Induction of linked suppression in addition to the donor H-2 class I-specific unresponsiveness in recipient T cells by transfusing class I plus class II-disparate, but not class I alone-disparate, bone marrow cells

This study was undertaken to determine whether bone marrow (BM) cells contain a cell population with the capacity to induce an unresponsiveness of T cells specific to the BM self-H-2 class I antigens in vivo, i.e., veto cell population. Recombinant or congenic mice were infused intravenously with H-2-incompatible BM cells. One to several weeks later, donor H-2-and irrelevant H-2-specific responses in mixed lymphocyte reaction cultures of recipient T cells were assessed. Transfusion of H-2-incompatible BM of C57BL/10 (B10) recombinant strains caused a long-lasting cytotoxic T lymphocyte (CTL) unresponsiveness to the donor class I antigens in recipient lymph node cells. When class I plus class II-disparate BM cells were transfused, an anti-donor class I CTL response and a response against a third-party class I antigen, which was presented on the stimulator cells coexpressing the donor class I and class II, were significantly suppressed. This linked suppression lasted for less than 2 weeks after transfusion. Transfusion of class I-alone-disparate BM induced the donor class I-specific CTL unresponsiveness, but not the linked suppression. The induction of linked suppression was prevented considerably by transfusing nylon wool-nonadherent BM or by treating recipients with cyclophosphamide 2 days before transfusion. An anti-third-party class I CTL response, stimulated in vitro with fully allogeneic spleen cells, was not hampered by the BM transfusion. Coculturing the lymph node (LN) cells obtained from the class I plus class II-disparate BM recipient with normal LN cells interfered with the generation of both anti-donor class I and anti-linked third-party class I CTL, whereas, coculturing LN cells from the class I alone-disparate BM recipient inhibited neither specificity of CTL generation. Transfusion of class I plus class II-disparate BM resulted in a significant suppression of the donor class II-specific proliferative response. In contrast, transfusion of class I alone-disparate BM did not suppress any proliferative responses, including even a "linked" third-party class II-specific response. Transfusion of bm 1, (B6 X bm 1)F1, or (bm 1 X bm 12)F1 BM to B6 did not induce unresponsiveness in bm 1-specific CTL responses. However, the transfusion resulted in a significant suppression of bm 1-reactive proliferative response of recipient LN cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Anti-bacterial immunity to Listeria monocytogenes in allogeneic bone marrow chimera in mice

Protection and delayed-type hypersensitivity (DTH) to the facultative intracellular bacterium Listeria monocytogenes (L.m.) were studied in allogeneic and syngeneic bone marrow chimeras. Lethally irradiated AKR (H-2k) mice were successfully reconstituted with marrow cells from C57BL/10 (B10) (H-2b), B10 H-2-recombinant strains or syngeneic mice. Irradiated AKR mice reconstituted with marrow cells from H-2-compatible B10.BR mice, [BR----AKR], as well as syngeneic marrow cells, [AKR----AKR], showed a normal level of responsiveness to the challenge stimulation with the listeria antigens when DTH was evaluated by footpad reactions. These mice also showed vigorous activities in acquired resistance to the L.m. By contrast, chimeric mice that had total or partial histoincompatibility at the H-2 determinants between donor and recipient, [B10----AKR], [B10.AQR----AKR], [B10.A(4R)----AKR], or [B10.A(5R)----AKR], were almost completely unresponsive in DTH and antibacterial immunity. However, when [B10----AKR] H-2-incompatible chimeras had been immunized with killed L.m. before challenge with live L.m., these mice manifested considerable DTH and resistance to L.m. These observations suggest that compatibility at the entire MHC between donor and recipient is required for bone marrow chimeras to be able to manifest DTH and protection against L.m. after a short-term immunization schedule. However, this requirement is overcome by a preceding or more prolonged period of immunization with L.m. antigens. These antigens, together with marrow-derived antigen-presenting cells, can then stimulate and expand cell populations that are restricted to the MHC (H-2) products of the donor type.     

Recipient micro-environment does not dictate the Igh-V restriction specificity of T cell suppressor inducer factor (TsiF) from allogeneic bone marrow chimera in mice

We have ascertained previously from a study of fully allogeneic irradiation chimeras in mice that the H-2 restriction of the suppressor factor (Ly-2 T suppressor factor) is determined by the post-thymic environment protected by the donor cells, rather than by the thymic environment of the recipient. In the present study, we analyzed differentiation influences that determine the Igh restriction specificities of the suppressor inducer T cell factor(s) (TsiF) that are produced by Ly-1+ splenic T cells in fully allogeneic bone marrow chimeras in mice. AKR mice that had been lethally irradiated and reconstituted with B10 marrow cells, [B10----AKR] chimeras, produced Ly-1 TsiF after hyper-immunization with sheep erythrocytes (SRBC) which suppressed antigen--specifically the primary antibody responses to SRBC that were generated in cells of the same Igh-Vb haplotype of donor strain and not those generated in cells of the recipient Igh-Va type. Similar results were obtained when Ly-1 TsiF from [B6----BALB/c] and [BALB/c----B6] chimeras were analyzed. Furthermore, the Ly-1 TsiF from [BALB/c----B6] chimeras suppressed the primary antibody responses of both BALB/c [H-2d, Igh-Va, Igh-Ca] and BAB-14 (H-2d, Igh-Va, Igh-Cb), but not those of CAL-20 (H-2d, Igh-Vd, Igh-Cd). These results demonstrate clearly that the Ly-1 TsiF from allogeneic bone marrow chimeras are donor Igh-V-restricted and are not influenced by the recipient micro-environment, presumably that provided by the thymuses of the recipient mice.     

Cyclosporine A and peripheral tolerance. Inhibition of veto cell-mediated clonal deletion of postthymic precursor cytotoxic T lymphocytes.

Veto cell-mediated suppression of CTL responses has been proposed as one mechanism by which self tolerance is maintained in mature T cell populations. We have reported that murine bone marrow cells cultured in the presence of high-dose IL-2 (activated bone marrow cells) mediate strong veto suppressor function in vitro and in vivo, and that such veto activity is effected through clonal deletion of cytotoxic T cell precursors. In our studies, we have determined that bone marrow cell populations from athymic NCr-nu mice (H-2d) mediate strong veto cell activity without exposure to exogenous IL-2 in vitro. To examine mechanisms by which these naturally occurring veto cell populations in BM suppress precursor CTL (pCTL) responses, we used as a responding cell population in MLC, spleen cells of transgenic mice expressing at high frequency TCR specific for H-2 Ld encoded Ag with stimulation by H-2d-expressing cells in culture. Flow cytometric analysis was performed by staining the responding MLC cell population with the mAb 1B2 specific for the transgene-encoded TCR and determined changes of 1B2+ T cells. Such experiments demonstrated that the anti-H-2d cytotoxic response by these cell populations was specifically suppressed by NCr-nu (H-2d) bone marrow, and that 1B2+ pCTL were in fact specifically deleted from the responding cell population by incubation with such naturally occurring veto cell populations expressing the appropriate target Ag. In addition, to further understand the interactions of pCTL and veto cells and possible contributions by the latter to peripheral tolerance, we evaluated the effect of cyclosporine A (CsA) on veto cell-mediated suppression of pCTL of the transgenic mice. CsA inhibited veto cell-mediated suppression of cytotoxic T cell responses, and this inhibition correlated with a lack of clonal deletion of pCTL by veto cells in the presence of CsA. Furthermore, CsA exerted its effect through pCTL and not through veto cells, indicating that pCTL may play an active role in their own deletion by veto cells.     

Two roles for CD4 cells in the control of the generation of cytotoxic T lymphocytes.

The generation of CTL against Qa-1 Ag in C57BL/6 (B6) (Qa-1b) and B6.Tlaa (Qa-1a) congenic strains requires in vivo priming with the Qa-1 alloantigen together with a helper Ag, such as H-Y. The primed precursors obtained from these female mice generate Qa-1-specific CTL activity upon culture in vitro. Although the presence of the H-Y helper Ag is not required for the in vitro sensitization, no response occurs in the absence of CD4 cells. Addition of unprimed B6.Tlaa CD4 cells from Qa-1 incompatible radiation bone marrow chimeras (B6.Tlaa----B6), that are presumably tolerant to Qa-1b, provide helper activity for Qa-1b-specific CTL. This indicates that although CD4 cells are obligatory for the Qa-1 response, they need not be specific for alloantigens on the APC to generate helper activity in in vitro cultures. Addition of unirradiated B6 CD8-depleted spleen cells to CD4-depleted B6.Tlaa anti-B6 cultures in the presence of either B6.Tlaa CD4 cells or rIL-2 prevents the generation of Qa-1 specific CTL. This inhibition is not due to an anti-idiotypic Ts cell since B6.Tlaa----B6 chimeric cells do not suppress an anti-Qa-1b response. Rather, this finding is consistent with that of a veto cell mechanism. To determine whether CD4 cells themselves exhibit veto activity, highly purified CD4 populations were tested for their ability to inhibit the generation of Qa-1-specific CTL. CD4 cells precultured for 2 to 3 days with Con A and rIL-2 specifically inhibit CTL activity whereas resting cells do not, similar to that noted for CD8 veto cells. The relative efficiency of activated CD4 cells is greater than that of resting NK cells but is less than that of activated CD8 or NK cells. Thus, CD4 cells not only provide helper activity for CTL precursors, but also act as veto cells to prevent the generation of CTL activity.     

Ia Restriction Specificity of KLH-Specific T Cells from Allogeneic Bone Marrow Chimeras Is Influenced by Histocompatibility at the H-2 and Minor Histocompatibility Loci

Ia restriction specificity involved in T cell proliferative responses to keyhole limpet hemocyanin (KLH) has been analyzed using a variety of allogeneic bone marrow chimeras. The chimeric mice were prepared by reconstituting irradiated AKR, SJL, B10.BR and B10.A(4R) mice with bone marrow cells from B10 mice. When such chimeric mice had first been primed with KLH in complete Freund's adjuvant (CFA), T cells from H-2 incompatible fully allogeneic chimeras showed significantly higher responses to KLH in the presence of antigen-presenting cells (APC) of donor strain (B10) than APC of recipient strain. However, in H-2 subregion compatible chimeras, [B10→B10.A(4R)], which were matched at the H-2D locus and at minor histocompatible loci, the T cells could mount vigorous responses to KLH with antigen-presenting cells (APC) of either donor or recipient type. The same results were obtained as well with chimeras that had been thymectomized after full reconstitution of lymphoid tissues by donor-derived cells. A considerable proportion of KLH-specific T cell hybridomas established from [B10→B10.A(4R)] chimeras exhibited both I-A^b and I-A^k restriction specificities. The present findings indicate that the bias to donor Ia type of antigen specific T cells is determined by donor-derived APC present in the extrathymic environment but that cross-reactivity to the recipient Ia is influenced to some degree by histocompatibility between donor and recipient mice, even though the histocompatible H-2D locus and minor histocompatibility loci seem not to be directly involved in the I-A restricted responses studied herein.     

Veto activity of activated bone marrow does not require perforin and Fas ligand

Veto cells suppress generation of CD8(+) T cell immune responses in an antigen-specific manner, with specificity dictated by antigens on the veto cell surface. Activated bone marrow (ABM) veto cells belong to the NK cell type lineage and veto by clonally deleting antigen-specific precursor cytotoxic T cell lymphocyte (CTL). In vitro cytotoxicity of ABM depends largely on the perforin/granzyme and Fas/Fas ligand pathways. Utilizing perforin-deficient and functional Fas ligand-deficient gld mice as a source of ABM and functional Fas-deficient lpr mice as a source of precursor CTL, we demonstrate in this study that ABM cells utilize a perforin- and Fas-independent pathway to veto allogeneic cell-mediated cytotoxic responses. We also show that ABM cells mediate perforin- and Fas-independent veto activity even in an 8-h clonal deletion assay. We conclude that ABM veto activity does not require the two primary pathways of cell-mediated death.     

Antiviral T cell competence and restriction specificity of mixed allogeneic (P1 + P2----P1) irradiation chimeras

Mixed irradiation bone marrow chimeras were prepared by reconstituting lethally irradiated C57BL/10 (B10) or B10.D2 mice with T cell-depleted bone marrow cells of B10 plus B10.D2 origin. These chimeras were healthy and survived well under conventional housing conditions and after experimental laboratory infections. Of a total of 17 chimeras tested, 2 died spontaneously or from the injected virus. Twelve of fifteen chimeras mounted a measurable cytotoxic T cell response to virus. Despite approximately equal percentages of B10 and B10.D2 lymphocytes in chimeras, cytotoxic T cell responses to vaccinia virus and lymphocytic choriomeningitis virus were mediated variably by either syngeneic or allogeneic donor lymphocytes; thus the H-2 type of effector T cells frequently did not correspond to the 50:50 distribution of spleen or peripheral blood lymphocytes. Cytotoxic responses were restricted exclusively to recipient H-2 type. All mixed chimeras examined were able to mount a good IgG response to vesicular stomatitis virus. These results confirm previous data suggesting that such mixed chimeras are healthy and immunocompetent and demonstrate strict recipient-determined restriction specificity of effector T cells; they also suggest that if T help is necessary for induction of virus-specific cytotoxic T cells, it does not require host-restricted interactions between helper T cells and precursor cytotoxic T cells.     

Mechanism of protection from graft-vs-host disease in murine mixed allogeneic chimeras. I. Development of a null cell population suppressive of cell-mediated lympholysis responses and derived from the syngeneic bone marrow component

Splenocyte populations from whole body-irradiated recipients of mixed T cell-depleted (TCD) syngeneic and allogeneic (complete H-2 disparity) bone marrow, or of TCD syngeneic marrow alone, contain cells with the ability to suppress the generation of cell-mediated lympholysis responses in vitro. This activity, which is present by 8 days after bone marrow transplantation and persists for several weeks, has been analyzed for possible veto-like or other specificity. Although reproducible patterns of suppression were observed, depending both on host strain and on the genetic combination of the response examined, the overall suppression in vitro most closely resembles that which has been ascribed to "natural suppressor" cells in other systems. The suppression appears to be mediated by a non-T cell, non-B cell, nonadherent, asialo GM1-negative population. Cold target inhibition and CTL activity of chimeric cells have been ruled out as factors contributing to the observed suppression. Significantly, in mixed chimeras, suppression was found to be mediated exclusively by cells which were syngeneic to the recipient in both recipient strains tested. The rapid development of this suppressive activity may explain the resistance to graft-vs-host disease conferred on whole body-irradiated mice by the addition of TCD syngeneic marrow to an allogeneic graft-vs-host disease-producing inoculum.     

Modified MHC restriction of donor-origin T cells in humans with severe combined immunodeficiency transplanted with haploidentical bone marrow stem cells

The choice of class II MHC determinants that serve as self-recognition elements for murine CD4+ T cells is thought to be determined by the environment in which T cells mature rather than their genotype. Patients with severe combined immunodeficiency (SCID) reconstituted with T cell depleted haploidentical parental stem cells provide an excellent model for studying this phenomenon in humans. After engraftment, the T cells that develop in these infants are all of donor origin. We sought to determine whether the successful immune reconstitution observed in two such SCID chimeras involved modification of the MHC restriction of Ag recognition by the genetically donor T cells as they matured to become competent T cells in the infants' microenvironment. A tetanus toxoid (TT)-specific T cell line and TT-specific T cell clones were established from the blood of two reconstituted SCID patients and from their maternal donors. T cell responsiveness was determined by [3H]thymidine incorporation after TT presentation by EBV-transformed B cell lines (EBV-B) from various donors. The TT-specific T cell line from patient 1 proliferated when presented Ag by patient, maternal donor, and paternal APC. A CD4+ donor origin clone that proliferated when presented TT by patient and paternal EBV-B, but not by maternal donor EBV-B, was isolated from each patient. TT recognition by these clones was shown to be restricted by the HLA DR determinant shared by patient and father, but not present in the donor. Four TT-specific clones isolated from maternal donors failed to proliferate when presented TT by the appropriate paternal EBV-B. These studies demonstrate that, in these human SCID bone marrow chimeras, engrafted donor-origin stem cells maturing to competent T cells in the recipient microenvironment are capable of utilizing recipient HLA determinants as restriction elements for Ag recognition. This suggests that human, as well as murine, MHC restriction patterns for Ag recognition by CD4+ T cells are environmentally determined.     

Induction of permanent mixed chimerism and skin allograft tolerance across fully MHC-mismatched barriers by the additional myelosuppressive treatments in mice primed with allogeneic spleen cells followed by cyclophosphamide

A pure method of drug (cyclophosphamide plus busulfan)-induced skin allograft tolerance in mice that can regularly overcome fully H-2-mismatched barriers in mice has been established. The components of the method are i.v. administration of 1 x 108 allogeneic spleen cells on day 0, i.p. injection of 200 mg/kg CP and 25 mg/kg busulfan on day 2, and i.v. injection of T cell-depleted 1 x 107 bone marrow cells from the same donor on day 3. Recipient B10 (H-2b; IE-) mice prepared with this conditioning developed donor-specific tolerance, and long-lasting survival of skin allografts was shown in almost of the recipient mice. In the tolerant B10 mice prepared with new conditioning, stable multilineage mixed chimerism was observed permanently, and IE-reactive Vbeta11+ T cells were reduced in periphery as seen in untreated B10.D2 (H-2d; IE+) mice. The specific tolerant state was confirmed by the specific abrogation against donor Ag in the assays of CTL activity and MLR and donor-specific acceptance in the second skin grafting. These results demonstrated that the limitation of standard protocol of cyclophosphamide-induced tolerance, which have been reported by us since 1984, can be overcome by the additional treatments with the myelosuppressive drug busulfan, followed by 1 x 107 T cell-depleted bone marrow cells. To our knowledge, this is the first report to induce allograft tolerance with a short course of the Ag plus immunosuppressive drug treatment without any kind of mAbs (pure drug-induced tolerance).     

Induction of alloreactive CD4 T cell tolerance in molecular chimeras: a possible role for regulatory T cells

Induction of molecular chimerism following reconstitution of mice with autologous bone marrow cells expressing a retrovirally encoded allogeneic MHC class I Ag results in donor-specific tolerance. To investigate the mechanism by which CD4 T cells that recognize allogeneic MHC class I through the indirect pathway of Ag presentation are rendered tolerant in molecular chimeras, transgenic mice expressing a TCR on CD4 T cells specific for peptides derived from K(b) were used. CD4 T cells expressing the transgenic TCR were detected in mice reconstituted with bone marrow cells transduced with retroviruses carrying the gene encoding H-2K(b), albeit detection was at lower levels than in mice receiving mock-transduced bone marrow. Despite the presence of CD4 T cells expressing an alloreactive TCR, mice receiving H-2K(b)-transduced bone marrow permanently accepted K(b) disparate skin grafts. CD4+CD25+ T cells from mice reconstituted with H-2K(b)-transduced bone marrow prevented rejection of K(b) disparate skin grafts when adoptively transferred into immunodeficient mice along with effector T cells, suggesting that induction of molecular chimerism leads to the generation of donor specific regulatory T cells, which may be involved in preventing alloreactive CD4 T cell responses that lead to rejection.     

The evolution of MHC restrictions in antigen recognition by T cells in a haploidentical bone marrow transplant recipient

We have longitudinally followed the major histocompatibility complex (MHC) restrictions that govern the response of T lymphocytes to specific Ag in a child with severe combined immunodeficiency who was successfully transplanted by using T cell depleted haploidentical maternal bone marrow cells and immunized shortly afterwards with tetanus toxoid (TT) Ag. In the first year post-transplant, monocytes were of both donor and recipient origin whereas T and B cells were of donor origin. Three years after transplant, all monocytes and T and B cells were of donor origin. T lymphocytes taken from the child at that time and depleted in vitro of alloreactivity to paternal Ag proliferated in response to TT presented by maternal as well as paternal monocytes. A TT-specific T cell line established from these cells in the presence of maternal monocytes cooperated with maternal but not with paternal monocytes, whereas a TT-specific T cell line established in the presence of paternal monocytes cooperated with paternal but not with maternal monocytes and with monocytes derived from a paternal uncle who shared the haplotype inherited by the recipient from her father. These results show that long-term memory T cells restricted to recipient MHC Ag not shared with the bone marrow donor continue to circulate long after the disappearance of accessory cells of recipient origin. These T cells could potentially participate in a secondary immune response because they were shown to recognize TT presented by recipient fibroblasts induced to express class II MHC molecules following treatment with IFN-gamma.     

Efficient natural defense mechanisms against Listeria monocytogenes in T and B cell-deficient allogeneic bone marrow radiation chimeras. Preactivated macrophages are the main effector cells in an early phase after bone marrow transfer

Radiation chimeras in the early phase after bone marrow transplantation are a good model to study the efficiency of the body's nonspecific defense system represented by macrophages (M phi), polymorphonuclear cells (PMN), and NK cells. These cell types are present in large numbers in spleen and liver at that time, whereas the specific immune system represented by T and B cells is functionally deficient. We previously reported enhanced activities in vitro of M phi (and PMN) from recipient animals in an early phase after allogeneic bone marrow transfer. We here demonstrate that these activities result in enhanced spontaneous resistance against Listeria monocytogenes in vivo: CFU of L. monocytogenes in spleen and liver 48 h after infection were about 1 or 2 to 4 log steps less than in untreated control mice of donor or host haplotype. This enhanced resistance decreased over the 4-mo period after marrow transfer. Preactivated M phi were identified as the most important effector cells. Isolated from spleen and peritoneal cavity, they performed enhanced killing of phagocytosed Listeria. Such preactivated M phi occurred in recipient animals after transfer of allogeneic but not of syngeneic bone marrow. The precise mechanism of M phi activation in the allogeneic radiation chimera in the complete absence of any detectable T cell function is not clear at present. However, these preactivated M phi display an important protective effect against L. monocytogenes: chimeras could eliminate Listeria without acquisition of positive delayed-type sensitivity when infected with 10(3) bacteria. An inoculum of 5 . 10(3) L. monocytogenes resulted either in prolonged survival compared with normal mice of the recipient haplotype or in definitive survival accompanied by a positive delayed-type sensitivity. We concluded that enhanced nonspecific immune functions can in part compensate for the defective specific immune system after bone marrow transfer.     

Essential requirement of I-A region-identical host bone marrow or bone marrow-derived cells for tumor neutralization by primed L3T4+ T cells

The antitumor activity of Meth A-hyperimmunized BALB/c mouse spleen cells (Meth A-Im-SPL) was assayed by the Winn test in H-2 incompatible bone marrow chimeras in closed colony CD-1 (nu/nu), inbred DDD/1(nu/nu) (H-2s), or inbred BALB/c(nu/nu) (H-2d) mice as recipients. We found that Meth A-Im-SPL suppressed Meth A growth in the chimera nude mice which were reconstituted with bone marrow cells of the H-2d haplotype (i.e., BALB/c, DBA/2 and B10.D2), but not in the chimeras which were reconstituted with bone marrow cells of the H-2a, H-2b, or H-2k haplotype (i.e., B10.A, B10, and B10.BR). These results suggested that H-2 restriction occurred between Meth A-Im-SPL and bone marrow or bone marrow-derived cells in tumor neutralization. Furthermore, Meth A-Im-SPL did not suppress Meth 1 tumors (antigenically distinct from Meth A tumors) in the presence or absence of mitomycin C-treated Meth A in a Winn assay. These results suggested that there is tumor specificity in the "effector phase" as well as in the "induction phase". The phenotype of the effectors in the Meth A-Im-SPL was Thy-1.2+ and L3T4+, because Meth A-Im-SPL lost their antitumor activity with pretreatment with anti-Thy-1.2 monoclonal antibody (mAb) and complement or anti-L3T4 mAb and complement, but not with anti-Lyt-2.2 mAb and complement or complement alone. Positively purified L3T4+ T cells from Meth A-Im-SPL (Meth A-Im-L3T4), obtained by the panning method, suppressed the tumor growth in the chimera nude mice which were reconstituted with bone marrow cells of B10.KEA2 mice (that were I-A region-identical with Meth A-Im-L3T4 cells but not others in H-2) as well as B10.D2 cells (that were fully identical with Meth A-Im-L3T4 cells in H-2). We conclude that Meth A-Im-SPL (L3T4+) neutralized the tumors in collaboration with I-A region-identical host bone marrow or bone marrow-derived cells, and the neutralization was not accompanied by the "bystander effect."     

Cloned cytolytic T cells can suppress primary cytotoxic responses directed against them

In vivo and in vitro, murine peripheral T cells can suppress or "veto" the activation of cytotoxic T lymphocytes directed against antigens presented by those T cells. This suppression is antigen-specific and H-2-restricted. The recognition event initiating this suppression appears to be unidirectional; precursors of cytotoxic T lymphocytes recognize the antigen-bearing veto cell and are thereby inactivated--the veto cell need not recognize the CTL precursor. We show here that 3/3 cytolytic T cell clones can exert veto activity in vitro on normal spleen cells which do not bear antigens the T cell clones can recognize. This suppression results in greatly diminished cytotoxic activity generated during a primary 5-day mixed lymphocyte culture against antigens which the veto cell expresses, but not against third-party antigens present in the same culture. In this same system, a noncytolytic T cell clone will not serve as a source of veto cells. Secondary cytotoxic responses are relatively resistant to the veto cell activity of cloned cytolytic T cells. The cloned veto cells do not suppress the generation of cytotoxic activity directed against antigens they recognize (and presumably carry over via antigen-specific receptors). Cold target competition during the cytotoxic assay has been eliminated as a possible mechanism for T cell clone-induced suppression, and suppression cannot be reversed by the addition to the mixed lymphocyte cultures of supernatants from concanavalin A-activated spleen cells. It is suggested that this mechanism of inactivating primary cytotoxic T lymphocyte responses could play an important role in the maintenance of self-tolerance and in the induction and maintenance of tolerance to allografts.     

Characterization of the Fas ligand/Fas-dependent apoptosis of antiretroviral, class I MHC tetramer-defined, CD8+ CTL by in vivo retrovirus-infected cells

C57BL/6 (B6; H-2(b)) mice mount strong AKR/Gross murine leukemia virus (MuLV)-specific CD8(+) CTL responses to the immunodominant K(b)-restricted epitope, KSPWFTTL, of endogenous AKR/Gross MuLV. In sharp contrast, spontaneous virus-expressing AKR.H-2(b) congenic mice are low/nonresponders for the generation of AKR/Gross MuLV-specific CTL. Furthermore, when viable AKR.H-2(b) spleen cells are cocultured with primed responder B6 antiviral precursor CTL, the AKR.H-2(b) cells function as "veto" cells that actively mediate the inhibition of antiviral CTL generation. AKR.H-2(b) veto cell inhibition is virus specific, MHC restricted, contact dependent, and mediated through veto cell Fas ligand/responder T cell Fas interactions. In this study, following specific priming and secondary in vitro restimulation, antiretroviral CD8(+) CTL were identified by a labeled K(b)/KSPWFTTL tetramer and flow cytometry, enabling direct visualization of AKR.H-2(b) veto cell-mediated depletion of these CTL. A 65-93% reduction in the number of B6 K(b)/KSPWFTTL tetramer(+) CTL correlated with a similar reduction in antiviral CTL cytotoxicity. Addition on sequential days to the antiviral CTL restimulation cultures of either 1) AKR.H-2(b) veto cells or 2) a blocking Fas-Ig fusion protein (to cultures also containing AKR.H-2(b) veto cells) to block inhibition demonstrated that AKR.H-2(b) veto cells begin to inhibit B6 precursor CTL/CTL expansion during days 2 and 3 of the 6-day culture. Shortly thereafter, a high percentage of B6 tetramer(+) CTL cocultured with AKR.H-2(b) veto cells was annexin V positive and Fas(high), indicating apoptosis as the mechanism of veto cell inhibition. Experiments using the irreversible inhibitor emetine demonstrated that AKR.H-2(b) cells had to be metabolically active and capable of protein synthesis to function as veto cells. Of the tetramer-positive CTL that survived veto cell-mediated apoptosis, there was no marked skewing from the preferential usage of Vbeta4, 8.1/8.2, and 11 TCR normally observed. These findings provide further insight into the complexity of host/virus interactions and suggest a fail-safe escape mechanism by virus-infected cells for epitopes residing in critical areas of viral proteins that cannot accommodate variations of amino acid sequence.     

Minor histocompatibility antigens on canine hemopoietic progenitor cells

Adoptive immunotherapy with CTL against minor histocompatibility Ags (mHA) provides a promising way to treat leukemia relapse in allogeneic chimeras. Here we describe the in vitro generation of CTL against mHA in the dog. We tested their inhibitory effect on the growth of hemopoietic progenitor cells stimulated by hemopoietic growth factors in a 4-day suspension culture. CTL were produced by coculture of donor PBMC with bone marrow-derived dendritic cells (DCs). These DCs were characterized by morphology, high expression of MHC class II and CD1a, and the absence of the monocyte-specific marker CD14. Characteristically these cells stimulated allogeneic lymphocytes (MLR) and, after pulsing with a foreign Ag (keyhole limpet hemocyanin), autologous T cells. CTL were generated either ex vivo by coculture with DCs of DLA-identical littermates or in vivo by immunization of the responder with DCs obtained from a DLA-identical littermate. In suspension culture assays the growth of hemopoietic progenitor cells was inhibited in 53% of DLA-identical littermate combinations. In canine families mHA segregated with DLA as restriction elements. One-way reactivity against mHA was found in five littermate combinations. In two cases mHA might be Y chromosome associated, in three cases autosomally inherited alleles were detected. We conclude that CTL can be produced in vitro and in vivo against mHA on canine hemopoietic progenitor cells using bone marrow-derived DCs.     

Study on the possible factors influencing the expression of H-2 restriction specificity and Ir phenotype of antigen-specific proliferative T cells with various types of radiation chimeras

To better understand the factors described previously as influencing the manifestation of H-2 restriction specificity and Ir phenotype of T cells from radiation bone marrow chimeras, we also examined H-2 restriction specificity (Ir phenotype) of antigen (DNP-OVA, (T, G)-A-L, (H, G)-A-L)-specific proliferative T cells generated in various types of H-2 incompatible radiation chimeras prepared under our specific-pathogen-free (SPF) condition. The results indicated the following: (a) T cells generated in F1----parent bone marrow chimeras preferentially manifested host-type H-2 restriction specificity and Ir phenotype, regardless of the radiation dose (8.70 vs 11.59 Gy); (b) T cells recovered from twice-reconstituted F1----(PA----PB) chimeras manifested primary host (PB)-type Ir phenotype; (c) T cells which were recovered from (B10.Thy-1.1 X B10.BR.Thy-1.1)F1----parent (Thy-1.2) bone marrow chimeras and treated with anti-Thy-1.2 plus complement to deplete host-derived T cells still manifested preferentially the restriction specificity for host-type H-2; (d) PA-derived T cells which had differentiated in a fully allogeneic host (PB) environment of (PA + PB)----PB chimeras manifested fully allogeneic host-type Ir phenotype; (e) T cells from F1----parent chimeras that were prepared with 13-day fetal liver cells also manifested host H-2-restricted Ir phenotype; and (f) host preference for Ir phenotype of antigen-specific proliferative T cells was observed even in the case of F1----parent bone marrow chimeras reconstituted with "intact" bone marrow cells. The data suggest that thymic APCs, surviving host T cells or the source of stem cells (adult bone marrow vs 13-day fetal liver), do not necessarily play a significant role in the manifestation of H-2 restriction specificity and Ir phenotype of T cells generated in H-2 incompatible radiation chimeras.