The Role Of Donor B Cells And Their Production Of Antibodies In Chronic GVHD Pathogenesis

Background and ObjectivesAllogeneic hematopoietic cell transplantation?allo-HCT?is a curative therapy for hematological malignancies and nonmalignant hereditary hematologic disorders.Graft-versus-host disease?GVHD?is a major cause of morbidity and mortality after allo-HCT and limits its wide spread clinical application.GVHD can be divided into acute GVHD?aGVHD?and chronic GVHD?cGVHD?.In the past,aGVHD is defined as GVHD that occurred before 100 days after allo-HCT and cGVHD is defined as GVHD that occurred beyond 100 days after allo-HCT.This definition has become outdated.The new definition from NIH consensus conference is:aGVHD includes classic acute GVHD?maculopapular erythematous rash,gastrointestinal symptoms,or cholestatic hepatitis?and also includes persistent,recurrent,or late-onset aGVHD occurring more than 100 days after transplantation.cGVHD includes classic chronic GVHD?bronchiolitis obliterans,scleroderma,lupus-like syndrome or hepatitis?and an overlap syndrome,which has diagnostic or distinctive chronic GVHD manifestations together with features typical of aGVHD.Over the past three decades,there has been little progress in prevention and treatment of cGVHD,due in part to the poor understanding of cGVHD pathogenesis.Unlike aGVHD,cGVHD shares characteristics with systemic autoimmune diseases,such as scleroderma and lupus-like syndrome,including elevated serum levels of IgG autoantibodies,sclerodermatous skin tissue damage,and systemic tissue collagen deposition and the immune pathology of cGVHD is more complex.Acute GVHD is mediated by alloreactive donor T cells.Conditioning regimen with irradiation or high-dose chemo-therapy causes tissue damage.The damaged tissues release proinflammatory cytokines?i.e.IL-lb and TNF-a?and chemokines as well as allow leakage of bacterial products?i.e.LPS?and pathogen-associated molecular patterns?PAMPs?into circulation;which leads to activation of host innate immune cells including antigen presenting cells?APCs?.Activated host APCs such as dendritic cells?DCs?migrated into draining lymph nodes where they activate alloreactive donor T cells.Activated donor CD4⁺ T cells can differentiatie into T helper 1?Thl?,T helper 2?Th2?and T helper 17?Th17?and CD8+ T cells into cytotoxic T cell 1?Tc1?,cytotoxic T cell 2?Tc2?,and cytotoxic T cell 17?Tc17?,but alloreactive T cells predominantly differentiate into Th1 and Tc1 cells due to the proinflammatory environment.The activated T cells migrate into GVHD target tissues?i.e.gut,liver,lung,and skin?,where T cells produce cytokines and recruit donor-type innate immune cells to mediate acute GVHD.Chronic GVHD is mediated by autoreactive CD4⁺ T cells and B cells.Chronic GVHD usually followed aGVHD.During aGVHD,activated T cells infiltrate thymus and damage thymic epithelial cells,which results in a defect in negative selection of denovo-generated autoreactive T cells.The GVHD-damaged thymus increases production of autoreactive pathogenic T cells but decreases production of Foxp3+natural Treg cells.The autoreactive T cells interact with donor-type DCs and B cells in the periphery.This interaction leads to the differentiation of autoracitve T cells into Thl,Th2 and Th17.This interaction also activates autoreactive B cells to produce autoantibodies.During acute GVHD,some alloreactive CD4⁺ T cells that have cross-reactive TCRs from CD4⁺ T cells in the transplant are expanded in the peripheral and they can also interact with donor-type DCs and B cells.As acute inflammation subsides,Thl cells contract,and Th17 and Th2 cells relatively expand.Autoreactive CD4⁺ T cells,B cells and their production of antibodies work together to mediate chronic GVHD.It has been proposed that during chronic GVHD pathogenesis,autoreactive CD4⁺ T cells release fibrogenic cytokines such as IL-2,IL-10 and transforming growth factor-?1?TGF?1?.Those cytokines activate macrophages that produce platelet-derived growth factor?PDGF?and TGF?1.These molecules induce the proliferation and activation of tissue fibroblasts and excessive production of collagen.Th 17 cells produce IL-17,IL-6,IL-21,IL-22 that cause skin damage.Acting as efficient APCs,donor B cells augment the clonal expansion of the residual autoreative donor CD4⁺T cells in the transplants and augment donor CD4⁺T differentiation into proinflammatory Th2 and Th17 cells.B cell Homeostasis is also proposed to be associated with cGVHD.There is an increase of CD27⁺ memory B cells and a decrease of naive B cells in cGVHD patients.And B cell-activating factor of the TNF family(BAFF is correlated with the development and severity of cGVHD.High levels of BAFF augment activation of autoreactive B cells that participate in the pathogenesis of chronic GVHD.However,the role of antibodies in the pathogenesis of chronic GVHD remains unclear.On the one hand,stimulatory antibodies to the platelet-derived growth factor receptor can be found in patients with chronic GVHD or systemic sclerosis but not in patients without GVHD,and anti-PDGFR antibodies are able to induce an increased production of collagen from human fibroblasts in vitro.Antibody responses to H-Y minor histocompatibility antigens also correlate with cGVHD.And donor B cell alloantibody deposition especially IgG2c are reported to be required for the development of bronchiolitis obliterans in a mouse model without cutaneous GVHD.In light of those findings,antibodies could therefore play an important roles in cGVHD pathogenesis.One the other hand,transfer of autoantibodies from mice with cGVHD to normal mice failed to cause autoimmune pathology.Some studies demonstrate that B cells contribute to SLE pathogenesis in an antibody-independent fashion.In fact,to date a direct involvement of antibodies in cGVHD pathology has not been conclusively demonstrated.And so far,apart from autoantibody-dependent autoimmune phenomena such as immune-mediated hemolysis or thrombocytopenia,no direct evidence for the causal relationship of autoantibodies or alloantibodies and the pathogenesis of organ manifestations of chronic GVHD in humans exists.Instead,the detection of alloreactive and autoreactive antibodies might just serve as a marker for the presence of antigen-specific B cells that contribute to disease pathology by mechanisms other than antibody production.To answer this question and to distinguish between the antibody-dependent and antibody-independent effects of B cells,we generated IgH_??l DBA/2 mice;these mice produce a B cell compartment consisting exclusively of B cells expressing the membrane form of IgM,with no IgM being detectable in the blood.B cells of those mice have APC function but cannot secrete antibodies.Using a cGVHD model of DBA/2 donor to MHC-matched BALB/c recipient,we explore the role donor B cells and their production of antibodies in the pathogenesis of cutaneous chronic GVHD.This study will provide new insights into chronic GVHD pathogenesis and provide new scientific basis for developing novel therapy for chronic GVHD.Method:1.Mice:DBA/2 and BALB/c mice were purchased from the National Cancer Institute animal production program?Frederick,MD?.IgH_??l DBA/2 mice were generated by backcrossing IgH_??l BALB/c mice to DBA/2 for 10 generations.The IgH_??l BALB/c mice were provided by Dr.Rajewski.The mice were screened with RT-PCR of IgH_??l with tail tissues.Homozygous IgH_??l DBA/2 mice are double-checked by ELISA of serum and flow cytometry analysis of B cell surface of IgM and IgD.The B cells of IgH_??l DBA/2 mice had only IgM hi IgD-B cells with no IgMloIgDhi B cells.Consistently,the IgH_??l DBA/2 mice had little IgM or IgG in the serum.B7H1-/-BALB/c mice were developed in the City of Hope Animal Resource Center?Duarte,CA?.Mice were maintained in a pathogen-free room at City of Hope Animal Research Center.2.We used a cGVHD model of DBA/2 donor to MHC-matched BALB/c recipient.BALB/c mice were exposed to 850 cGy total body irradiation?TBI?with the use of a[137Cs]source 8-10 hours before transplantation.Recipients were injected intravenously i.v.with CD25⁺T cell-depleted spleen cells?75 X 10⁶?or?25X 10⁶?and T cell-depleted BM?TCD-BM??5 X 10⁶?from either wild-type?WT?DBA/2 or IgH_??l DBA/2.Control recipients were injected i.v.with TCD-BM?5X10⁶?alone.CD25 depletion for the spleen cells and T cell depletion for the BM cells were accomplished using first antibody of biotin-conjugated anti-CD25?spleen?or biotin-conjugated anti-CD4 and biotin-conjugated anti-CD8 mAb,and then with streptavidin Microbeads?Miltenyi Biotec,Germany?,followed by passage through an autoMACS cell sorter?Miltenyi Biotec,Germany?.The purity of depletion was>99%.The recipients were monitored for clinical signs of GVHD including hunched back,raffled fur,diarrhea,weight-loss,hair-loss,proteinuria,and death.3.Tissue harvest and single cell suspension preparation:Mice were killed using CO2 asphyxiation.The spleens,peripheral lymph node?PLN?,thymus,skin,liver and lung were collected for lymphocyte analysis.Spleens,PLN,thymus were mashed using a 70-mm filter.Skin samples were cut into small pieces,and digested with 2-3 Mg/ml Dispase in PBS on a shaker at 37? for 45 mins.Then skin samples were digested with P/S,66 ?g/ml Liberase TM;100ug/mg DNASE 1,0.5 mg/ml hyaluronidase,2%FBS in RPMI on a shaker at 37? for 90 minutes.The digestion was stoped by adding cold PBS containing 2%BSA and EDTA.Lung tissues were first injected with 25ul collagenase D and 25ul DNASE 1 in complete media and incubated for 30-45 mins.Liver samples were first treated with PBS containing 2%BSA and EDTA.The tissues were mashed through a 70?m filter,and lymphocytes were separated using Lympholyte-M.And mashed through a 70?m filter,and lymphocytes were separated using Lympholyte-M.Flow cytometry analysis were performed with a 4-laser CyAn,and data were analyzed with Flow Jo software.4.Tissue harvest and Histopathology:skin,liver,lung,salivary gland were collected for histopathology.For skin tissue collection,remove hair and cut a section?3x 5 cm?of skin from the mouse's upper back and neck.Place the skin centered,muscle-side down,on a biowrap.Gently spread the skin out on the biowrap.Put the skin in the cassette,and then put the cassette in formalin.For liver and salivary tissue collection,the liver and salivary tissues were put in the cassette directly,and then the cassette was embedded in formalin.For lung collection,3ml formalin was injected into lung through the trachea to fill the lung completely before harvest.The formalin-fixed tissues were embedded in paraffin blocks.Slides were stained with Hematoxylin and Eosin?H&E?.H&E slides were examined at original magnification 200-400 and visualized with an Olympus and a Pixera?600CL?cooled charge-coupled device camera5.Tissue harvest and immunofluorescent staining:Frozen tissue of skin and spleen were embedded in optimal cutting temperature compound?OCT compound?,snap frozen in dry ice,and store in-80?.Frozen tissue of thymus were put in PFA over night in 4?,then transferred to sucrose over night in 4?.48 hours later,embedded in OCT gel,frozen in dry ice and store in-80?.For Antibody deposition,skin and thymus tissue were stained with FITC labeled anti-mouse-IgG.Antibody deposition was quantified by the intensity of IgG staining in whole skin or thymus.For thymic epithelial cells staining,thymus tissue were stained with UEA-1,Anti-Cytokeratin 8?Troma-1?and DAPI.For splenic B cell germinal center formation,spleen tissue were stained with rhodamine-peanut agglutinin?PNA?,Anti-B220,Anti-CD3.For IL23 staining,skin tissue were stained with anti-CD 11c,anti-IL23,DAPI.For B7H1 staining,skin paraffin sections were stained with Anti-B7H1.And samples were visualized with an Olympus BX51 fluorescent microscope.Color composite images were generated using Adobe Photoshop 7.0 software.6.Real-time RT-PCR:Primers used are as follows:CCL20,forward,5,-CACTCGGTCGTCATGGGT-3',reverse,5'-CATCTTCTTGACTCTTAGGCT-3';IL23,forward,5'-TATCCAGTGTGAAGATGGTTGTG-3',reverse,5'-CACTAAGGGCTCAGTCAGAGTTG-3';IL6,forward,5'-AAGAAATGATGGAT GCTACC-3',reverse,5'-GAAGGACTCTGGCTTTGTC-3';B7H1,forward,5'-CAGAAGCTGAGGTAATCTGGA-3',reverse,5'-TGAGTCCTGTTCTGTGGAG G-3';IFN-?,forward,5'-ACAGCAAGGCGAAAAAGGATG-3',reverse,5'-TGGTGACCACTCGGATGA-3';GAPDH,forward,5'-TCACCACCATGGAGAAGG C-3',reverse,5'-GGTAAGCAGTTGGTGCA-3'.Relative expression levels of genes were normalized within each sample to the house keeping gene GAPDH.7.Statistical analysis:All data was analyzed using GraphPad Software?Prism Version 5.0;GraphPad Software,San Diego,C.A?.Clinical cutaneous damage scoring,Proteinuria incidences and survival in different groups were compared by using the log-rank test.Comparison of different group was analyzed using one way-ANOVA.Multiple comparision was analyzed using LSD test.P-value<0.05 was considered to be statistically significant.Result1.Establishment of IgH_??l DBA/2 mice.After backcrossing IgH_??l BALB/c mice to DBA/2 for 10 generations,we successfully obtain homozygous IgH_??l DBA/2 mice,which produce a B cell compartment consisting exclusively of B cells expressing the membrane form of IgM without IgD,and the mice have no detectable IgM in the serum.2.Recipients transplanted with IgH_??l donor transplants develop only transient skin cGVHD.TBI-conditioned BALB/c recipients were transplanted with high-dose?75 x10⁶?and low-dose?25x10⁶?CD25⁺ T-depleted?Treg cells-depleted?spleen cells and TCD-BM cells from wild-type or IgH_??l DBA/2 donors.While the recipients given TCD-BM alone appeared to be heathy,the recipients transplanted with high and low dose spleen cells from WT-donors developed severe proteinuria and cutaneous cGVHD.The high-dose group all died from proteinuria and ascites by 35 days after HCT.75%of the low-dose recipients survived from proteinuria for more than 60 days but they all developed cutaneous GVHD.In contrast,none of the recipients given high and low dose spleen cells from IgH_??l DBA/2 donors showed proteinuria,and they all survived for more than 60 days.Although the recipients given low-dose of IgH_??l DBA/2 donor spleen cells showed little signs of chronic GVHD,the recipients given high dose of IgH_??l donor cells showed cutaneous GVHD around 40 days after HCT and then recovered by 60 days after HCT.And although 75 x10⁶ IgH_??l 25--SPL cells induced no tissue damage in the lung,the cells did induce tissue pathology in the liver,salivary gland,and skin,despite of no antibody deposition in those tissues.And those tissue damage have been disappeared at 60 days after HCT.These results suggest that,except lung,IgG antibodies from donor B cells are not required for initiating cGVHD tissue damage,but the antibodies are required for persisting tissue damage.3.Donor antibodies contribute to destruction of germinal center and lymphatic follicles.At 40 and 60 days after transplantation,in the spleen of recipients given high or low dose IgH_??l donor spleen cells,there were both complete lymphatic follicles and germinal center.However,in the cGVHD recipients given low-dose WT donor cells,no germinal center or lymphatic follicles were observed.Consistently,at 40 and 60 days after transplantation,there are significant difference in percentage of T follicular helper cells of spleen among threee groups?F=10.19,P=0.0049;F=28.48,P=0.0001?.Percentage of T follicular helper cells in spleen of low does WT group are much lower than low dose IgH_??l group?P=0.0017;P=0.0005?and high dose IgH_??l group?P=0.0155;P<0.0001?.There are also significant difference in percentage of follicular B cells of spleen spleen among threee groups?F=11.24,P=0.0036;F=19.18,P=0.0006?.Percentage of follicular B cells in spleen of low does WT group are also much lower than low dose IgH_??l group?P=0.0030;P=0.0004?and high dose IgH_??l group?P=0.0024;P=0.0005?.In addition,at 40 days and 60 days after transplantation,the yield of total CD4⁺ T cells in the spleen and PLN of recipients transplanted with high dose and low dose IgH_??l donor spleen cells were also much higher than that of recipients given WT donor spleen cells?P=0.0345;P=0.0096 and P=0.0102;P=0.0110?.These results indicate that IgG antibodies from donor B cells contribute to destruction of lymphatic follicles and lymphopenia.4.Donor antibodies contribute to thymic damage.At 40 and 60 days after transplantation,the percentage of CD4⁺CD8+ thymocyte of the recipients transplanted with high-dose and low dose IgH_??l spleen cells were similar to that of recipients transplanted with TCD-BM alone,that is,>80%.In contrast,the percentage of CD4⁺CD8+ thymocyte in the recipients transplanted with low-dose WT spleen cells were less than 20%.And at 40 and 60 days after transplantation,the yield of CD4⁺CD8+ thymocyte in the recipients transplanted with low-dose WT spleen cells were lower than recipients transplanted with high-dose IgH_??l spleen cells?P=0.0149;P=0.0002?and recipients transplanted with low-dose IgH_??l spleen cells?P=0.0071;P<0.0001?.Immunofluorescent staining of mTEC showed that transplantation of IgH_??l donor spleen cells did not cause damage of thymic mTEC but transplantion of WT spleen cells did.These results indicate that antibodies from donor B cells contribute to thymic mTEC damage in chronic GVHD.5.Donor antibodies contribute to Th17 expansion in the lymphoid and GVHD target tissues.At 40 and 60 days after transplantation,percentage of donor Th17 cells in the spleen of recipients given IgH_??l or WT donor spleen cells was not significantly different.In contrast,At 40 days and 60 days after transplantation,percentage of Th17 cells in the peripheral lymph nodes of recipients given low-dose of IgH_??l donor spleen cells was much lower than that of recipients given low-dose of WT donor spleen cells?P=0.0164;P=0.0003?.Althoguh there were few Th17 cells in the skin tissues at 40 days after HCT in both recipients,at 60 days after HCT,the percentage of Th17 cells in the skin tissue of recipients given WT donor spleen cells was much higher than that of recipients given high dose IgH_??l donor spleen cells?P=0.0002?and that of recipients given low dose IgH_??l donor spleen cells?P=0.0001?.These results indicate that antibodies from donor B cells contribute to expansion of Th17 in lymphoid tissue early after onset and skin tissue later after onset.6.We observed that 40 days after HCT,there was higher percentage of CD11b^-CD11c⁺DCs in the PLN and skin tissues of GVHD recipient given 25 ×10⁶ WT 25--SPL cells as compared to non-GVHD recipients given 25 x10⁶ IgH_??l 25--SPL cells?P<0.05?.Histoimmunofluoresent staining also showed increase of CDllc⁺ cells in the PLN and skin tissues of recipients given 25 x10⁶ WT.It is of interest that DCs in the PLN tissues and SKIN tissue of GVHD recipients given WT 25--SPL cells secrete more IL-23,which was?2 folder higher than that of non-GVHD recipients given 25 x10⁶ IgH_??l 25--SPL cells?P<0.05?.These results indicate that antibodies from donor B cells stimulate DCs in the PLN and skin tissues to secret IL-23 that augments Th17 expansion.7.We found that 40 days after HCT,as compared to GVHD recipients given 25 x10⁶ WT 25-SPL cells,skin tissue expression of B7H1 in non-GVHD recipients given 25 x10⁶ IgH_??l 25--SPL cells was augmented as judged with immunofluoresent staining.Consistently,skin tissue expression of mRNA of B7H1 of latter recipients was elevated?P<0.01?.Furthermore,we found that DC cells in the skin of recipients given IgH_??l,CD25-SPL cells upregulated expression of B7H1?P<0.01?.And the IL-6 expression levels in the skin tissues of cGVHD recipients given WT-CD25-SPL cells was-3 fold higher than that of non-GVHD recipients given IgH_??l-25-SPL.In addition,there was a marked increase of CD11b+CD11c-monocyte in the skin tissue of GVHD recipients given WT-25--SPL cells.These results suggest that antibodies from donor B cells may contribute to down-regulation of tissue expression of B7H1.Conclusion1.IgG antibody tissue deposition is associated with damage of thymus and peripheral lymphopenia,as well as cutaneous GVHD2.Tissue infiltration of Thl cells is associated with early stage cutaneous cGVHD,and infiltration of Th17 cells is associated with later-stage and persistent cutaneous cGVHD.3.Th17 infiltration of skin tissues is associated with infiltration of IL-6-producing monocytes,skin tissue release of CCL20,skin tissue DC release of IL-23,and down-regulation of skin tissue expression of B7H1 in recipient with donor B cell antibody secretion.