB/c mice; H-2d) or syngeneic heart transplants (hearts from B6 mice) were performed on day 0. A, The total number of peripheral blood CD19⁺ cells was determined 2 weeks later between groups. No difference in aCD20-treated naïve, allo or syngeneic transplants were observed (P >0.05). B, Total number of splenic naïve B cell counts, CD19⁺IgD⁺ by flow cytometry, after aCD20- or control IgG treatment with or without syngeneic or allogeneic heart transplantation (* P <0.05, ** P <0.0001). C, There was no difference in serum concentration of alloantibody in allograft recipients that received aCD20- or control IgG treatment two weeks after transplantation (P = 0.75 by unpaired t test). D, Antigen-independent inflammation is sufficient to inhibit the efficacy of aCD20-mediated depletion of naïve B cells. B6 mice were treated with aCD20 or control IgG on day -1 and then they received PBS or inflammatory stimuli the next day. The number of splenic naïve B cells (CD19⁺IgD⁺) from these mice was measured one and four weeks later. There were ≥ 4 mice per group at each time-point. Unless indicated, ANOVA with Bonferroni's post-test comparing aCD20-treated groups was used for statistically assessment. LOD: limit of detection.

Inflammation alters the pharmacokinetics (PK) of aCD20-mediated B cell depletion

We compared the aCD20 serum concentration among mice that receive the drug and no inflammatory stimulus and mice that were treated with aCD20 followed by transplantation or a TLR ligand (Fig. 2). Stimuli that caused accelerated B cell repopulation, such as transplantation or LPS administration, led to lower serum aCD20 concentrations one week after treatment. We calculated the effect of transplantation and LPS administration on the half-life and drug exposure (AUC) of aCD20 and found that inflammation lowers both measures by 30%. Therefore, these studies indicate that the PK of aCD20 is altered by immune activation and that antigen-independent immune activation is sufficient to cause this effect.

Fig. 2. Inflammation causes aCD20-resistance by altering the PK of the drug.

Serum aCD20 concentration was measured at 3 days, 1 and 2 weeks after mice were treated with aCD20 or control IgG (day -1) under inflammatory stimuli, transplantation or PBS (day 0). aCD20 half-life and exposure (AUC) measures are listed on the plot. There were ≥3 mice in every group at each time-point. Significance was measured vs. aCD20/PBS. ** P < 0.01 by ANOVA with Bonferroni's post-test. LOD: limit of detection.

Inflammation alters the pharmacodynamics (PD) of aCD20-mediated B cell depletion

The lower aCD20 half-life observed in mice treated with inflammatory stimuli could be caused by enhanced aCD20 consumption through target-specific (binding to CD20) mechanisms. Because inflammation accelerates the egress of immature B cells from the bone marrow (21), we hypothesized that an increase in peripheral CD20-expressing B cell precursors could lead to enhanced consumption of aCD20 and subsequent reduction of aCD20 levels. We tested this hypothesis by counting the number of immature B cells in the spleen after aCD20 treatment and inflammatory challenge. One week after these interventions and prior to an appreciable recovery of mature naïve B cells, splenic T1 transitional B cell counts (CD19⁺CD20⁺CD23^negCD93⁺IgD^negIgM⁺) were higher under conditions that resulted in aCD20 depletion-resistance (transplantation and LPS) compared to controls (PBS) (Fig. 3A). Therefore, the inflammation-driven increase in aCD20-targets is one mechanism for the accelerated recovery of B cells. Since BAFF (B cell activating factor) is associated with B cell development and maintenance beyond the T1 stage (22) and has been shown to contribute to B cell repopulation following Rituximab (23), we measured serum BAFF levels. While BAFF levels did not correlate with B cell depletion resistance after aCD20 in LPS or syngeneic transplant groups (Fig. 3B), it was significantly elevated in allogeneic transplantation, and thus may be an additional factor contributing to depletion-resistance seen in alloimmunity.

Fig. 3. Splenic transitional B cell counts, not serum BAFF concentration, predict accelerated B cell reconstitution after aCD20 treatment.

Mice were treated with aCD20 (day -1) and given LPS or a cardiac transplant (day 0). One week later, splenic T1 transitional B cells (CD19⁺CD20⁺CD23^negCD93⁺IgD^negIgM⁺) were counted (A), and serum BAFF concentrations were measured (B). Significance was measured among the aCD20-treated groups. * P < 0.05, by ANOVA with Bonferroni's post-test.

Co-administration of IVIG and aCD20 lowers aCD20 concentration and causes depletion-resistance

IVIG is often co-administered with aCD20 (Rituximab) for the prevention and treatment of antibody-mediated rejection (24). Among its mechanisms of action, IVIG increases IgG catabolism (25). This mechanism of action of IVIG would be expected to decrease the serum concentration of Rituximab, an IgG1 antibody, and may further reduce the efficacy of aCD20 depletion in transplant recipients. To test this hypothesis, we measured serum concentrations of aCD20 in mouse recipients of a transplant and found that those who received aCD20 and IVIG concurrently had a lower concentration of aCD20 one week later (Fig. 4A). The combined therapy led to accelerated recovery of naïve B cells (Fig. 4B). The effect of IVIG on aCD20 PK/PD is independent of an inflammatory response, as multiplex cytokine analysis of serum from recipients showed no difference between mice treated with IVIG alone, aCD20 alone, or in combination (data not shown). Therefore, these experiments provide clinically relevant evidence that co-administration of aCD20 with IVIG can alter aCD20 PK and further cause accelerated recovery of the B cell population.

Figure 4. IVIG treatment lowers aCD20 concentration and accelerates B cell recovery.

Mice were treated with control IgG, aCD20/PBS, or aCD20/IVIG and one week later aCD20 concentration was measured (A). One and three weeks after these treatments, splenic naïve B cells (CD19⁺IgD⁺) were counted (B). Each symbol represents one mouse. * P =0.007, ** P <0.0001 comparing aCD20-treated groups by unpaired t test. LOD: limit of detection.

Repeated dosing of aCD20 overcomes naïve B cell depletion-resistance and prolongs allograft survival

Prior studies using B cell depleting antibody to prevent acute graft rejection have generally used only 1-2 doses of the drug (8, 18). However, based on the accelerated recovery of B cells seen with a single dose of aCD20, we presumed we would have to give frequent doses of aCD20 to maintain drug exposure in the setting of the persistent alloimmune response. In addition, we knew we would have to inhibit the alloreactive T cell response because T cells are sufficient to reject allografts (26). We chose to inhibit T cells with a standard clinical immunosuppressant, cyclosporine A (CSA), dosed to levels used in patients. Because cyclosporine alone can cause allograft tolerance in B6 allograft recipients(27), we used BALB/c mice as transplant recipients in these experiments.

As with B6 mice, one dose of aCD20 depleted B cells in otherwise untreated BALB/c mice (data not shown) and a single dose of aCD20 alone failed to prolong B6 graft survival in BALB/c recipients (MST 9 days on both groups, p=0.51). In combination with CSA, single-dose aCD20 failed to extend the survival of allografts transplanted into CSA-treated BALB/c recipients (P = 0.80). However, repeated dosing of aCD20 (every 3 days beginning at day -4 prior to transplant) did prolong graft survival over CSA treatment alone (P = 0.002, Fig. 5A), and this corresponded with sustained B cell depletion (Fig. 5B) and reduced alloantibody formation (Fig. 5C). Four out of 6 recipients in the single dose had detectable antibody compared with 2 out of 8 recipients who received repetitive aCD20. Although alloantibody levels were lower in aCD20-treated recipients, splenic allospecific IgG⁺ ASCs were similar between single and repeated aCD20 dosing (Fig. S2).

Figure 5. Repeated dosing of aCD20 improves B cell depletion and prolongs allograft survival.

A, Graft survival after single or repeated dosing of aCD20 in BALB/c recipients of B6 cardiac allografts. Mice treated with repeated doses of aCD20 received 200 mcg on experiment days -4, -1, 3, 7, and then weekly for the life of the allograft. CSA treatment was initiated on day 0 and continued daily for the life of the graft. Naïve B cells on spleens (B) and alloantibody concentration (C) were determined on recipients 2 weeks after an allogeneic heart transplant. D, Graft survival in IgMi BALB/c recipients of B6 cardiac allografts in combination with CSA and repeated dosing of aCD20 as above. Significance was measured by log-rank test comparing single versus multiple aCD20 dosing (* p<0.05; ** P <0.005).

B cells have now been implicated in chronic allograft vasculopathy independent of their ability to affect antibody (8). To test whether B cell depletion protects against graft rejection in the absence of allo-antibody formation, we used IgMi mice, BALB/c mice with B cells but no secreted antibody, as recipients of cardiac allografts. Untreated IgMi allograft recipients rejected their transplants at a tempo similar to untreated BALB/c mice (P = 0.096, Fig. 5D). CSA treatment significantly prolonged allograft survival in IgMi mice compared to untreated IgMi graft recipients (P = 0.004) and compared to CSA-treated BALB/c mice (P = 0.023). Survival of allografts in IgMi mice treated with CSA/repeated-aCD20 was longer than graft survival in BALB/c treated with CSA/repeated-aCD20 (P = 0.021) (Fig. 5D). These data show that repeated dosing of aCD20 can prolong graft survival in mice treated with immunosuppression currently used in the clinic. They also confirm that B cells also promote rejection through antibody-independent mechanisms, and extend these prior findings to show that B cell depletion can protect against these mechanisms.

Discussion

Rituximab is a chimeric mouse/human anti-CD20 monoclonal antibody of the IgG1 subtype whose binding to CD20 on B cells leads to their cell death. The dosing strategy currently used in clinical practice in transplantation is largely based upon the initial studies using rituximab as induction therapy in the treatment of non-Hodgkin's lymphoma. In these PK/PD studies, a single dose of 375mg/m2 given intravenously showed a long half-life of ∼21 days (28). However, in patients with SLE, the pharmacokinetics of rituximab are highly variable (28) and its half-life in patients with renal disease is relatively short at ∼12 days (29, 30). Notably, the extent of B cell depletion in target tissues (eg. synovium in rheumatoid arthritis; (31)) and in peripheral blood (32, 33) can also vary significantly between individuals receiving a standard dose. In rheumatoid arthritis (RA), standard dosing consists of 1000 mg intravenously for two doses given two weeks apart, which correlates to ∼525 mg/m² in men and ∼625 mg/m² in women per dose (assuming average body surface areas of 1.9 m2 and 1.6 m2 respectively). Several trials using a 500 mg dose have shown the lower dose may also be effective in RA, but clinical responsiveness is dose-dependent and correlates with the degree of B cell depletion (33), and incomplete B cell depletion is associated with worse outcome (32). Furthermore, in mouse models of autoimmunity, mouse strains that are more prone to SLE show greater resistance to anti-CD20-mediated depletion in part because of greater degrees of inflammation (34-36). In transplant, most current protocols use a single dose of 375 mg/m² for induction therapy and two doses of 375 mg/m² given two weeks apart for treatment of AMR. In a recent prospective multicenter randomized placebo-controlled double-blinded trial, Sautenet et al. tested the benefits of adding rituximab (375 mg/m2) on day 5 of AMR treatment (plasmapheresis with IVIG) in comparison to placebo. The primary composite endpoint of graft loss or no improvement in renal function at day 12 was similar between groups (3). To our knowledge, in-depth PK/PD studies have not been done in transplant patients. Given the variability in half-life seen in various clinical diseases and our data demonstrating that the robust alloimmune response and IVIG reduce serum concentrations of anti-CD20 mAb and mitigate its B cell-depleting effects, we suggest that optimal dosing in transplantation remains unclear.

Furthermore, many clinical studies have used monitoring of B cell populations in the peripheral blood as a surrogate marker for rituximab's efficacy. However, models of autoimmune disease have questioned the validity of circulating B cell numbers as an accurate biomarker, particularly under inflammatory conditions (34, 36, 37). Additionally, the re-emergence of B cell populations first appear within secondary lymphoid organs, and the rapidity by which this occurs appears to also be context and disease-specific (38, 39). In our study, we did observe a rapid resurgence of T1 transitional B cells as an early indicator of B cell depletion-resistance. The few human studies that have been done examining rituximab's effect on B cell populations following transplantation have primarily relied upon monitoring of peripheral B cell population with rituximab given prior to transplant and assessment of lymph nodes only at time of transplant, before inflammation has occurred (6, 29, 40). In a small study assessing grafts undergoing chronic rejection (n=2), rituximab was effective in depleting peripheral B cells but not at depleting B cells on ectopic lymphoid structures in the rejecting allografts (41). BAFF expression on the allografts was increased both at genetic and protein level, suggesting that this survival factor could in part contribute to the B cell accelerated repopulation. Our data confirms that peripheral blood B cell depletion does not reflect tissue B cell depletion by anti-CD20 in transplant recipients. We further extend those findings to show that inflammatory stimuli lowers serum anti-CD20 concentration and causes early re-emergence of B cells, whereas maintenance of drug exposure by repeated dosing is capable of delaying their re-population and promoting graft survival. BAFF has been suggested to modulate B cell repopulation in kidney transplantation (42). While BAFF was increased in the periphery in the allo-transplants in our study, it was not increased in syngeneic transplant nor LPS-treated mice, indicating that it is unlikely to be the primary mechanism to explain accelerated B cell recovery in inflammation, but may be an additional contributing factor in reducing the efficacy of aCD20 in the alloimmune response.

Since peripheral blood monitoring overestimates the extent of depletion in tissues, monitoring of anti-CD20 concentrations may be a better functional marker for anti-CD20-mediated B cell depletion. By extension, studies claiming failure of rituximab to alter the clinical course of disease despite “complete” peripheral B cell depletion must be re-evaluated with this in mind. Unfortunately, it is not currently feasible to routinely and non-invasively determine the extent of cellular depletion in secondary lymphoid tissue of treated patients. Further studies will be needed to evaluate whether serum anti-CD20 concentrations also correlate with tissue depletion in human transplant recipients, and thus be used as a biomarker of depletion efficacy in future studies. In cancer, the clinical response to rituximab has been correlated with serum trough concentrations (43) and utilized to create individualized dosing schedules (44).

Rituximab triggers B cell lysis through antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity or apoptosis induction (45). Macrophages are the predominant cells involved in Fc-dependent B cell depletion (46, 47). Thus, apart from insufficient dosing, reduced efficacy of antibody-mediated depletion could result from defects in Fc-receptor dependent and/or complement-dependent clearance mechanisms (35, 48-50). Furthermore, Beers et al. have shown that CD20 internalization upon rituximab binding to B cells may shorten the kinetics of aCD20 Ab, which could be overcome by either redosing or by using a different aCD20 Ab (tositumomab) that is associated with reduced CD20/mAb internalization and degradation (51).

Defective anti-CD20 mediated depletion has been shown in lupus and viral infection models (35, 50). However, these models required logarithmically greater aCD20 dosing to achieve initial depletion. This is different from our studies that mimic dosing levels used in patients and responses in transplant recipients. We observe efficient B cell depletion at early time-points and that repeated doses of aCD20 are able to prevent accelerated B cell reconstitution, making it less likely that our findings are caused by Fc-receptor dysfunction. Supporting this hypothesis, Wieland et al. have shown that chronic infection per se in mice does not lead to a defect in FcγR-mediated antibody effector functions, though immune complex generated against the virus may compromise Fc-receptor dependent function (50). Therefore, we propose that rather than an inherent failure to deplete B cells, it is the quick repopulation of B cells driven by inflammation that shortens the efficacy of rituximab in alloimmunity. In accordance with our findings, Fairchild's groups has recently shown that repetitive dosing of anti-huCD20 mAb also promotes long-term graft survival and reduced DSA titers in B6.huCD20/CCR5^-/- mice recipients of A/J renal allografts (52).

The amount of IgG present in the circulation has been shown to significantly affect B cell depletion in a dose-dependent manner (35), with excess IgGs being capable of dramatically inhibiting effector mechanisms that rely on low or intermediate affinity FcγRs (53, 54) because of direct competition between free plasma IgGs and rituximab. Thus, macrophage and neutrophil IgG-dependent phagocytosis and activation are mitigated when high IgG levels are present (53, 55). In accordance, we showed co-administration of IVIG mitigated the ability of aCD20 to achieve complete B cell depletion. Additionally, IVIG can also increase IgG catabolism (25), which could theoretically lower anti-CD20 serum concentrations. Indeed, we found that mice that received aCD20 and IVIG concurrently had a lower concentration of anti-CD20 mAb one week later and early accelerated recovery of naive B cells. This supports the need to reevaluate dosing strategies in protocols for the treatment of AMR or in desensitization protocols that involve the concomitant use of IVIG and Rituximab.

Finally, our data do not eliminate the possibility that inflammation could accelerate the non-specific elimination of the exogenous antibody. Once IgG is in the circulation, it is distributed to the body tissues through convection and diffusion. Thereafter, IgG may interact with Fc receptors. FcgRI, which is readily upregulated in inflammatory conditions (48, 49), binds free IgG with high affinity and leads to its uptake and proteolytic degradation, thereby limiting the amount of mAb available to bind to its target (CD20) on B cells (56). On the other hand, interactions with the non-specific neonatal FcRn receptor result in its stability and increased half-life in vivo(57, 58). Binding of IgG to FcRn protects the antibody from lysosomal degradation following cellular uptake, and allows for recycling of IgG. Alterations in either FcgRI or FcRn expression/function can therefore affect non-specific elimination of antibody. We were unable to test this directly because of an inability to detect exogenously supplied murine antibody in vivo. However, experiments using a rat isotype antibody hint that inflammation may also increase non-specific antibody turnover (Fig. S3). Regardless, monitoring of serum drug concentrations would also account for the variability in non-specific mAb elimination between individuals, and provide better assessment of rituximab bioavailability in patients.

Given the importance of B cells in the pathogenesis of AMR and emerging evidence implicating its involvement in chronic allograft vasculopathy, targeting B cells remains of clinical interest. However, much remains to be learned about the mechanisms involved in these effects and the optimal way to target and administer therapy. It is still not clear how best to utilize rituximab to either prevent or treat rejection, how long to maintain B cell depletion, and the dose required to achieve complete tissue depletion. Furthermore, modifying the Fc portion may allow improvement of the half-life of rituximab in inflammatory conditions (59). Our present studies identify a complicating factor that may contribute to the null findings in some clinical studies, but does not rule out the possibility that rituximab may be ineffective because it does not deplete memory B cells or antibody-producing plasma cells which do not express CD20. Anti-CD20-mediated depletion has also been shown to deplete regulatory B cell populations, and absent or dysfunctional Breg leads to accelerated rejection (8, 9). Thus, indiscriminate B cell depletion may paradoxically enhance immune responsiveness in certain scenarios. However, regardless of the final verdict on rituximab, our findings have implications that are applicable in assessing the efficacy of other humanized monoclonal antibody therapies in transplantation.

Supplementary Material

Supplementary Figures

Fig. S1. aCD20 treatment results in depletion of B cells in spleen, blood and bone marrow of B6 mice. Representative flow cytometry plots and quantification of naïve B cells on spleens (A), peripheral blood (B) and bone marrow (C) according to aCD20 dosing and over time with single dose of aCD20 200 mcg (** P<0.005 by t test).

Fig. S2. Repeated aCD20 dosing does not prevent allospecific ASC differentiation. BALB/c received a B6 cardiac allograft on day 0. Cyclosporine was given daily in combination with single dose of aCD20 on day -1 or with repeated dosing of aCD20 (days -4, -1, 3, 7). Mice were euthanized on day 14. Allospecific ASC (CD138⁺intracellular-IgG⁺H-2Kb were measured by flow cytometry (t test).

Fig. S3. LPS treatment decreases serum concentration of isotype antibody. We were unable to detect exogenous isotype murine IgG control antibody in mice. Therefore, we used an ELISA to measure rat IgG2b isotype antibody one week after dosing (200 mcg IV) followed by treatment with PBS or LPS. LPS-treated mice had a lower concentration of this irrelevant antibody than PBS-treated mice, supporting a role for inflammation-induced non-specific turnover of antibody. * P = 0.03 using t test.

Abbreviations

ADCC

antibody-dependent cellular cytotoxicity

AMR

antibody-mediated rejection

ASC

antibody-secreting cells

BM

bone marrow

DSA

donor-specific antibody

LPS

lipopolysaccharide

MST

mean survival time

PD

pharmacodynamics

PK

pharmacokinetics