Mixed Leukocyte Culture Responses Research Articles

Long-Term Tolerance and Skin Allograft Survival in CD200tg Mice After Autologous Marrow Transplantation.

CD200 overexpression in transgenic recipients, or by tissue allografts, increases skin and cardiac graft survival in mice receiving rapamycin, in association with increased Foxp3(+)Treg graft infiltration. However, less than 50% of skin grafts persist beyond 25 days. We investigated whether immune ablation using busulphan and cyclophosphamide, followed by autologous marrow reconstitution, would permit long-term survival in a greater percentage of recipients and induce tolerance, allowing for withdrawal of immunosuppressive drugs. C57BL/6 wild type (BL/6 WT) or CD200(tg) mice received BALB/c skin grafts with rapamycin (1 mg/kg/36 hr) for 7 days. Thereafter, subgroups received busulphan or cyclophospamide for 6 days, followed by BL/6 marrow transplantation (BMTx), whereas controls were maintained on rapamycin. Beginning 7 days after marrow transplantation, mice receiving BMTx also received rapamycin for a further 7, 14, or 21 days (to a maximum of 42 days after transplantation) after which times, all immunosuppressions were withdrawn. Graft survival was monitored throughout, and mixed leukocyte cultures (MLCs) (using peripheral blood leukocytes) were performed for all groups at 60 days after transplantation. Gene expression was performed in grafts harvested at 80 days. Although all WT mice rejected grafts by 16 days, survival in CD200(tg) was approximately 40% at 60 days, with antigen-specific decreased MLC responses to BALB/c. After BMTx, survival in WT mice was greater than 40% at 60 days, and greater than 90% at 60 days in CD200(tg), with corresponding attenuated MLC responses. Long-term surviving grafts were infiltrated by Tregs of both host and donor marrow origins, as defined using CD45 congeneic donors or recipients. Survival was increased by infusion of an antibody directed to TNFRSF25 on Tregs. Autologous BMTx improves graft survival and promotes graft tolerance.

Effect of CD200 and CD200R1 expression within tissue grafts on increased graft survival in allogeneic recipients

In transgenic mice over-expressing CD200 (CD200tg) graft survival is associated with increased intra-graft expression of mRNAs for genes associated with altered T cell subset differentiation (Foxp3; TGFβ; IL-10). Grafts are rejected in recipients lacking the inhibitory receptor for CD200, CD200R1.We compared grafts of C57BL/6 skin taken from control, CD200KO, CD200tg, CD200R1KO or CD200tg.CD200R1KO C57BL/6 donor mice transplanted to control or CD200tg BALB/c recipients. Animals received either low-dose rapamycin (0.5mg/kg), which only enhanced survival in CD200tg mice, or high dose rapamycin (1.5mg/kg) which increased graft survival in all recipients. Recipient draining lymph nodes (DLNs) were analyzed at 14days post grafting in mixed leukocyte cultures (MLCs) with irradiated BL/6 or C3H/HeJ stimulator cells, assaying antigen-specific CTL at day 5. MLC responses were correlated with changes in mRNA gene expression in skin tissue harvested from the same recipients, focusing on genes altered in “graft-accepting” CD200tg recipients. Tissue histology was used to assess graft infiltrating Foxp3+ Tregs, mast cells (MCs) and their degranulation.CD200tg grafts were accepted in control but not CD200KO/CD200R1KO recipients, along with decreased degranulation in graft MCs, diminished DLN MLC responses, and augmented intragraft Foxp3, TGFβ, IL-10 and mast cell gene expression. Skin grafts from either CD200KO or CD200R1KO donors to control mice were rejected, with no change in DLN MLC responses, no altered graft gene expression from that seen using control skin grafts, and pronounced graft MC degranulation. Our data highlight a role for both graft and host CD200/CD200R expression in increased allograft survival.

Persistence of Gene Expression Profile in CD200 Transgenic Skin Allografts Is Associated With Graft Survival on Retransplantation to Normal Recipients

Expression of CD200 increases allograft survival by suppressing inflammation and acquired immunity. Increased allograft survival in transgenic mice overexpressing CD200 (CD200) occurs in association with increased intragraft expression of messenger RNAs (mRNAs) for genes associated with altered T-cell differentiation. We investigated whether donor CD200 BL/6 skin grafts taken from primary control or CD200 recipient BALB/c mice persisted after retransplantation at 14 days to control (nontransgenic) secondary BALB/c recipients, with or without transfer of splenocytes from autologous primary recipients. Splenocytes from primary and secondary recipients were analyzed 14 days after grafting, using in vitro mixed leukocyte cultures (MLCs) incubated with irradiated BL/6 (or third-party C3H/HeJ) stimulator cells and assayed for antigen-specific cytotoxic T lymphocyte. Cytotoxic T lymphocyte responses were correlated with changes in the mRNA gene expression profile observed in the skin tissue harvested from primary or secondary recipients on day 14 after grafting, using real-time polymerase chain reaction to compare quantitative mRNA expression in the graft tissue of primary/secondary recipients. Adoptive transfer of tolerance in MLC to BL/6 grafts was most evident when both skin and splenocytes were transferred from primary BALB/c recipients, although there was an attenuation of MLC responses after graft transfer alone. Adoptive transfer of tolerance occurred concomitant with persistent overexpression of genes encoding Foxp3, transforming growth factor β, interleukin 10, and PD-1 (and PD-L1/PD-L2) in tolerant skin grafts and increased expression of mRNAs for indoleamine 2,3-dioxygenase and the subunits encoding interleukin 35. Infusion of anti-CD4 or anti-transforming growth factor β to secondary recipients on retransplantation abolished increased graft survival, suggesting the importance of each to the final outcome.

Immune Inhibitory Ligand CD200 Induction by TLRs and NLRs Limits Macrophage Activation to Protect the Host from Meningococcal Septicemia

Macrophage activation is essential for protection against bacterial pathogens but needs to be regulated to prevent damage to the host. We show a key role for the immune inhibitory receptor CD200R and its ligand CD200 in the context of infection with the Gram-negative human pathogen Neisseria meningitidis. N. meningitidis induced CD200 but downregulated CD200R on macrophages in a manner dependent on Neisserial lipopolysaccharide, Toll-like receptor-4 (TLR-4), and the MyD88 pathway but independent of a known Neisserial receptor, scavenger receptor A (SR-A). Agonists of the pattern-recognition receptors nucleotide oligomerization domain 2 (NOD2) and NACHT-LRR protein 3 (NALP3) also induced CD200. The NF-κB member c-Rel was essential for TLR-, NOD2-, and NALP3-mediated induction of CD200. CD200(-/-) animals showed higher lethality in response to experimental meningococcal septicemia, induced higher levels of proinflammatory cytokines, and recruited increased numbers of activated leukocytes, despite comparable bacterial clearance. Thus CD200 is induced by TLR-, NOD2-, and NALP3-mediated pathways, limiting their function and protecting the host from excessive inflammation.

Mice Lacking CD200R1 Show Absence of Suppression of Lipopolysaccharide-Induced Tumor Necrosis Factor-α and Mixed Leukocyte Culture Responses by CD200

CD200:CD200R interactions deliver immunoregulatory signals. A family of CD200Rs (CD200R1-5) has been described, and engagement of CD200R1 by its ligand CD200 suppresses LPS-induced macrophage cytokine production, decreases alloimmune responses in vivo and in vitro, and suppresses collagen-induced arthritis. We generated C57BL/6 mice lacking the genomic exons encoding the extracellular domains of the CD200R1 molecule using transformation of ES cells and explored cell subtypes and immune responses in these mice. Myeloid cells/splenocytes from CD200R1(-/-) mice were not stained in FACS by anti-CD200R1 mAb. Stimulation of splenic tumor necrosis factor-alpha production by lipopolysaccharide was enhanced relative to control (+/+) mice and was not suppressed by addition of exogenous CD200Fc. Modulation of alloreactivity in mixed leukocyte cultures by CD200Fc depended upon CD200R1+ stimulatory cells, although maximal immunoregulation by CD200Fc occurred only when CD200R1+ T responder cells also were used. CD200Fc failed to suppress graft rejection in CD200R1(-/-) mice. CD200:CD200R1 plays an immunoregulatory role in vivo.

Stable trichimerism after marrow grafting from 2 DLA-identical canine donors and nonmyeloablative conditioning

Although hematopoietic cell transplantation (HCT) is generally accomplished using a single donor, multiple donors have been used to enhance the speed of engraftment, particularly in the case of umbilical cord blood grafts. Here we posed the question in the canine HCT model whether stable dual-donor chimerism could be established using 2 DLA-identical donors. We identified 8 DLA-identical littermate triplets in which the marrow recipients received 2 Gy total body irradiation followed by marrow infusions from 2 donors and postgrafting immunosuppression. All 8 dogs showed initial "trichimerism," which was sustained in 5 dogs, while 2 dogs rejected one of the allografts and remained mixed chimeras, and 1 dog rejected both allografts. Immune function in one trichimeric dog, as tested by mixed leukocyte culture response and antibody response to sheep red blood cells, was found to be normal. Five dogs received kidney grafts from one of their respective marrow donors at least 6 months after HCT without immunosuppressive drugs, and grafts in 4 dogs are surviving without rejection. In summary, following nonmyeloablative conditioning, simultaneous administration of marrow grafts from 2 DLA-identical littermates could result in sustained trichimerism, and immunologic tolerance could include a kidney graft from one of the marrow donors.

DNA associated with the cell membrane is involved in the inhibition of the skin rejection response induced by infusions of photodamaged alloreactive cells that mediate rejection of skin allograft.

Cell membrane DNA (cmDNA) is a form of DNA located on the surface of human and murine T-cells. It has recently been characterized as a target for photomodification by 8-methoxypsoralen (8-MOP) and long-wave ultraviolet light (UV-A). Whereas 8-MOP itself is biologically inert, photoactivated 8-MOP is covalently bound to pyrimidine bases in DNA. We have investigated the possible involvement of cmDNA photomodification in the induction of the suppression of skin allograft rejection in BALB/c mice preimmunized with 8-MOP/UV-A photodamaged alloreactive cells which mediates this allograft rejection. This suppression is demonstrated by inhibition of delayed-type hypersensitivity (DTH) and mixed leukocyte culture (MLC) responses. Splenocytes from BALB/c mice undergoing rejection of CBA/j skin graft which contained an expanded population of the effector T lymphocytes that mediate the rejection were treated with DNAse to remove cmDNA before or after treatment with 8-MOP and UV-A prior to infusion into naive BALB/c recipients. Mice that received pretreated effector cells were tested for MLC responses to CBA/j or B10 alloantigens before and after the DTH response. The DTH response of all groups of pretreated BALB/c mice to the relevant alloantigen was specifically suppressed as compared with the response of control mice. However, adoptive transfer of the suppression of the DTH response was optimally demonstrable only in syngeneic recipients of cells from donor mice treated with photodamaged alloreactive cells. Also, splenocytes from BALB/c mice immunized with photodamaged alloreactive cells demonstrated highly significant hyporesponsiveness and suppression of the MLC response of naive mice to the relevant alloantigen in the case of the primary MLC response, and to both alloantigens in the secondary MLC response which was totally eliminated by prior pretreatment of these effector cells with DNAse. Therefore, it appears that the suppression of the DTH response can be induced by pretreatment of the effector cells with DNAse and/or 8-MOP and UV-A but is adoptively transferable optimally only from mice which are recipients of photodamaged alloreactive cells. Moreover, the effectiveness of this treatment is decreased by prior removal of cmDNA from these cells. The presence of cmDNA is necessary for induction of suppression of the primary and secondary MLC responses in mice treated with photodamaged cells of allograft rejection.

Inhibition of Antiskin Allograft Immunity Induced by Infusions with Photoinactivated Effector T Lymphocytes (PET Cells). Is In Vivo Cell Transferrable?

We previously reported producing donor-specific tolerance to alloantigens by intravenous exposure to pretreated antidonor T cells. The current study extends that work by adoptively transferring the donor-specific tolerance into naive syngeneic recipients. Eight days after BALB/c mice received histoincompatible CBA/j skin grafts, their splenocytes which included an expanded population of cells mediating rejection were treated with 100 ng/ml 8-methoxypsoralen (8-MOP) photoactivated by 1 Joule/cm2 of ultraviolet A (UVA) light prior to infusion into naive BALB/c recipients. Whereas 8-MOP itself is biologically inert, photoactivated 8-MOP crosslinks DNA by covalently binding to pyrimidine bases. Recipient BALB/c mice which had been previously demonstrated to be hyporesponsive to CBA/j alloantigens in mixed leukocyte culture (MLC), cytotoxicity (CTL) and in vivo delayed type hypersensitivity (DTH) assays were the donors of spleen cells for the adoptive transfer experiments. Fifty to one hundred million viable spleen cells from these pretreated BALB/c mice were transferred into naive syngeneic recipients which then were tested for DTH response and allograft survival to the relevant and irrelevant antigens. The radiosensitivity of this transferrable suppression was evaluated by exposing the adoptively transferred cell population to 3200 rads of C-irradiation prior to cell transfer. The phenotype of the cells transferring this suppressive response was performed by depleting specific populations of cells with monoclonal antibodies prior to cell transfer. In vivo the DTH response of the pretreated BALB/c mice was specifically suppressed to the relevant alloantigen, correlating with retention of CBA/j skin grafts for up to 42 days post engraftment without visual evidence of rejection, in comparison to control mice complete rejection of the skin graft in less than 8 days. In vitro, splenocytes from BALB/c recipients of pretreated syngeneic splenocytes containing large numbers of BALB/c anti-CBA/j T cells proliferated less in MLC and generated lower cytotoxic T cell responses to CBA/j alloantigens than did controls and suppressed the naive and sensitized BALB/c MLC and CTL responses to CBA/j alloantigen. This specific suppressive response to alloantigen was optimally transferred into syngeneic naive recipients when the adoptive transfer was performed on the sixth day after the last infusion received by the spleen cell donor mice. The adoptive transfer of this suppressive response was abrogated by the prior X-irradiation of the donor spleen cells and significantly abolished by the depletion of Thy-1+, Lyt-2+, L3T4- T lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

INHIBITION OF ANTI-SKIN ALLOGRAFT IMMUNITY INDUCED BY INFUSIONS WITH PHOTOINACTIVATED EFFECTOR T LYMPHOCYTES—THE CONGENIC MODEL

We have previously reported the capacity to produce donor-specific tolerance to alloantigens by intravenous exposure to pretreated antidonor T cells. The current study has refined this system by using congenic mice differing only at the H-2 major histocompatibility complex genetic loci. Twelve days after B10 mice received MHC-incompatible B10.D2 skin grafts, their splenocytes that included an expanded population of cells mediating rejection were treated with 100 ng/ml 8-methoxypsoralen (8-MOP) photoactivated by 1 J/cm2 of ultraviolet A prior to infusion into naive B10 recipients. Whereas 8-MOP itself is biologically inert, photoactivated 8-MOP crosslinks DNA by covalently binding to pyrimidine bases. Recipient B10 mice were tested for tolerance to B10.D2 alloantigens in mixed leukocyte culture (MLC), cytotoxicity (CTL), and to in vivo delayed type hypersensitivity assays and challenged with a fresh B10.D2 graft. In vivo, the DTH response of the pretreated B10 mice was specifically suppressed to the relevant alloantigen, correlating with retention of B10.D2 skin grafts for up to 22 days postengraftment without visual evidence of rejection, in comparison to control complete rejection of the skin graft in less than 12 days. In vitro, splenocytes from B10 recipients of pretreated syngeneic splenocytes containing large numbers of B10 anti-B10.D2 T cells proliferated less in MLC and generated lower cytotoxic T cell responses to B10.D2 alloantigens than did controls and suppressed the B10 MLC and CTL responses to B10.D2 alloantigen. These results reveal that, in a highly defined congenic transplantation system, infusions of photoinactivated effector cells resulted in selective inhibition of the in vivo responses that correlated with allograft rejection and permitted prolonged retention of histoincompatible skin grafts. This approach may have significant practical applicability for treatment of human disorders caused by aberrant T cells.

Alloactivated HLA class II-positive T-cell lines induce IL-2 reactivity but lack accessory cell function in mixed leukocyte culture

Recently, much interest has focused on the role of HLA class II antigens in T cell-T cell interactions. We have studied the stimulatory capability in the primary mixed leukocyte reaction and the primed lymphocyte reaction of 11 alloactivated, HLA-DR- or -DP-reactive CD4-positive T-cell lines (Ta). From 70 to 90% of the Ta were HLA class II-positive as judged by the reactions with HLA class II-reactive monoclonal antibodies, and the Ta carried the DR allospecificities of the original T-cell donor when typed in the microcytotoxic test using DR-specific alloantisera. Neither irradiated nor nonirradiated Ta stimulated primed lymphocytes directed against the relevant HLA class II antigens on the Ta. Interferon-γ, recombinant interleukin 1, phorbol myristate acetate, calcium ionophore, and adherent cells had no effect on the stimulatory capability of Ta. The ability of irradiated Ta to stimulate in the primary mixed leukocyte reaction {median counts per minute (cpm) 5.5 × 10 3} was significantly lower than that of peripheral blood mononuclear cells (cpm: 44.0 × 10 3). The stimulation by Ta was almost only seen when the Ta were specifically directed against the class II antigens of the responder peripheral blood mononuclear cells (i.e., in combinations with “backstimulation”) (median cpm: 21,000). In mixed leukocyte reaction combinations without backstimulation, significantly weaker reactions were seen (median cpm: 1,000). This observation may explain previous controversies concerning the stimulatory capacity of Ta. Recombinant interleukin 2 significantly enhanced the very low mixed leukocyte culture responses induced by class II-incompatible Ta in combinations without backstimulation but had no significant effect on cultures with Ta autologous to the responder peripheral blood mononuclear cells. Thus, allogeneic class II-psoitvie Ta can induce interleukin 2 responsiveness but lack accessory cell function(s) necessary for the induction of interleukin 2 production in primed and unprimed T cells.

Inhibition of antiskin allograft immunity by infusions with syngeneic photoinactivated effector lymphocytes

Induction of tolerance for skin allotransplantation requires selective suppression of the host response to foreign histo-compatibility antigens. This report describes a new approach that employs pretreatment of effector cells with 8-methoxy-psoralen (8-MOP) and ultraviolet A light (UVA) to render the effector cells of graft rejection immunogenic for the syngeneic recipient. Reinfusion of photodamaged cells resulted in an immunosuppressive host response that permitted prolonged retention of histoincompatible skin grafts and specifically inhibited in vitro and in vivo responses that correlate with allograft rejection.Eight days after BALB/c mice received CBA/j skin grafts, their splenocytes served as a source of alloreactive effector cells. The splenocytes were treated with 100 ng/ml8-MOP and 1 J/cm2 UVA before reinfusion into naive BALB/c recipients. Recipient mice were tested for tolerance to alloantigens in mixed leukocyte culture (MLC), cytotoxicity (CTL), delayed type hypersensitivity assays (DTH), and challenge with a fresh CBA/j graft.Splenocytes from BALB/c recipients of photoinactivated splenocytes containing the effector cells of CBA/j alloantigen rejection proliferated poorly in MLC and generated lower cytotoxic T cell responses to CBA/j alloantigens in comparison with sensitized and naive controls. Splenocytes from these hyporesponsive mice suppressed the MLC and CTL response to alloantigen from sensitized and naive BALB/c mice. In vivo the DTH response was specifically suppressed to the relevant alloantigen in comparison with controls. Moreover, BALB/c mice treated in this fashion retained a CBA/j skin graft for up to 42 d posttransplantation without visual evidence of rejection. These results indicate that the in vivo and in vitro response to alloantigen can be attenuated by pretreating the host with photoinactivated splenocytes containing the effector cells of alloantigen rejection.

Inhibition of Antiskin Allograft Immunity by Infusions With Syngeneic Photoinactivated Effector Lymphocytes.

Induction of tolerance for skin allotransplantation requires selective suppression of the host response to foreign histocompatibility antigens. This report describes a new approach that employs pretreatment of effector cells with 8-methoxy-psoralen (8-MOP) and ultraviolet A light (UVA) to render the effector cells of graft rejection immunogenic for the syngeneic recipient. Reinfusion of photodamaged cells resulted in an immunosuppressive host response that permitted prolonged retention of histoincompatible skin grafts and specifically inhibited in vitro and in vivo responses that correlate with allograft rejection. Eight days after BALB/c mice received CBA/j skin grafts, their splenocytes served as a source of alloreactive effector cells. The splenocytes were treated with 100 ng/ml 8-MOP and 1 J/cm2 UVA before reinfusion into naive BALB/c recipients. Recipient mice were tested for tolerance to alloantigens in mixed leukocyte culture (MLC), cytotoxicity (CTL), delayed type hypersensitivity assays (DTH), and challenge with a fresh CBA/j graft. Splenocytes from BALB/c recipients of photoinactivated splenocytes containing the effector cells of CBA/j alloantigen rejection proliferated poorly in MLC and generated lower cytotoxic T cell responses to CBA/j alloantigens in comparison with sensitized and naive controls. Splenocytes from these hyporesponsive mice suppressed the MLC and CTL response to alloantigen from sensitized and naive BALB/c mice. In vivo the DTH response was specifically suppressed to the relevant alloantigen in comparison with controls. Moreover, BALB/c mice treated in this fashion retained a CBA/j skin graft for up to 42 d posttransplantation without visual evidence of rejection. These results indicate that the in vivo and in vitro response to alloantigen can be attenuated by pretreating the host with photoinactivated splenocytes containing the effector cells of alloantigen rejection.

Primate cardiac allo-and xenotransplantation: modulation of the immune response with photochemotherapy.

Survival of heterotopic allo- and xenografts was studied in a primate cardiac transplantation model. Initially, animals were presensitized to donor xenoantigens by blood transfusion and treated with strategies designed to elicit pretransplant immunosuppression. These animals underwent rapid rejection whether treated with ciclosporin and conventional immunosuppression, autologous sera, sensitized lymphocytes, or photochemotherapy. In a nonsensitized xenograft study, xenografts were maintained for prolonged periods with a combination of ciclosporin and photochemotherapy. All animals treated with photochemotherapy demonstrated periodic suppression of the mixed leukocyte culture response to their donor. Inhibition of lymphocytotoxic antibody was achieved only in animals treated with both ciclosporin and photochemotherapy. Xenosensitized animals were secondarily exposed to donor alloantigens. These animals were successfully allografted. Ciclosporin therapy was terminated and the animals maintained by photochemotherapy alone. This preliminary trial indicates that a combination of ciclosporin and photochemotherapy may be of value in maintaining concordant xenografts and preventing antibody mediated rejection.

Functional activities of enriched Fc receptor bearing T lymphocytes in Hodgkin's disease.

Our earlier studies have demonstrated that the peripheral blood mononuclear cells (PBMNC) of patients with Hodgkin's disease (HD) have reduced percentage of T mu cells and increased percentage of T gamma cells, while the splenic MNC showed the reversed proportions. In this paper, the functional abilities of subsets with these phenotypes have been investigated. T gamma and T mu cells were enriched from theophylline-sensitive and -resistant populations of lymphocytes obtained from PBMNC and splenic MNC of untreated HD patients and normal healthy donors. The effect of addition of these enriched populations on PHA and mixed leukocyte culture (MLC) responses of corresponding autologous MNC has been studied. The results showed that both PHA and MLC responses of normal as well as of HD lymphocytes could be suppressed by T gamma cells, while T mu cells failed to show an appreciable helper activity. The T gamma population in HD, therefore, appears to maintain suppressor function, thereby influencing the peripheral blood T cell responses.

DNA ASSOCIATED WITH THE CELL MEMBRANE IS INVOLVED IN THE INHIBITION OF THE SKIN REJECTION RESPONSE INDUCED BY INFUSIONS OF PHOTODAMAGED ALLOREACTIVE CELLS THAT MEDIATE REJECTION OF SKIN ALLOGRAFT

AbstractCell membrane DNA (cmDNA) is a form of DNA located on the surface of human and murine T‐cells. It has recently been characterized as a target for photomodification by 8‐methoxypsoralen (8‐MOP) and long‐wave ultraviolet light (UV‐A). Whereas 8‐MOP itself is biologically inert, photoactivated 8‐MOP is covalently bound to pyrimidine bases in DNA. We have investigated the possible involvement of cmDN A photomodification in the induction of the suppression of skin allograft rejection in BALB/c mice preimmunized with 8‐MOP/UV‐A photodamaged alloreactive cells which mediates this allograft rejection. This suppression is demonstrated by inhibition of delayed‐type hypersensitivity (DTH) and mixed leukocyte culture (MLC) responses. Splenocytes from BALB/c mice undergoing rejection of CBA/j skin graft which contained an expanded population of the effector T lymphocytes that mediate the rejection were treated with DNAse to remove cmDNA before or after treatment with 8‐MOP and UV‐A prior to infusion into naive BALB/c recipients. Mice that received pretreated effector cells were tested for MLC responses to CBA/j or B10 alloantigens before and after the DTH response. The DTH response of all groups of pretreated BALB/c mice to the relevant alloantigen was specifically suppressed as compared with the response of control mice. However, adoptive transfer of the suppression of the DTH response was optimally demonstrable only in syngeneic recipients of cells from donor mice treated with photodamaged alloreactive cells. Also, splenocytes from BALB/c mice immunized with photodamaged alloreactive cells demonstrated highly significant hyporesponsiveness and suppression of the MLC response of naive mice to the relevant alloantigen in the case of the primary MLC response, and to both alloantigens in the secondary MLC response which was totally eliminated by prior pretreatment of these effector cells with DNAse. Therefore, it appears that the suppression of the DTH response can be induced by pretreatment of the effector cells with DNAse and/or 8‐MOP and UV‐A but is adoptively transferable optimally only from mice which are recipients of photodamaged alloreactive cells. Moreover, the effectiveness of this treatment is decreased by prior removal of cmDNA from these cells. The presence of cmDNA is necessary for induction of suppression of the primary and secondary MLC responses in mice treated with photodamaged cells of allograft rejection.

Response against single minor histocompatibility antigens. I. Functional and immunogenetic analysis of cloned cytolytic T cells.

A clonal approach was used to investigate the cellular basis of a T cell response to single minor histocompatibility antigens (miHA). This analysis was performed by functional and immunogenetic characterization of a large number of clones derived from short-term mixed leukocyte culture (MLC) populations generated against the miHA, H-1.3. Forty-nine clones isolated from such MLC were specifically cytolytic for H-1.3-bearing, H-2Db-compatible target cells. Thirty-seven of the 49 cytolytic clones were driven to proliferate when stimulated by spleen cells bearing the H-1.3 alloantigen in the absence of added T cell-derived growth factor(s) (GF). The remaining 12 clones proliferated only when GF was added. A strong positive correlation was observed between antigen-induced proliferation and the production of interleukin 2 (IL 2) activity. A similar correlation was observed when comparing the ability of both antigen and concanavalin A to induce IL 2 activity from the clones. These data suggest that i) antigen-driven or helper T cell-independent cytolytic T cells (HITc) are frequent components of an MLC response to a single miHA, and ii) the ability of HITc to undergo antigen-driven proliferation is related to their ability to produce antigen-induced GF.

Suppression of the immune response in tumor-bearing mice. III. Induction of functionally suppressive antigen-driven macrophages.

A functionally suppressive antigen-driven subpopulation of macrophages has been demonstrated in mice-bearing tumors produced by MSV-transformed BALB/3T3 cells. The suppressor macrophages depressed markedly both allogeneic and syngeneic mixed leukocyte culture responses and functioned even when assayed at low concentrations, suggesting that high concentrations of normal macrophages were not responsible for the suppression. Studies also showed that suppressor macrophages acted to suppress the proliferation of hyperreactive Lyt 1+,2- cells found in the splenic cell suspensions of tumor-bearing mice after the removal of the adherent suppressor cells. Finally, the suppressive activity of macrophages induced in tumorous mice could be augmented by KiMSV or MMSV but not by MuLV antigen. These results suggest that suppression might be a direct response to transformation-associated determinants expressed on the tumor cells as well as on the MSV virions or the result of cellular interactions between suppressor macrophages and other antigen-specific spleen cell populations.

Inhibition of mixed leukocyte culture responses in cadmium-treated mice

Previous studies have documented inhibition of antibody-mediated immunity by exposure of experimental animals to heavy metals. To date, very little is known of possible effects of heavy metal ions on cellular-mediated immunity. The mixed leukocyte culture (MLC) assay was used in this study to evaluate the effects on lymphocyte blastogenic response of chronic exposure of mice to cadmium (2 mg CdCl 2/kg body wt/day). Spleen cells from two strains of mice treated with CdCl 2 were unable to respond as effectively as control normal spleen cells to stimulation with allogeneic leukocytes. Furthermore, X-irradiated splenocytes from the cadmium-stressed animals were less effective than splenocytes from control normal mice as stimulator cells in the MLC reaction. This comparative decrease in T-lymphocyte reactivity could not be attributed to loss of lymphocyte viability or to decrease in ability of lymphocytes from cadmium-treated animals to incorporate tritiated thymidine. Although spleen cells from cadmium-intoxicated mice responded poorly to stimulation with allogeneic leukocytes, they were capable of responding to a mitogenic stimulus. Possible explanations for these observations are discussed.

Studies of the response in mixed leukocyte culture of cells from patients with aplastic anemia to cells from HLA-identical siblings.

We have studied the mixed leukocyte culture (MLC) reactions of 64 patients with severe aplastic anemia. Their peripheral blood mononuclear cells showed an increased relative response (RR) to cells from HLA-identical siblings as compared to cells from normal HLA-identical siblings, confirming the results reported in an earlier series of 34 patients. Elevated RRs were associated with patient antidonor lymphocyte antibodies as detected by the antibody-dependent cell-mediated cytotoxicity assay, but were not associated with antidonor complement-dependent cytotoxic antibodies or with antidonor cytotoxic T lymphocytes. Among 98 patients receiving marrow grafts from HLA-identical sibling donors, those with elevated RRs before transplantation were more apt to reject the transplant than those without (P less than 0.0001). There was no elevation of the RR in 10 untransfused patients, although positive RRs were noted in 2 patients within 12 to 24 hr of their first transfusions. Five patients with identical twins were also tested, and elevated RRs were noted in three. Although blood transfusion appears to be responsible for the increased RRs observed in some aplastic patients, genetic differences between donor and recipient were not always necessary for eliciting an increased MLC response, suggesting that mechanisms other than alloimmunization are involved.

Modulation of the in vitro lymphocyte response by rabbit blastocysts

Modulation by preimplantation (day 6 post coitum) rabbit blastocysts or by blastocoel fluid of the phytohemagglutinin (PHA)-stimulated autologous splenocytes as well as mixed leukocyte cultures (MLC) of rabbit allogeneic splenocytes in vitro was studied. Results showed that in the absence of the blastocyst or blastocoel fluid, specific incorporation (S.I.) of PHA activated cultures was 7.11 ± 1.19 (mean ± S.E., n = 11), whereas the addition of 1 ( n = 9), 2 ( n = 8) or 3 ( n = 7) blastocysts significantly ( P < 0.001) reduced the S.I. (0.56 ± 0.09, 0.47 ± 0.09 and 0.27 ± 0.05, respectively). In the absence of the blastocyst or blastocoel fluid, 3057 ± 1763 cpm were recorded as MLC response; the addition of 1 or 3 blastocysts showed 83 and 87% suppression, respectively ( n = 11, P < 0.001). The addition of blastocoel fluid also suppressed PHA or MLC responses ( P < 0.05). The effect was not due to the crowding or cytotoxic effects induced by the blastocysts in the cocultures, since spleen cell viability was > 89%. Rabbit blastocysts around the time of implantation have a great capacity of modulating PHA-induced splenocyte proliferation as well as allogeneic recognition in vitro.