H-2 complementation in anti-H-Y cytotoxic T-cell responses can occur in chimeric mice.

Cytotoxic T-cell responses against the H-Y antigen in mice are under the control of major histocompatibility complex genes. Not only must cytotoxic T cells recognize both H-Y antigens and "self" H-2K/D molecules to lyse male target cells, but also "appropriate association" between H-Y antigens and H-2K/D antigens is required to induce such cytotoxic responses. Furthermore, it is suggested that appropriate association with H-2-I antigens may also be required to generate H-Y specific helper cells for the cytotoxic response. BALB/c(KdIdDd) mice are nonresponders against syngeneic H-Y antigens, because they lack appropriate associative H-2K/D antigens. This results in the failure of generation of anti H-Y cytotoxic cells, although helper cells may be induced. F1 hybrid mice (BALB/c X C3H/He)F1 or H-2 recombinant mice C3H-OH(KdIdDk) are responders, because H-2Dk (and H-2Kk in the F1) molecules offer appropriate association to H-Y antigens. We here report that allophenic chimeras (H-2d reversible H-2k) and irradiation bone marrow chimeras [H-2d + H-2k leads to F1(H-2d X H-2k)] generate anti-H-Y cytotoxic responses but that cells of the BALB/c(H-2d) genotype comprise most if not all of the cytotoxic cells. A working model is proposed to account for major histocompatibility complex control over anti-H-Y cytotoxic T-cell responses.     

Studies of immunologic tolerance to host minor histocompatibility antigens following allogeneic bone marrow transplantation in mice

We showed previously that transplantation of 10(7) unmanipulated C57BL/6 marrow cells to irradiated LP mice yields healthy (B6-LP) chimeras showing no signs of rejection or graft-versus-host disease (GVHD). The aim of this work was to gain more insight into the mechanism(s) responsible for tolerance to host minor histocompatibility antigens following allogeneic bone marrow transplantation (BMT). (B6-LP) chimeras showed very good immune reconstitution when studied in vitro for proliferative response to mitogens and alloantigens and generation of T cell cytotoxic activity. In co-culture experiments their spleen cells showed no natural suppressor activity. When used as cell donors, their capacity to initiate GVHD in four strains of mice presenting H-2 differences was normal when compared to C57BL/6 donors. However, they provoked no GVHD in the three strains of H-2 compatible mice studied. Re-irradiated (B6-LP) chimeras rapidly died of GVHD following injection of C57BL/6 marrow + spleen cells. (B6-LP.R111) chimera cells appeared tolerant to LP minor antigens presented in the context of H-2r or H-2b. No anamnestic anti-idiotypic suppressor response was noted when stable (B6-LP) chimeras were stimulated with naive C57BL/6 cells. These findings suggest that in BMT chimeras transplanted across minor histocompatibility barriers: (1) both host and donor-derived antigen-presenting cells can present host antigens to donor T cells whose numbers in the marrow inoculum will determine if GVHD or tolerance will ensue, (2) GVHD can be triggered by only a limited number of 'dominant' minor antigens, and (3) we found no evidence for the presence of natural suppressors, veto cells or anti-idiotypic suppressor T cells.     

Generation of the alloreactive T-cell repertoire: interaction of T-cell genotype and maturation environment.

The influences of the T-cell genotype and the T-cell maturation environment on generation of the T-cell alloreactive repertoire were evaluated in cytotoxic T-lymphocyte responses to Kb mutant determinants expressed by the strains B6. C-H-2bm1 and B6-H2-bm6. By constructing bone marrow chimeras using either H-2b of H-2d mice as the source of donor cells and either H-2d or H-2b irradiated mice as recipients, it was first determined whether the T-cell major histocompatibility complex genotype alone determines the alloreactive repertoire. The results of such experiments indicated that H-2b T cells that have matured in a normal H-2b environment (C57BL/6N, C57BL/10Sn) or H-2d T cells that have matured in a chimeric H-2b environment (B10.D2 leads to C57BL/10Sn) are responsive to Kbm1 and Kbm6 determinants while H-2b T cells that have matured in an H-2d chimeric environment (C57BL/10Sn leads to B10.D2) have a diminished responsiveness to H-2bm1 and are completely unresponsive to H-2bm6. These findings showed that T-cell genotype alone does not determine the alloreactive repertoire to mutant Kbm1 and Kbm6 determinants and suggested that the T-cell maturation environment plays a critical role in this process. Further studies were carried out to determine whether the T-cell maturation environment alone determines this repertoire, such that maturation in an H-2b but not in an H-2d environment is both necessary and sufficient to generate reactivity to Kbm6. Experiments in which either H-2d responder populations neonatally made tolerant to H-2b or unlabeled target blocking of normal H-2d responders were used provided evidence that T cells specific for Kbm6 mutant determinants are present in the repertoire of H-2d T cells that have matured in an H-2d environment. These findings suggest that the alloreactive T-cell repertoire is not determined by the T-cell major histocompatibility complex genotype alone or by the T-cell maturation environment alone but rather than it is the product of unique interactions between the two.     

Host stem cells can selectively reconstitute missing lymphoid lineages in irradiation bone marrow chimeras

The regulatory elements governing the process of lymphopoiesis from pluripotential stem cells to mature lymphocytes are not well understood. In this study we found that in bone marrow chimeras made by reconstituting lethally irradiated normal mice with bone marrow taken from genetically B-cell-deficient animals (microMT.B6 --> F1) the B-cell compartment is reconstituted with host-derived B cells. Similarly, in animals reconstituted with bone marrow taken from mice with genetic deficiencies in the development of T cells (TCR-/- --> F1) or both B and T cells (RAG-/- --> F1), the missing lymphocyte lineage(s) was specifically reconstituted from host-derived cells. In all chimeras, all other blood lineages were generated from donor-derived stem cells. Control chimeras (B6 --> F1) had only donor-derived hematopoietic cells as expected. The reconstituted, host-derived lymphoid compartments contained normal functional cell populations as determined by the presence of T cells expressing all 16 common TCR Vbeta families, and the presence of all antibody isotypes in the serum. Reconstituted TCR-/- --> F1 chimeras were also able to mount T-cell proliferative responses to foreign antigens equal to those of control animals. This observation would seem to suggest that during lymphopoietic reconstitution, missing lymphoid lineages can dictate their own reconstitution.     

Significant MLR but not CTL responses against recipient antigens generated in T cells from bone marrow chimeras recovered from acute GVHD

Lethally irradiated AKR mice received BMT from H-2D and minor lymphocyte stimulatory (Mls)-1 disparate B10.A mice. No GVHD signs were detected in AKR recipients of T cell-depleted BM cells (1 x 10(7)) alone ([B10.A --> AKR] T-). When B10.A splenic T cells (1 x 10(5)) were injected in addition to T cell-depleted BM cells ([B10.A --> AKR] T+), overt GVHD was observed. [B10.A --> AKR] T+ chimeras recovered from the GVHD 8 weeks after BMT. In T cells from these [B10.A --> AKR] T+ chimeras, a substantial population of Mls-1a-reactive Vbeta6+ T cells was present, whereas the Vbeta6+ cells were deleted in [B10.A --> AKR] T- chimeras. T cells from [B10.A --> AKR] T+ chimeras showed considerable MLR but no CTL response against AKR cells (split tolerance). Upon stimulation with AKR stimulators or anti-CD3 MoAb, T cells from [B10.A --> AKR] T+ chimeras produced significantly more IL-4 but significantly less IFN-gamma compared with those from [B10.A --> AKR] T- chimeras or unmanipulated B10.A mice. The serum level of IgG1 in [B10.A --> AKR] T+ chimeras was also significantly higher than that in [B10.A --> AKR] T- or B10.A mice. The present findings suggest that the split tolerance observed in BMT chimeras recovered from GVHD is attributable to the Th2 dominant state.     

Thymus: a direct target tissue in graft-versus-host reaction after allogeneic bone marrow transplantation that results in abrogation of induction of self-tolerance.

Graft-versus-host reaction (GVHR) following allogeneic bone marrow (BM) transplantation was investigated by analyzing expression of antigen receptors on T cells specific for recipient antigens. GVHR chimeras were prepared by transplanting mixtures of splenic T cells and T-cell-depleted BM cells from B10 (I-E-, Mls-1b) or B10.AQR (I-E+, Mls-1b) mice into lethally irradiated AKR/J (I-E+, Mls-1a) recipients. Increased proportions of V beta 6+ T cells reactive to recipient antigens (I-E and Mls-1a) were observed in thymuses from such chimeras 1 or 5 wk after BM transplantation. V beta 6+ T cells observed 1 wk after BM transplantation were derived from mature T cells that had been inoculated into recipients. These cells responded to recipient antigens expressed in the thymus. After 5 wk, thymocytes brightly positive for V beta 6+ were shown not to descend from mature T cells but to differentiate from precursor cells present in the BM inocula. Since V beta 6+ T cells were eliminated in thymuses from non-GVHR chimeras 5 wk after BM transplantation using T-cell-depleted BM cells alone, it appears that GVHR occurring in the thymus at an early stage abrogates thymic stromal functions essential to induction of self-tolerance in the T-cell repertoire. These findings propose a mechanism (autoimmunity) to explain in part the pathogenesis of chronic GVHR.     

H-2-incompatible bone marrow chimeras produce donor-H-2-restricted Ly-2 suppressor T-cell factor(s).

To study adaptive-differentiation phenomena of T lymphocytes, suppressor T-cell factors (TsF) produced by Ly-2+ splenic T cells from fully allogeneic mouse bone marrow chimeras were analyzed. AKR mice irradiated and reconstituted with B10 marrow cells (B10----AKR chimeras) produced an Ly-2+ TsF after hyperimmunization with sheep erythrocytes. The TsF suppressed primary antibody responses (to sheep erythrocytes) generated with spleen cells of mice of H-2b haplotype but not those of H-2k haplotype. Thus, this suppressor factor was donor-H-2-restricted. The immunoglobulin heavy chain variable region gene (Igh-V)-restricting element was not involved in this form of suppression. Similar results were obtained when TsF from B6----BALB/c and BALB/c----B6 chimeras were analyzed. The TsF from B10----AKR chimeras suppressed responses of B10.A(3R) and B10.A(5R) mice but not those of B10.A(4R). This finding showed that identity between the factor-producing cells and target spleen cells is required on the left-hand side of the E beta locus of the H-2 region and that the putative I-Jb locus is not involved in this form of suppression. The present results support the postulate that post-thymic differentiation in the presence of continued or repeated stimulation with antigen and donor-derived antigen-presenting cells generates donor-H-2-restricted T-cell clones that may predominate within the repertoire of the specific antigen being presented.     

Marrow transplantation from tolerant donors to treat and prevent autoimmune diseases in BXSB mice.

Autoimmune-prone BXSB male mice were supralethally irradiated and transplanted with CBA/H bone marrow cells. A complete and long-term chimerism was established when donor mice had been induced to develop tolerance of BXSB male antigens by combined treatment with BXSB male spleen cells and cyclophosphamide. Such chimeras did not express autoimmune phenomena or develop lethal autoimmune manifestations. Nor did the recipient mice develop the wasting syndrome or evidence of persistent immunodeficiencies that have been seen in other strains of autoimmune-resistant mice that had been transplanted with bone marrow cells across major histocompatibility complex barriers following an initial purging of the bone marrow of Thy-1+ cells using anti-Thy-1+C.     

Graft-host tolerance in bone marrow transplant chimeras. Absence of graft-versus-host disease is associated with unresponsiveness to minor histocompatibility antigens expressed by all tissues

Because bone marrow (BM) transplantation is used with increasing frequency, it is important to elucidate the mechanisms involved in the establishment of tolerance to host minor histocompatibility antigens (MiHA) in recipients transplanted with T-cell-undepleted marrow grafts. We have previously shown that BM chimeras transplanted across MiHA barriers showed specific unresponsiveness to MiHA expressed on recipient-type concanavalin A blasts. Because expression of many MiHA is tissue-specific, we wanted to determine if chimera T lymphocytes would be tolerant to MiHA expressed by all host tissues and organs. To investigate this issue, we measured in vivo proliferation of lymphoid cells from normal C57BL/10 (B10) mice and (B10-->LP) chimeras in tissues and organs of lethally irradiated syngeneic and allogeneic recipients. Donor B10 cells were either untreated, or depleted with anti-Thy-1.2, anti-CD4, or anti-CD8 antibodies. Transplantation of B10 cells in LP recipients triggered an important T-cell-dependent 125I- dUrd uptake in several organs that involved both CD4+ and CD8+ cells. Using Thy-1-congeneic mice we showed that in long-term chimeras practically all CD4+ and CD8+ T lymphocytes were derived from hematopoietic progenitors and not from mature T cells present in the BM graft. When (B10-->LP) BM chimera cells were injected to secondary recipients, no proliferation was observed in any organ of LP hosts whereas normal proliferation was seen in H-2k allogeneic hosts. Thus, in these BM chimeras, tolerance encompasses MiHA expressed by all organs.     

Bone marrow-derived thymic antigen-presenting cells determine self-recognition of Ia-restricted T lymphocytes.

We previously have demonstrated that in radiation-induced bone marrow chimeras, T-cell self-Ia restriction specificity appeared to correlate with the phenotype of the bone marrow-derived antigen-presenting (or dendritic) cell in the thymus during T-cell development. However, these correlations were necessarily indirect because of the difficulty in assaying thymic function directly by adult thymus transplant, which has in the past been uniformly unsuccessful. We now report success in obtaining functional T cells from nude mice grafted with adult thymuses reduced in size by treatment of the thymus donor with anti-thymocyte globulin and cortisone. When (B10 Scn X B10.D2)F1 nude mice (I-Ab,d) are given parental B10.D2 (I-Ad) thymus grafts subcutaneously, their T cells are restricted to antigen recognition in association with I-Ad gene products but not I-Ab gene products. Furthermore, thymuses from (B10 X B10.D2)F1 (I-Ab,d)----B10 (I-Ab) chimeras transplanted 6 months or longer after radiation (a time at which antigen-presenting cell function is of donor bone marrow phenotype) into (B10 X B10.D2)F1 nude mice generate T cells restricted to antigen recognition in association with both I-Ad and I-Ab gene products. Thymuses from totally allogeneic bone marrow chimeras appear to generate T cells of bone marrow donor and thymic host restriction specificity. Thus, when thymus donors are radiation-induced bone marrow chimeras, the T-cell I-region restriction of the nude mice recipients is determined at least in part by the phenotype of the bone marrow-derived thymic antigen presenting cells or dendritic cells in the chimeric thymus.     

Influence of H-2 antigen expression on killer T cell specificity, differentiation, and induction.

Murine cytotoxic T lymphocytes (CTL) and helper cells are H-2 antigen restricted in their specificity: recognition of foreign antigen by these cells requires the concomitant recognition of self-H-2 molecules. Which H-2 antigens T cells treat as "self" is determined by the particular H-2 antigens expressed on radioresistant cells of the thymus in which these T cells mature. Using tetraparental [(P1 + P2) leads to F1] radiation chimeras with in situ F1 thymuses, we have found that the H-2 genotype of the stem cells does not influence their H-2 restriction specificity. This has allowed us to use tetraparental chimeras that have been thymectomized and grafted with parental (P1, P2, or both) thymus lobes to study the requirements for H-2-restricted T--T interactions during CTL ontogeny and induction. In animals that have received thymus grafts of both parental origins, CTL display no preference for maturation within a syngeneic thymus graft, a finding that is not compatible with a suggested requirement for intrathymic H-2-restricted T--T interactions in the maturation of precursor CTL. We have also grafted thymectomized tetraparental radiation chimeras with thymus grafts from only one parent to compare the induction of P1 and P2 CTL in environments in which peripheral (extrathymic) T cell interactions are restricted to one H-2 haplotype. Again, we find no evidence for preferential induction of CTL precursors syngeneic to the thymus graft, contrary to expectation if CTL induction requires that T helper cells restricted to thymic H-2 antigens interact directly with precursor CTL. In those animals with one parental thymus graft, there is variability in the ratios of P1 and P2 cells induced with several antigens, a finding that may be indicative of an H-2-restricted suppression mechanism operating in the periphery.     

Enhanced antibody affinity in sublethally irradiated mice and bone marrow chimeras.

Sublethally irradiated mice primed with dinitrophenyl (Dnp)-keyhole limpet hemocyanin immediately after irradiation or 30 days later and subsequently boosted with a second injection of antigen displayed a secondary response to Dnp characterized by antibody affinity greater than that in unirradiated controls. Also, in radiation chimeras primed with Dnp-keyhole limpet hemocyanin 120 days after syngeneic or allogeneic bone marrow transplantation the antibodies against Dnp produced after boosting were of higher affinity than the antibodies raised in normal mice. These findings are tentatively attributed to lack of suppressor thymus-derived lymphocytes (T cells) in sublethally irradiated mice and bone marrow chimeras, in which the enhanced ability to produce antibodies of high affinity may compensate for quantitative defects of the immune system.     

Tolerance of thymic cytotoxic T lymphocytes to allogeneic H-2 determinants encountered prethymically: evidence for expression of anti-H-2 receptors prior to entry into the thymus.

This study has assessed the possibility that anti-H-2 receptors are expressed on T-cell precursors prior to their entry into the thymus. Parental strain A thymus was transplanted into either normal or thymectomized (A x B)F1 mice which were then irradiated and reconstituted with strain A bone marrow. The cells repopulating the engrafted strain A thymus were shown to be of donor bone marrow origin. Thus, strain A thymocytes were differentiating within a syngeneic thymus, after exposure to allogeneic strain B major histocompatibility complex (MHC) determinants of the irradiated F1 host. The cells repopulating the engrafted thymus were assessed for their ability to generate alloreactive cytotoxic T lymphocyte responses and were found to be specifically tolerant to allogeneic strain B MHC determinants. This tolerance existed in the absence of detectable suppression and in the absence of detectable strain B MHC determinants intrathymically. These data are most consistent with the concept that precursor T cells express anti-MHC receptors prior to their entry into the thymus and that exposure to MHC determinants prethymically results in their functional inactivation.     

Mechanism for cotolerance in nonlethally conditioned mixed chimeras: negative selection of the Vbeta T-cell receptor repertoire by both host and donor bone marrow-derived cells

Bone marrow (BM) chimeras prepared by complete recipient ablation (A-- >B) exhibit donor-specific tolerance, yet survival is often limited by graft-versus-host disease (GVHD). Negative selection of potentially donor-reactive T cells, as assessed by relative T-cell receptor (TCR)- Vbeta expression, is dependent on donor BM-derived deleting ligands. Mixed chimerism and tolerance for both donor and host antigens can be achieved using partial recipient myeloablation with 500 cGy total-body irradiation (TBI) before transplantation followed by cyclophosphamide (CyP) on day +2. To examine the influence of residual host elements on negative selection, the peripheral TCR-Vbeta repertoire was analyzed in partially ablated C57BL/10SnJ (B10) recipients reconstituted with BM from major histocompatibility complex (MHC)-disparate B10.BR/SgSnJ or MHC, Hh-1 and Mls-disparate BALB/cByJ donors, which delete Vbeta5+ and 11+ or Vbeta3+, 5+, and 11+ TCR subsets, respectively. As in myeloblated recipients, donor-reactive subfamilies were deleted in B10.BR-->B10 and BALB/c-->B10 chimeras, suggesting that donor I-E and minor lymphocyte-stimulating (Mls) antigens contribute to the deleting ligands in the nonmyeloablated host. In striking contrast to completely ablated B10-->B10.BR chimeras, partially ablated recipients showed intramedullary I-E expression in the thymus and deleted host-reactive Vbeta5+ and Vbeta11+ subfamilies. These data demonstrate that efficient clonal deletion occurs after partial myeloablation and that both donor and host ligands contribute to TCR repertoire selection.     

Age-dependent appearance of non-major histocompatibility complex-restricted helper T cells.

T cells which recognize antigen in association with self major histocompatibility complex (MHC) molecules are positively selected within the thymus. This results in skewing of the T-cell repertoire toward the recognition of antigenic peptides presented by self MHC molecules. While the thymus gland involutes at a relatively young age, bone marrow function and the size of the peripheral T-cell pool are maintained with age. We have investigated the MHC restriction of helper T-cell function for B-cell production of specific antibody in mice of different ages. Splenic helper T cells from 2- to 3-month old mice immunized with phosphocholine-keyhole limpet hemocyanin conjugate were MHC-restricted as defined by their capacity to induce phosphocholine-specific antibody synthesis by syngeneic but not by allogeneic B cells. In contrast, splenic T cells from immunized 18- to 20-month-old mice induced specific anti-phosphocholine antibodies by both syngeneic and allogeneic B cells. No evidence of polyclonal immunoglobulin synthesis was detected. The ability of T cells from old mice immunized with phosphocholine-keyhole limpet hemocyanin to induce phosphocholine-specific antibody synthesis by B cells from allogeneic mice was inhibited by T cells from immunized young mice. These findings suggest that non-MHC-restricted T-cell helper activity in old mice results from the loss of T cells, present in young mice, which suppress non-MHC-restricted helper cells.     

Transplantation analysis of B cell destruction in (NOD x CBA)F1 mouse bone marrow chimeras

Summary F1 hybrids produced by outcross of non-obese diabetic (NOD) mice with diabetes resistant strains are also diabetes resistant. This resistance is abrogated if F1 hybrids are lethally irradiated and then haematopoietically reconstituted with NOD bone marrow. This model was employed to determine whether T lymphocyte recognition and elimination of pancreatic B cells in NOD mice is restricted by the MHC haplotype of the target B cell. Diabetes resistant (NOD/Lt x CBA/J)F1 hybrids were lethally irradiated and reconstituted with NOD/Lt bone marrow. Following haematopoietic reconstitution, donor matched NOD/Lt or CBA/J pancreatic islet and anterior pituitary grafts were grafted under a renal capsule to determine whether effector cells derived from NOD/Lt marrow progenitors would reject islet grafts in a MHC restricted fashion. The H-2^k haplotype expressed by CBA/J mice differs from all known loci of the unique H-2 haplotype of NOD; therefore, if NOD/Lt T lymphocytes eliminate pancreatic B cells in a MHC restricted fashion, NOD islet grafts would be eliminated in these chimeras while CBA islet grafts would be retained. Overt diabetes developed in 80% of the female and 40% of the male F1 hybrids following reconstitution with NOD/Lt marrow, while no hybrids reconstituted with CBA/J marrow became diabetic through a year of age. The retention of CBA/J skin and pituitary grafts in NOD/Lt marrow reconstituted F1 hybrids confirmed that the F1 thymic environment imparted tolerance to CBA/J alloantigens. Nonetheless, responses to a T cell dependent model antigen were restricted to the unique MHC haplotype of NOD. This was associated in the hyperglycaemic chimeras with rejection (8–21 days post-implantation) of both CBA/J and NOD/Lt islet grafts. This indicated generation of islet specific effectors that were not restricted by the MHC haplotype of the target B cell. Islet specific effectors were less active in those chimeras remaining normoglycaemic following NOD/Lt marrow reconstitution, since both NOD/Lt and CBA/J islet grafts remained intact 21 days post-implantation. Islet and pituitary grafts of each genotype remained free of leucocytic involvement in CBA/J marrow reconstituted F1 hybrids. The generation of B cell specific, but not genotype specific, effectors elicited from NOD/Lt marrow was age-dependent. Following priming with porcine insulin in vivo, T lymphocytes from 8-weekold NOD male mice failed to respond to porcine insulin in vitro, while primed T lymphocytes isolated from 16-weekold mice proliferated vigorously. This suggested that as pancreatic insulitis progressed in aging NOD/Lt mice, T lymphocytes capable of responding to B cells in a tissue specific, but not genotype specific fashion, were activated. Thus, we propose that immunological effectors derived from NOD/Lt, but not CBA/J marrow, are capable of eliminating islet grafts in non-MHC restricted fashion. However, this population could be activated as a consequence of B cell erosion initated in the pancreas by another class of effectors which may be restricted by the MHC haplotype of the target B cell.     

Reconstitution of Ir genes, Ia antigens, and mixed lymphocyte reaction determinants by gene complementation.

By using clones of murine T cells reactive with alloantigens as well as soluble antigens, it has been possible to demonstrate that Ia antigens, Ir gene phenomena (defined as the ability of immune T cells to recognize antigen), and mixed lymphocyte reaction (MLR)-stimulating determinants encoded within the I-A subregion are different manifestations associated with same product. These studies utilized the I-Ab mutant mouse B6.C-H-2bm (bm12), whose defect in the normal expression of the cell surface products of the I-Ab subregion can be partially circumvented through trans-complementation by deriving hybrids between bm12 and mice expressing normal I-A subregion products. Such mice [e.g., (bm12 X B10.A)F1] express several serologically normal I-Ab products but have defects in certain I-A subregion gene products normally expressed on cells of H-2a X H-2b heterozygote mice that are used as restricting elements for antigen recognition by antigen-reactive T cell clones or recognized as alloantigens by alloreactive T cell clones. This defect in cell surface expression of certain normal I-Ab products on the mutant bm12 cells has allowed us to suggest that there exist trans-complementing products on H-2a X H-2b heterozygote mice consisting of Ab alpha Ak beta and Ak alpha Ab beta that restrict antigen recognition by cloned T cells, stimulate MLR responses by cloned T cells, and react with certain anti-Ia antisera.     

Local irradiation enhances congenic donor pluripotent hematopoietic stem cell engraftment similarly in irradiated and nonirradiated sites

Long-term multilineage chimerism is achieved in CD45 congenic mice receiving high bone marrow doses with or without mediastinal irradiation (MI). Increased donor chimerism results in MI-treated compared with nonirradiated animals, suggesting that MI makes "space" for engraftment of donor pluripotent hematopoietic stem cells (PHSCs). We have now examined whether space is systemic or whether increased engraftment of donor marrow in locally irradiated mice is confined to the irradiated bones. While increased donor chimerism was observed in irradiated bones compared with nonirradiated bones of MI-treated animals 4 weeks following bone marrow transplantation (BMT), these differences were minimal by 40 weeks. MI-treated chimeras contained more adoptively transferable donor PHSCs in the marrow of both irradiated and distant bones compared with non-MI-treated chimeras. Similar proportions of donor PHSCs were present in irradiated and nonirradiated bones of locally irradiated mice at both 4 and 40 weeks. Irradiated bones contained more donor short-term repopulating cells than distant bones at 4 weeks, but not 40 weeks, after BMT. Our study suggests that local proliferation of donor PHSCs in mice receiving local irradiation rapidly leads to a systemic increase in donor PHSC engraftment.     

Suppressor T cells in allogeneic bone marrow chimeras

Immunosuppression is believed to play a role in the maintenance of stable bone marrow (BM) chimeras. This study investigates the nature and specificity of the suppression that lymphocytes from allogeneic BM chimeras exert upon the alloreactivity of donor and recipient lymphocytes. Lethally irradiated CBA/J (H-2k) mice were infused with 10(7) unseparated (WBM) or T cell-depleted BM (TDBM) cells of B10.BR mice (H-2k, disparate at minor histocompatibility antigens). Mixtures consisting of spleen cells (SC) from BM chimeras and SC from either normal donor, recipient, or third party (C3H, H-2k) mice, were sensitized with irradiated BALB/c (H-2d) leukocytes, then assayed for proliferative and anti-H-2d cytotoxic activity and compared with those of appropriate control cultures. The alloreactivity of all three types of normal SC was non-specifically suppressed by SC from both WBM and TDBM chimeras taken 2 weeks post-BM transplantation (BMT). In contrast, at 4 weeks post-BMT, SC from both chimeras suppressed the alloreactivity of recipient-type cells whereas only SC from WBM, but not from TDBM chimeras, suppressed normal donor-type response, and neither could suppress the response of normal third party cells. The suppression of donor-type alloreactivity diminished with time, while that exerted on recipient-type lasted for at least 10 weeks post-BMT. The suppression of donor alloreactivity was mediated by radioresistant Thy1.2+, Lyt1+2+ cells while that exerted upon recipient's alloreactivity was mediated by radiosensitive Thy1.2+, Lyt1+2- cells. Both types of suppressor cells were of donor origin. The potential biological role of the suppressive activity in the engraftment of allogeneic BM is discussed.     

Generation of primary antigen-specific human cytotoxic T lymphocytes in human/mouse radiation chimera

Severe combined immunodeficient (SCID) mice are increasingly used as hosts for the adoptive transfer of human lymphocytes. Human antibody responses can be obtained in these xenogeneic chimeras, but information about the functionality of the human T cells in SCID mice is limited and controversial. Studies using human peripheral blood lymphocytes (PBL) injected intraperitoneally (IP) into SCID mice (hu-PBL-SCID mice) have shown that human T cells from these chimeras are anergic and have a defective signaling via the T-cell receptor. In addition, their antigenic repertoire is limited to xenoreactive clones. In the present study, we tested the functionality of human T cell in a recently described chimeric model. In this system, BALB/c mice are conditioned by irradiation and then transplanted with SCID bone marrow, followed by IP injection of human PBL. Our experiments demonstrated that human T cells, recovered from these hu-PBL-BALB mice within 1 month posttransplant, proliferated and expressed activation markers upon stimulation with anti-CD3 monoclonal antibody. A vigorous antiallogeneic human cytotoxic T-lymphocyte (CTL) response could be generated in these mice by immunizing them with irradiated allogeneic cells. Moreover, anti-human immunodeficiency virus type 1 (HIV-1) Net- specific human CTLs could be generated in vivo from naive lymphocytes by immunization of mouse-human chimeras with a recombinant vaccinia-nef virus. This model may be used to evaluate potential immunomodulatory drugs or cytokines, and could provide a relevant model for testing HIV vaccines, for production of antiviral T-cell clones for adoptive therapy, and for studying human T-cell responses in vivo.