Blood transfusion-induced suppression of delayed-type hypersensitivity to allogeneic histocompatibility antigens

In this study delayed-type hypersensitivity against histocompatibility antigens in mice was suppressed by a single donor-specific blood transfusion. Whole blood as well as purified white blood cells and purified red blood cells were capable of inducing suppression. White cells appeared more potent in inducing suppression than red cells. Suppression was dose-dependent, still detectable after administration of as little as 0.001 ml of whole blood and maximal at a dose of 0.1 ml. The suppression was already present a few hours after transfusion and proved to be long-lasting. The suppressive effect could be transferred to naive recipients by Thy-1+, L3T4-, Lyt-2+ spleen cells. This suppressor T cell population was of recipient origin--which excludes the possible involvement of "veto cells" and suppresses the afferent phase as well as the efferent phase of the DTH response.     

Immunobiology of the graft-versus-host reaction. I. Symptoms of graft-versus-host disease in mice are preceded by delayed-type hypersensitivity to host histocompatibility antigens.

During initiation of an acute graft-versus-host reaction by injection of C57BL/Rij spleen cells into lethally irradiated (C57BL/Rij X CBA/Rij)F1 hybrid mice, a state of delayed-type hypersensitivity (DTH) against host histocompatibility (H) antigens occurs. This was demonstrated by means of transfer of host spleen and lymph node cells into C57BL/Rij recipients, which received a challenge with CBA/Rij spleen cells. Initiation and transfer of the graft-versus-host-related DTH reactivity was highly dependent on Thy-1.2+ cells. The development of DTH reactivity started between 8 and 24 hr after semiallogeneic spleen cell transplantation and increased during the days thereafter. In the spleen maximal DTH reactivity was found on day 4, whereas in the lymph nodes maximal reactivity occurred on day 5 after irradiation and reconstitution. Thereafter, the reactivity decreased until there was no further DTH reactivity demonstrable on day 13. The specificity of the DTH reactivity for host H antigens was proved by no reactivity to a challenge of DBA/2 and Swiss spleen cells, which are H-2-incompatible with CBA cells.     

Antigenicity of passively acquired major histocompatibility antigens on T cells

T cell blasts and lines passively acquire MHC molecules in vitro. To determine the role of these molecules in immunoregulatory reactions, we examined whether T cell lines grown on irradiated F1 spleen cells were able to supply allogeneic MHC antigens for the stimulation of T cell proliferation. Immunofluorescence analysis demonstrates that autoreactive T cell lines grown with irradiated F1 spleen cells acquire allogeneic class II molecules and subsequently lose the MHC molecules within 4 days of coculture with syngeneic cells. The proliferative response of (H-2k x H-2d)F1T cells stimulated by a T cell line grown on (H-2k x H-2d)F1 cells is inhibited by the addition of hybridoma-culture supernatants containing anti-IAd as well as anti-IEk antibodies. The proliferation of the F1 T cells to the T cell line grown on H-2k spleen cells is only affected by supernatants containing anti-IEk antibodies. To investigate the role of acquired class I MHC antigens, we examined their ability to serve as antigens for cytotoxic cells. Anti-H-2k cytotoxic T cells are generated when H-2b T cells are cultured with an H-2b-derived T cell line, only if the line has been grown on (H-2k x H-2b)F1 cells. An H-2b-derived T cell line exposed to (H-2k x H-2b)F1 cells can be lysed by anti-H-2k cytotoxic T cells from a primary MLR. Similarly, an H-2k anti-H-2b cytotoxic T cell clone will kill an H-2k-derived T cell clone grown on (H-2k x H-2b)F1 spleen cells. These results demonstrate that passively acquired class I molecules can stimulate the generation of cytotoxic T cells that lyse cells expressing the class I antigens and that passively acquired class I molecules expressed on T cells serve as the target for cytotoxic T cells.     

Immune mechanisms in organ allograft rejection. II. T helper cells, delayed-type hypersensitivity, and rejection of renal allografts

The cellular requirements for renal allograft rejection have been assessed by adoptive transfer studies in a rat model. Sublethally irradiated (780 rads) LEW (RT1l) recipients of WF (RT1u) renal allografts were selectively reconstituted with spleen cells from specifically sensitized donors. In some experiments the reconstituting inocula were depleted of SIg+ cells by passage over anti-Ig columns or subjected to additional depletion of cytotoxic T cells (Tc) and their precursors reactive with monoclonal antibody OX8. WF renal allografts underwent acute rejection in the unmodified LEW recipient with day 7 serum creatinines of 6.8 +/- 0.9 mg/dL (mean +/- SD; n = 7), graft histology characterized by marked mononuclear cell infiltration and evidence of a brisk humoral response (complement-dependent cytotoxicity (CDC) titer greater than 2(6] and generation of Tc demonstrable by in vitro monitoring. Sublethally irradiated recipients mounted no detectable immune response, and day 7 serum creatinine and graft histological findings were not significantly different from those obtained in isograft controls. Renal allografts were, however, rejected in sublethally irradiated recipients reconstituted with unfractionated immune spleen cells, as evidenced by functional and histologic criteria (day 7 serum creatinine of 5.5 +/- 1.2 mg/dL; histology characterized by extensive interstitial hemorrhage, fibrinoid necrosis of blood vessels, and polymorphic infiltration). Neither antibody nor Tc appear, in this model, to be required to effect rejection, because recipients reconstituted with inocula depleted of SIg+ cells (day 7 CDC titer less than 2l) or subjected to additional depletion of Tc and their precursors (day 7 lymphocyte-mediated cytotoxicity assay: % specific chromium release at 100/1 E/T ratios less than 5%) underwent acute rejection with a day 7 serum creatinine of 5.0 +/- 1 and 4.3 +/- 1.5 mg/dL, respectively, and histological findings were characterized by marked mononuclear cell infiltration and a paucity of hemorrhage.     

Tolerance to non-H-2 histocompatibility antigens. Transplantation tolerance to the H-4 and H-7 histocompatibility antigens.

There have been several reports of observations which suggest that transplantation tolerance may be a result of positive immunoregulation rather than simply unresponsiveness attributable to a lack of competent effector cells. In particular, several investigators have reported that tolerance of the H-Y and H-1 histocompatibility antigens is mediated by a population of thymus-derived lymphocytes. In a companion report, we have presented evidence that supports the existence of a suppressor cell to the H-Y antigen. Furthermore, we have observed that female mice rendered tolerant of the H-Y antigens by neonatal exposure to male lymphoid cells or by multiparity accept male skin grafts indefinitely, but inactivate male peritoneal exudate cells (PEC) in a second-set fashion. This observation has led us to investigate whether tolerance of other non-H-2 antigens is controlled by a similar mechanism. Using mice congenic with C57BL/10 at the H-4 and H-7 loci, we have shown that mice rendered tolerant of the H-7a and H-4b antigens by neonatal exposure to histoincompatibe lymphoid cells are incapable of rejecting either skin or peritoneal cell allografts, suggesting that identical histocompatibility antigens are present on skin and peritoneal cells. Tolerance induced in neonatal mice to the H-4b and H-7a antigens could not be adoptively transferred to syngeneic recipients. These results suggest that tolerance involving the H-4 and H-7 antigens is most likely because of a clonal inactivation of alloantigen-reactive cells as a consequence of neonatal exposure to antigen.     

Allograft immunity to histocompatibility and organ-specific antigens.

An experimental model has been developed in the dog in which a renal allograft was placed in the neck, leaving one of the dog's own kidney in situ. Five nonimmunosuppressed pairs of dogs have been studied by using the leucocyte migration test (LMT) as an in vitro measure of cell-mediated immunity. Antigen preparations from leucocytes, kidney, liver, and skeletal muscle from both the kidney donor and the recipient were used in the LMT in order to study responses against transplantation and organ-specific antigens. Inhibition of migration with donor-specific leucocyte and kidney antigens was detectable prior to clinical evidence of rejection, which was confirmed histologically. Concurrently, inhibition was also observed with autologous kidney antigen and histological damage was noted in the recipient's own nontransplanted kidney, accompanied by increasing proteinuria. Autologous serum withdrawn daily and added to the test culture medium abolished the inhibition of migration, thus suggesting the development of blocking factor.     

Antigen competition in cytotoxic T cell response to minor histocompatibility antigens

Two populations of class-I-restricted CTL are generated from BDF1 mice against multiple minor-HA when BALB.B cells carrying multiple minor-HA are used as immunogen, whereas one population of Db-restricted CTL is generated when H-25.3 antigen on B6.C-H-25c cells is used as antigen. CTL generated against H-25.3 antigen on B6.C-H-25c cells are cytolytic to B6.C-H-25c and BALB.B targets, whereas CTL generated in the same manner with multiple minor-HA on BALB.B cells kill BALB.B targets only. This must be an instance of antigen competition, because H-25.3 antigen is immunostimulatory when used on its own, but not when accompanied by other minor-HA. This competition effect is not an artefact seen during the generation of CTL in MLC cultures in vitro, because it is also seen during the generation of CTL in vivo. We have not identified the immunodominant antigen(s) on BALB.B cells. We have studied this form of antigen competition in some detail. First, BDF1 spleen primed in vivo to multiple minor-HA on BALB.B cells do not respond in vitro to restimulation by H-25.3 antigen, suggesting that antigen competition is not mediated by suppressor cells, but that BALB.B cells do not prime BDF1 spleen cells against H-25.3 antigen in vivo. Second, BDF1 spleen cells primed to the same multiple minor-HA carried on F1(BALB/c X B6.C-H-25c) cells respond to restimulation by H-25.3 antigen in vitro. Third, BDF1 spleen cells primed in vivo to H-25.3 antigen on B6.C-H-25c cells do not respond in vitro to restimulation by multiple minor-HA on BALB.B cells, but they do respond to F1(BALB/c X B6.C-H-25c) cells that carry the same multiple minor-HA as BALB.B cells, and generate Db-restricted anti-H-25.3 CTL. Fourth, BDF1 spleen cells primed in vivo and boosted in vitro with B6.C-H-25c cells readily develop CTL to H-25.3 antigen, whereas when the BDF1 mice are exposed to H-25.3 antigen in vivo by repeated footpad injections of B6.C-H-25c cells, CTL to H-25.3 antigen do not develop. However, anti-H-25.3 CTL do develop after H-25.3 antigen-bearing B6.C-H-25c cells and antigen-specific Th cells are injected into preprimed BDF1 mice. These results are discussed with respect to possible mechanisms of antigen competition.     

Regulation of cytotoxic reactivity to minor histocompatibility antigens by administration of Corynebacterium parvum

B10.BR(H-2k) mice were primed with H-2-identical allogeneic CBA/J(H-2k) spleen cells and restimulated in vitro 14 days later to generate specific secondary cytotoxic lymphocytes. A single intravenous injection of primed mice with 700 micrograms of Corynebacterium parvum 7 days after alloimmunization markedly inhibited the subsequent secondary in vitro generation of cytotoxic cells. In addition, regulatory spleen cells were detected in alloimmunized C-parvum-injected mice that prevented the restimulation of primed control spleen cells. Suppressive activity could not be abrogated by treating regulatory cells with anti-theta antibody and complement or by removing phagocytic cells, but it was overcome by treatment with mitomycin C. Unfractionated regulatory cells suppressed responses in an antigen nonspecific fashion. However, cells remaining after carbonyl and iron treatment (nonphagocytic) could no longer suppress responses to third party alloantigens while maintaining significant suppression of anti-CBA responses. These data suggest the generation of two distinct suppressor cell types that can control the cytotoxic response to minor histocompatibility antigens: an antigen-nonspecific phagocytic cell and an antigen specific nonphagocytic, non-theta-bearing cell.     

Alloreactive T suppressor cells in the rat. I. Evidence of three distinct subsets of splenic suppressor T cells resistant to cyclosporine

In this study we examined the effect of cyclosporine on three distinct subsets of T suppressor (Ts) cells identified in a rat renal allograft model. Ts inducer (Ts1) cells having the CD4 marker are found in the spleens of DA rats undergoing acute rejection of LEW kidneys. Transducer (Ts2) and effector (Ts3) cells both carry the CD8 marker and are found in the spleens of long-term surviving DA rats bearing LEW kidney allografts made tolerant by donor-specific blood transfusions or by cyclosporine (in most cases). These latter cells are distinguished by their susceptibility to cyclophosphamide (CY), Ts2 cells being resistant while Ts3 cells are sensitive to CY. When Ts cells from DA rats undergoing acute graft rejection of LEW kidneys or bearing long-term-surviving LEW kidneys that had been treated with cyclosporine (10mg/kg/day) for 2 or 10 days, respectively, were adoptively transferred into lightly irradiated DA recipients, these cells were still able to specifically induce long-term survival of LEW kidneys. LEW kidney survival was not prolonged in DA rats given no cells or cells from rats treated with cyclosporine for 10 days. Thus it would appear that the three functional subsets of Ts cells demonstrated in this renal allograft model by adoptive transfer of spleen lymphocytes are not inhibited by cyclosporine, suggesting that this resistance of Ts cells to cyclosporine may be partly responsible for the immunosuppressive effect of this agent.     

T cell subsets required for in vivo and in vitro responses to single and multiple minor histocompatibility antigens.

The requirement for helper T cells in the in vivo and in vitro T cell response to a diverse panel of minor histocompatibility antigens in mice was investigated. Target H antigens included: (a) multiple antigens distinguishing H-2-matched, inbred strains, and (b) single H antigens, including H-4, H-3, and the male-specific (H-Y) antigen. The involvement of helper T cells in skin allograft rejection and CTL priming was evaluated by pretreating graft recipients with anti-CD4 antibody. Anti-CD4 treatment had no effect on rejection of H-3- and H-4-incompatible skin grafts, but slowed rejection of male and BALB.B skin grafts. Comparable pretreatment with anti-CD4 antibody in vivo eliminated the priming of H-4-specific CTL, multiple B10.BR anti-CBA/J CTL, and all but a minor C57BL/6 anti-BALB.B CTL population. However, CTL specific for the two classes of H antigens, single and multiple minor H antigens, differed in their in vitro requirements for CD4+ helper T cells: (a) multiple antigen-specific CTL required CD4+ helper T cells for optimal expansion, and (b) CTL specific for single H antigens expanded in the absence of helper T cells. The CTL specific for all tested H antigens were CD8+ T cells. These results suggest that CD4+ helper T cells are not always required for effective skin allograft rejection or CTL expansion in vitro; the requirement for helper T cells is apparently dependent upon the identity of the stimulatory minor H antigen(s). This variable dependency contrasts with the evident requirement for helper T cells in the in vivo priming of CTL specific for minor H antigens.     

Human suppressor T cells induced in vitro with an autologous renal allograft-derived T cell line. II. Activity and specificity of a soluble suppressor factor

Human suppressor T cells induced by autologous mixed lymphocyte reaction (AMLR) using fresh responder PBL from a renal transplant recipient and an autologous irradiated antidonor CTL line (EE-1) established from a biopsy of the patient's own allograft were studied for the production of suppressor factors. The suppressor cell lines propagated (designated TsEE) were capable of inhibiting the in vitro generation of proliferative and cytotoxic responses of responder cells from the recipient or other individuals who shared HLA-B7 with TsEE cells, regardless of the stimulatory cell phenotype. Coculture of TsEE cells with the autologous irradiated EE-1 inducer cell line in vitro yielded a soluble factor (designated TsEEF) capable of inhibiting the generation of MLR and CTL responses, as well as mitogen-induced proliferative responses to PHA and PWM in an HLA-unrestricted manner. TsEEF also inhibited the replication of lymphoblastoid T cell lines (Molt-4 and HSB) but not B cell lines (SB and JC-EBV) or PBL stimulated with the B cell mitogen LPS. Control supernatants obtained from each of the cells used to generate TsEE in AMLR (i.e., EE-PBL and the EE-1 line) cultured alone or together for 48 hr demonstrated no suppressive activity in any of these test systems. TsEEF was nontoxic for lymphoid cells, was nondialyzable (greater than 12kDa), did not act by interfering with IL-1 or IL-2 utilization, and was negative for TNF and IFN-gamma activity. Functionally, the suppressive activity of TsEEF was dose-dependent, did not shift MLR kinetics, and could be absorbed by T cells. T cells incubated with TsEEF for 4 hr were unresponsive to subsequent mitogen or MLR stimulation. These findings indicate that, whereas T suppressor cell lines propagated from the circulation of a stable renal transplant recipient demonstrate class I HLA restriction, the activity of their soluble products is not HLA-restricted, and functionally inhibits T cell proliferation.     

Effector mechanisms in graft-versus-host disease in response to minor histocompatibility antigens. II. Evidence of a possible involvement of proliferative T cells

To study helper T cell activation against minor histocompatibility (mH) antigens of the host after HLA-identical bone marrow transplantation, patients' lymphocytes collected longitudinally after transplantation were tested in a primed lymphocyte test using PBL from patients and donors as stimulator cells. Sixteen patients were studied between 1 and 25 months after grafting. Antihost Th cells were detected in 10 patients. Optimum levels of antihost activity were generally reached within the first 3 months, thereafter two patterns were identified; in some patients the antihost Th cell activity persisted for at least 2 years, whereas in other patients a decline was observed with time. Antihost Th cell activity developed in each of 5 patients with acute GVHD, in 3 out of 5 patients with chronic GVHD, but in only 2 out of 6 patients without GVHD. The average antihost Th cell activity in patients with acute GVHD was significantly higher than in patients without GVHD (P = 0.036) and was also higher, although not significantly, than in patients with chronic GVHD. These findings indicate that, in man, as was shown in studies in mice, helper T cells do participate in the response to mH antigens. Although other mechanisms may also be involved, we here propose that mH antigen-specific Th cells may be a risk factor for acute GVHD.     

Major histocompatibility complex and non-major histocompatibility complex antigens on mouse ectoplacental cone and placental trophoblastic cells.

The expression of major histocompatibility complex and non-major histocompatibility complex antigens on mouse trophoblast cultured from two defined stages of development was investigated by the sensitive in vitro mixed haemadsorption assay. Outgrowths obtained 3 to 5 days after explanation of 7 1/2-day ectoplacental cones contained a mixed population of cells. Those with a giant cell morphology showed no haemadsorption with congenic H-2 antisera and were reactive with non H-2 antiserum only in the CBA strain. Other, smaller cells were reactive for both H-2 and non-H-2 in all strains examined except for C57BL, where the cells were nonreactive for H-2. Monolayer cultures of 13 to 14-day placental suspensions tested 24 hr after preparation were strongly reactive for both H-2 and non-H-2. The identity and alloantigenic status of the cells are discussed in relation to their function in maternal-foetal immunological interactions.