Supplementary Figures 1-6 from Concomitant Tumor and Minor Histocompatibility Antigen–Specific Immunity Initiate Rejection and Maintain Remission from Established Spontaneous Solid Tumors

Clinical bone marrow transplantation started in 1957 at a time when remarkably little was known about hematopoietic stems cells, immune responses to transplants or the identity of transplant antigens. This review will delineate the substantial increase in knowledge about these three areas gained between then and 1992 when the Ceppellini School course on Bone Marrow Transplantation was held, along with the progress made in clinical application, as well as the stumbling blocks that remained to be overcome by further research to advance knowledge. It will outline the significant progress made between 1992 and the present year, 2019, and the remaining problems.

Memory T cells are the very essence of adaptive immunity with their rapid and efficient response to antigen rechallenge and long-term persistence. However, it is becoming increasingly evident that when primed with self or transplanted tissue, these cells play a key role in causing and perpetuating tissue damage. Furthermore, current treatments, which efficiently control the naive response, have limited effects on primed T cells. We have used a treatment based on a combination of antibodies specific for molecules expressed by activated T lymphocytes to selectively remove these cells. This approach, which we termed multi-hit therapy, leads to cumulative binding of antibodies to the target T cells and a striking prolongation of skin graft survival in presensitized recipients in a stringent skin transplant model. The findings are consistent with the depletion of graft-specific CD4+ and CD8+ T cells, although other modes of action, such as T-cell regulation and altered migration could play a role. In conclusion, our therapeutic strategy controls primed T cells which are a major driving force in the pathology of many autoimmune diseases and in transplant rejection.

H-Y antigen is expressed in mammals only by males, so that grafts of male tissue are rejected by females within certain highly inbred strains. H-Y antigen appears to be a simple, non-polymorphic antigen and the genetic control of anti-H-Y responses has been extensively studied. In this article Elizabeth Simpson discusses the many insights obtained.

The major histocompatibility complex (MHC) plays an important role in the regulation of the immune response in vertebrates. The MHC in man (HLA) and the mouse (H-2) is the most thoroughly studied case of a complex of linked genes (see [I] for a review). The H-2 locus maps on the murine chromosome 17 (Fig. 1). Three different types of proteins are encoded by the H-2 region. The class I molecules regulate the killing of virus-infected cells. The virus antigen is recognized in association with class I proteins. This is known as MHC restriction of T-cell recognition [2]. The bestcharacterized class I molecules are the classical transpantation antigens found on virtually all cells, encoded by the H-2K, H-2D, and H-2L loci. Class I antigens are also encoded by the TL complex, adjacent to the classical H-2 loci. The Qa and Tla proteins are lymphoid differentiation antigens. Class I molecules are intrinsic membrane proteins with molecular weights (MW) of 40,000-45,000. They are associated with a smaller polypeptide, ß, microglobulin, MW 12,000, not encoded by chromosome 17. The class I1 genes were originally discovered as immune-response genes. They regulate the interaction of T-helper cells and B cells to induce antibody prod~ction [3]. The class I11 molecules encoded by the H-2 region are complement components. One of the most remarkable properties of the H-2 antigens is their genetic polymorphism. In fact, about 50 alleles at both the H-2K and H-2D loci have been detected. In this aspect, they differ sharply from almost all other genes. We are particularly interested in how the H-2 polymorphism is generated. We think it likely that the analysis of the class I genes by recombinant DNA techniques will provide some clues to the nature of the polymorphism.

There is evidence showing that high avidity CTLs can be more effective than low avidity CTLs for adoptive tumor immunotherapy. Because many T cell-recognized tumor antigens are nonmutated self-proteins, tolerance mechanisms are likely to render high avidity T cells unresponsive or cause T cell elimination by clonal deletion. We recently used the allo-restricted strategy to circumvent immunologic tolerance to a ubiquitously expressed tumor-associated protein, MDM2, and raised high avidity CTLs in humans and in mice. In this study, we investigated whether high avidity MDM2-specific CTLs can mediate tumor protection without causing damage to normal tissues in mice. Although the CTLs prolonged survival of tumor-bearing mice without causing damage to normal tissues, tumor protection was incomplete. We show that tumor growth occurred despite the continued presence of MDM2-specific CTLs and the continued susceptibility of tumor cells to CTL killing. However, analysis of the CTLs revealed that they had been rendered unresponsive in vivo because they did not produce interferon gamma in response to antigen-specific stimulation. These experiments suggest that induction of unresponsiveness may be an important mechanism limiting the efficacy of adoptive CTL therapy.

Vox SanguinisVolume 87, Issue s2 p. 11-14 Minor histocompatibility antigens and stem cell transplantation R. Laylor, R. Laylor Imperial College London, UKSearch for more papers by this authorL. Cannella, L. Cannella Imperial College London, UKSearch for more papers by this authorE. Simpson, E. Simpson Imperial College London, UKSearch for more papers by this authorF. Dazzi, Corresponding Author F. Dazzi Imperial College London, UKFrancesco Dazzi, Imperial College London, Commonwealth Building, Hammersmith Hospital, Du Cane Road, W12 ONN, UK E-mail: [email protected]Search for more papers by this author R. Laylor, R. Laylor Imperial College London, UKSearch for more papers by this authorL. Cannella, L. Cannella Imperial College London, UKSearch for more papers by this authorE. Simpson, E. Simpson Imperial College London, UKSearch for more papers by this authorF. Dazzi, Corresponding Author F. Dazzi Imperial College London, UKFrancesco Dazzi, Imperial College London, Commonwealth Building, Hammersmith Hospital, Du Cane Road, W12 ONN, UK E-mail: [email protected]Search for more papers by this author First published: 22 June 2004 https://doi.org/10.1111/j.1741-6892.2004.00445.xCitations: 6Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume87, Issues2July 2004Pages 11-14 RelatedInformation

and in 1971 moved to the Basle Institute for Immunology, under the direction of Niels K. Jerne

Injection of female C57BL/6 mice with immature female bone marrow-derived dendritic cells (BMDC) pulsed with a single immunodominant HY(Db) Uty peptide, WMHHNMDLI, induces prolonged survival of syngeneic male skin grafts. In contrast, injection of immature female BMDC pulsed with a single MHC class I-restricted HY(Ab) Dby peptide, NAGFNSNRANSSRSS, causes immunization similar to that following injection of male cells. Tolerance induced by HY(Db) Uty peptide pretreatment is not characterized by clonal deletion: long-term tolerant mice maintain circulating HY(Db) Uty tetramer(+) T cells which expand following exposure to male cells in vivo or in vitro. Tolerance to male skin grafts can be adoptively transferred into neonatal females with splenocytes from tolerant donors. Tolerance is specific-third-party skin grafts are rejected. We propose that tolerance in this model is initiated by cognate interaction of HY(Db) Uty-specific CD8(+) T cells with their ligand, presented either on the injected immature BMDC or on recipient DC. This interaction leads to incomplete activation of the CD8(+) T cells resulting in diminished responsiveness of CD4(+) and CD8(+) T cells specific for HY peptide epitopes subsequently presented on the male graft.

Genes controlling both testis determining and expression of the male-specific transplantation antigen, HY, are located on the short arm of the mouse Y chromosome, and on the X and Y-linked translocation, Sxr(a). A mutation of Sxr(a) was discovered in a cross between an Sxr carrier male and a T16H/X female. This was designated Sxr(b) and found to affect both the expression of HY and spermatogenesis, but not testis differentiation, thereby disproving Ohno's hypothesis that HY controlled testis determination. Molecular genetic analysis showed the mutation to be caused by fusion of two duplicated genes, Zfy1 and Zfy2, deleting the intervening DNA. This deletion interval, deltaSxr(b), contained a number of genes, each a candidate HY gene. Expression cloning with HY-specific T cell clones identified Smcy, Uty and Dby as encoding peptide epitopes of this transplantation antigen. The human homologues SMCY and UTY likewise express HY antigens and these are targets of damaging graft-versus-host (GVH) responses and potentially therapeutic graft-versus-leukaemia (GVL) responses following bone marrow transplantation (BMT). Knowledge of the peptide identity of HY epitopes allows monitoring of immune responses following BMT, using fluorescent tetramers, and also offers the possibility of inducing immunological tolerance.

We have applied MHC class I tetramers representing the two H2(b) MHC class I-restricted epitopes of the mouse male-specific minor transplantation Ag, HY, to directly determine the extent of expansion and immunodominance within the CD8(+) T cell compartment following exposure to male tissue. Immunization with male bone marrow (BM), spleen, dendritic cells (DCs) and by skin graft led to rapid expansion of both specificities occupying up to >20% of the CD8(+) T cell pool. At a high dose, whole BM or spleen were found to be more effective at stimulating the response than BM-derived DCs. In vivo, immunodominance within the responding cell population was only observed following chronic Ag stimulation, whereas epitope immunodominance was established rapidly following in vitro restimulation. Peptide affinity for the restricting MHC molecule was greater for the immunodominant epitope, suggesting that this might be a factor in the emergence of immunodominance. Using tetramers, we were able to directly visualize the cross-primed CD8(+) HY response, but we did not find it to be the principal route for MHC class I presentation. Immunization with female spleen or DCs coated with the full complement of defined HY peptides, including the A(b)-restricted CD4(+) Th cell determinant, failed to induce tetramer-reactive cells.

BACKGROUND:At present, it is not clear whether xenogeneic MHC molecules are recognized by T cells directly or indirectly through self-MHC-restricted presentation in a transplantation setting.METHODS:We have transplanted skin from HLA-A2 transgenic (B6.A2) to nontransgenic C57BL/6 (B6) mice and investigated the subsequent mouse T-cell responses to HLA molecules, in vivo and in vitro.RESULTS:Skin transplanted from transgenic B6.A2 to B6 mice was rejected rapidly, in 12-16 days. Although naive B6 mice did not respond to B6.A2 splenocytes in vitro, spleen cells from mice that underwent transplantation showed strong proliferative responses. An anti-B6.A2 T-cell line from mice that underwent transplantation made proliferative responses to B6.A2 splenocytes but did not recognize HLA-A2 on human cells or transfected allogeneic mouse cells. The indirect, self-H-2-restricted recognition of HLA-A2 implied by this was confirmed by the finding that lysates of HLA-A2-positive, but not HLA-A2-negative, human B cells were stimulatory when pulsed onto syngeneic antigen-presenting cells and by inhibition of anti-B6.A2 proliferation with both anti-mouse MHC class I and class II antibodies.CONCLUSION:Our results suggest that indirect recognition of xenogeneic MHC antigen plays a predominant role in graft rejection.

In this review, we describe the evidence from which the existence of non-MHC histocompatibility (H) antigens was deduced, the clinical setting of bone marrow transplantation in which they are important targets for T-cell responses, and the current understanding of their molecular identity. We list the peptide epitopes of the human and murine minor H antigens now identified at the molecular level, their MHC restriction molecules and the genes encoding them. Identification of the peptide epitopes allows T-cell responses to these antigens following transplantation of MHC-matched, minor H-mismatched tissues to be enumerated using tetramers and elispot assays. This will facilitate analysis of correlations with host-versus-graft (HVG), graft-versus-host (GVH) and graft-versus-leukaemia (GVL) reactions in vivo. The potential to use minor H peptides to modulate in vivo responses to minor H antigens is discussed. Factors controlling immunodominance of T-cell responses to one or a few of many potential minor H antigens remain to be elucidated but are important for making predictions of in vivo HVG, GVH and GVL responses and tailoring therapy after HLA-matched bone marrow transplantation and donor lymphocyte infusion.

Minor or histocompatibility (H) antigens are recognized by CD4+ and CD8+ T lymphocytes as short polymorphic peptides associated with MHC molecules. They are the targets of graft versus host and graft versus leukemia responses following bone marrow transplantation between HLA-identical siblings. Several genes encoding class I-restricted minor H epitopes have been identified, but approaches used for these have proved difficult to adapt for cloning class II-restricted minor H genes. We have combined the unique antigen-presenting properties of dendritic cells and high levels of episomal expression following transfection of COS cells to identify a Y chromosome gene encoding two HY peptide epitopes, HYAb and HYEk.