B Cells and Transplantation: An Educational Resource

Trudy N. Small,1 William H. Robinson,2 David B. Miklos2

INTRODUCTION neic HCT as they contribute to (1) vaccine-induced antimicrobial immunity, (2) autoimmune responses, Allogeneic hematopoietic cell transplantation (HCT) and (3) allogeneic antibody responses. We will discuss offers a unique opportunity to monitor the develop- a B cell role in chronic graft-versus-host diease ment of immune reconstitution using the patient as (cGVHD) pathogenesis, review anti-B cell cGVHD his or her own control. Furthermore, the donor therapy using rituximab, and, finally, consider the serves as an HLA-identical normal control and pathogenic role of agonistic antibodies targeting plate- source of adoptive immune transfer. Donor hemato- let-derived growth factor receptor (PDGFR). poietic stem cells (HSCs) differentiate and prolifer- ate to repopulate all blood lineages providing normal cell numbers of red blood cells, platelets, Normal B Cell Ontogeny and neutrophils. Successful immune reconstitution B cell development is schematically depicted in and protection from infection requires antimicrobial Figure 1. Progenitor B cells receive signals from essen- B cell and antibody development. Studies of B cell tial bone marrow stromal cells via cell-cell contacts and reconstitution after HCT have primarily examined secreted signals. Stem cell factor (SCF) on stromal cell immunoglobulin concentration, B cell quantification, membranes binds ckit (CD117) on the lymphocyte and antimicrobial antibody development in relation membrane, and secreted cytokines, especially interleu- to donor/recipient serologic status or vaccination. kin (IL)-7, promote B cell development [7-9]. B cells Following HCT, humoral immunity has 3 distinct bind antigen with varying affinity through B cell recep- contributions. First, recipient antibody persists with an tors that gain diversity through intrachromosomal var- average half-life of 30-60 days, and some recipient iable (V) and constant (C) region recombination [10].B plasma cells persist for years following allogeneic cell positive selection requires tonic signaling through HCT [1] providing protective antimicrobial humoral membrane pre-B receptor and membrane IgM expres- immunity [2]. Some recipient antidonor alloimmune sion for the B cell to survive. Mouse knock-out exper- responses are detrimental contributring to primary iments expressing null alleles of the heavy chain graft rejection [3,4] and prolonged red cell aplasia transmembrane exon, Iga or Igb genes, or their ITAMs when donors and recipients are ABO major mis- prevents B cell development [11,12]. Likewise, produc- matched [5,6]. Second, donor grafts contain naı¨ve tive somatic recombination leads to allelic exclusion for and memory B cells that have already undergone both heavy and light chains in each individual B cell. B positive and negative selection in the HLA-identical cells recognizing self antigens are negatively selected donor and contribute adoptive antimicrobial and allor- before emerging from the bone marrow. eactive B cells. Third, B cells reconstituting from do- Accumulating data suggests the BCR affinity nor HSCs recognizing disparate recipient antigens as ‘‘threshold’’ is influenced by cytokine tumor necrosis ‘‘self’’ will be clonally deleted, preventing alloreactive factor (TNF) family member B cell-activating factor responses, but remain capable of responding to infec- (BAFF; also termed BLyS). Three receptors have tious challenges and vaccinations. This educational been identified that bind to BAFF: transmembrane ac- session will consider B cell responses following alloge- tivator, calcium modulator, and cyclophilin ligand in- teractor (TACI); B cell maturation Ag (BCMA); and BAFF-R. Baff-R(2/2) mice mount significant, but re- From the 1Department of Pediatrics and Clinical Laboratory Med- duced, Ag-specific Ab responses [13]. BAFF and its re- icine, Memorial Sloan Kettering Cancer Center, New York, ceptors, play a crucial role in peripheral B cell selection New York; and 2Stanford University, Stanford, California. and survival, by dictating the set point for the number Financial disclosure: See Acknowledgments on page 110. of mature primary B cells and adjusting thresholds Correspondence and reprint requests: David B., Miklos, MD, PhD, for specificity-based selection during transitional dif- Stanford University, 450 Serra Mall, Stanford, CA 94305 (e-mail: [email protected]). ferentiation [14,15]. Transgenic models demonstrate 1083-8791/09/151S-0001$36.00/0 that antigen-induced anergy and exclusion from follic- doi:10.1016/j.bbmt.2008.10.016 ular niches of autoreactive B cells depends on the

104 Biol Blood Marrow Transplant 15:104-113, 2009 B Cells and Transplantation 105

IgM Antigen encounter Plasmablast IgD

Plasma cell IgM CLP Progenitor Pre B cell Immature Transition Mature B B cell B cell B cell cell Plasma cell

centroblast CD27

Memory B cell CD19 CD79a CD10 CD34 CD20 CD38 CD138 IgM and/or IgD

Figure 1. B cell maturation profile. presence or absence of a diverse B cell pool [16]. In ad- were of recipient origin for the first year post-HCT dition, B cell reconstitution and homeostasis after [2,39,40]. Persistent high-titer IgG responses persisted myeloablation requires the B survival factor BAFF when recipients were seropositive and donors seroneg- [17]. Limiting amounts of BAFF are required for on- ative, and likewise recipients remained seronegative going B cell turnover and avoidance of B cell autoreac- even when the donor was seropositive unless viral tivity [18]. This is because in the setting of a limited B infection erupted [41]. Deliberate vaccination of donors cell pool, excess BAFF promotes the survival of autor- pretransplant with a protein recall antigen, such as eactive B cells [19]. These BAFF homeostatic demands tetanus toxoid [42], or neoantigen KLH [43], could suggest a paradigm that unites peripheral negative and demonstrate adoptive B cell transfer but antibody de- positive selection with the maintenance of mature B velopment still required recipient revaccination after cell numbers [20,21] that probably have an impact on transplant. post-HCT reconstitution. Plasma BAFF levels are markedly elevated following myeloablative condition- Donor and Recipient Antibody Contribution ing and decrease as lymphocyte numbers recover. Ele- Can Be Distinguished by Allotype vated BAFF has been associated with cGVHD [22] and Allotype-specific monoclonal antibodies (mAbs) autoimmune diseases [23-25]. specifically bind single amino acid polymorphisms located in the immunoglobulin constant fragment of Antibody Reconstitution after HCT specific IgG isotypes [44]. If either the donor or recip- Early studies showed IgG and IgM return to nor- ient is homozygous null for an allotype then the HCT mal concentrations 3-4 months after allogeneic HCT pair is informative allowing antibody origin to be deter- [26,27], whereas B cells are quantitatively deficient mined. For example, if the recipient is null for IgG1 during the first month and persists in some patients detection using allotype reagent, then the development for more than a year after allo-HCT [28-30]. Antibody of donor derived IgG1 is allotype detected. Such allo- assessment is complicated by blood product support type reagents provided the first evidence of donor B transferring significant immunoglobulin and antibody cell reconstitution after HCT [45], and that some re- half-life extending 30-60 days. Nonetheless, vaccina- cipient plasma cells persist for years after myeloablative tion with neoantigens phage 4X174 and keyhole lim- HCT. In a pediatric study, 8 of 13 informative pairs pet hemocyanin (KLH) demonstrated effective IgG showed some persistent recipient IgG more than responses 6 months after HCT, but antibody response a year and some 8 years after myeloablative allogeneic lagged in patients with cGVHD or those who received HCT [1]. More recently, IgG allotype studies showed antithymocyte globulin (ATG) [31]. A reduced immu- recipient antimicrobial IgG predominates following noglobulin repertoire persists for at least 2 years after reduced-intensity conditioning (RIC) HCT, and DNA HCT with oligoclonal dominance [32,33]. The diver- chimerism confirmed that the majority of bone marrow sity of heavy IgG VDJ gene rearrangement reflects a plasma cells remained recipient derived 1 year after broad adult m-chain useage arguing against a recapitula- RIC allo-HCT [46]. In support of this recipient hu- tion of fetal ontogeny [34]. Post-HCT IgH repertoire moral immunity predominance, a study of 87 patients is characterized by decreased somatic hypermutation randomized to receive either marrow or peripheral [35], delayed class-switching [36], and oligoclonal blood stem cells (PBSCs) showed vaccine-specific anti- dominance [37,38]. body responses in the first year after allo-HCT re- Analysis of herpes simplex virus (HSV) and cyto- flected primarily the recipient’s pretransplant titer megalovirus (CMV) clarified that antiviral Ab responses [47]. Thus, humoral immunity is predominately 106 T. N. Small et al. Biol Blood Marrow Transplant 15:104-113, 2009 recipient derived for the first year after RIC allogeneic months (n 5 22). At Memorial Sloan Kettering Cancer HCT, and this persistent recipient derived antimicro- Center (MSKCC), 96% of 219 allogeneic HCT recip- bial IgG may benefit RIC allo-HCT patient and con- ients responded to a series of 3 IPV when administered tribute to their decreased treatment related mortality following acquisition of minimal milestones of im- (TRM). mune competence (CD4 .200/mL, PHA within 10% of normal, IgG .500 mg/dL) [56]. There was no dif- Immunogenicity of Vaccines following HCT ference in response in related or unrelated HCT recip- ients, or those who received a T cell-depleted or T Vaccine preventable infections, such as pneumo- cell-replete HCT. The range of responses in these 3 coccus [48], influenza [49], and varicella [50] remain studies may reflect in part variable levels of immune a significant cause of morbidity, rehospitalization, competence at the time of revaccination. and mortality after successful HCT. Despite the fact that influenza infections remain a significant cause of serious upper and lower respiratory tract infections Vaccine efficacy varies by glycosylation and post-HCT, many patients do not receive their yearly conjugation influenza immunization. Most studies report a 30% Although most transplant patients respond well to to 40% incidence of reactivation of varicella zoster vi- vaccines containing bacterial toxoids, response to pure rus (VZV) following HCT. In a retrospective study of polysaccharide vaccines has been poor [57-60]. Barra 100 consecutive allogeneic HCT recipients, 41% de- et al. [57] demonstrated that only 4 of 20 (25%) adult veloped a VZV infection at a median of 227 (range: allogeneic transplant recipients immunized with a sin- 45–346) days posttransplantation [50]. Forty percent gle nonconjugated H flu vaccine developed a specific of patients required admission to the hospital, with IgG response compared to 11 of 20 patients recipients a mean stay of 7.2 days. Postherpetic neuralgia and pe- 1 H flu conjugate vaccine (Hib) (P\.05). Most studies ripheral neuropathy developed in 68% of patients [18]. have also shown that \25% of HCT recipients are ca- As such, revaccinating HCT patients prevents signifi- pable of responding to the polyvalent polysaccharide cant morbidity and mortality, and, in addition, well- vaccine, PPV23 [57-59], as well as no benefit of donor designed vaccine studies functionally assess B cell vaccination with PPV23 prior to stem cell donation immune reconstitution. [60]. Limited response to polysaccharide antigens post-HCT may be because of the predominance of Protective Immunity Dissipates after immature B cells expressing CD1c, CD5, CD38, Myeloablative HCT 1 CD23 , B cells similar to the circulating B cells de- Multiple studies have demonstrated that in the ab- tected in cord blood and young children [29]. Limited sence of revaccination, both autologous and allogeneic responses may also reflect deficits in CD271 memory transplant recipients lose seroprotection to pathogens B cells, a subset that may take years to recover even they were immunized against during childhood (re- in children following HCT [61]. Nonetheless, heavy- viewed in [51,52]). Although there is some variability chain IgG sequence analysis of Hib-specific B cells in the time to protective titer loss among different collected after Hib revaccination 9 months after transplant groups, loss of pneumococcal, H flu, and myeloablative allogeneic BMT showed unique pat- tetanus titers usually occur by 2 years post-HCT [53]. terns of hypermutation, suggesting 90 of 121 (74%) Residual titers against measles, mumps, and rubella were derived from only 16 precursors, and 12 of these persist longer after HCT [54]. These vaccine studies clones were identified in the donor [62]. Thus, this showing the loss of humoral immunity studied myeloa- single-patient study suggests memory B cells specific blative HCT patients, therefore, RIC studies are to Hib were transferred from the donor, persisted 9 needed. months, and contributed the majority of Hib-specific repertoire. Vaccine Lessons In contrast to the poor response to polysaccharide vaccines, response to protein-conjugated polysaccha- Repetitive vaccination improves efficacy ride vaccines is significantly better, and can be en- Following an HLA matched related bone marrow hanced by donor vaccination prior to the stem cell transplant (BMT), Ljungman et al. [55] showed that harvest [63,64]. Meisel et al. [65] showed a 74% re- 42%, 36%, and 21% of patients immunized with 1 in- sponse to the 7 serotypes contained in PCV7 in 43 pa- activated polio virus vaccine (IPV) developed a 4-fold tients \17 years of age immunized with 3 PCV7 rise in titer against serotypes 1, 2, and 3, respectively. starting at 6 months following a related or unrelated Following 3 doses, 50% of patients responded to all HCT. A retrospective study of PVC7 and Hib re- 3 serotypes. In contrast, Parkkali et al. [53] reported sponses in 127 patients immunized at MSKCC also a 100% rate following 3 IPV whether administered demonstrated a decline in PCV7 response with ad- at 6, 8, and 14 months (n 5 23) or at 18, 20, and 26 vancing age [66]. Forty-five of 51 patients \18 years Biol Blood Marrow Transplant 15:104-113, 2009 B Cells and Transplantation 107 of age responded to PCV compared to 34 of 76 adults Table 1. Consensus Vaccination Guidelines \ (P .001). Although PCV7 response was adversely af- Vaccine CDC EBMT fected by older age (P \ .001), individuals $50 years old responded significantly better if vaccinated follow- Td, IPV, Hib 2 doses Started 12m 3 doses Started 6-12m PPV23 12 m, 24 m 12m ing acquisition of specific minimal milestones of im- HepB Optional Recommended mune competence, CD4 .200/mL, IgG .500 mg/ MMR 24 months 24 months dL, or PHA within 60% lower limit of normal (11 of Varivax NO NO 19 versus 0 of 8, P \ .006). A similar trend was ob- served in patients with limited cGVHD. Each of these search (CIBMTR) guidelines will soon be issued. Pro- studies demonstrates that PCV7 is immunogenic in spective trials documenting the efficacy of these HCT patients, including older adults, but suggest guidelines to protect the growing numbers of trans- that vaccination timing might depend on immune plant survivors are needed. competence for best vaccine response. B cell Reconstitution following Allogeneic HCT Vaccine efficacy is predicted by recipient immune reconstitution status Quantitative B cell deficiency in peripheral blood has long been noted in the first month after HCT Revaccination against hepatitis B is mandatory for and can persist for years being worsened by cGVHD reentry to school and certain workplaces. Machado and/or the treatment of cGVHD [28-30,70]. Figure 2 et al. [67] reported a 100% seroconversion rate in 50 shows that peripheral blood CD191 B cell reconstitu- HCT recipients immunized at least 1 year after trans- tion in pediatric and adult recipients has similar kinet- plant. Despite this excellent initial response, 60% of ics but varies by graft source with T cell depleted bone patients failed to sustain titers for more than 1 year marrow products preceeding unmanipulated, T cell after vaccination. MSKCC evaluated the response of replete bone marrow grafts (T. Small data). 267 allogeneic transplant recipients immunized with Peripheral blood B cell development is currently rHBV following acquisition of minimal milestones of characterized by 4 surface markers: CD19, IgD, immune competence [56]. Sixty-four percent of pa- CD38, and CD27 immunophenotyping. The results tients seroconverted, including 73% of 99 children of normal donor CD191 gated B cells are presented and 59% of 168 adults (P 5 .02). In multivariate anal- as 2-dimensional comparison of IgD versus CD38 in yses, response was adversely affected by age .18 years Figure 3. The IgD versus CD38 comparison provides (P \ .01) and history of prior cGVHD (P \ .0001). the Bm1-Bm5 classification that identifies: (a) virgin Eighty-two percent of 99 evaluated patients remained naı¨vecells (IgD1CD382), (c) pregerminal center cells seropositive 5 years following their last vaccine. This or transitional (IgD1CD3811), (b) postgerminal cen- greater proportion of patients with sustained hepatitis ter memory cells (IgD2CD381/2), and (d) germinal B titers in the MSKCC (82%), compared to Machado center plasmablasts (IgD2CD3811), reviewed [71]. and colleaugue’s study (40%), suggests qualitative and/ The IgD vervus CD27 classification provides meaning or quantitative differences in the circulating memory because CD27 is the universal marker of ‘‘memory’’ T and/or B cells present at the time of vaccination. and thus distinguishes between CD272IgD1 naı¨ve Surrogate markers of immune competency may be B cells, CD271IgD1 ‘‘class able to switch’’ memory necessary in determining the need and timing of booster immunizations to maintain durable protective TCD related titers following vaccination. TCD unrelated unmanipulated unrelated 1000 Vaccine guidelines The above data demonstrates that unlike vaccina- tion in healthy individuals, vaccination post-HCT 100 does not ensure seroprotection, emphasizing the need to document pre- and postvaccine titers to deter- mine response. Although vaccination of patients with 10 limited or no cGVHD will likely respond well to im- l/usllec+91DC munization starting 6 months post-HCT, how best <18 years, n=98 >18 years, n=159 to protect patients requiring steroids 6 other immu- 1 nosuppressive agents, is currently not known. Table 1 0-2m 2-4m 4-6m 6-9m 0-2m 2-4m 4-6m 6-9m 9-12m 9-12m presents the consensus vaccination guidelines of the 12-18m18-24m 12-18m18-24m Center for Disease Control (CDC) [57] and the Euro- Months post HCT pean Blood and Marrow Transplant [58], and Center Figure 2. Median number of CD191 B cells/mL peripheral blood after for International Blood and Marrow Transplant Re- allogeneic HCT. Kindly provided by Trudy Small. 108 T. N. Small et al. Biol Blood Marrow Transplant 15:104-113, 2009

103 103

102 102 a c

101 101 IgD CD19 100 100

100 101 102 103 b d ssLog 100 101 102 103 CD38

a c Naive Transitional vs. Pre-GC

Memory Plasmablast b d

Figure 3. Peripheral B cell immunophenotyping based on the expression of IgD and CD38 shows normal donor peripheral blood B cells are mostly nay¨ve. cell, and CD271IgD2 ‘‘isotype switched’’ memory switched’’ (IgD2CD271) B cells when compared to cells. Increased CD27 expression on human B cells is age-matched controls [63]. Even at 18 to 24 months known to correlate with commitment to the plasma after HCT there was a significant number of patients cell lineage [72]. B cells develop in bone marrow with CD271 memory cells less than the fifth percentile and the immunophenotype of the earliest bone of normal controls. As predicted by the lack of memory marrow emigrant to the peripheral blood is the B cells, an in vitro T cell-independent polyclonal stim- IgD1CD381CD272 ‘‘transitional’’ cell, which is the ulation revealed reduced IgG production, which has precursor to the ‘‘naı¨ve’’ mature IgD1CD382 cell. also been reported in adults [70]. Neither B cell subset Progression then moves to pregerminal center founder distribution nor stimulated Ig production correlated (Pre-GC) CD271 and postgerminal center memory with source of stem cells, type of conditioning, immune cell. Both the pre-GC and post-GC can become suppression, or the development of acute GVHD IgD2 isotype switched plasmablasts. The pre-GC are (aGVHD) or cGVHD. Avanzini et al. [63] propose IgD1CD38HiCD271 cells are typically found in hu- that germinal center B cell reactions are disturbed man tonsils [73], and have been shown to circulate post-HCT possibly because of lymph node histoarch- with increased frequency in systemic lupus erythema- itectural damage. Additional B cell immunophenotyp- tosis (SLE) patients [74,75]. Sarantopoulos et al. [76] ing studies may identify abnormal developmental analyzed peripheral CD271 B cell subsets in 57 post- immunophenotypes especially in the setting of elevated HCT patients and healthy controls by performing 4 BAFF possibly accounting for autoantibody produc- color-flow cytometry analyses. CD271 was increased tion and functional immune deficiencies after HCT. on all IgD versus CD38 populations in patients with cGVHD compared to patients without cGVHD. Pre-GC B cells were more prevalent in patients B Cells and cGVHD with active cGVHD than those without cGVHD Mouse studies suggest a role for B cells in the (P 5 .06) [76]. development of scleroderma and cGVHD. B lym- A longitudinal study of 139 pediatric HCT recipi- phocytes have been implicated in cutaneous sclerosis ents undergoing myeloablative (119 allogeneic) HCT in both murine and human studies. The tight skin showed recovering B cells at 3, 6, and 12 months after (Tsk/1) mouse has a tandem duplication within the fi- HCT are predominately naı¨ve (IgD1CD271), and brillin-1 (FBN1) gene and heterozygous mice exhibit consequently, a significantly lower percentage of both increased collagen and other matrix protein deposits ‘‘class able to switch’’ (IgD1CD271) and ‘‘class in their skin [77]. If bone marrow and splenocytes are Biol Blood Marrow Transplant 15:104-113, 2009 B Cells and Transplantation 109 transplanted from Tsk/1 donors into normal mice, were undetectable in all peripheral blood samples for a scleroderma phenotype and autoantibodies develop 9 to 12 months; median serum IgG levels fell 37%, [78,79]. B cell depletion using an antimouse CD20 but protective IgG antibody responses against epstein mAb before or 3 days after birth suppressed skin fibro- barr virus (EBV) EBNA1 and tetanus toxoid remained sis by 43% and autoantibody production [80]. Studies unchanged in 17 out of 18 patients. Antibody titers in minor histocompatibility antigen mismatched against H-Y antigens decreased after rituximab ther- mouse models of cGVHD support involvement of B apy and remained low for a year after treatment. All cells in its pathogenesis [81-83]. 4 patients with demonstrable H-Y antibodies had In humans, Miklos et al. [84] demonstrated alloge- clinical responses to rituximab therapy [93]. neic antibodies against at least 1 Y chromosome en- Zaja et al. [94] reported a 65% overall response rate coded protein (H-Y antigen) developed in 52% of in 38 patients with steroid-refractory cGVHD treated female / male recipient HCT tested by ELISA with rituximab. Skin was the most likely to respond against a 5 H-Y antigen panel: DDX3y, UTY, ZFY, (63%) followed by mouth (48%), eyes (43%), lung RPS4Y, and EIF1AY. In the presence of antibodies (38%), and liver (25%). Infections were the major to at least 1 H-Y protein, the cumulative incidence of complication. Mohty et al. [95] treated 15 severe or cGVHD reached 89% at 5 years after transplantation steroid refractory cGVHD patients with rituximab compared to only 31% in the absence of H-Y anti- weekly for 4 weeks, and responders received 1 to 2 bodies (P \ .0001) [85]. The concordant development courses of maintenance rituximab. With a median fol- of allogeneic H-Y antibodies and chronic GVHD pro- low-up of 118 days from first rituximab infusion, no vided early evidence for an important B cell role in major toxicities were ascribed to rituximab. Overall, chronic GVHD pathogenesis. 10 patients (66%) responded, and 3 achieved complete A possible role for elevated soluble BAFF in responses. Four patients did not respond and died of cGVHD is plausible because high BAFF levels are refractory cGVHD. found in patients with autoimmune diseases [25,86], As shown schematically in Figure 1, CD20 (devel- including scleroderma [87], and murine models of B opmental expression of CD20 shown in red) is first ex- cell autoimmunity. In mice, both normal and autoreac- pressed on B cells after they have undergone heavy- tive B cell development depends on the relative balance and light-chain recombination and express IgM BCR of B cell receptor (BCR) and BAFF signaling [88-92]. on their cell surface, a stage called ‘‘immature B cells.’’ In the setting of a limited B cell pool, excess BAFF pro- CD20 continues to be expressed until the cell becomes motes the survival of autoreactive B cells [19]. Saranto- an immunoglobulin secreting plasma cell. T cell-de- poulos et al. [22] studied BAFF levels in 104 allogeneic pendent human plasma cells in secondary lymphoid HCT patients and showed BAFF levels were signifi- tissue are CD201 [96]. Thus, rituximab treatment cantly higher in patients with active cGVHD, com- postallogeneic HCT may potentially eliminate pared to those without disease (P 5 .0002). Serial CD201 alloreactive cells while leaving early pre- and testing of 24 HCT patients showed BAFF levels pro-B cells to develop into nonalloreactive mature B were high in the first 3 months following HCT, but cells. Rituximab therapy is well tolerated with rela- subsequently decreased in 13 patients who never devel- tively few infectious complications [93] because long- oped cGVHD. In contrast, BAFF remained elevated in lived host plasma cells continue to secrete protective 11 patients who developed cGVHD. BAFF .10 ng/ antimicrobial antibodies [97]. mL 6 months after allogeneic HCT was strongly asso- ciated with subsequent cGVHD development [22]. Autoantibodies Are Associated with Systemic One complication of this analysis is that BAFF levels Sclerosis are suppressed in patients receiving more than 30 mg Systemic sclerosis involves progressive fibrosis of prednisone daily, and prednisone is almost always with obliteration of small artery lumens as well as used to treat aGVHD and cGVHD. humoral immunologic dysregulation associated with hypergamaglobulinemia and antinuclear antigen anti- Rationale for Rituximab Treatment of cGVHD bodies (ANA) [98]. These autoantibodies include Rituximab, a humanized IgG1 antibody against antitopoisomerase I (Scl70), anticentromere, anti- CD20, depletes B cells and is effective in treating pa- RNA polymerase III, anti-U3-fibrillarin, and others tients with steroid-refractory cGVHD. Cutler et al. [99]. However, despite their association with sclero- [91] reported a phase I clinical trial testing Rituximab derma, these ANA have not been shown to be patho- in 21 patients with steroid-refractory cGVHD. Ritux- genic. Other autoantibodies targeting extracellular imab (375 mg/m2/week  4 weeks) was given with an matrix proteins include: antifibrillin-1 (anti-FBN1) option for a second course for non- or partial re- [100] and antimatrix metalloproteinases (MMP), such sponders. Rituximab was well tolerated, and objective as MMP1 (interstitial collagenase) [101] and MMP3 responses were noted in 13 of 20 patients (70%, pri- (stromelysin) [102]. FBN1 is a 350-kDa glycoprotein marily cutaneous and rheumatologic). CD191 B cells that is the major constituent of microfibrils in the 110 T. N. Small et al. Biol Blood Marrow Transplant 15:104-113, 2009 extracellular matrix and they sequester transforming Transplant Meeting in April 2008 [114]. They re- growth factor (TGF)-b. Anti-FBN-1 antibodies are ported an overall response of 86% with a follow-up present in the majority of scleroderma patients, and of 8 months. Aspects of cGVHD that responded to im- some authors suggest anti-FBN1 antibodies may atinib included sclerodermatous disease, chronic bron- release TGF-b, which can then activate fibroblasts chiolitis, and osteomyalgia. Toxicity was evaluated at 3 promoting fibrosis [103,104]. and 6 months. Four of 15 subjects experienced minor Because the clinical manifestations of cGVHD extrahematologic toxicity and 1 of 15 experienced share many features with scleroderma [105], cGVHD grade 3-4 toxicity. Taken together, the frequent asso- patients have been repeatedly examined for autoanti- ciation of cGVHD and anti-PDGFR antibody and re- body development with inconsistent results. Some ported imatinib safety and tolerability in cGVHD studies suggest autoantibodies develop in association patients supports further imatinib efficacy trials to with cGVHD [106,107], whereas others show autoan- determine cGVHD response rate and confirm its tibodies develop as frequently as 25% after allogeneic mechanism of action. transplantation but occur equally in patients with and without cGVHD [108]. Future Directions Antibodies against PDGFR Associate with Protective antimicrobial immunity and allogeneic Systemic Sclerosis and cGVHD immune responses ultimately results from the recon- Fibroblasts have been extensively investigated as stitution of donor lymphocytes with diverse T and B the target of autoantibodies in patients with sclero- cell repertoires following allogeneic HCT. Nonethe- derma [109]. PDGF receptors are upregulated in the less, humoral immunity immediately post-HCT is skin and bronchoalveolar lavage fluid in scleroderma predominately recipient derived. Over the past 10 [110]. In 2006, Svegliati et al. [111] reported 46 patients years, RIC regimens have decreased treatment-related with systemic sclerosis had stimulatory antibodies mortality (TRM) and extended HCT to older patients. against PDGFR. These antibodies were absent in 20 The success of these RIC allogeneic HCT relies pri- healthy controls and another 55 patients with a variety marily on developing beneficial allogeneic immune of other rheumatologic conditions. In another study, responses, graft-versus-leukemia/lymphoma (GVL), agonistic PDGFR antibodies were detected in 22 and effective protective immune reconstitution. Al- allogeneic HCT patients with extensive cGVHD. though RIC regimens have succeeded in decreasing Anti-PDGFR antibodies were not detected in 17 TRM and aGVHD incidence, cGVHD remains prob- HCT patients without cGVHD and 20 normal con- lematic. One key difference between high-dose condi- trols [112]. These PDGFR-a antibodies induced tyro- tioning and RIC is the dynamic balance between the sine phosphorylation, accumulation of reactive oxygen decreasing host-versus-graft (HVG) resistance to en- species (ROS), and type 1 collagen gene expression graftment and the developing graft-versus-host (GVH) through the Ha-Ras-ERK1/2-ROS signaling pathway, immune responses. Although myeloablative HCT all processes implicated in inflammation and fibrosis causes rapid conversion to full donor T and B cell chi- [113]. PDGFR antibodies are relatively long lived merism, RIC patients’ progress through a transient compared to the 15-minute half-life of PDGF itself. mixed chimerism extending weeks to months, and the Thus, anti-PDGFR agonistic antibodies may be pace of transition differs by conditioning regimen the pathogenic cause of sclerosis in some cGVHD pa- [115]. Thus, we believe an improved understanding of tients, and drug inhibition of PDGFR signaling is B cells and serologic immune responses following a promising treatment for sclerosis. This might be RIC HCT in relation to antimicrobial immunity, achieved through either direct tyrosine kinase inhibi- GVL, and GVHD are critical. Future vaccine studies tion of PDGFR or anti-B cell therapy that eliminates will functionally asses B cell immune reconstitution the agonistic anti-PDGF antibody. Drs. Lorinda following RIC allogeneic HCT thereby decreasing Chung and Bill Robinson have initiated a phase I clin- patients’ infectious complications and revealing B cell ical trial of imatinib treatment of systemic sclerosis transplant biology. B cell immunophenotyping, heavy- with promising preliminary results (Chung et al, man- chain IgG repertoire analysis, and B cell functional uscript submitted). Immunohistochemical analysis of assays combined with multiplexed serologic antigen serial skin biopsies obtained before and 1 month after binding assays promises to identify allogeneic antibody imatinib 200 mg/day treatment show decreased anti- and B cell contributions to both GVL and GVHD. phospho-PDGFR antibody staining in the setting of clinical improvement. Extending this treatment strat- ACKNOWLEDGMENTS egy to cGVHD, the Italian bone marrow transplant cooperative group, GITMO, reported a safety and tol- Financial disclosure: Dr. Miklos is the PI of Novartis erability study of imatinib (100-200 mg daily) for sponsored Investigator Initiated Trial of Imatinib for cGVHD as an abstract at the European Bone Marrow steroid refractory chronic GVHD. Dr. Miklos is Biol Blood Marrow Transplant 15:104-113, 2009 B Cells and Transplantation 111 supported by an ASBMT Young Investigator Award, 19. Thien M, Phan TG, Gardam S, et al. Excess BAFF rescues self- ASH scholar Award, and this work was supported by reactive B cells from peripheral deletion and allows them to en- ter forbidden follicular and marginal zone niches. Immunity. NIH/NCI P01 CA049605; LLS 6204-06.The remain- 2004;20:785-798. ing authors have nothing to disclose. 20. Cancro MP. Living in context with the survival factor BAFF. Immunity. 2008;28:300-301. 21. De Falco M, Oliva G, Ragusa M, et al. Surgical treatment of REFERENCES differentiated thyroid carcinoma: a retrospective study. G Chir. 2008;29:152-158. 1. van Tol MJ, Gerritsen EJ, de Lange GG, et al. The origin of 22. Sarantopoulos S, Stevenson KE, Kim HT, et al. High levels of IgG production and homogeneous IgG components after allo- B-cell activating factor in patients with active chronic graft- geneic bone marrow transplantation. Blood. 1996;87:818-826. versus-host disease. Clin Cancer Res. 2007;13:6107-6114. 2. Wimperis JZ, Brenner MK, Prentice HG, Thompson EJ, 23. 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