Tolerance to host minor histocompatibility antigens after allogenic bone marrow transplantation. Specific donor-host unresponsiveness is maintained by peripheral tolerizing cells.

The development of graft-host tolerance after allogeneic bone marrow (BM) transplantation is more demanding than the acquisition of self-tolerance because both donor-derived mature T cells and immature thymocytes encounter host Ag. The mechanism involved in tolerization of mature T cells, contained in unmanipulated BM, remains undefined. In previous experiments, we showed in vivo unresponsiveness to host minor histocompatibility Ag (MiHA) in immunocompetent chimeras obtained after MiHA-incompatible BM transplantation. In this study, we wanted to determine: first, what was the specificity of this graft-host unresponsiveness, and second, whether peripheral cells were involved in tolerization? LP recipients were irradiated (9, 5 Gy), injected with 10(7) undepleted BM cells from B10 donors and studied 100 to 150 days later. (B10-->LP) chimeras were immunized in vivo and restimulated in vitro with cells displaying one or multiple incompatible MiHA. In bulk culture experiments, chimeras demonstrated specific CTL unresponsiveness to host MiHA but responded normally to third party MiHA. In limiting dilution analysis conditions, chimeras showed a profound deficit, but not a complete absence of anti-host CTL precursor. Studies with congenic stimulators/targets showed that graft-host tolerance was induced against both immunodominant (e.g., H-3.2) and nonimmunodominant (e.g., H-8.2) MiHA although at the CTL precursor level, it was more complete against the former. Furthermore, chimera spleen cells inhibited the generation of CTL activity against host- and donor-type MiHA but not against third party Ag. This specific suppressor activity was not T cell dependent, and was mediated by radiosensitive cells that are not found in freshly explanted organs from normal mice. Taken together, our results suggest that peripheral tolerization can be a remarkably efficient process to maintain tolerance to MiHA after BM transplantation. Thus, peripheral tolerizing mechanisms may contribute not only to the induction of tolerance to Mls superantigens or to the product of transgenes (if expressed at high levels) but also to a wide array of MiHA.     

Intrathymic T cell differentiation in radiation bone marrow chimeras and its role in T cell emigration to the spleen. An immunohistochemical study

Immunohistochemical studies were made on the regeneration of T cells of host- and donor-type in the thymus and spleen of radiation bone marrow chimeras by using B10- and B10.BR-Thy-1 congenic mice. Both the thymic cortex and the medulla were first repopulated with thymocytes of irradiated host origin, restoring the normal histologic appearance by days 11 to 14, regardless of the H-2 compatibility between the donor and the host. In Thy-1 congenic chimeras, thymocytes of donor bone marrow origin, less than 100 cells in one thymic lobe, were first recognized at day 7, when the thymus involuted to the smallest size after the irradiation. The thymocytes of donor-type then proliferated exponentially, showing a slightly faster rate when higher doses of bone marrow cells were used for reconstitution, reaching a level of 100 million by day 17 and completely replacing the cortical thymocytes of host origin by day 21. The replacement of cortical thymocytes started from the subcapsular layer in a sporadic manner. The replacement of medullary thymocytes from host- to donor-type occurred gradually between days 21 and 35, after the replacement in the cortex was completed. In the spleen, about 1 million survived cells were recovered at day 3 after the irradiation, and approximately 60% of them were shown to be host-type T cells that were observed in the white pulp areas. The host-type T cells in the spleen increased gradually after day 10, due to the influx of host-type T cells from the regenerating thymus. Thus a pronounced increase of T cells of host-type was immunohistochemically observed in the splenic white pulp between days 21 and 28, when thymocytes of host-type were present mainly in the thymic medulla. These host-type T cells were shown to persist in the spleen for a long time, as long as 420 days after the treatment. Phenotypically, they were predominantly Lyt-1+2+ when examined at day 28, but 5 mo later, they were about 50% Lyt-1+2+ and 50% Lyt-1+2-. Donor-type T cells in the spleen began to appear at about day 14 in chimeras that were transplanted with a larger dose of bone marrow cells, whereas this was slightly delayed in those grafted with a smaller dose of bone marrow cells, starting at about day 28.(ABSTRACT TRUNCATED AT 400 WORDS)     

Duration of alloantigen presentation and avidity of T cell antigen recognition correlate with immunodominance of CTL response to minor histocompatibility antigens

CD8 T lymphocytes (CTL) responsive to immunodominant minor histocompatibility (minor H) Ags are thought to play a disproportionate role in allograft rejection in MHC-identical solid and bone marrow transplant settings. Although many studies have addressed the mechanisms underlying immunodominance in models of infectious diseases, cancer immunotherapy, and allograft immunity, key issues regarding the molecular basis of immunodominance remain poorly understood. In this study, we exploit the minor H Ag system to understand the relationship of the various biochemical parameters of Ag presentation and recognition to immunodominance. We show that the duration of individual minor H Ag presentation and the avidity of T cell Ag recognition influence the magnitude and, hence, the immunodominance of the CTL response to minor H Ags. These properties of CTL Ag presentation and recognition that contribute to immunodominance have implications not only for tissue transplantation, but also for autoimmunity and tumor vaccine design.     

Soluble suppressor of T cell proliferation in primary in vitro response to murine minor histocompatibility antigens

Normal mouse lymphocytes are not capable of mounting a primary cytotoxic T cell response to Mls encoded, non H-2, allodeterminants, although a strong lymphoproliferative response is observed in primary MLR between Mls incompatible cells. In this study it is reported that in the supernatant of primary cultures between AKR macrophages and CBA/H lymphocytes (H-2 identical, incompatible for Mls and other minor antigens) a suppressor of T cell proliferation in MLR is detected. By contrast, a suppressor is not detected in supernatants from primary cultures between BALB/C macrophages and CBA/H lymphocytes (H-2 incompatible, Mls identical), B10.BR macrophages and CBA/H macrophages and CBA/H lymphocytes (syngeneic) suggesting that the production of the suppressor factor occurs only when an Mls incompatibility exists. The suppressive activity of the Mls incompatible culture supernatant upon MLR between incompatible macrophages and lymphocytes is neither antigen specific nor Mls or H-2 restricted, nor is it due to an irreversible toxic effect on T lymphocytes or macrophages. The inhibition of T cell proliferation could be explained by inhibition of IL 2 production, by blocking its union to T cells or by a combination of both effects. Our findings could help explain previous observations that lymphocytes from mice preimmunized with Mls incompatible cells have a depressed proliferative response as well as depressed cytotoxicity against alloantigens.     

Clonal deletion of self-reactive T cells at the early stage of T cell development in thymus of radiation bone marrow chimeras

Sequential appearance of T cell subpopulations occurs in the thymocytes of irradiated C3H/He mice (H-2k, Mls-1b2a, Thy-1.2) after transplantation with bone marrow cells of AKR/J mice (H-2k, Mls-1a2b, Thy-1.1) (AKR----C3H chimeras). The donor-derived thymocytes of AKR----C3H chimeras on day 14 after bone marrow transplantation (BMT) contained a large number of blastlike CD4+CD8+ cells which represent relatively immature thymocytes, whereas those on day 21 after BMT consisted of small sized CD4+,CD8+ cells which represent a great part in normal thymocytes. To define the developmental stage at which clonal deletion of self-reactive T cells occurs in adult thymus, we followed the fate of V beta 6- or V beta 11-bearing T cells in the donor-derived thymocytes at the early stage of AKR----C3H chimeras. Mature thymocytes expressing high intensity of V beta 6 or V beta 11, which are involved in recognition of Mls-1a or MHC I-E gene products, respectively, were deleted from the donor-derived thymocytes on day 21. Immature thymocytes expressing low intensity of V beta 6 in CD3low thymocyte fraction decreased in proportion, whereas those expressing low intensity of V beta 11 rather increased in proportion in the donor-derived thymocytes of AKR----C3H chimeras from day 14 to day 21 after BMT. These results suggest that the clonal deletion of V beta 6-positive cells occurs just at the stage of immature CD3lowCD4+CD8+ cells, whereas the clonal deletion of V beta 11-positive cells may begin at the transitional stage from CD3lowCD4+CD8+ cells to CD3high single positive cells. Timing of negative selection of thymocytes may vary in distinct T cells capable of recognizing different self-Ag.     

The human CD1-restricted T cell repertoire is limited to cross-reactive antigens: implications for host responses against immunologically related pathogens

The repertoires of CD1- and MHC-restricted T cells are complementary, permitting the immune recognition of both lipid and peptide Ags, respectively. To compare the breadth of the CD1-restricted and MHC-restricted T cell repertoires, we evaluated T cell responses against lipid and peptide Ags of mycobacteria in leprosy, comparing tuberculoid patients, who are able to restrict the pathogen, and lepromatous patients, who have disseminated infection. The striking finding was that in lepromatous leprosy, T cells did not efficiently recognize lipid Ags from the leprosy pathogen, Mycobacterium leprae, or the related species, Mycobacterium tuberculosis, yet were able to efficiently recognize peptide Ags from M. tuberculosis, but not M. leprae. To identify a mechanism for T cell unresponsiveness against mycobacterial lipid Ags in lepromatous patients, we used T cell clones to probe the species specificity of the Ags recognized. We found that the majority of M. leprae-reactive CD1-restricted T cell clones (92%) were cross-reactive for multiple mycobacterial species, whereas the majority of M. leprae-reactive MHC-restricted T cells were species specific (66%), with a limited number of T cell clones cross-reactive (34%) with M. tuberculosis. In comparison with the MHC class II-restricted T cell repertoire, the CD1-restricted T cell repertoire is limited to recognition of cross-reactive Ags, imparting a distinct role in the host response to immunologically related pathogens.     

Helper T cell lines to major and to minor histocompatibility antigens are equally effective in the regulation of B cell responses in vivo

Long-term lines of helper T (Th) cells, reactive to minor histocompatibility (minor-H) antigens, were grown by antigen restimulation in the absence of exogenous interleukin-2. These lines were antigen specific and H-2b restricted. When introduced in vivo by adoptive transfer, these Th cells helped syngeneic B cells in an antibody response to other alloantigens. Linked recognition was required for effective help to occur, this suggests B cell presentation of antigen to Th cells in vivo. Parallel titration experiments performed with long-term cultured Th lines to MHC and to minor-H antigens showed that, on a per cell basis, they are equivalent in their ability to help in vivo B cell responses. This shows that any inability to produce antisera to minor-H antigens is not due to a Th or APC defect, but results from either a B cell defect or from suppression.     

Effects of T cell depletion in radiation bone marrow chimeras. I. Evidence for a donor cell population which increases allogeneic chimerism but which lacks the potential to produce GVHD

The opposing problems of graft-vs-host disease (GVHD) and failure of alloengraftment present major obstacles to the application of bone marrow transplantation (BMT) across complete MHC barriers. The addition of syngeneic T-cell-depleted (TCD) bone marrow (BM) to untreated fully allogeneic marrow inocula in lethally irradiated mice has been previously shown to provide protection from GVHD. We have used this model to study the effects of allogeneic T cells on levels of chimerism in recipients of mixed marrow inocula. The results indicate that T cells in allogeneic BM inocula eliminate both coadministered recipient-strain and radioresistant host hematopoietic elements to produce complete allogeneic chimerism without clinical GVHD. To determine the role of GVH reactivity in this phenomenon, we performed similar studies in an F1 into parent combination, in which the genetic potential for GVHD is lacking. The presence of T cells in F1 marrow inocula led to predominant repopulation with F1 lymphocytes in such chimeras, even when coadministered with TCD-recipient-strain BM. These results imply that the ability of allogeneic BM cells removed by T cell depletion to increase levels of allochimerism may be mediated by a population which is distinct from that which produces GVHD. These results may have implications for clinical BM transplantation.     

Proliferative responses of T cells from SJL----F1 and F1----SJL bone marrow chimeras to SJL lymphoma cells

RCS tumor cells induce marked proliferation of syngeneic SJL T cells in vivo and in vitro. Certain F1 hybrids of SJL mice give high proliferative responses to gamma-RCS, while other F1 hybrids give low responses. SJL----"non-responder" F1 and "non-responder" F1----SJL semiallogeneic bone marrow chimeras were prepared to study how the host environment affects the ability of T cells to give a proliferative response to gamma-RCS. The results indicate that T cells educated in an SJL host become responsive to RCS cells, while T cells educated in an (SJL X BALB/c)F1 host become unresponsive. This finding applies to both thymus and lymph node T cells. The unresponsiveness in F1 mice is not due to suppressor cells, since added F1 cells do not affect the proliferative response of SJL cells to gamma-RCS. Instead, it appears that RCS-specific T cells are either deleted in (SJL X BALB/c)F1 mice, or expanded in SJL mice as they develop. These findings are discussed in relation to the specificity of the responding T cells, for LPS activated syngeneic B cell blasts as well as RCS cells, and to the presence of a "leaky" thymus barrier in SJL mice for B cells.     

Contrasting feature in the repopulation of host-type T cells in the spleens of F1----P and P----F1 radiation bone marrow chimeras

The regeneration and persistence of host- and donor-derived T cells were examined in the thymus as well as the spleen of mouse radiation bone marrow chimeras of two semiallogeneic combinations (F1----P, P----F1) with different Thy-1 markers on T cells of donor and host origins. An unexpectedly large number of host-type T cells were recovered from the spleens of F1----P chimeras, amounting to as high as 45 and 25% of total T cells at 6 and 14 weeks after bone marrow transplantation (BMT), respectively. To the contrary, the residual host-type T cells in the spleens of P----F1 chimeras disappeared quickly, resulting in less than 0.1% of total T cells at 6 weeks after BMT. It was also revealed that the number of host-type T cells in the spleens of F1----P chimeras decreased in proportion to increase of radiation dose given to the recipients.     

Induction of peripheral T cell tolerance by antigen-presenting B cells. I. Relevance of antigen presentation persistence

Various mechanisms of peripheral T cell tolerization have evolved to avoid responses mediated by autoreactive T cells that have not been eliminated in the thymus. In this study, we investigated the peripheral conditions of Ag presentation required to induce T cell tolerance when the predominant APCs are B cells. We show that transient Ag presentation, in absence of inflammation and in a self-context, induces CD4(+) T cell activation and memory formation. In contrast, chronic Ag presentation leads to CD4(+) T cell tolerance. The importance of long-lasting Ag presentation in inducing tolerance was also confirmed in the herpes stromal keratitis autoimmune disease model. Keratogenic T cells could be activated or tolerized depending on the APC short or long persistence. Thus, when APCs are B cells, the persistence of the Ag presentation itself is one of the main conditions to have peripheral T cell tolerance.     

Anti-recipient cytotoxic T lymphocyte precursors are present in the spleens of mice with acute graft versus host disease due to minor histocompatibility antigens

A model for bone marrow transplantation across minor histocompatibility barriers was developed by using mouse strains that were H-2 identical and mutually non-reactive in MLC. Acute graft-vs-host disease was induced only when donor lymphoid cells were included in the marrow inoculum, in both C57BL/6 recipients of LP cells and BALB/c recipients of B10.D2/nSN cells. GVHD was prevented by treating the lymphoid cells with anti-Thy 1.2 and C before transplantation. Spleen cells from mice with acute GVHD were not directly cytotoxic to recipient strain target cells. However, when spleen cells from mice with GVHD were boosted in vitro to recipient strain stimulator cells they generated a specific anti-recipient cytotoxic response. Spleen cells from mice without GVHD did not generate a cytotoxic response in vitro. The cytotoxic effector cells and their precursors were shown to be T lymphocytes. This model and the in vitro method described may be useful in further studies of the immunobiology of GVHD due to minor histocompatibility antigens and of transplantation tolerance.     

Functional T cell deficits after bone marrow transplantation across minor histocompatibility barriers: effects of graft-vs-host disease on precursor frequency of reactive cells

We have studied T cell responses in mice transplanted with bone marrow from H-2-identical, minor histocompatibility loci-nonidentical donors (B10.BR----CBA) in which graft-vs-host disease is induced by the addition of donor T cells. T cell responses to mitogen were examined both in high density, conventional bulk cultures and by limiting dilution analysis. Long-lasting deficits in the frequency of functional T cells were observed, for both IL 2-producing and cytotoxic cells, in proportion to the severity of the graft-vs-host disease induced. These deficits did not reflect a corresponding loss of Thy-1+ cells nor a loss of function in conventional cultures in mice studied at later times after bone marrow transplantation. These deficits in reactive cells are not completely correctable with IL 2, and provide further insight into the nature of T cell reconstitution of the immune system after bone marrow transplantation.     

Induction of peripheral T cell tolerance by antigen-presenting B cells. II. Chronic antigen presentation overrules antigen-presenting B cell activation

Ag presentation in the absence of danger signals and Ag persistence are the inductive processes of peripheral T cell tolerization proposed so far. Nevertheless, it has never been definitively shown that chronic Ag presentation per se can induce T cell tolerance independent of the state of activation of APCs. In the present work, we investigated whether chronic Ag presentation by either resting or activated B cells can induce tolerance of peripheral Ag-specific T cells. We show that CD4(+) T cells that re-encounter the Ag for a prolonged period, presented either by resting or activated Ag-presenting B cells, become nonfunctional and lose any autoimmune reactivity. Thus, when the main APCs are B cells, the major mechanism responsible for peripheral T cell tolerization is persistent Ag exposure, independent of the B cell activation state.     

Modification of the cytotoxic T cell repertoire in neonatal tolerance. Evidence for preferential survival of cells with low avidity for tolerogen

Clonal deletion of developing lymphocytes with potential reactivity for self is thought to play a crucial role in the establishment of self tolerance. One prediction of the clonal deletion hypothesis is that cells bearing receptors with high affinity for self are more likely than cells with low affinity receptors to be deleted from the repertoire. Experimental models of B cell tolerance have provided evidence for the preferential survival of low affinity cells with specificity for tolerogen in tolerant animals, but no comparable evidence exists for T cells. To examine this issue in T cells, cytotoxic T cell lines specific for the Kb mutant class I H-2 molecule, bm1, were generated from C57BL/6 mice rendered neonatally tolerant of bm1 and compared with anti-bm1 lines generated from normal mice. Compared with normal lines, those from tolerant mice differed in five ways: 1) they grew more slowly; 2) they were less efficient at lysing bm1 targets; 3) they showed different patterns of lysis against a panel of third party targets; 4) their cytotoxic activity against bm1 could be increased in the presence of leukoagglutinin, whereas the activity of normal lines was not increased by leukoagglutinin; and 5) their cytotoxic activity was more susceptible to inhibition by anti-Lyt-2 antibody. Taken together, these results demonstrate that the repertoire of the remaining tolerogen-specific cytotoxic T cells in neonatally tolerant mice is different from the normal C57BL/6 anti-bm1 repertoire, and the results are consistent with the idea that the remaining tolerogen-specific cells are low avidity cells that have preferentially escaped the clonal deletion process.     

Chromosome aberrations in peripheral lymphocytes and radiation dose to active bone marrow in patients treated for cancer of the cervix

An international study of cervical cancer patients reported a doubling of the risk for leukemia following radiotherapy. To evaluate the extent of residual chromosome damage in circulating T-cell lymphocytes in this population, approximately 200 metaphases were examined from each of 96 irradiated and 26 nonirradiated cervical cancer patients treated more than 17 years ago (average 23 years). Radiation dose averaged over the total red bone marrow was estimated to be 8.1 Gy. The type and frequency of stable and unstable chromosome aberrations were quantified in 24,117 metaphases. Unstable aberrations did not differ significantly between irradiated and nonirradiated patients (P greater than 0.5). Stable aberrations (i.e., translocations, inversions, or chromosomes with deleted segments), however, were significantly higher among irradiated (2.8 per 100 cells) compared to nonirradiated (0.7 per 100 cells) women (P less than 10(4). The frequency of these stable aberrations was found to increase significantly with increasing dose to the bone marrow. These data indicate that a direct relationship between radiation dose and extent of damage to somatic cells persists in populations and can be detected many years after partial-body radiation exposure. The stable aberration rate in irradiated cervical cancer patients was 50 to 75% lower than those observed 25 years or more after radiation exposure in atomic bomb survivors and in ankylosing spondylitis patients treated with radiotherapy. The average marrow dose was only 1 Gy in the examined atomic bomb survivors and 3.5 Gy in the ankylosing spondylitis patients. It appears, then, that a very high dose delivered to the pelvic cavity in fractionated doses resulted in far fewer persistent stable aberrations than lower doses delivered either in acute whole-body exposure or in fractionated doses to the spinal column and sacroiliac joints. The higher radiation dose and the concentration of that dose in a smaller area of the body appear to be responsible for the lower rate of persistent aberrations observed in cervical cancer patients.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. IV. Partial tissue distribution and mapping of immunodominant antigens

The immunization of C57BL/6 responder mice with spleen cells from H-2-matched BALB.B donors, which differ by multiple non-H-2 histocompatibility (H) antigens, results in the generation of cytotoxic T lymphocytes (CTL) that are specific for only a limited number of immunodominant antigens. Previous analysis of the genes encoding these dominant antigens has not mapped these genes to any of the non-H-2 H loci defined by congenic strains. It would have been expected that the histogenetic techniques employed for congenic strain selection would have preferentially identified the "strongest" H antigens. Therefore, we have investigated the possibility that immunodominant antigens do not belong to the class of non-H-2 H antigens encoded by genes mapping to H loci defined and mapped by congenic strains. The first experiments were aimed at identifying antigens that were expressed by independently derived inbred strains and were cross-reactive with the immunodominant cytotoxic T cell target (CTT-1) antigen of BALB.B. Strong cross-reaction with the C3H.SW (H-2b) strain was observed; the C3H gene encoding this antigen was mapped with BXH recombinant inbred strains. Contrary to the mapping of the CTT-1 gene to chromosome 1 in BALB.B, the C3H gene was shown to map to either chromosome 4 or chromosome 7. This result indicates that identical, or at least extensively cross-reactive, non-H-2 antigens may be encoded by genes mapping to independently segregating loci in different inbred strains. The tissue distribution of immunodominant antigens was approached by determining the reactivity of CTL specific for these antigens with either lymphoid-derived or fibroblast-derived targets. These CTL effectively lysed lymphoblast and lymphoid tumor targets but did not lyse an SV40-transformed fibroblast line that was shown to be efficiently lysed by CTL specific for non-H-2 H antigens defined by congenic strains. Therefore, it was concluded that immunodominant antigens detected by B6 anti-BALB.B CTL have a restricted tissue distribution in comparison to non-H-2 H antigens defined by congenic strains. The implications of these results for our understanding of the origin and heterogeneity of non-H-2 cell-surface antigen recognized by effector T cells are discussed.     

Effect of selective T cell depletion of host and/or donor bone marrow on lymphopoietic repopulation, tolerance, and graft-vs-host disease in mixed allogeneic chimeras (B10 + B10.D2----B10)

Reconstitution of lethally irradiated mice with a mixture of T cell-depleted syngeneic plus T cell-depleted allogeneic bone marrow (B10 + B10.D2----B10) leads to the induction of mixed lymphopoietic chimerism, excellent survivals, specific in vivo transplantation tolerance to subsequent donor strain skin grafts, and specific in vitro unresponsiveness to allogeneic donor lymphoid elements as assessed by mixed lymphocyte reaction (MLR) proliferative and cell-mediated lympholysis (CML) cytotoxicity assays. When B10 recipient mice received mixed marrow inocula in which the syngeneic component had not been T cell depleted, whether or not the allogeneic donor marrow was treated, they repopulated exclusively with host-type cells, promptly rejected donor-type skin allografts, and were reactive in vitro to the allogeneic donor by CML and MLR assays. In contrast, T cell depletion of the syngeneic component of the mixed marrow inocula resulted in specific acceptance of allogeneic donor strain skin grafts, whether or not the allogeneic bone marrow was T cell depleted. Such animals were specifically unreactive to allogeneic donor lymphoid elements in vitro by CML and MLR, but were reactive to third party. When both the syngeneic and allogeneic marrow were T cell depleted, variable percentages of host- and donor-type lymphoid elements were detected in the mixed reconstituted host. When only the syngeneic bone marrow was T cell depleted, animals repopulated exclusively with donor-type cells. Although these animals had detectable in vitro anti-host (B10) reactivity by CML and MLR and reconstituted as fully allogeneic chimeras, they exhibited excellent survival and had no in vivo evidence for graft-vs-host disease. In addition, experiments in which untreated donor spleen cells were added to the inocula in this last group suggest that the presence of T cell-depleted syngeneic bone marrow cells diminishes graft-vs-host disease and the mortality from it. This system may be helpful as a model for the study of alloresistance and for the identification of syngeneic cell phenotypes, which when present prevent engraftment of allogeneic marrow.