Unlinked Memory Helper Responses Promote Long-Lasting Humoral Alloimmunity

This information is current as Thomas M. Conlon, Jennifer L. Cole, Reza Motallebzadeh, of September 29, 2021. Inês Harper, Chris J. Callaghan, Eleanor M. Bolton, J. Andrew Bradley, Kourosh Saeb-Parsy and Gavin J. Pettigrew J Immunol 2012; 189:5703-5712; Prepublished online 16

November 2012; Downloaded from doi: 10.4049/jimmunol.1202257 http://www.jimmunol.org/content/189/12/5703

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Unlinked Memory Helper Responses Promote Long-Lasting Humoral Alloimmunity

Thomas M. Conlon, Jennifer L. Cole, Reza Motallebzadeh, Ineˆs Harper, Chris J. Callaghan, Eleanor M. Bolton, J. Andrew Bradley, Kourosh Saeb-Parsy, and Gavin J. Pettigrew

Essential help for long-lived alloantibody responses is theoretically provided only by CD4 T cells that recognize target alloantigen, processed and presented by the allospecific . We demonstrate that in an alloresponse to multiple MHC disparities, cognate help for class-switched alloantibody may also be provided by CD4 T cells specific for a second “helper” alloantigen. This response was much shorter-lived than when help was provided conventionally, by Th cell recognition of target alloantigen. Nevertheless, long- lasting humoral alloimmunity developed when memory against the helper alloantigen was first generated. Costimulatory blockade abrogated alloantibody produced through naive Th cell recognition of target alloantigen but, crucially, blockade was ineffective when help was provided by memory responses to the accessory helper alloantigen. These results suggest that memory Th Downloaded from cell responses against previously encountered graft alloantigen may be the dominant mechanism for providing help to generate new specificities of alloantibody in transplant patients receiving immunosuppression. The Journal of Immunology, 2012, 189: 5703–5712.

llograft rejection has long been considered the culmination enables linked cognate interaction with the CD4 TCR is essential of cellular alloimmunity, but recent clinical studies indi- for effective delivery of help. According to this tenet, alloreactive A cated that alloantibody directed against mismatched donor direct-pathway CD4 T cells (that cannot interact with the MHC http://www.jimmunol.org/ MHC Ags may be at least as important in effecting the rejection class II/peptide complex of the allospecific B cell) are unable to of kidney (1, 2) and heart allografts (3, 4). Two main histological provide help; additionally, help is restricted to indirect-pathway variants of alloantibody-mediated rejection are described (5), with CD4 T cells that recognize the same target alloantigen that binds rodent studies confirming that alloantibody can contribute to acute the BCR. In support, recent analysis of the humoral response to graft rejection (6) and to progression of chronic transplant arteri- vaccinia virus emphasized that exquisite T–B cell linkage opathy (TA) (7, 8). As with conventional protein Ags, help from also pertains to protein subunits of complex pathogens (19). CD4 T cells is essential for generating alloantibody against MHC Nevertheless, Hunziker et al. (20) highlighted that presentation alloantigens (6, 9, 10), but the complexities of CD4 T cell allorec- of processed lymphocytic choriomeningitis virus by nonviral spe- ognition have prevented elucidation of how precisely this help is cific B cells can derive help for induction of bystander hyper- by guest on September 29, 2021 provided. CD4 T cells recognize alloantigen through two distinct gammaglobulinemia, although the mechanism responsible was not pathways (11–14). In the direct pathway, which is unique to trans- assessed. This raises the possibility that, in the context of a com- plantation, alloantigen is recognized intact on the surface of donor plex immune response targeted typically against multiple dispa- APCs, whereas in the indirect pathway, alloantigen is processed and rate alloantigens, indirect-pathway CD4 T cells specific for one presented as peptide fragments by recipient APCs for self-restricted alloantigen may provide B cell help for production of alloantibody recognition. One study suggested that alloreactive B cells can re- directed against a different graft alloantigen. ceive help from direct-pathway CD4 T cells (15), but this has re- Confirmation of this mechanism would have important con- mained controversial (16), and we demonstrated recently that only sequences for our understanding of the helper arm of humoral indirect-pathway CD4 T cells act as T follicular helper (TFH)cells alloimmunity, because it represents an “unlinked” or dissociated for generating long-lasting, class-switched alloantibody (17). system whereby Th cell activation occurs independently of the The provision of help by indirect-pathway CD4 T cells conforms alloreactive B cell response. Hence, it would be possible for to the fundamental tenet, first proposed by Noelle and Snow (18), memory CD4 T cells generated from prior exposure to one helper that B cell presentation of internalized, processed target Ag that alloantigen to provide help to naive B cells responding to a second, newly encountered target alloantigen: help that may be quantita- tively and qualitatively different from that delivered conventionally Department of Surgery, University of Cambridge, Cambridge CB2 0QQ, United Kingdom by those naive CD4 T cells specific for the target alloantigen. To Received for publication August 13, 2012. Accepted for publication October 13, formally examine this concept of dissociated help, we developed 2012. murine heart transplant models that incorporated monoclonal This work was supported by a British Heart Foundation project grant and by the populations of alloreactive T and B to limit helper National Institute for Health Research Cambridge Biomedical Research Centre. CD4 T cell allorecognition to a single alloantigen distinct from the C.J.C. and K.S.-P. were supported by Wellcome Trust/Academy of Medical Sciences starter grants. R.M. and I.H. were supported by clinical research training fellowships B cell target. from The Wellcome Trust and Raymond and Beverly Sackler Scholarships. Address correspondence and reprint requests to Mr. Gavin J. Pettigrew, University of Cambridge, Box 202, Level E9, Addenbrookes Hospital, Cambridge CB2 0QQ, U.K. Materials and Methods E-mail address: [email protected] Animals Abbreviations used in this article: HEL, hen egg lysozyme; TA, transplant arteri- 2/2 b b opathy; TFH, T follicular helper. Tcrbd B6 mice (H-2 ) (21) and H-2 MHC class II-deficient mice (22) were purchased from The Jackson Laboratory (Bar Harbor, ME). Tcrbd2/2 Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 mice were crossed with MHC class II–deficient mice to create mice that www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202257 5704 UNLINKED MEMORY HELPER RESPONSES were TCR and MHC class II deficient. TCR-transgenic Rag22/2 TEa mice lution curve was plotted, and the area under the curve was calculated (33). b b (H-2 ), specific for I-A –restricted I-Ea52–68 peptide (23), were gifted The area under the curve of an experimental sample was expressed as the by Prof. A. Rudensky (University of Washington, Seattle, WA). TCR- percentage of positive control pooled serum. transgenic Rag22/2 Mar mice (H-2b), specific for I-Ab–restricted H-Y pep- Single anti–H-2Kd IgG Ab–secreting cells were detected by ELISPOT tide (24), were gifted by Dr. D. Scott (Imperial College, London, U.K.). assay, as described (17). TCR-transgenic Rag12/2 TCR75 mice (H-2b), specific for I-Ab–restricted d d H-2K 54–68 peptide (25) and B6.K mice, which express the full sequence Flow cytometry of H-2Kd (26), were gifted by Prof. P. Bucy (University of Alabama, b a FITC-conjugated anti-mouse CD3 (clone 145-2C11), FITC-conjugated Birmingham, AL). B6 mice that lack I-A , but express I-E (B6.I-E) (27), anti-mouse CD44 (clone IM7), FITC-conjugated anti-mouse I-Ab (clone were gifted by Prof. C. Benoist (Joslin Diabetes Center, Boston, MA). b AF6-120.1), PE-conjugated anti-mouse CD19 (clone 1D3), PE-conjugated BCR-transgenic SWHEL mice (H-2 ) specific for hen egg lysozyme (HEL) k b anti-mouse TCR Vb6 (clone RR4-7), PE-conjugated anti-mouse I-E protein (28) and B6.mHEL mice (H-2 ) that express membrane-bound (clone 14-4-4S), biotinylated anti-mouse H-2Kd (clone SF1-1.1), biotinylated HEL (29) were gifted by Prof R. Brink (Centenary Institute of Cancer anti-mouse CD62L (clone MEL-14), and biotinylated and allophycocyanin- Medicine and Cell Biology, Newtown, Australia). B6 (H-2b) and BALB/c d conjugated anti-mouse CD4 (clone GK1.5) were purchased from BD mice (H-2 ) were purchased from Charles River Laboratories (Margate, Pharmingen (San Diego, CA). HEL protein was biotinylated using the U.K.). All animals were maintained in specific pathogen–free facilities, ImmunoProbe biotinylation kit (Sigma). Peripheral blood (depleted of and experiments were approved by the U.K. Home Office Animal (Sci- erythrocytes by incubating with 0.17 M NH Cl red cell lysis buffer) and entific Procedures) Act 1986. 4 splenic single-cell suspensions were blocked with anti-mouse CD16/CD32 Generation of bone marrow chimeras (clone 2.4G2) before staining with the relevant Abs and dead cell exclusion dye 7-aminoactinomycin (both from BD Pharmingen). Biotinylated Abs To create mice that lacked MHC class II expression only on their B cells and were detected by allophycocyanin-conjugated streptavidin (Invitrogen) or 2/2 2/2 PE-Cy7–conjugated streptavidin (BD Pharmingen), and all cells were were T cell deficient (B.CII ), Rag2 mice were sublethally irradiated Downloaded from (2 Gy) and reconstituted with 2 3 107 bone marrow cells obtained from analyzed on a FACSCanto II flow cytometer with FACSDiva software Tcrbd2/2 MHC II–deficient mice. Control chimeric mice (B.CII+/+) were (Becton Dickinson U.K., Oxford, U.K.). created by reconstituting sublethally irradiated Rag22/2 mice with 2 3 107 Tcrbd2/2 bone marrow cells. Chimerism was confirmed by flow cyto- CFSE cell proliferation metric analysis of peripheral blood 4 wk after reconstitution. Single-cell suspensions of splenocytes obtained from Mar or TEa mice were stained with 5 mM CFSE (Invitrogen) in the dark for 5 min and Generating mosaic mice then quenched with PBS + 5% FCS. A total of 2–5 3 106 CFSE-stained http://www.jimmunol.org/ Eight-celled embryos were harvested from B6.I-E and B6.Kd mice, ag- splenocytes was injected i.v., spleens were harvested 7 d later, and flow gregated as described (30), and implanted into pseudopregnant (B6 3 cytometry was performed using allophycocyanin-conjugated anti-CD4 b CBA/CaJ) F1 females. Flow cytometric analysis of peripheral blood cells plus PE-conjugated anti-TCR V 6 to identify Mar or TEa cells. taken from the offspring was undertaken, and mice demonstrating equiv- Statistical analysis alent numbers of cells expressing either I-E or H-2Kd were selected for use as heart donors. Alloantibody responses and luminal stenosis measurements were compared using two-tailed unpaired t tests. Statistical analyses were performed using Skin and heterotopic heart transplantation GraphPad Prism version 5.02 (GraphPad Software, San Diego, CA). Full-thickness tail skin was sutured as 1-cm2 grafts onto the recipient’s back. Cardiac allografts were transplanted intra-abdominally. Tcrbd2/2 Results by guest on September 29, 2021 2/2 3 7 and Rag2 mice were reconstituted by i.v. injection of 1 10 Help for class-switched anti–MHC class I alloantibody can be splenocytes from Rag-deficient TCR75, TEa, or Mar mice. Grafts were excised 50 d after transplantation and cut transversely; one half was stored provided by CD4 T cells that recognize unrelated alloantigen at 280˚C, and the other was fixed in 10% buffered formalin. The ability of CD4 T cells that recognize additional graft allo- Adoptive transfer of primed, allospecific T and B lymphocytes to provide help for anti–MHC class I alloantibody pro-

2/2 duction was examined using a model similar to that used recently SWHEL.Rag2 mice were challenged with B6.mHEL or B6.mHEL 3 B6. 2/2 I-E F1 hearts. Four days later, splenic B cells were harvested, purified with to assess allospecific TFH cells (17). C57BL/6 Tcrbd mice, anti-mouse CD19 MicroBeads (Mitenyi Biotec, Bergisch Gladbach, Ger- which lack all T cells yet retain relatively normal B cell homeo- many) using an autoMACS Separator (Mitenyi Biotec), and injected i.v. stasis and compartmentalization (34), were challenged with 6 2/2 2/2 (3 3 10 ) into Tcrbd mice. Tcrbd mice simultaneously received BALB/c (H-2d) heart allografts. Helper function was provided by primed TEa CD4 T cells by i.v. injection of 5 3 106 splenocytes harvested m a adoptive transfer of monoclonal populations of TCR-transgenic fromTEamiceimmunized5dpreviouslywith50 g I-E 52–68 peptide 2/2 emulsified in CFA. CD4 T cells. Thus, as described previously (17), Tcrbd mice reconstituted with Rag12/2 TCR75 CD4 T cells specific for I-Ab– d Costimulation blockade restricted H-2K 54–68 allopeptide, which provide classical, linked CD40–CD40L signaling was blocked by administering 0.5 mg anti-CD154 helper recognition of target MHC class I alloantigen (Fig. 1A), mAb i.p. (clone MR-1; Bio X Cell, West Lebanon, NH) on the day prior to mounted a robust anti–H-2Kd IgG alloantibody response (Fig. 1B) transplant and 0.5 mg i.v. on the day of transplant. that was even stronger than that observed in wild-type B6 recip- Histology and immunohistochemistry ients (Fig. 1B); we reported recently that this is associated with germinal center development and deposition of long-lived plasma Formalin-fixed hearts were paraffin mounted and stained using Weigert’s cells in the bone marrow (17). This strong alloantibody response Elastin van Gieson method to delineate the internal elastic lamina, and the severity of TA was assessed morphometrically, as recently reported (31). resulted in rapid rejection of the heart allografts within 7 d. Seven-micrometer cryostat sections of OCT-embedded hearts were ex- To examine whether recognition of additional mismatched graft amined for complement C4d deposition, as described previously (32). alloantigen generates help for anti–MHC class I IgG alloantibody responses, female Tcrbd2/2 mice were reconstituted with Rag22/2 Determining circulating alloantibody and B cell ELISPOT 2 2 TEa (specific for I-Ab–restricted I-Ea allopeptide) or Rag2 / assay 52–68 Mar (specific for I-Ab–restricted H-Y peptide) CD4 T cells and Serum was analyzed for the presence of anti–H-2Kd and anti-HEL IgG challenged with female or male BALB/c heart allografts, respec- alloantibody by ELISA. In brief, 96-well ELISA plates (Immulon 4HBX; tively (Fig. 1C). Both groups developed early, albeit weak and Thermo, Milford, MA) were coated with recombinant conformational d d H-2Kd [generated in-house (17)] or HEL (Sigma, Poole, U.K.) at 5 mg/ml short-lived, anti–H-2K IgG responses (Fig. 1D). No anti–H-2K in Na2CO3-NaHCO3 buffer (pH 9.6), and both ELISAs were performed IgG alloantibody was generated when the Mar-reconstituted mice as described previously (17). For each sample, an absorbance versus di- received female BALB/c heart grafts (Fig. 1D). The Journal of Immunology 5705 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. H-2Kd–specific B cells can receive effective help from CD4 T cells that recognize unrelated alloantigen. (A) Diagrammatic representation of classical linked help provided to H-2Kd–specific B cells by H-2Kd peptide–specific TCR75 CD4 T cells. (B) Serum levels (mean 6 SD)ofanti–H-2Kd IgG alloantibody, after challenge with BALB/c heart allografts, at day 14 in B6 (n =3),Tcrbd2/2 (n = 3), and Tcrbd2/2 recipients adoptively transferred with 107 splenocytes from Rag12/2 TCR75 mice (monoclonal CD4 T cells specific for I-Ab–restricted H-2Kd peptide) (n =3).(C) Diagrammatic representation of possible unlinked help provided to H-2Kd–specific B cells by either TEa CD4 T cells (specific for self-restricted I-E peptide) or Mar CD4 T cells (specific for self-restricted H-Y peptide). (D) Serum levels of anti–H-2Kd IgG alloantibody, following transplantation of male or female BALB/c heart allografts into Tcrbd2/2 recipients adoptively transferred with 107 splenocytes from Rag22/2 TEa or Mar mice as a source of monoclonal CD4 T cells. One group of Mar CD4 T cell–reconstituted Tcrbd2/2 recipients of a female BALB/c graft were immunized additionally with 107 male B6 Rag22/2 splenocytes to provide unlinked activation of the Mar CD4 T cells. Data are the values for each individual mouse, with mean values indicated. (E) Representative flow cytometry plots (of two independent experiments) of CFSE-labeled Mar CD4 T cells 7 d after adoptive transfer into unchallenged B6 and Tcrbd2/2 mice or Tcrbd2/2 mice that received a male B6 heart graft. The indicated levels of cell division refer to the percentage division of the parent population, as calculated using FlowJo software. *p # 0.01, two-tailed unpaired t test.

To exclude the possibility that activation of the transferred CD4 CD4 T cells that recognize additional alloantigen interact with T cells simply provided bystander help to the H-2Kd–specific B cell MHC class II 2/2 B cells, female Tcrbd mice were reconstituted with Mar CD4 Given the requirement for cognate interaction between the B and T T cells and challenged simultaneously with male B6 splenocytes helper lymphocytes in generating class-switched Ab to conven- 2/2 from Rag2 mice (to provide a source of male Ag without tional protein Ag, we hypothesized that help from CD4 T cells contaminating T and B cells) and a female BALB/c heart graft. No specific for additional alloantigen would depend similarly upon d anti–H-2K IgG alloantibody was generated (Fig. 1D), suggesting presentation of their appropriate allopeptide epitope by the MHC that help provided through T cell recognition of additional allo- class I–specific B cell. To test this, bone marrow chimeric mice antigen requires coexpression of the B cell target and helper Ag on were created in which the B cells lacked MHC class II expression the allograft. (expression on non-B cell APCs was maintained) and were T cell The provision of help through recognition of additional graft deficient (B.CII2/2, Fig. 2A). Although lacking MHC class II alloantigens was not a facet of homeostatic proliferation of the expression on B cells, CD4 T cell alloimmunity in these mice was helper CD4 T cell population following transfer into an empty not impaired, because adoptively transferred TEa T cells re- niche (35), because Mar CD4 T cells did not undergo division sponded to challenge with H-2d alloantigen with comparable vigor 2 2 upon transfer into naive female Tcrbd / mice (Fig. 1E). In con- to control B.CII+/+ chimeric mice that retained B cell MHC class II trast, robust proliferation was observed following challenge with expression (Fig. 2B). This mirrors our recent finding that division of amaleB6heartgraft(Fig.1E). TCR75 CD4 T cells is comparable in control and experimental bone 5706 UNLINKED MEMORY HELPER RESPONSES

transplanted into TEa-reconstituted Tcrbd2/2 recipients, in the expectation that recipient H-2Kd–specific B cells would be unable to simultaneously acquire donor I-E Ag and, thus, would not re- ceive help from TEa CD4 T cells for production of anti–MHC class I H-2Kd alloantibody (Fig. 3B). Compared with the response to BALB/c heart allografts, in which I-E and H-2Kd alloantigens are coexpressed on donor cells, anti–H-2Kd alloantibody produc- tion following transplantation with mosaic I-E/Kd hearts was re- duced (Fig. 3C). Nevertheless, the mosaic hearts provoked a weak response that was greater than background observed in naive mice. This might be explained by the observation that confinement of H-2Kd and I-E expression to different cells in the mosaic mice is not absolute; a small percentage (4.1 6 0.75%, mean 6 SD) of PBMCs in mosaic mice express both H-2Kd and I-E (Fig. 3A), Downloaded from http://www.jimmunol.org/ FIGURE 2. CD4 T cells that recognize additional helper alloantigen interact with MHC class II expressed on allospecific B cells. (A) Repre- sentative flow cytometric plots depicting CD19 and I-Ab staining of live peripheral blood cells from T cell–deficient, female bone marrow chimeric mice (B.CII2/2)(right panel), highlighting that MHC class II was not expressed on B cells (upper right quadrant) but was maintained in a pro- portion of non–B cell APCs (upper left quadrant). Control bone marrow chimeric mice (B.CII+/+) are shown for comparison (left panel). (B) Representative plots of TEa CD4 T cell division in chimeric B.CII+/+ or by guest on September 29, 2021 B.CII2/2 mice following i.v. challenge with 107 BALB/c splenocytes. Di- vision in similarly challenged Tcrbd2/2 mice is shown for comparison. The indicated levels of cell division were calculated using FlowJo soft- ware. (C) Serum anti–H-2Kd IgG alloantibody following challenge of Mar CD4 T cell–reconstituted (using 107 splenocytes from Rag22/2 Mar mice, as Fig. 1) B.CII+/+ and B.CII2/2 mice with a male BALB/c heart allograft. Anti–H-2Kd alloantibody responses to a female BALB/c heart allograft in Mar CD4 T cell–reconstituted B.CII+/+ recipients simultaneously chal- lenged i.v. with male APCs are also depicted. Data are the values for each individual mouse, with mean values indicated. The p values were calcu- lated using a two-tailed unpaired t test. Values from naive mice are shown for comparison. marrow chimeras (17). Despite this, when reconstituted with Mar CD4 T cells, female B.CII2/2 recipients of male BALB/c heart grafts failed to mount an anti–H-2Kd IgG alloantibody response (Fig. 2C); in contrast, modestly strong responses developed in similarly challenged control B.CII+/+ recipients (Fig. 2C). This FIGURE 3. Helper and target alloantigen must be expressed on the same mirrors our recent demonstration that B cell MHC class II expres- graft cell. (A) Tetra-parental mosaic mice were created for use as heart d sion is required for delivery of conventional help to allospecific donors; their APCs would express either I-E or H-2K but not both. Representative flow cytometric plots of gated live peripheral blood cells B cells (17) and indicates that help from CD4 T cells that recognize d additional graft alloantigen is similarly reliant upon cognate inter- demonstrating the percentage of I-E– and H-2K –expressing cells from B6.IE, B6.Kd, B6.IE 3 B6.Kd F1, and mosaic mice. The number of cells action with the MHC class II/peptide complex of the B cell. expressing both I-E and H-2Kd in the gated region is depicted as the Allospecific B cells present acquired helper alloantigen as percentage of total live cells. (B) Because individual graft cells from processed allopeptide mosaic I-E.Kd heart allografts express either I-E or H-2Kd alloantigens, but not both, presentation of I-E peptide by recipient H-2Kd–specific B cells is We hypothesized that the above requirement for recognition of not expected. (C) Serum anti–H-2Kd IgG alloantibody in TEa CD4 T cell– B cell MHC class II reflected acquisition and processing of ad- reconstituted Tcrbd2/2 recipients, following challenge with BALB/c (n = ditional, adjacent alloantigens on the donor cell by the class I– 6) or mosaic (n = 5) heart allografts. Data represent the values for each b specific B cell. This was examined by creating tetra-parental H-2 individual mouse, with mean values indicated. The p values were calcu- d mosaic mice, whose cells expressed either I-E or H-2K on their lated using a two-tailed unpaired t test. Values from naive mice (n = 8) are surface but not both (Fig. 3A). Hearts from these mice were shown for comparison. The Journal of Immunology 5707 presumably reflecting exchange of membrane proteins through with membrane HEL Ag expressing B6 (B6.mHEL) 3 B6.Kd F1 trogocytosis (36) prior to heart graft procurement. Unfortunately, heart grafts provoked an anti-HEL IgG Ab response that was 2/2 this exchange prevents definitive interpretation of the results ob- comparable to similarly treated SWHEL.Tcrbd mice (Fig. 4B). tained using mosaic mice as donors. To examine the presentation of processed helper alloantigen by One possible explanation for how allospecific B cells acquire the allospecific B cell, HEL-specific B cells were purified from 2/2 graft alloantigen other than that recognized by their BCR is that SWHEL.Rag2 mice 4 d after challenge with either a B6.mHEL 3 small numbers of B cells express a second BCR specific for ad- B6.I-E F1 heart or a B6.mHEL heart allograft and adoptively ditional alloantigen, either because of incomplete receptor editing 2/2 2/2 transferred into Tcrbd mice. The primary SWHEL.Rag2 (37) or because of bystander acquisition of the second receptor recipients did not develop an anti-HEL alloantibody response from activated B cells (38). The possibility of dual-receptor ex- (data not shown), reflecting the absence of a helper T cell pop- pression was examined using T cell–deficient mice that contained ulation; the helper arm was instead provided in the secondary only a single, monoclonal population of B cells, which were Tcrbd2/2 mice by adoptive transfer of CD4 T cells from Rag22/2 created by crossing SW BCR transgenic mice onto a Rag22/2 HEL TEa mice, primed 5 d previously by with I-Ea background (Fig. 4A). SW mice were originally created using 52–68 HEL peptide. Anti-HEL IgG alloantibody was detectable in the sec- homologous recombination to target a HEL-specific IgH V region ondary Tcrbd2/2 mice that received SW B cells from SW . gene to the endogenous IgH locus (28); therefore, the BCR trans- HEL HEL Rag22/2 3 genic anti-HEL B cell population can class-switch. However, recipients of B6.mHEL B6.I-E F1 heart allografts; on the Rag22/2 background, receptor editing through further VDJ however, crucially, transfer of SWHEL B cells from recipients recombination of endogenous sequences is not possible, and all challenged with a B6.mHEL heart, which could not have en- Downloaded from mature B cells express a single HEL-specific BCR (Fig. 4A). countered helper I-E alloantigen, did not provoke a response (Fig. 2/2 4C). Thus, these experiments provide definitive confirmation that SWHEL mice were also crossed onto a Tcrbd background; such mice lack T cells but maintain a polyclonal population of B cells, B cells with a single receptor specific exclusively for one allo- within which the transgenic HEL-specific B cell population is antigen can receive effective help from CD4 T cells that recognize 2/2 nested (Fig. 4A). Notably, reconstitution of SWHEL.Rag2 mice exclusively another graft alloantigen and that this help is depen-

with a helper population of TCR75 CD4 T cells and challenge dent upon B cell presentation of the helper Ag (Fig. 4D). http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Allospecific B cells present acquired helper alloantigen as processed allopeptide. (A) Representative flow cytometric graphs of gated live 2/2 2/2 2/2 2/2 lymphocytes from SWHEL BCR–transgenic mice crossed onto the Rag2 (SWHEL.Rag2 )orTcrbd (SWHEL.Tcrbd ) background. Both strains 2/2 lack CD3 T cells (left panel; analysis of SWHEL mouse included for comparison); although only a select population of SWHEL.Tcrbd B cells binds HEL, 2/2 all B cells in SWHEL.Rag2 mice are HEL specific (middle panel and right panel, which shows percentages from individual mice). (B) Serum anti-HEL d 2/2 IgG Ab responses following transplantation of B6.mHEL 3 B6.K F1 heart allografts into TCR75 CD4 T cell–reconstituted SWHEL.Tcrbd mice or 2/2 2/2 similarly reconstituted SWHEL.Rag2 mice. (C) Circulating anti-HEL IgG Ab in Tcrbd mice following adoptive transfer of TEa CD4 T cells, purified 2/2 2/2 from primed Rag2 TEa mice, and splenic B cells, purified from SWHEL.Rag2 mice that had received either a B6.mHEL heart graft or a B6.mHEL 3 B6.I-E F1 heart graft 4 d earlier. The p values were calculated using a two-tailed unpaired t test. (D) Diagrammatic representation of unlinked help provided by TEa CD4 T cells to HEL-specific SWHEL B cells that present I-E peptide following challenge with a B6.mHEL 3 B6.I-E F1 (mHEL.I-E) heart allograft. 5708 UNLINKED MEMORY HELPER RESPONSES

Memory helper CD4 T cell recognition of additional was associated with the presence of long-lived plasma cells within alloantigen bone marrow (Fig. 5C). The above models, wherein the Ags for Th cell recognition are Memory CD4 T cell responses differ characteristically from dissociated from those acting as B cell targets, theoretically enable naive responses in their reduced requirements for costimulation, memory T cell responses from prior alloantigen exposure to greater proliferation, and secretion of a different spectrum provide help to naive B cells that recognize additional, newly (39, 40). We hypothesized that, in clinical transplantation, the encountered, graft alloantigen. To examine how help provided from principal pathway for providing help for alloantibody responses recognition of additional alloantigen differs according to memory against newly encountered alloantigen—naive Th cell recognition status, Mar CD4 T cell–reconstituted female Tcrbd2/2 mice were of the processed target Ag—is likely inhibited by concurrent challenged with a male B6 skin graft and further challenged 6 wk immunosuppression administration; instead, memory CD4 T cell later with a male BALB/c heart graft, and the anti–H-2Kd allo- responses against additional graft alloantigen becomes the domi- response was quantified. In this model, help for alloan- nant mechanism for delivery of help. This was tested by com- tibody production can only be provided by Ag-experienced Mar paring the impact of blocking CD40–CD40L signaling on the CD4 T cells, whereas the H-2Kd–specific B cells remain in a naive alloantibody response to a heart allograft in recipients with state until a heart allograft is introduced. Male skin grafting memory and naive helper CD4 T cell populations. Blockade was resulted in marked expansion of the Mar CD4 T cells and ac- achieved by administering anti- CD154 mAb (41), a protocol that quisition of memory phenotype (Fig. 5A); notably, the anti–H-2Kd abrogated the robust anti–H-2Kd alloantibody response to BALB/c alloantibody response to the BALB/c heart allograft was markedly heart allografts generated when help was provided conventionally, different from that observed in control mice challenged with by TCR75 T cells (Fig. 5D). When help was provided by naive Downloaded from a female B6 skin graft: circulating alloantibody was readily de- Mar CD4 T cells, anti-CD154 mAb treatment was similarly ef- tectable at termination of the experiment on day 50 (Fig. 5B) and fective at blocking anti–H-2Kd alloantibody responses; however, http://www.jimmunol.org/ FIGURE 5. Memory CD4 T cell recognition of additional helper alloantigen results in long-lived alloantibody production that is costimulation inde- pendent. (A)NumberofcirculatingMarCD4 T cells, as a percentage of total lymphocytes in Tcrbd2/2 mice reconstituted with 107 splenocytes from Rag22/2 Mar mice, 6 wk after receiving either female (naive Mar, n = 7) or male (memory Mar, n = 4) B6 skin grafts (left panel, mean 6 SD; naive B6 mice are included as comparison). Representative by guest on September 29, 2021 (of three mice/group) flow cytometry dot plots of CD44 and CD62L staining on live splenic CD4 T cells from the same groups. Values indicate numbers of CD44highCD62Llow memory CD4 T cells within gated area, expressed as the per- centage of total live CD4 population. (B) Serum levels of anti–H-2Kd IgG alloantibody, following transplantation of male BALB/c heart allografts, into Tcrbd2/2 recipients reconstituted with naive or memory Mar CD4 T cells, as described above, with or without anti-CD154 mAb treatment at the time of transplant. Data are values for each individual mouse, with mean values indicated. (C) Number of H-2Kd–specific IgG-secreting Ab-secreting cells (ASCs) per 106 plated cells within spleen and bone marrow 50 d after transplantation of male BALB/c heart allografts into Tcrbd2/2 recipients, recon- stituted as above with naive or memory Mar CD4 T cells, with or without anti-CD154 mAb treatment at the time of transplant. Values for naive Tcrbd2/2 mice are included for comparison. Data represent a minimum of three mice/group (mean 6 SD). (D) Serum levels of anti–H-2Kd IgG alloantibody in TCR75-CD4 T cell–reconstituted Tcrbd2/2 recipi- ents that received a BALB/c heart allograft with (n = 5) or without (n = 5) concurrent treatment with anti- CD154 mAb. Data are the values for each individual mouse, with mean values indicated. Values for the TCR75 group that did not receive anti-CD154 treatment were published previously (17). *p , 0.05, **p , 0.005, two-tailed unpaired t test. ns, Not significant. The Journal of Immunology 5709 in contrast, it failed to inhibit the development of the long-lasting treatment ameliorated arteriopathy development in recipients with responses aided by memory Mar CD4 T cells (Fig. 5B), with a naive Mar helper population (58.2 6 19.2% versus 21.9 6 seeding of H-2Kd–specific, long-lived plasma cells to the bone 11.5% mean luminal stenosis 6 SD, p = 0.013), the same treat- marrow preserved (Fig. 5C) but reduced. ment had less impact on the memory group (90.4 6 4.7% versus 73.4 6 10.0% mean luminal stenosis 6 SD, p = 0.057, Fig. 6A). Recognition of additional helper alloantigen contributes to TA The heightened TA in the memory group is unlikely to simply Alloantibody responses that develop in human heart transplant reflect greater activation of an effector Mar T cell population recipients are associated with early graft loss (3, 4), and although that mediates vasculopathy development autonomously, because 2 2 BALB/c heart grafts in the Mar T cell–reconstituted recipients did memory T cell generation in Rag2 / recipients did not alter the not reject acutely, the contribution of alloantibody to chronic graft severity of arteriopathy (38.6 6 15.0% versus 43.0 6 15.1% mean failure was assessed by quantifying the severity of TA. In the luminal stenosis 6 SD, p = 0.720) (Fig. 6A). In support, endo- absence of a humoral arm, indirect-pathway Mar CD4 T cells can thelial C4d complement deposition was readily detectable in autonomously effect development of modest TA in male allo- allografts in which memory CD4 T cells generated long-lasting grafts, because their adoptive transfer into T and B cell–deficient alloantibody, irrespective of the administration of costimulation Rag22/2 recipients of male BALB/c hearts provoked more severe blockade (Fig. 6B). In contrast, C4d deposition was not detectable 2 2 TA than observed in similarly reconstituted Tcrbd2/2 recipients of in Rag2 / recipients that did not develop alloantibody or in mice female hearts, even when the Mar population in the latter was with short-lived alloantibody responses triggered by naive Mar activated by simultaneous immunization with male B6 spleno- CD4 T cell help (Fig. 6B). Thus, these experiments demonstrate cytes (Fig. 6A). TA was slightly greater in the Mar-reconstituted that memory indirect-pathway CD4 T cells generate more severe Downloaded from Tcrbd2/2 recipients of male BALB/c heart grafts than in Mar- TA, most likely as a consequence of their ability to promote long- reconstituted Rag22/2 recipients (Fig. 6A), although this differ- lasting alloantibody responses, and that costimulation blockade ence was not statistically significant. Thus, transient alloantibody fails to prevent this damage. responses promoted by unlinked, naive Th cell responses to a second alloantigen do not appear to contribute to TA develop- Discussion ment. In contrast, when long-acting alloantibody responses were The experiments were designed to determine the alloantigen http://www.jimmunol.org/ generated by providing help via memory Mar CD4 T cells, TA was specificity of CD4 T cells that provide help for alloantibody much more severe (Fig. 6A). Furthermore, although anti-CD154 production. We demonstrated that CD4 T cells specific for one by guest on September 29, 2021

FIGURE 6. Alloantibody contributes to the development of allograft arteriopathy. (A) TA (% luminal stenosis) was assessed in male BALB/c donor hearts excised at day 50 from Rag22/2 recipients that contained naive or memory Mar CD4 T cells at the time of transplant, Tcrbd2/2 recipients that contained naive or memory Mar CD4 T cells at the time of transplant, and Tcrbd2/2 recipients that contained either naive or memory Mar CD4 T cells at the time of transplant and received concurrent anti-CD154 costimulation blockade. Female Tcrbd2/2 recipients challenged with a female BALB/c heart and 107 male Rag22/2 splenocytes (B6 APC) are shown for comparison. Data are means 6 SD of at least three mice/group. (B) Photomicrographs depicting representative (of at least three hearts/group) immunohistochemical analysis of arterial complement C4d deposition (brown staining) within male BALB/c heart allografts excised 50 d after transplantation from Rag22/2 recipients reconstituted with naive Mar CD4 T cells (i), Tcrbd2/2 recipients reconstituted with naive Mar CD4 T cells (ii), Tcrbd2/2 recipients reconstituted with memory Mar CD4 T cells (iii), and Tcrbd2/2 recipients reconstituted with memory Mar CD4 T cells and additionally administered anti-CD154 costimulation blockade (iv). Counterstained with Harris hematoxylin. Scale bar, 100 mm. *p , 0.001, **p = 0.720, ***p = 0.102, {p = 0.019, {{p = 0.013, {{{p = 0.057, two-tailed unpaired t test. 5710 UNLINKED MEMORY HELPER RESPONSES alloantigen can provide help to B cells whose allospecificity is for is internalized via the BCR, fragments of donor cell (most likely a different alloantigen expressed by cells within the graft. More- the surrounding membrane portions) that contain additional helper over, CD4 T cell memory responses against additional graft alloantigen are also captured. Real-time visualization of B cell alloantigens are able to provide costimulation-independent B cell synapse formation revealed that spreading and then contraction help for new specificities of alloantibody that can contribute to graft of the BCR concentrates target Ag to the synapse, while simul- failure. These findings might be clinically relevant, because the taneously excluding other membrane proteins (44), perhaps indi- problems associated with long-lasting humoral alloimmunity pose cating that nonselective acquisition is unlikely. Nevertheless, one of the major challenges for clinical transplantation. “Sensi- Suzuki et al. (45) recently demonstrated effective acquisition of tized” patients on the waiting list for kidney transplantation have follicular surface protein by B cells as they acquire preformed alloantibody that often results in either excessively their target Ag; similarly, CD8 T cells can acquire additional long waiting times or precludes transplantation entirely. In addi- membrane proteins from target cells during TCR-mediated inter- tion, the development of donor-specific alloantibodies after organ nalization of MHC class I complex (46). transplantation is associated with early graft failure. Alloantibody Long-lasting humoral is a defining trait of the germi- responses often develop in transplant recipients who are seem- nal center response, help for which is limited to a select, highly ingly adequately immunosuppressed with stable initial graft specialized subset of CD4 TFH cells (47, 48). The durable al- function, indicating that their development is relatively resistant to loantibody responses generated when T cell help is provided conventional immunosuppressive treatment. Our demonstration through conventional recognition of target alloantigen are simi- that, memory CD4 T cells that recognize one graft alloantigen can larly characterized by germinal center formation and estab- deliver CD40-independent help to naive alloreactive B cells that lishment of a bone marrow niche (17); we further Downloaded from recognize another, may explain how these alloantibody responses confirmed that indirect-pathway CD4 T cells that recognize occur. target alloantigen acquire a signature TFH cell phenotype (17). In Our findings appear to contravene the maxim of “linked rec- comparison, help provided through naive recognition of additional ognition,” whereby, as exemplified by recent analysis of humoral “helper” alloantigen is less effective, with the transient Ab pro- responses to complex viral Ag (19), help for T-dependent Ab duction and absence of bone marrow plasma cell deposition

responses is restricted to CD4 T cells specific for the same Ag that suggesting that short-lived extrafollicular, and not germinal center, http://www.jimmunol.org/ is recognized by the BCR. However, it has long been appreciated, foci are responsible. Nevertheless, the dissociated nature of our as demonstrated readily by haptenated systems, that Th cell model, in which the target for Th cell recognition is distinct from are not necessarily derived from the same region of target that for alloreactive B cell recognition, theoretically enables cel- Ag as the B cell epitope; indeed, shared helper recognition has lular memory responses against one alloantigen to provide help to been proposed as the mechanism responsible for simultaneous naive B cells responding to another; our experiments highlight development of against several different, yet struc- that, in this situation, the alloantibody response against newly turally complexed, autoantigens (42). Nevertheless, our work encountered MHC class I alloantigen differs markedly. The ability differs, in that although the inability of Mar CD4 T cells to pro- of H-Y–specific memory CD4 T cells to provide help for pro- vide help for alloantibody responses against a female heart allo- duction of long-lasting anti–MHC class I alloantibody and for by guest on September 29, 2021 graft (despite simultaneous challenge with male Ag) suggests formation of a bone marrow plasma cell niche suggests that the a necessity for coexpression of the target and helper alloantigen on memory Mar CD4 T cells perform TFH cell function for devel- the same cell, the nature of this linkage is much less obvious and opment of germinal center responses. However, the outcomes of appears simply a facet of tethering within a common membrane extrafollicular and germinal center responses may differ less dis- (either surface or intracellular). The possibility that recognition of tinctly than thought previously (49), and formal assessment of minor Ags could provide help for alloantibody secondary follicle development, which was not performed in this responses was suggested two decades ago (43); however, this study, will be the subject of future studies. concept was largely ignored and only became verifiable more Despite the reported requirement for CD40/CD154 signaling recently, with the availability of monoclonal populations of allo- in germinal center formation (50), its blockade did not prevent specific B and T helper cells. memory cellular responses against additional helper alloantigen With regard to how T cell help is provided by recognition of from mediating the development of durable alloantibody re- additional graft alloantigen, our experiments highlighting the sponses. Presumably, CD40 costimulation is not essential for pro- importance of MHC class II–restricted presentation indicate that ductive interaction between the B and helper T , but cognate interaction with the CD4 TCR is still a fundamental re- is instead necessary for initiating T cell activation upon cognate quirement. Although the additional helper alloantigen could the- encounter with dendritic cells; once activated, CD40-independent oretically be acquired through non-Ag–specific mechanisms that B cell help that results in long-lasting alloantibody production is operate in professional APCs, these pathways are generally inef- possible. In support, agonistic CD28 Ab promotes germinal cen- fective in B cells. It is also unlikely that B cells express the rel- ter development in CD154-deficient mice (51), and additional evant helper determinant following capture of MHC class II/ ligands, such as complement C4B-binding protein (52), were allopeptide complexes from other APCs that have already pro- noted to signal through the B cell CD40 complex. In contrast, the cessed alloantigen, because no alloantibody was produced in the robust and long-lived humoral alloresponses that were mediated bone marrow chimeric recipients that lacked MHC class II se- by conventional, naive helper recognition of target alloantigen lectively on B cells. Instead, the requirement for coexpression of were completely inhibited by anti-CD154 treatment; although the target and helper alloantigen on the same donor cell suggests unproven, this suggests that memory recognition of additional that BCR-mediated internalization is responsible. If so, the ex- alloantigen may be the dominant pathway for developing new periments incorporating B cell–transgenic recipients, in which specificities of alloantibody in human transplant patients. This all B cells express a single specificity of BCR, confirm that ac- ability of memory helper CD4 T cells to provide costimulation quisition of helper Ag occurs through BCR recognition of blockade–resistant help to naive B cells with a different Ag target alloantigen and not, for example, through capture of a sec- specificity may hold wider relevance. For example, in patients ond BCR specific for the helper Ag (38). Presumably, as target Ag with humoral autoimmune disease, unlinked helper recognition The Journal of Immunology 5711 may enable, despite concurrent immunosuppression, memory 7. Russell, P. S., C. M. Chase, H. J. Winn, and R. B. Colvin. 1994. Coronary atherosclerosis in transplanted mouse hearts. II. Importance of humoral immu- CD4 T cells specific for one autoantigen to deliver effective help nity. J. Immunol. 152: 5135–5141. to naive B cells that recognize a different autoantigen, thereby 8. Hancock, W. W., R. Buelow, M. H. Sayegh, and L. A. Turka. 1998. Antibody- providing a mechanism by which intermolecular diversification induced transplant arteriosclerosis is prevented by graft expression of anti- oxidant and anti-apoptotic genes. Nat. Med. 4: 1392–1396. occurs (53). 9. Lovegrove, E., G. J. Pettigrew, E. M. Bolton, and J. A. Bradley. 2001. Epitope Although our study examined additional graft alloantigen as the mapping of the indirect T cell response to allogeneic class I MHC: sequences focus for delivery of help, the corollary of our findings is that help shared by donor and recipient MHC may prime T cells that provide help for alloantibody production. J. Immunol. 167: 4338–4344. theoretically could be provided by concurrent T cell recognition of 10. Callaghan, C. J., F. J. Rouhani, M. C. Negus, A. J. Curry, E. M. Bolton, nontransplant Ag, provided that the Ag is expressed on the allo- J. A. Bradley, and G. J. Pettigrew. 2007. Abrogation of antibody-mediated al- lograft rejection by regulatory CD4 T cells with indirect allospecificity. J. graft. Of perhaps the most relevance, this provides a mechanism by Immunol. 178: 2221–2228. which viral-specific memory CD4 T cells responding to CMV 11. Benichou, G. 1999. Direct and indirect antigen recognition: the pathways to infection of the allograft could trigger alloantibody production. allograft immune rejection. Front. Biosci. 4: D476–D480. 12. Gould, D. S., and H. Auchincloss, Jr. 1999. Direct and indirect recognition: the Animal and human transplant studies highlighted a strong asso- role of MHC in graft rejection. Immunol. Today 20: 77–82. ciation between CMV infection and graft failure (54), with early 13. Jiang, S., O. Herrera, and R. I. Lechler. 2004. New spectrum of allorecognition progression to TA (55). Although a correlation between infection pathways: implications for graft rejection and transplantation tolerance. Curr. Opin. Immunol. 16: 550–557. and humoral alloimmunity has not been observed, endothelial 14. Bolton, E. M., J. A. Bradley, and G. J. Pettigrew. 2008. Indirect allorecognition: CMV infection can trigger antiviral CD4 T cell memory responses not simple but effective. Transplantation 85: 667–669. 15. Kelly, C. M., A. M. Benham, G. J. Sawyer, R. Dalchau, and J. W. Fabre. 1996. A (56); similarly, latent allograft infection can promote intragraft three-cell cluster hypothesis for noncognate T-B collaboration via direct T cell lymphoid neogenesis (57), a process that is likely to favor aug- recognition of allogeneic dendritic cells. Transplantation 61: 1094–1099. Downloaded from mented cellular responses. 16. Steele, D. J., T. M. Laufer, S. T. Smiley, Y. Ando, M. J. Grusby, L. H. Glimcher, and H. Auchincloss, Jr. 1996. Two levels of help for B cell alloantibody pro- Finally, our results add to the literature documenting a role for duction. J. Exp. Med. 183: 699–703. alloantibody in chronic allograft rejection (7, 8) and highlight the 17. Conlon, T. M., K. Saeb-Parsy, J. L. Cole, R. Motallebzadeh, M. S. Qureshi, additional distinction that duration of alloantibody exposure is S. Rehakova, M. C. Negus, C. J. Callaghan, E. M. Bolton, J. A. Bradley, and G. J. Pettigrew. 2012. Germinal center alloantibody responses are mediated important; severe TA only developed when memory T cells pro- exclusively by indirect-pathway CD4 T follicular helper cells. J. Immunol. 188: voked long-lasting humoral alloimmunity. The TA in this group 2643–2652. http://www.jimmunol.org/ 18. Noelle, R. J., and E. C. Snow. 1990. Cognate interactions between helper T cells is unlikely to be a consequence of heightened effector cellular and B cells. Immunol. Today 11: 361–368. responses mediated by an aggressive memory T cell population, 19. Sette, A., M. Moutaftsi, J. Moyron-Quiroz, M. M. McCausland, D. H. Davies, because similar memory responses in B cell–deficient Rag22/2 R. J. Johnston, B. Peters, M. Rafii-El-Idrissi Benhnia, J. Hoffmann, H. P. Su, et al. 2008. Selective CD4+ T cell help for antibody responses to a large viral recipients resulted in only modest graft arterial disease. However, pathogen: deterministic linkage of specificities. Immunity 28: 847–858. B cells are required for generating optimal CD4 T cell memory 20. Hunziker, L., M. Recher, A. J. Macpherson, A. Ciurea, S. Freigang, (58), and it is possible that the memory Mar T cell response H. Hengartner, and R. M. Zinkernagel. 2003. Hypergammaglobulinemia and 2/2 autoantibody induction mechanisms in viral infections. Nat. Immunol. 4: 343–349. generated in the Rag2 recipients was less robust than in the 21. Mombaerts, P., A. R. Clarke, M. A. Rudnicki, J. Iacomini, S. 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