Secondary Mixed Leukocyte Culture Research Articles

CD4 +CD25 +Foxp3 +IFN-γ + human induced T regulatory cells are induced by interferon-γ and suppress alloresponses nonspecifically

Interferon-γ (IFN-γ)-producing CD3 +CD4 +CD25 +Foxp3 + peripheral blood lymphocytes (PBL) are more frequently detectable in patients with good than in patients with impaired long-term kidney graft function, suggesting an immunoregulatory role of this induced T regulatory (iTreg) subtype. Herein, the in vitro function of separated CD3 +CD4 +CD25 +Foxp3 +IFN-γ + PBL that were induced by phorbol 12-myristate 13-acetate (PMA)/ionomycin or alloantigenic stimulation was investigated using cell coculture techniques and flow cytometry. CD4 +CD25 +Foxp3 + PBL with intracellular IFN-γ production increased to 26% in cell cultures stimulated with PMA/ionomycin for 6 hours. Recombinant IFN-γ augmented and anti-IFN-γ monoclonal antibody blocked induction of CD4 +CD25 +Foxp3 +IFN-γ + PBL, suggesting their IFN-γ-dependent induction. In addition, CD4 +CD25 +Foxp3 +IFN-γ + PBL produced immunosuppressive interleukin (IL)-10, transforming growth factor-β, and IL-4 intracellularly and expressed both IFN-γ and IFN-γ receptors (CD119) on the cell surface, allowing separation of CD4 +CD25 +IFN-γ + PBL with 98% purity. Addition of enriched CD4 +CD25 +IFN-γ + PBL to autologous PMA/ionomycin stimulated PBL decreased blast formation ( p < 0.05), indicating suppression of cell proliferation by CD4 +CD25 +IFN-γ + PBL. CD4 +CD25 +IFN-γ + PBL separated from primary mixed leukocyte cultures (MLC) and added to autologous or third-party secondary MLC suppressed allogeneic T-cell activation nonspecifically ( p < 0.05). We conclude that CD4 +CD25 +Foxp3 +IFN-γ + PBL are induced by IFN-γ, making them sensors for IFN-γ and initial immune responses. Circulating CD4 +CD25 +Foxp3 +IFN-γ + PBL could suppress allogeneic T-cell responses in patients and may be involved in inhibition of the posttransplant alloresponse.

Cord Blood Nucleated Cells Induce Delayed Proliferative and Cytotoxic T Cell Alloreactivity.

Transplantation of HLA-mismatched nucleated cells from cord blood (CB) has reduced risks of graft rejection and severe acute graft-versus-host disease. In this study we analyzed the in-vitro alloantigen presenting capacity of cord blood nucleated cells. CB mononuclear cells (MNCs) or immunomagnetically-selected CD34+ cells, or CD14+ monocytes, were irradiated and tested as stimulators of allogeneic blood T cells in primary (stimulator:responder ratio = 1:1) or secondary (stimulator:responder ratio = 1:2) mixed leukocyte culture (MLC), or in cytotoxic T-lymphocytes (CTL) assays. CB-MNCs failed to induce allogeneic T cell proliferation in 6-days primary MLC, whereas CD34+ or CD14+ cells stimulated brisk T cell responses. A suppressive effect of CB-MNCs was ruled out since CD3+ cell-depletion of CB-MNCs did not restore CB immunogenicity and the addition of increasing doses of CB-MNCs did not inhibit T cell alloreactivity to CD34+ cells. Despite allogeneic T cells were unresponsive to CB-MNCs after primary and secondary MLC, T cell anergy was ruled out since T cells that were unresponsive after primary MLC proliferated potently in secondary MLC stimulated with CB CD34+ cells, and even more with CB monocyte-derived dendritic cells (Mo-DC) generated in-vitro with GM-CSF and IL-4. Interestingly, after co-culture with irradiated allogeneic T cells for 6 days, CB-MNCs showed a greater proportion of CD86+ cells and elicited allo- T cell proliferation. In addition, allo-CTL activity was induced by CB-MNCs only after restimulating effector cells for 3–4 weeks (26±7% lysis of antigen-specific PHA-blast at 50:1 E:T ratio), and was comparable to CTL activity induced after 1 week by Mo-DC generated from the same CB. When T cell effectors were stimulated by combining two incompatible cord blood MNCs mixed together, CTL activity was then detected after 4 weeks against both of them regardless of the CB:CB cell ratio. These results show an impaired allo-APC activity of CB-MNCs, without suppressive or tolerogenic activity. These findings might partially explain the initial engraftment of combined HLA mismatched CB grafts in vivo, however they also suggest that a delayed T cell response may occur due to CB-derived APCs activating CTLs.

Impaired Alloantigen Presenting Activity of Cord Blood Nucleated Cells.

Transplantation of HLA-mismatched cord blood (CB) nucleated cells has limited risk of severe acute graft-versus-host disease and graft rejection. This may depend on naïve T cells not yet exposed to many antigens and on immature antigen-presenting cells (APC) not delivering appropriate signals to allogeneic T cells. In order to test the APC activity of human circulating CB cells in-vitro, we initially used irradiated CB mononuclear cells (MNC) or immunomagnetically selected CD34+ cells, CD133+ cells, or CD14+ monocytes to stimulate the proliferative response of incompatible blood T cells in mixed leukocyte culture (MLC). CB MNC failed to induce allogeneic T cell proliferation, while CD34+ and CD133+ progenitors or CD14+ monocytes induced potent T cell alloresponses. Nevertheless, since allogeneic T cell response was not restored after depletion of CD3+ cells in the CB, nor the add-back of irradiated CB MNC to CD34+ or CD14+ stimulators inhibited allo-T cells, a direct suppressive effect of CB MNC was excluded. Allogeneic peripheral blood cytotoxic T-lymphocyte (CTL) responses were not induced after 7 days of stimulation with irradiated CB MNC, although after 4 weekly rechallenges with CB MNC, on average a 23% lysis of antigen-specific CB PHA-blasts was observed at the highest effector:target ratio (50:1). To test the tolerogenic potential of CB MNC, T cells initially exposed to CB MNC were rechallenged in secondary MLC with CB MNC, or CD34+ cells, or monocyte-derived dendritic cells (Mo-DC) generated in liquid culture with GM-CSF and IL-4. Allogeneic T cells were still unresponsive upon rechallenge with CB MNC, but proliferated upon 3 days of restimulation with CD34+ cells or Mo-DC from the same CB. Surprisingly, the supernatant of these latter MLCs did inhibit completely a 3rd party MLC. Instead, the supernatant of blood T cells that had been activated by CB CD34+ cells or Mo-DC both in primary and secondary MLC did not. These results show an impaired allo-APC activity of CB MNC but not CB CD34+ cells, and suggest that T cells releasing immunosuppressive cytokines may be activated by CB MNC and then expanded by a second more potent stimulation with professional APC. This hypothesis could explain the sustained engraftment of HLA-mismatched CB stem cell transplants in humans. Based on these results, the in-vivo or ex-vivo downregulation of T cell alloreactivity induced by CB MNC will be tested in experimental models of stem cell, as well as solid organ transplantation.

Bop: a new T-cell-restricted gene located upstream of and opposite to mouse CD8b.

In the course of transient expression studies undertaken to determine the location of the mouse CD8b gene promoter, two additional promoter activities were detected within 600 nucleotides upstream of the gene. One activity directs transcription in the same direction as CD8b but fails to transcribe the CAT reporter gene due to an apparent transcription-blocking element lying between it and the gene. The second activity directs transcription opposite to that of the CD8b gene. Northern hybridization with a probe consisting of nucleotides -875 to -550 relative to the site of CD8b transcription initiation revealed hybridizing species of 4 kilobases (kb) and 1.8 kb in poly-A-selected RNA from mouse thymus but not from any other tissues. Similar RNA species were detected in poly-A+ RNA from concanavalin A-stimulated spleen cells and several long-term CTL lines but not from the EL4 or BW5147 T-cell lines or the J558L myeloma. The mRNA species were most abundant in cells of a secondary mixed leukocyte culture which were greater than 95% CD8(+). Northern hybridizations using single-stranded unidirectional probes indicated that these mRNAs represent transcription opposite to the CD8b gene. The tissue and cell type distribution of this newly-discovered gene (designated Bop for CD8b opposite) are consistent with T-cell-specific and possibly CD8-positive T-cell-specific expression. The head-to-head arrangement of the Bop and CD8b genes is reminiscent of the arrangement of the Tap1 and Lmp2 genes, and the expression of the Bop gene in CD8-positive cells raises the possibility that these genes are involved in the same functional pathway.

Activation of CD4+ T cells in the presence of a nondepleting monoclonal antibody to CD4 induces a Th2-type response in vitro.

In vitro experiments using purified rat CD4+ T cells in primary and secondary mixed leukocyte cultures (MLC) have been carried out to explore the mechanism of inhibition of cell-mediated autoimmune disease in the rat by a nondepleting monoclonal antibody (mAb) to CD4. Previous work has shown that W3/25, a mouse anti-rat CD4 mAb of immunoglobulin G1 isotype, completely prevents the development of the paralysis associated with experimental allergic encephalomyelitis (EAE) in Lewis rats, but does so without eliminating the encephalitogenic T cells. The in vitro experiments described in this study have shown that when CD4+ T cells were activated in the presence of the anti-CD4 mAb in a primary MLC, the synthesis of interferon (IFN) gamma, but not interleukin (IL) 2, was completely inhibited. After secondary stimulation, now in the absence of the mAb, the synthesis of IL-4 and IL-13 mRNA was greatly enhanced compared with that observed from CD4+ T cells derived from primary cultures in which the mAb was omitted. As IL-4 and IL-13 are known to antagonize cell-mediated immune reactions, and as EAE is cell-mediated disease, the data suggest that the W3/25 mAb controls EAE by modifying the cytokine repertoire of T cells that respond to the encephalitogen. The capacity for the mAb to suppress IFN-gamma synthesis provides, in part, an explanation for this change in cytokine production. These findings are discussed in terms of what is known of the factors that determine which cytokine genes are expressed on T cell activation. Possible implications for the evolution of T cell responses in human immunodeficiency virus infection are also discussed.

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.

Evidence that protein kinase C regulates allosensitized T lymphocyte function

The role of second messengers in the response of alloactivated T cells to growth factors (e.g., interleukin-2) is unknown. We have previously found that intracellular calcium may be an important T-cell alloactivation marker. To determine whether protein kinase C (PKC) is also involved in allosensitized T cell function, we studied the effects of the PKC agonists phorbol 12-myristate 13-acetate (PMA), mezerein (MEZ), and 1-oleoyl-2-acetylglycerol (OAG) and the PKC antagonists phloretin and d-sphingosine on the in vitro proliferation and migration of allosensitized cells from a C57BL/6 anti-DBA/2J mixed leukocyte culture (MLC). At 0.01–1.0 μg/ml, PMA, a potent stimulant of PKC, exerted profound stimulatory effects on secondary MLC supernatant (2° SN)-induced proliferation of allosensitized T cells (132–200% increase, P < 0.01). Both MEZ and OAG are less potent activators of PKC than PMA but also stimulated T cell proliferation (132–152% of control, P < 0.01). The PKC antagonists phloretin and d-sphingosine both inhibited proliferation by >50% at 1.0–10.0 μ M ( P < 0.01). Further experiments showed that these agents exert similar effects on in vitro T cell locomotion in a Boyden chamber assay. These data indicate that PKC activation may be an important component of allosensitized T cell proliferation and locomotion and may constitute a pathway by which T cell function may be modified in the allograft response.

Response of resident murine peritoneal macrophages to in vivo administration of granulocyte-macrophage colony-stimulating factor.

The effect of s.c. inoculation of purified recombinant derived granulocyte-macrophage (GM)-CSF on resident murine peritoneal macrophages was assessed in this study. From 18 to 24 h after s.c. administration of GM-CSF to normal mice, the resident peritoneal macrophages were harvested and the levels of membrane-bound IL-1, FcR, Mac-1 cell-surface Ag, and class II MHC expression were assessed. Peritoneal cells from GM-CSF-inoculated mice had significantly greater levels of membrane-bound IL-1 than did control mice. In addition when resident peritoneal macrophages from normal mice were purified by adherence and grown in the presence of GM-CSF, they produced greater levels of both membrane-bound and secreted IL-1. The peritoneal cells from GM-CSF-inoculated mice did not differ from controls in the expression of class II MHC-encoded Ag. This observation was confirmed by the finding that GM-CSF was unable to induce class II MHC expression on P388D1 cells, whereas a secondary mixed leukocyte culture supernatant was. Peritoneal cells from GM-CSF-inoculated mice also exhibited greater levels of expression of FcR and the Mac-1 cell-surface Ag. This resulted in an increase in their ability to phagocytose opsonized SRBC in vitro.

In vitro differentiation of mouse embryonic yolk sac cells

Abstract The embryonic yolk sac is the first site in the mammalian embryo in which cells are found that can carry out cell-mediated immune functions, yet the relation of cells of this primitive hematopoietic organ to the development of the mature immune system has not been established. We have initiated a series of experiments to determine the potential of cells of the mouse yolk sac to differentiate in vitro, in order to get an insight into the development of immunocompetence in this primary population of hematopoietic stem cells. The present paper describes the conditions promoting stem-cell differentiation and provides an initial characterization of cell surface phenotypes of the cell lineages established in vitro. Yolk sac cells obtained from 10- to 13-day mouse embryos were maintained in culture for more than 18 months, giving rise to a variety of cell types belonging to the hematopoietic lineages and culminating in the establishment of long-term cell lines. Supernatants of secondary mixed leukocyte cultures were found to be an effective source of growth factors promoting the initial differentiation as well as the maintenance of these cells. Flow-cytometric analysis showed that, in contrast to freshly obtained yolk sac cells, which had no detectable Thy 1 antigen, cells expressing significant levels of Thy 1 were obtained after 1 week or more of culture. Ly 1 and Lyt 2 antigens were detected only rarely and the L3T4 (GK 1.5) antigen was never expressed. In contrast, the Ly 5 (T200) antigen, detectable on 0%–3% of freshly isolated yolk sac cells, was expressed on the majority of cultured yolk sac cells within 1 week and was present on virtually all long-term cultured yolk sac cells. Newly isolated yolk sac cells did not express any surface-associated immuno-globulin (sIgM), but sIgM was detected on a significant number of cells in 1- to 8-week yolk sac cultures. On the other hand, no antibody binding was observed with the pre-B-cell reagents 14.8 or 6C2 (GP 220) at any time. The presence of angiotensin converting enzyme (ACE), a marker of macrophage and endothelial cells, was determined both by tripeptide cleavage tests and by immunofluorescence using a monoclonal antibody against ACE. The enzyme was limited to the adherent cell population present in yolk sac cultures. Similarly, only the adherent cell population bound acetyl low density lipoprotein (acLDL) as measured by dilacLDL fluorescence. Transformed cells appeared in at least three separate cell lines after 6–8 months in culture, and they all expressed Thy 1 and Ly 1 antigens. Both Ly 5 (T200) and Ly 9 (T100) were expressed, but neither the B220 antigen nor surface immunoglobulin could be detected. On transfer to irradiated recipients, these cells developed both as solid lymphomas at the site of injection (intramuscular or subcutaneous) and as ascites tumors following intraperitoneal injection, with maintenance of the Ly 5-possitive, Thy 1.2-positive, Ly 9-positive, Ly 1-positive pheno-type. Yolk sac cells obtained from 13-day-old mouse embryos were fused with BW 5147.3 T-lymphoma cells (Thy 1.1) to generate stable yolk sac/lymphoma hybrids. Flow-cytometric analysis demonstrated the expression of Thy 1.2, Ly 1, and Lyt 2 antigens in many of these hybrids. Following selection and cloning, various clonotypes were isolated that differed in the expression of cell surface antigens. Functional and molecular analysis of the embryonic yolk-sac cell lines, tumors, and hybrids that we have established should provide new insight into the events underlying establishment of the immune defense system in the mammalian embryo.

A new enzymatic pathway of citrullinogenesis in murine hemopoietic cells

Citrullinogenesis is demonstrated when murine bone marrow cells are incubated with dialyzed secondary mixed leukocyte culture supernatant. The identity of citrulline in bone marrow cell supernatants has been established by gas chromatographic mass spectrometric analysis. It is shown that, in our model, citrulline synthesis proceeds directly from arginine without intermediate ornithine production, ruling out the involvement of ornithine transcarbamylase (EC 2.1.3.3.). Moreover, none of the other enzymatic activities described for catalyzing citrullinogenesis, i.e. arginine deiminase or peptidyl arginine deiminase can be demonstrated. The generation of oxygen radicals is necessary for this enzymatic reaction. It is induced by a thermolabile protein produced during the antiallograft immune response with a molecular weight of about 150,000.

Evidence that secondary mixed leukocyte culture supernatant mediates changes in cellular recruitment, blood flow, and vascular permeability.

Lymphokines produced as a result of allostimulation may play an important role in allograft rejection, mediating changes in cellular infiltration and migration, regional blood flow, and vascular permeability in a manner similar to that of delayed type hypersensitivity reactions. Changes in cellular recruitment (CR), regional blood flow (RBF), and vascular permeability (VP) were studied in paired healed subcutaneous urethane sponge grafts inoculated with secondary mixed leukocyte culture supernatant (2 degrees MLC SN), a rich source of a variety of lymphokines secreted in response to allostimulation. Intravenous injection of Indium-111-labeled unsensitized lymphocytes (UL), Rubidium-86-chloride, and Iodine-125-labeled albumin were used to assess CR, RBF, and VP, respectively. An increase in CR (P less than 0.001), RBF (P less than 0.05), and VP (P less than 0.001) could be demonstrated at the site of injection of allogeneically restimulated 2 degrees MLC SN compared with the syngeneically restimulated 2 degrees MLC SN. The quantitative response and the kinetics of CR using 2 degrees MLC SN were similar to previous studies in which specifically sensitized lymphocytes were injected with target cells bearing the sensitizing alloantigen, but the increase in RBF and VP were less. Injection of purified human interleukin, 2 serotonin, histamine, and bradykinin had no significant effect on CR. Injection of purified human interleukin 1 resulted in a moderate increase in CR. These results are consistent with previous studies suggesting that the in vivo elaboration of lymphokines during allogeneic cellular interactions leads to an increase in CR, RBF, and VP. The small changes in RBF and VP in these experiments, however, do not account for the greater magnitude of the changes in CR. It is likely that other specific or nonspecific chemoattractants or inhibitors of cell migration also play a significant role in CR in vivo.

A mitogenic factor for endothelial cells obtained from mouse secondary mixed leukocyte cultures.

Abstract Supernatants from secondary mixed leukocyte cultures (MLC) are capable of stimulating both endothelial cell proliferation and endothelial cell chemokinesis. The proliferation factor(s) has been subject to further biochemical characterization. At least one factor is precipitable by 30% ammonium sulfate, a procedure that separates this factor from the chemokinesis-inducing factor(s) and most other growth-stimulating lymphokines. The partially purified lymphokine preparation can stimulate growth of microvascular endothelial cells, yet fails to induce proliferation of large vessel endothelium and a variety of other cell types. Addition of indomethacin to the MLC medium leads to an increase in endothelial cell mitogenic activity. Chromatography on S-200 Sephacryl suggests a m.w. greater than 200,000 when eluted in isotonic saline, whereas the apparent m.w. is less than 30,000 when the material is eluted from the same column with 0.5 M ammonium acetate. The proliferative activity is stable to acid (pH 2) but not to heat treatment at 50 degrees C. Although the factor is resistant to trypsin digestion, it is sensitive to digestion with pronase, suggesting that it is a protein. The potential importance of endothelial cell-specific lymphokines in the vascular responses accompanying inflammation and disease-associated neovascular reactions is discussed.

Characterization of the major histocompatibility complex genes and their products

Since the independent discoveries by Gorer and Snell of the murine major hist6compatibility complex (MHC), H2, monumental strides have been taken in defining both the structure and function of MHC genes and their products. First incorrectly regarded as a blood group locus (12), the H-2 was later observed as a locus controlling allogeneic organ grafts (14). The MHC is currently ascribed the crucial role of orchestrating cell-cell interactions of the immune response. Although the MHC encompasses class I, II, and III genes, as well as several enzyme-encoding genes (16), the class I and II genes and their products are the focus of this paper. It is in the context of these latter MHC gen e products that immunocompetent cells discriminate self from non-self. Initially examined in murine models, the MHC was found to have multiple loci, many of which are polymorphic. The murine MHC genetic map was recently condensed from its previous 5 division H-21 region (class II), omitting the subregions l-B, l-C, and l-J (18). The H-2 presently contains the class I loci of K, D, L, Qa, and Tla and the class II loci of IAs, I-AI3, I-Ea, I-EI31, and I-El32. Following the abovementioned work, the human MHC (Fig. 1) received recognition when Dausset described the first human leukocyte antigen (HLA) in the early 1950s (8). The 9th International Histocompatibility Testing Workshop convened this year and the HLA nomenclature committee is currently composing a final draft of HLA nomenclature revisions for 1984 (13). Their changes include formal recognition of three polymorphic class I loci A, B, and C, and an expansion of the HLA-D/DR region to include DR, DQ, and DP, formerly known as DR, DC (MB or DS), and SB, respectively (Table 1). All class I and some class II gene products are defined serologically using various HLA-A, B, C, and DRand DQ-specific antisera, while Dw region specificities are defined solely by mixed leukocyte culture (MLC). These MLC-defined determinants are as yet considered distinct from the DR determinants, yet their loci are probably genetically linked. A secondary MLC technique, termed primed lymphocyte typing (PLT), is used to detect the elusive specificities of the DP (SB) locus (10, 1 I, 19). These assays are now approaching worldwide standardization in histocompatibility testing laboratories. The gene products of the class I and II genes have been biochemically characterized and compared by numerous methods, such as: (a) extraction from membranes via papain and detergent In T h i s I s s u e

Lymphokine-mediated induction of cytolytic activity in a T cell hybridoma.

Functionally inducible CTL hybridomas were constructed by fusing alloantigen-specific T cells (C57BL/6 alpha-DBA/2) with cells from the rat thymoma line W/FU (C58NT)D. A cloned hybridoma line (KSH.4.13.6) that was specifically cytolytic in the presence of activated rat spleen cell supernatant fluid (rat Con A SN) lost activity when transferred to normal medium. However, a cytolytic activity could be reinduced by culturing KSH.4.13.6 cells in medium containing rat Con A SN or secondary mixed leukocyte culture SN. By using various sources of SN, it was found that cytolytic induction required two different factors. PMA-induced EL-4 SN and SN from antigen-activated cloned T cells, neither of which were capable of inducing cytolytic activity alone, were able to synergize in the cytolytic induction of KSH.4.13.6 IFN-gamma and IL 1 failed to induce cytolytic activity even in the presence of EL-4 SN. Furthermore, this hybridoma produced macrophage activating factor (MAF) upon culture in rat Con A SN, although MAF production could not be induced by either specific antigen or lectins. The kinetics of induction and loss of cytolytic activity mediated by rat Con A SN were similar to those of the induction of MAF production. However, EL-4 SN, which by itself was incapable of inducing cytolytic activity, was able to induce MAF production in the KSH.4.13.6 hybrid to an extent similar to that induced by rat Con A SN. These results suggest that the induction of cytolytic activity and of MAF production in this cloned hybridoma cell line are regulated by different mechanisms. Such a functionally inducible T cell hybrid may provide a tool for biochemical and molecular analysis of T cell function and regulation, and of the characterization of cytokines required for CTL differentiation.

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.

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures.

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.

Three different signals are required for the induction of cytolytic T lymphocytes from resting precursors.

The requirement for the signals in induction of cytolytic T lymphocytes (CTL) has been investigated. C57BL/6 X CBA/T6 F1 spleen cells stimulated with the lectin leukoagglutinin (L-A) failed to show CTL activity in a PHA-facilitated assay, although L-A-activated splenic T cells were able to respond to T cell growth factor (TCGF). Concanavalin A (Con A) on the other hand was able to induce cytolytic activity from CTL-P, as well as to render splenic T cells responsive to TCGF. Furthermore, L-A-activated splenic T cells could generate cytolytic activity upon subsequent culture in secondary mixed leukocyte culture supernatant (2 degrees MLC SN). In contrast, EL-4-derived SN (EL-4 SN) was unable to induce cytolytic activity from L-A-activated spleen cells. In addition, proliferation of L-A-activated spleen cells cultured in EL-4 SN was similar to those cultured in 2 degrees MLC SN. Nonactivated spleen cells were totally unresponsive to both SN in proliferation and generation of CTL. Analysis of T cell clones for the production of a factor necessary for induction of cytolytic activity revealed that both cytolytic and noncytolytic T cell clones were able to produce a factor(s) for the generation of cytolytic activity from L-A-activated T cells. On the other hand, SN from certain antigen-stimulated T cell clones produced factors capable of inducing cytocytic activity by L-A-activated T cells only in the presence of EL-4 SN. Neither EL-4 SN nor cloned T cell SN alone had such a capacity. The nature of the necessary lymphokines in the SN from the clone cells or from the EL-4 is unknown. In the case of the EL-4 SN, it is not known whether the presence of TCGF plays a role or whether that role is perhaps more differentiative than proliferative. This study provides evidence that the induction of CTL from CTL-P can be dissociated into activation, which is required to render T cells responsive to second signals, and differentiation, which is mediated by two different factors.

Heterogeneity of an anti-H-2 I-A response as determined by cloned T cell reactivity.

T lymphocytes activated against the I-Aq gene product in primary mixed leukocyte culture (MLC) exhibit an unusually strong cross-reactivity against all third-party stimulating strains tested in secondary MLC. Through the development of anti-I-Aq clones, this cross-recognition has been shown to result from the activation of a heterogeneous T lymphocyte population bearing receptors that recognize the I-Aq gene product differently, yet specifically. The determination of functional activities of the anti-I-Aq cloned cells, e.g., lymphokine production and ability to induce lethal graft-vs-host disease, suggested even further heterogeneity in T cells grouped according to their similar cross-reactive pattern.

Relationship between expression of Fcγ receptors or la antigens and cytolytic activities of alloactivated human T cells

The relationship between expression of Ia antigens or Fcγreceptors (FcγR) and different cytolytic activities of mixed leukocyte culture (MLC)-activated T-cell populations was studied. Lymphocytes mediating specific lysis of target cells bearing the stimulating alloantigens (CTL) could clearly be distinguished from cells mediating antibody-dependent cellular cytotoxicity (ADCC) in that CTL were FcγR − while cells mediating ADCC were FcγR +. In contrast, MLC-generated natural killer (NK) cells were distributed in both the FcγR − and FcγR + cell fractions. The presence or absence of Ia antigens did not correlate with any of the cytolytic activities studied. In addition, FcγR − and not FcγR + cells responded in the secondary MLC. Thus, although FcγR + cells are generated in large numbers in MLC, they do not appear to play a direct role in specific cytotoxicity nor do they proliferate in response to secondary stimulation. However, their ability, following MLC generation, to mediate NK activity and ADCC, both of which may contribute to in vitro and in vivo lysis of allogeneic cells, might explain their appearance following allogeneic stimulation.

Functional reactivities of lymphocytes primed in murine graft-versus-host reactions.

Secondary mixed leukocyte culture (MLC) responses of T lymphocytes effecting graft-versus-host (GVH) reactivity have been compared with the response patterns of T lymphocytes primed in vitro. GVH reactions were initiated in donor-recipient combinations selected to involve genetic differences at the entire H-2 complex or individual K/D and I regions. GVH-primed lymphocytes, whether derived from spleen, liver, or lymph nodes, exhibited unexpectedly specific proliferative reactivity against I region-encoded gene products. In combinations involving both K/D plus I region differences, cytotoxic T lymphocytes were generated against the K/D antigens. These response patterns contrasted to those exhibited by MLC-primed T cells. MLC-primed cells elicited proliferative responses against both K/D and I region antigens and revealed a high cross-reactivity among I region gene products of various haplotypes. The different response patterns for in vivo versus in vitro primed cells do not appear to be artifactual but a result of the priming environment.