Potentiation of a primary in vivo antibody response by alloantisera against gene products of the I region of the H-2 complex.

Mice were immunized intravenously with suboptimal numbers (3.5-5 X 10(5)) of sheep erythrocytes together with various anti-Ia antisera or with sheep erythrocytes alone, and the primary IgM and IgG plaque-forming cell responses were assayed 6 days later9 A/J (H-2a) mice given 5 X 10(5) sheep erythrocytes together with as little as 0.4 mul of a (129 X A.TH)F1 anti-A.TL (anti-Iak) antiserum developed 2-3 times as many IgM and IGG plaque-forming cells as mice injected with antigen alone or together with various antisera not containing anti-Ia antibodies. Similar results were obtained with BALB/c (H-2d) mice and a (C3H X LG/Ckc)F1 anti-C3H. OH (anti-Iad) antiserum plus sheep erythrocytes. In the case of the anti-Iad antiserum, the potentiating activity could be absorbed with C3H. OH (Id) but not C3H(Ik) spleen cells, demonstrating that the active antibodies were specific for the Id region. Antiserum to I-Jk subregion-coded determinants was tested in A/J (I-Jk) mice and found to also potentiate 2- to 3-fold the plaque-forming cell response to suboptimal erythrocyte immunization. This antiserum [(BIO.A(3R) X DBA/2)F1 anti-B10.(5R)] failed to potentiate responses in BALB/c (I-Jd) mice, as expected on a genetic basis. The potentiating antibodies could be removed by absorption with B10.BR (I-Jk) but not B10 (I-Jb) spleen cells, also confirming the I-J specificity of the activity. The interference of anti-I-J antibodies with T lymphocyte suppressor mechanisms is prposed as a possible explanation for this phenomenon.     

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.     

Suppressor T-cell factor(s) display an altered pattern of Igh (immunoglobulin heavy chain locus) genetic restriction when developed in an Igh-congeneic host.

Suppressor T-cell factor(s) (TsF1) inhibit the in vivo priming of azobenzenearsonate-specific cytotoxic T-cell responses. The activity of TsF1 is restricted by genes linked to Igh-1 allotypic markers. TsF1 obtained from B6.Igh-1n mice was unable to suppress the immune response in B6.Igh-1b mice and vice versa. However, TsF1 prepared from B6.Igh-1n T cells "parked" in an Igh-congeneic B6.Igh-1b environment displays an additional restriction specificity of the host. Thus, TsF1 prepared from these Igh-chimeric mice suppressed immune responses in both B6.Igh-1n (donor) and B6.Igh-1b (recipient) mice but not in mice of the unrelated strain BALB/c.Igh-1a. The results indicate that the establishment of the suppressor T-cell repertoire is dependent not only upon the genetic background of the individual T cell but also upon the influence of Igh-linked determinants present when T-cell clones are selected during the response.     

Chronic treatment with rabbit anti-mouse mu-chain antibody alters the characteristic immunoglobulin heavy-chain restriction of murine suppressor T-cell factors.

Prolonged treatment of mice, starting at birth, with rabbit anti-mouse mu-chain antibodies resulted in the elimination of immunoglobulin-bearing B cells in these animals. The ability of these animals to elicit antigen-specific delayed-type hypersensitivity or cytotoxic T-cell responses to azobenzenearsonate-coupled spleen cells was not impaired. The effect of anti-mu treatment on the restriction by immunoglobulin heavy-chain genes (Igh) of suppressor T cells was investigated. We found that first-order suppressor T-cell factor ( TsF1 ) obtained from anti-mu treated animals expresses an Igh restriction pattern distinct from that observed with TsF1 from normal untreated mice. Furthermore, TsF1 prepared from anti-mu treated animals did not express the major crossreactive idiotypic determinants normally present in TsF1 . The significance of these findings in relation to the role of immunoglobulin on the T-cell repertoire is discussed.     

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.     

Microenvironmental immunoregulation: possible role of contrasuppressor cells in maintaining immune responses in gut-associated lymphoid tissues.

The addition of Peyer's patch T cells from most strains of mice to spleen cells in primary Mishell-Dutton cultures either has no effect or augments the spleen cells' response to sheep erythrocytes. However, if the Peyer's patch T cells are treated with an anti-I-J antiserum and complement to remove contrasuppressor-inducer cells, the remaining Ly-2 cells (T cells that express Ly-2 but not Ly-1) are highly suppressive. This "latent" suppressor cell activity also can be revealed by removing contrasuppressor-acceptor (transducer) cells from the splenic assay population with either an anti-I-J or anti-Ly-2 antiserum. These findings, taken together with previous work showing that orally administered antigen leads to systemic tolerance, give experimental support to the notion that contrasuppression may be important in allowing microenvironmental immune responses (in this case the gut-associated lymphoid tissue) to take place while systemic immunity is suppressed.     

Impact of pretransplant conditioning and donor T cells on chimerism, graft-versus-host disease, graft-versus-leukemia reactivity, and tolerance after bone marrow transplantation

Graft rejection, mixed chimerism, graft-versus-host disease (GVHD), leukemia relapse, and tolerance are interrelated manifestations of immunologic reactivity between donor and host cells that significantly affect survival after allogeneic bone marrow transplantation (BMT). In this report, a mouse model of BMT, in which the donor and host were compatible at the major histocompatibility complex (MHC), was used (1) to examine the interrelationship of pretransplant conditioning and T-cell content of donor BM with regard to lymphoid chimerism and GVHD and (2) to determine how these factors affected graft-versus-leukemia (GVL) reactivity and donor-host-tolerance. AKR (H-2k) host mice were administered optimal or suboptimal total body irradiation (TBI) as pretransplant conditioning followed by administration of BM cells from B10.BR (H-2k) donor mice with or without added spleen cells as a source of T lymphocytes. Transplanted mice were injected with a supralethal dose of AKR leukemia cells 20 and 45 days post-BMT to assess GVL reactivity in vivo. The pretransplant conditioning of the host and T-cell content of the donor marrow affected the extent of donor T-cell chimerism and the severity of GVH disease. GVL reactivity was dependent on transplantation of mature donor T cells and occurred only in complete chimeras. Transplantation of T-cell-deficient BM resulted in the persistence of host T cells, ie, incomplete donor T-cell chimerism, even when lethal TBI was used. Mixed chimerism was associated with a lack of GVL reactivity, despite the fact that similar numbers of donor T cells were present in the spleens of mixed and complete chimeras. In this model, moderate numbers of donor T cells facilitated complete donor T-cell engraftment, caused only mild GVHD, and provided a significant GVL effect without preventing the subsequent development of tolerance after conditioning with suboptimal TBI. In contrast, severe, often lethal, GVHD developed when the dose of TBI was increased, whereas tolerance and no GVH/GVL reactivity developed when the T-cell content of the marrow was decreased.     

Resistance to Listeria monocytogenes in mice: genetic control by genes that are not linked to the H-2 complex.

After mice of several inbred strains were injected with Listeria monoyctogenes, two parameters of resistance, the 50% lethal dose and the suppression of bacterial proliferation in spleen, were determined. The strains of mice tested could be segregated into two groups: the resistant C57BL/10Sn mice and the sensitive A/J and DBA/2J mice. Congenic resistant strains of mice were used because they would express the H-2 haplotype of the sensitive strains (H-2a or H-2d) on the background of a resistant strain, C57BL/10Sn. Both the B10.A/SgSn (H-2a) and the B10.D2/Sn (H-2d) mice were as resistant as mice from their background strain and were significantly more resistant than the strains that donated their H-2 locus (A/J or DBA/2J). Therefore, the resistance of mice to Listeria, although genetically controlled, is not controlled by gene (s) linked to the H-2 haplotype. On the other hand, the level of specific immunity to listeria antigens (as indicated by the footpad reaction) was higher in the C57BL/10Sn (H-2b) mice than in either the A/J and B10.A/SgSn (H-2a) mice or the DBA/2J and B10.D2/Sn (H-2d) mice. This observation suggests an H-2 linkage of specific immunity to Listeria.     

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.     

Reciprocal stimulation of negatively selected high-responder and low-responder T cells in virus-infected recipients.

After depletion of alloreactive potential, immunologically naive T cells from C57BL/6J (Kb-Db) mice (B6) can be induced to respond to vaccinia virus in the context of both H-2Kk and H-2Db when stimulated in B10.A(4R) (Kk-Db) recipients. However, negatively selected B10.A(2R) (Kk-Db) T cells respond to H-2Db-vaccinia virus but not to H-2Kb-vaccinia virus when primed in an irradiated B6 environment. The B6 mouse strain is a high responder to vaccinia virus associated with H-2Db, whereas the B10.A(2R) and B10.A(4R) recombinants are low responders. Responsiveness in the context of H-2Db is thus recognized when the only homology between T cell and recipient is at the H-2D locus and is suppressed when H-2Kk is also present in both situations. The fact that negatively selected H-2Kb-Db T cells can be induced to recognize H-2Kk-vaccinia virus may reflect the existence of an "altered self" complex which is recognized via a single receptor, perhaps drawn from an alloreactive T-cell repertoire. At least in some instances, patterns of T-cell responsiveness are not totally constrained by the spectrum of H-2 antigens encountered in thymus.     

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.     

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.     

Genetic and serological analysis of the expression of crossreactive idiotypic determinants on anti-p-azobenzenarsonate antibodies and p-azobenzenarsonate-specific suppressor T cell factors.

Data are reported on the genetic control and structure of antigen-specific suppressive molecules obtained from azobenzenearsonate (ABA)-specific suppressor T cells (Ts). Ts-derived suppressor factors (TsFs) were previously shown to bear determinants encoded by genes of the I-J subregion of the H-3 major histocompatibility complex and structures encoded by VH (variable region of heavy chains) genes linked to the Igh locus. To further characterize TsF we have made use of a K light (L) chain variable region (VK) genetic marker linked to the locus governing expression of a crossreactive idiotype on anti-ABA antibodies. The experiments evaluated the possible contribution to TsF of structures under control of the VK locus. TsF was prepared in strains of mice with known Igh and VK genes. Mice carrying Igh-1e genes produced TsF that was retained by an immunoadsorbent containing bound anti-idiotype antibodies; the genetic constitution at the VK locus was not relevant. By using a panel of anti-idiotypic antibodies prepared against the antigen-binding site of antibody carrying the idiotype, against determinants outside the site or against VH structures, we determined that idiotypic structures on the TsF are associated with V-region elements associated with the H chain and not with the binding site formed by a VH-BL combination.     

Synergistic effects of in vivo depletion of Ly-49A and Ly-49G2 natural killer cell subsets in the rejection of H2(b) bone marrow cell allografts

Subsets of murine natural killer (NK) cells exist that express the Ly-49 family of molecules that recognize different major histocompatibility complex (MHC) determinants. Bone marrow transplantation studies were performed to examine the in vivo functions of 2 of these subsets. Subsets of Ly-49A and Ly-49G2 NK share specificity for the same MHC class 1 ligand, D(d), binding of which results in an inhibitory signal to the NK cell but allows them to lyse H2(b) targets in vitro. We therefore examined the ability of these subsets to reject H2(b) bone marrow cell allografts in lethally irradiated mice. Surprisingly, depletion of Ly-49A(+) NK cells in BALB/c or B10.D2 mice (both H2(d)) had no effect on the rejection of H2(b) BMC. However, Ly-49A depletion did partially abrogate the ability of B10.BR (H2(k)) mice to reject H2(b) allografts. Although depletion of either Ly-49A(+) or Ly-49G2(+) NK cells alone had no effect on the ability of B10.D2 mice to reject H2(b) BMC, depletion of both subsets dramatically and synergistically abrogated rejection. Studies with various B10 congenic mice and their F(1) hybrids indicate that this synergy between Ly49A and Ly4G2 depletion occurs in every instance. Thus, Ly-49A(+) NK cells appear to play a role in the rejection H2(b) bone marrow allografts, but, in most strains of mice studied, Ly-49G2(+) NK cells must also be eliminated. The putative roles of these NK cell subsets in clinical transplantation remains to be elucidated. (Blood. 2000;95:3840-3844)     

Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells

The cell surface molecule Ly-6A/E provides a convenient marker for primitive stem cells in the hematopoietic tissues of both fetal and adult mice. However, previous studies have shown that Ly-6A/E expression by lymphocytes is variable depending on the haplotype of the Ly-6 locus. Therefore, strain-specific variation in Ly-6A/E expression by bone marrow (BM) cells was investigated. The results show that Ly-6a mice have, on average, 50% of the number of BM cells expressing Ly-6A/E relative to that for Ly-6b mice. Furthermore, among the 5% of BM cells that do not express antigens characteristic of mature T, B, myeloid, or erythroid lineages, which include the primitive hematopoietic stem cell compartment, Ly-6a mice have, on average, more than fivefold fewer Ly- 6A/E+ cells relative to that for Ly-6b mice. Isolation of Ly-6A/E- and Ly-6A/E+ cells from mice of both haplotypes showed that, whereas 99% of the marrow repopulating activity (MRA) of C57BL/Ka (Ly-6b) mice could be recovered in the Ly-6A/E+ fraction, only about 25% of the MRA of BALB/c (Ly-6a) was recoverable in the same population. On a per-cell basis, the Ly-6A/E+ cells that were isolated from BALB/c mice were essentially equivalent in MRA to those isolated from C57BL/Ka mice. Thus, whereas a large percentage of the hematopoietic stem cells of Ly- 6a mice do not express the Ly-6A/E molecule, the antigen may be used to isolate a subset of stem cells from these mice. These results show that hematopoietic stem cell phenotype can vary between mouse strains and imply that caution should be exercised in the identification of human stem cell antigens such as CD34, because a similar variability may occur between individual humans. To further explore the influence of Ly- 6 haplotype on Ly-6A/E expression by specific cell subsets, lymph-node lymphocytes from a panel of mouse strains were analyzed by multiparameter flow cytometry for correlated expression of Ly-6A/E, CD4, and CD8. All Ly-6a strains examined had less than 20% Ly-6A/E+ cells, and those cells were predominantly CD8+ T lymphocytes. In contrast, the Ly-6b strains had greater than 30% Ly-6A/E+ cells, and those cells included CD4+, CD8+, and B lymphocytes.     

The mechanism of graft-host-tolerance in murine radiation chimeras transplanted across minor histocompatibility barriers

A better understanding of the mechanism(s) involved in graft-host-tolerance following allogeneic bone marrow transplantation is needed to develop new strategies to prevent graft-versus-host disease (GVHD). Based on previous studies, mainly in MHC-mismatched donor-recipient pairs, three hypotheses have been proposed: clonal deletion, active suppression and lack of adequate antigen-presenting cells. Our goal was to identify the mechanism(s) by which tolerance is achieved and maintained in radiation chimeras transplanted across minor histocompatibility barriers. Healthy (B6----LP) chimeras were obtained following injection of 10(7) C57BL/6 marrow cells to irradiated (9.5 Gy) LP hosts and used experimentally 100 days after chimerization. The tolerance state of (B6----LP) chimeras could not be abrogated after i.v. transfer of 5 x 10(7) donor-type spleen cells alone or with repeated i.p. injection of host-type antigen-presenting cells. No GVHD was observed when 10(7) marrow cells plus 5 x 10(7) spleen cells from (B6----LP) chimeras were injected to irradiated LP recipients. Chimera spleen cells suppressed GVHD when adoptively transferred to LP recipients of a C57BL/6 graft. These results suggest that in this model the presence of suppressor cells is both necessary and sufficient to maintain graft-host-tolerance.     

Treatment of donor mice with an alpha beta T-cell receptor monoclonal antibody induces prolonged T-cell nonresponsiveness and effectively prevents lethal graft-versus-host disease in murine recipients of major histocompatibility complex (MHC)-matched and MHC-mismatched donor marrow grafts

The purpose of this study was to determine whether the administration of high doses of an anti-T-cell receptor (TCR) monoclonal antibody (H57- 597) to donor animals could induce a state of T-cell nonresponsiveness and prevent the development of graft-versus-host disease (GVHD) in murine recipients of major histocompatibility complex (MHC)-matched (B10.BR[H-2k] --> AKR/J[H-2k]) and mismatched (B10.BR[H-2k] --> DBA/2[H- 2d]) marrow grafts. Transplantation of H57-597-treated B10.BR T cells into irradiated AKR or DBA mice resulted in protection from GVHD, which was otherwise lethal in transplanted recipients receiving untreated T cells. The administration of H57-597-treated T cells did not compromise alloengraftment in either strain combination and was found to accelerate donor T-cell reconstitution in recipients of MHC-matched marrow grafts. Optimal protection for GVHD was dependent on the duration of antibody exposure in donor mice. T cells from donor exposed to antibody for only 1 day caused lethal GVHD, whereas exposure for at least 4 days was necessary to abrogate graft-versus-host reactivity. The ability of antibody treatment to protect against the development of GVHD could not be ascribed to the antibody-induced production of Th2 cytokines, the induction of a T- or non-T-suppressor cell population, or the preferential depletion of CD4+ T cells by H57-597. Donor T cells exposed to H57-597 antibody were detectable in recipients for up to 5 weeks after transplantation, indicating that these cells were not eliminated in the host immediately after bone marrow transplantation and contributed to enhanced donor T-cell reconstitution. Moreover, in B10.BR --> DBA chimeras that did not have any clinical evidence of GVHD, potentially MIs-reactive donor-derived Vbeta6+ T cells were present in the spleens of recipients at comparable numbers to normal mice but appeared functionally nonresponsive in vivo. These data strongly suggested that protection from GVHD was due to the fact that antibody treatment resulted in a state of prolonged T-cell anergy that persisted despite the presence of potential costimulatory signals in the recipient. This observation is of potential clinical significance in that it shows that the prevention of GVHD can be accomplished without posttransplantation immunosuppression or the need for in vitro or in vivo T-cell depletion.     

Immunosuppression by Friend leukemia virus is H-2 restricted by alloreactive T lymphocytes.

Friend leukemia virus suppresses mitogen-responsive cells in vitro by activating thymus-dependent suppressor cells. The interaction between T suppressor and mitogen-responsive cells is H-2D restricted by a third cell type, called an interfering cell. The interfering cells could be characterized as alloreactive T cells that functionally mature in the spleen at 2 weeks of age and that can be functionally inhibited by mitomycin C, irradiation, and cortisol. Interfering cells are stimulated by H-2D (and not H-2L) alloantigens of the mitogen-responsive cells. H-2D differences between interfering and T suppressor cells are unimportant. Induction of "tolerance" to H-2 alloantigens in semi-allogeneic radiation marrow chimeras resulted in the specific loss of interfering cell function. It is possible that interfering or similar cells participate in other forms of H-2 restriction.     

Prevention of graft-versus-host disease by induction of immune tolerance with ultraviolet B-irradiated leukocytes in H-2 disparate bone marrow donor

Transfusions (Tx) of Ultraviolet B (UVB)-irradiated peripheral blood mononuclear leukocytes (MNL) have been shown to induce humoral immune tolerance to major histocompatability complex (MHC) antigens (Blood 88:4375, 1996). To determine whether cellular immune tolerance to MHC antigens can be induced by the same approach, transplantation of bone marrow and spleen cells from tolerant donors across the H-2 barrier was conducted to study its effect on prevention of graft-versus-host disease (GVHD). After immune tolerance induction by four weekly Tx of UVB-irradiated BALB/c (H-2(d)) peripheral blood MNL into CBA/HT6 (H-2(k)) mice, bone marrow cells (BMC) and spleen MNL from tolerant or naive CBA mice were transplanted into lethally irradiated BALB/c mice. The transplanted mice were followed by measuring body weight, peripheral leukocyte counts, GVHD, survival, and cytokine response. All BALB/c recipient mice were fully engrafted with H-2(k) CBA donor cells after transplantation. The severity of GVHD was significantly attenuated in BALB/c mice transplanted with BMC and spleen MNL from tolerant CBA donor mice. The recovery of peripheral leukocyte and lymphocyte counts were faster and more complete in mice transplanted with cells from the tolerant donors. The serum cytokine profile after transplantation with tolerant donor cells showed increased interleukin-4 and reduced gamma interferon that are consistent with a polarized Th2 response. The results pooled from three separate experiments showed that BALB/c mice transplanted with 5 x 10(6) BMC and 4 x 10(5) spleen MNL from tolerant CBA donors had better overall survival than the control group (72% v 17%, P =.018). The findings show that transplantation with bone marrow and spleen cells from tolerant H-2 disparate donor mice is associated with significant attenuation of GVHD and better outcomes. The results also support that transfusions of UVB-irradiated leukocytes may induce cellular immune tolerance.     

Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells.

Striated muscle is the predominant site of gene expression after i.m. immunization of plasmid DNA, but it is not clear if myocytes or professional antigen-presenting cells (APCs) of hematopoietic origin present the encoded antigens to class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL). To address this issue, CTL responses were assessed in mice engrafted with immune systems that were partially MHC matched with antigen-producing muscle cells. Spleen cells (sc) from immunocompetent F1 H-2bxd mice were infused into H-2b or H-2d mice carrying the severe combined immunodeficiency (scid) mutation, creating F1sc-->H-2b and F1sc-->H-2d chimeras, respectively. Immunization with DNA plasmids encoding the herpes simplex virus gB or the human immunodeficiency virus gp120 glycoproteins elicited antiviral CTL activity. F1sc-->H-2d chimeras responded to an H-2d-restricted gp120 epitope but not an H-2b restricted gB epitope, whereas F1sc-->H-2b chimeras responded to the H-2b but not the H-2d restricted epitope. This pattern of epitope recognition by the sc chimeras indicated that APCs of recipient (scid) origin were involved in initiation of CTL responses. Significantly, CTL responses against epitopes presented by the mismatched donor class I molecules were elicited if F1 bone marrow cells and sc were transferred into scid recipients before or several days to weeks after DNA immunization. Thus, bone marrow-derived APCs are sufficient for class I MHC presentation of viral antigens after i.m. immunization with plasmid DNA. Expression of plasmid DNA by these APCs is probably not a requirement for CTL priming. Instead, they appear to present proteins synthesized by other host cells.