Mixed Leukocyte Culture Reaction Research Articles

Expression analysis of Carassius auratus-leukocyte-immune-type receptors (CaLITRs) during goldfish kidney macrophage development and in activated kidney leukocyte cultures.

Carassius auratus leukocyte immune-type receptors (CaLITRs) were recently discovered immunoregulatory receptors in goldfish that have diverse immunoglobulin-like (Ig-like) ectodomains and intracellular signaling motifs. Genomic analysis shows that CaLITR-types are also located as distinct gene clusters across multiple goldfish chromosomes. For example, CaLITR1 (unplaced) is a functionally ambiguous receptor having two Ig-like domains, a transmembrane domain (TM), and a short cytoplasmic tail (CYT) devoid of any recognizable signaling motifs. CaLITR2 (Chr47) is a putative inhibitory receptor containing four Ig-like domains, a TM, and a long CYT with an immunoreceptor tyrosine-based inhibition motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). A putative activating receptor-type, CaLITR3 (Chr3), has four Ig-like domains, a TM, and a short CYT containing a positively charged histidine residue and CaLITR4 (ChrLG28B) is a receptor with putative multifunctional signaling potential as well as five Ig-like domains, a TM, and a long tyrosine-motif containing CYT region. The variable genomic locations of the CaLITRs suggest that they are likely under the influence of different cis- and/or trans-regulatory elements. To better understand the transcriptional activities of select CaLITRs from variable genomic regions, we used an RT-qPCR-based approach to examine the expression of CaLITR1, CaLITR2, CaLITR3, and CaLITR4 during goldfish primary kidney macrophage (PKM) development and in mixed leukocyte reaction cultures (MLRs) of the goldfish. Our results showed that the select CaLITRs are differentially expressed during PKM development and in goldfish MLRs exposed to T-cell mitogens/immunosuppressive drugs, supporting that the transcription of these CaLITRs is likely regulated by distinct cis- and/or trans-regulatory elements.

Alloreactive T cell proliferation is suppressed by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) independent of AMP-activated protein kinase

Abstract The benefits of hematopoietic stem cell transplantation, a therapy used to treat a variety of hematologic and immunologic disorders, are often outweighed by the risks of acute graft-versus-host disease (GVHD), where donor T cells attack and destroy host tissues. Our lab has previously shown that targeting lymphocyte metabolism is a promising and novel strategy for the treatment of GVHD. More recently, we have found that AMP-activated protein kinase (AMPK), a major cellular energy sensor, plays a significant role in T-cell proliferation during disease pathogenesis. To explore this relationship further, we treated alloreactive T cells generated during a mixed leukocyte reaction (MLR) with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMP mimetic that strongly activates AMPK. AICAR treatment of MLR cultures strongly repressed T cell proliferation, with a 70% decrease in total cells by 96 hours. Interestingly this decrease in T cell proliferation was independent of AMPK, as similar results were obtained using AMPK−/− cells. Mechanistically AICAR repressed AKT signaling in stimulated T cells, with a 50% decrease in AKT phosphorylation when cells were concurrently treated with 100 μM AICAR. Cytokine driven STAT5 activation was also significantly blunted by simultaneous AICAR exposure. In summary, despite its wide use as an AMPK agonist, we found that AICAR exerts significant inhibitory effects on T cell allo-activation independent of AMPK. This inhibition occurs, at least in part, through decreased phosphorylation of AKT and down-regulation of STAT signaling, making AICAR a potential therapy to combat GVHD and similar inflammatory disorders.

Synthetic peptides from the N-terminal regions of CD200 and CD200R1 modulate immunosuppressive and anti-inflammatory effects of CD200-CD200R1 interaction

A series of 15-mer peptides were synthesized defining continuous sequences of the extracellular region of the murine and human CD200 molecule. In addition, peptides mapping to the presumptive CDR1, CDR2 and CDR3 of the human and mouse CD200R1 molecules were synthesized. The ability of these various molecules to block the interaction of CD200 with CD200R1 was studied in a competitive ELISA using plate-bound CD200R1Fc and biotinylated CD200Fc, and by FACS using FITC-conjugated CD200Fc binding to 24-h LPS-activated adherent cells. Results from these data were compared with the functional ability of the same peptides to suppress the inhibition of generation of allo-specific CTL in vitro following inclusion of CD200Fc in mixed leukocyte culture reactions. Peptides defining discrete regions in the N terminal regions of CD200 and CD200R1 were functionally active in these different assays. Moreover, infused in vivo, the same mouse-specific peptides suppressed protection from graft rejection afforded by injection of soluble immunosuppressive CD200Fc. Used alone in vitro, these peptides enhanced alloimmunity.

Additive efficacy of CTLA4Ig and OX40Ig secreted by genetically modified grafts.

The use of systemic immunosuppressive drugs have been paramount in the success in transplantation, but there are serious deleterious effects. Genetic modification of grafts to secrete immunomodulators locally may be a way to reduce the need for systemic immunosuppression. An insulinoma cell line, NIT, having the nonobese diabetic (NOD) genotype but also expressing the SV40 large T Ag, was transfected with CTLA4Ig or OX40Ig in an attempt to block signals in the costimulatory/adhesion pathways. The extracellular domains of these molecules have been fused to the Fc of IgG2c derived from the NOD mouse strain. This resulted in secreted and dimerized proteins. SV40 T Ag is potent at inducing graft rejection. Test and control transfectants were transplanted subcutaneously into young NOD mice to determine whether secretion of CTLA4Ig and OX40Ig would promote survival of the insulinoma graft. In immunodeficient mice, cell growth was similar for all transfectants. However, in immunocompetent NOD mice, the survival/growth of test grafts was significantly better than that of controls. By combining test transfectants, we found that graft survival was enhanced in an additive and significant fashion. In vitro, there was a significant reduction in immune responses-compared with control-when purified fusion proteins were added to mixed leukocyte reaction cultures. We conclude that blockade of individual costimulatory/adhesion signals by graft manipulation can contribute to transplantation success and that blockade of combinations of signals in these pathways enhances this success. Successful immunomodulation by the graft itself can be achieved.

Endogenously produced interleukin 6 is an accessory cytokine for dendritic cell hematopoiesis.

The aim of this study was to investigate the role of interleukin 6 (IL-6) in normal dendritic cell (DC) hematopoiesis. We used an enzyme-linked immunosorbent assay to quantitate IL-6 levels in CD34+ progenitor cell cultures favoring monocyte (mono) development versus those supporting mono-DC growth, and studied the neutralizing effects of alphaIL-6 antibody on DC hematopoiesis. IL6 levels in mono cultures (GM-CSF alone) were detected by day 4 and remained constant (approximately 100+ pg/ml) for 18 days. In mono-DC cultures, higher IL-6 levels correlated with DC content and development. Short-term mono-DC cultures initiated with GM-CSF + tumor necrosis factor (TNF) + stem cell factor (SCF) exhibited increases in IL-6 level until day 11 (peak DC growth). By day 18, the levels had declined and cells expressing typical DC features were no longer present. Long-term mono-DC cultures sustained with GM-CSF + TNF + SCF contained the highest IL-6 levels (671 pg/ml) on day 11. In these cultures, DCs and higher IL-6 levels persisted beyond 18 days. Anti-IL-6 profoundly inhibited cell proliferation associated with DC hematopoiesis when added on days 0, 2 and 5 to GM-CSF + TNF + SCF cultures, indicating that various stages of mono-DC development rely on IL-6. There was no reduction in the T cell response when alphaIL-6 was added to mixed leukocyte reaction cultures containing mature DCs as stimulators. Thus, alphaIL-6 appears to downregulate developmental processes associated with optimal mono-DC growth, but not the effector functions of mature DCs. These studies substantiate the importance of IL-6 as a secondary cytokine during DC development and provide insight into another control point in the DC pathway.

Towards a structural understanding of alloreactivity

One of the most thoroughly documented, yet paradoxical responses in immunology occurs in what is known as the mixed leukocyte culture (MLC). Typically, peripheral blood mononuclear cells proliferate vigorously when presented with allogeneic PBL (peripheral blood lymphocytes), but not with autologous or syngeneic cells. MLC reactivity as an in vitro measure of histocompatibility can be correlated with graft rejection or graft versus host disease. The paradoxical aspect of alloreactivity is manifested in light of recent experiments, which demonstrate that T cells normally respond to antigens in the context of autologous MHC molecules (Zinkernagel and Doherty, 1979; Rosenthal and Shevach, 1973; Shevach and Rosenthal, 1973). T-cell recognition is thus said to be self-restricted. Furthermore, we also know from these same experiments that allogeneic antigen-presenting (APC) cells are essentially incapable of presenting antigen to appropriately primed T ceils. How then can T cells, which normally see antigen in a self-restricted fashion, also recognize allogeneic MHC molecules in the MLC ? Numerous theories have sought to describe alloreactivity as somehow qualitatively or quantitatively different from self-restricted recognition. However, we would like to contend that allorecognition is fundamentally and qualitatively no different from self-restricted recognition at the structural level. In this context, we argue that an appreciation of the structural basis of allorecognition is a prerequisite to developing effective, antigen-specific therapeutic strategies for ameliorating graft rejection and graft versus host disease.

Human retrovirus-related synthetic peptides inhibit T lymphocyte proliferation.

Immunosuppression frequently accompanies infections with the human retroviruses HTLV-1 and HIV. Previous studies have shown that UV-inactivated and detergent-disrupted preparations of either virus can produce immune dysfunction in vitro although the active component of such preparations has not yet been identified. We now report that synthetic peptides corresponding to a conserved sequence within the transmembrane envelope proteins of both HTLV-1 (CS-1) and HIV (CS-3) are capable of suppressing the in vitro proliferative responses of human T lymphocytes to mitogenic or allogeneic stimulation. CS-1 inhibits proliferation in response to stimulation by anti-CD3 antibody by up to 57% a concentration of 3 nmol per culture well. CS-3 inhibits by up to 49% at a similar concentration. CS-1 and CS-3 inhibit two-way mixed leukocyte culture reactions by 81 and 88%, respectively, at the 3 nmol per well concentration. The inhibition by such human retrovirus-related peptides appears to be T cell-specific in that B cell proliferation in response to stimulation with anti-IgG is not affected by the CS-3 peptide. The release of such immunosuppressive peptides may thus play a role in the pathogenesis of human exogenous retroviral disease such as AIDS.

Rearrangement and expression of T cell receptor genes in cloned murine natural suppressor cell lines

Naturally occurring suppressor cells of the in vitro mixed leukocyte culture reaction and of in vivo graft-vs.-host disease have been identified in the spleens of neonatal mice (1) and of adult mice recovering from total lymphoid irradiation (2), whole-body irradiation (3), and syngeneic marrow transplantation (4), or cyclophosphamide therapy (5). Using both positive and negative selection procedures, the suppressors were reported to be null lymphocytes that did not express mature macrophage surface markers, nor differentiate into mature macrophages in vitro, nor demonstrate natural killer (NK) activity (1). Subsequently, cloned lines of these natural suppressor (NS) cells were derived from either adult mice given total lymphoid irradiation (TLI) (2) or from neonates (6). The cloned NS cell lines expressed a surface phenotype (2, 6) similar to that reported previously for cloned NK cells (Thy-1(+), asialo-GM1(+), Ig(-), Lyt-1(-), Lyt-2(-), Ia(-), MAC-1(-)) (7-9). However, the NS cells did not show NK activity in the standard assay with YAC-1 target cells. The cloned NS lines suppressed the proliferation of responder cells and the generation of cytolytic cells in the mixed leukocyte reaction (MLR), and suppressed lethal graft-vs.-host disease in vivo (10, 11). In view of the unusual function and surface phenotype of the cells, the lineage of these cells remained unclear. To determine the lineage of the cloned NS cells, we searched for expression and rearrangement of the alpha and beta chain genes of the T cell antigen receptor, as well as that of the gamma chain gene. Studies of the phenotypically similar NK cell yielded conflicting results. Thus, cloned lines of murine NK cells were reported to have rearrangements of the beta chain genes, and to express mRNA for all three chains (12). In contrast, freshly purified rat or human large granular lymphocytes (LGL) were shown to express only the 1.0 kb mRNA species of the beta chain gene (13), indicative of D-J joining (14). Thus, some but not all cells with NK function express the T cell receptor and are members of the T cell lineage. The current report shows that the NS lines express full-length mRNA transcripts for the a and beta chain of the T cell receptor, as well as the gamma chain gene.

Capacity of B cells to function as stimulators of a primary mixed leukocyte reaction.

The capacity of B cells to serve as stimulator cells for a primary mixed leukocyte reaction (MLR) was evaluated. Percoll-fractionated B cells were stimulated with lipopolysaccharide and dextran sulfate (L/D) or a B cell stimulatory factor (BSF-1)-containing culture supernatant, and then were fixed before being used as stimulator cells to more precisely define the state of activation associated with MLR stimulatory capacity. It was found that unstimulated B cells or B cells stimulated for 1 day with L/D or BSF-1 were incapable of initiating a primary MLR, whereas B cells incubated for 3 days in L/D were potent stimulators. The differential activity of 1 day L/D- and BSF-1-activated B cells compared with 3 day L/D-activated B cells was not related to the amount of the relevant MHC class I or class II alloantigens on these cell populations, because all three groups had large increments in MHC class II expression in the following order: BSF-1 greater than 3 day L/D greater than 1 day L/D, and had little difference in MHC class I expression. Also, all three populations were capable of stimulating both MHC class I- and class II-specific T cell hybrids. It was concluded that the capacity of 3 day L/D-activated cells to stimulate a primary MLR was due to the elaboration of necessary co-stimulator molecules. We evaluated whether interleukin 1 (IL 1) was the co-stimulator involved. That this was not the case was indicated by two findings. First, 3 day-activated L/D cells failed to express IL 1 activity as measured by a highly sensitive IL 1 assay that utilizes the T cell line D10.G4.1. Second, recombinant IL 1 added to MLR cultures containing 1 day L/D- or BSF-1 activated B cells failed to function as a co-stimulator. In contrast, the phorbol ester PMA was a potent co-stimulator in this system. We conclude from these experiments that appropriately activated B cells can function as stimulators of a primary MLR, and that they elaborate critical co-stimulator molecules, distinct from IL 1, that enable them to function in this regard.

Recruitment of unrelated persons as bone marrow donors. A preliminary experience.

Seven hundred and sixty-three blood donors previously typed for HLA-A and B antigens were asked to consider donating bone marrow to unrelated patients for whom no HLA-matched sibling was available. Eighty-eight percent (668) indicated willingness to donate marrow, and of these, 615 (81%) participated in testing and 53 (7%) were unable to participate at the time of the request. Donors matched for HLA-A, B, C, and DR antigens and compatible in the mixed-leukocyte culture reaction were identified for 12 patients and eight transplants were performed. Engraftment was successful in all eight patients. In this article the factors considered in planning the program, the protocol eventually developed, the problems encountered, and critical issues of informed consent and donor confidentiality are reviewed.

IMPAIRED REJECTION OF MINOR-HISTOCOMPATIBILITYANTIGEN–DISPARATE SKIN GRAFTS AND ACQUISITION OF TOLERANCE TO THYMUS DONOR ANTIGENS IN ALLOTHYMUS-IMPLANTED, THYMECTOMIZED XENOPUS

We examine whether J strain Xenopus laevis and X laevis/gilli (LG) isogeneic hybrids, thymectomized (Tx) at 7 days and implanted with larval or postmetamorphic thymus (either normal or irradiated) in late larval life, can be used to explore the role of the developing thymus in the education of minor histocompatibility (H) antigen-reactive T cells and in the acquisition of tolerance to MHC (major histocompatibility complex) antigens. Tx LG Xenopus implanted with isogeneic thymus display fully restored ability to reject both MHC-incompatible and minor H-disparate grafts. Allothymuses (both MHC- and minor-H-disparate) restore normal rejection of skin grafts with MHC incompatibility to thymus donor and Tx (J or LG) host, but reactivity to grafts with putative minor H disparity is impaired. Defective minor-H-disparate skin graft rejection occurs even when the Tx host has received a thymus of the "correct" MHC haplotype (i.e., the same MHC as the assay skin graft). This defect may be attributable to inadequate establishment of minor-H-reactive T lymphocytes, to sharing of minor antigens, or to "nonspecificity" of tolerance induction to minor antigens, since such tolerance is demonstrated here following perimetamorphic grafting of skin to control LG clones. Skin grafts from Xenopus isogeneic to the donors of the MHC-incompatible larval and adult thymus implants are always tolerated by Tx hosts. In contrast, in vitro one-way MLC (mixed leukocyte culture) reactivity of splenocytes to thymus donor MHC antigens occurs when certain donor/host combinations are used but not when others are tested. Postmetamorphic thymus implants appear to promote in vitro MLC tolerance more effectively than larval thymus implants.

Alteration of the T cell self-specificity repertoire by treatment with anti-Ia antibody during embryonic life.

The effect of anti-Ia antibody on the development of murine neonatal self-Ia-reactive T lymphocytes in the thymus was studied. A C57BL/6 (B6) mouse pregnant with (B6 X C3H/He)F1 (B6C3F1) embryos was injected with monoclonal anti-I-Ek (m alpha I-Ek) antibody every other day starting from the 12th day of gestation. The mixed leukocyte culture reaction (MLR) responses of thymocytes from m alpha I-Ek-treated neonatal mice were assayed against adult B6 (H-2b), C3H/H3 (H-2k) or BALB/c (H-2d) splenic stimulator cells. The syngeneic MLR response of m alpha I-Ek-treated B6C3F1 neonatal thymocytes against C3H stimulator cells was augmented compared to that of untreated neonatal thymocytes. Experiments utilizing recombinant inbred strains of mice as well as monoclonal anti-Ia antibody blocking experiments suggested that the antigen(s) responsible for this augmentation of syngeneic MLR resided in the I-Ek molecule. The MLR response against B6 or BALB/c stimulator cells was not influenced by m alpha I-Ek treatment. We speculate that m alpha I-Ek antibody blocked the expression of I-Ek molecules in B6C3F1 embryos and this resulted in the alteration of T cell self-specificity repertoire in the thymus. Our results support the concept that Ia molecules play important roles for the elimination of self-reactive T lymphocyte clones in the thymus.

COEXISTENCE OF STOMACH CANCER AND RENAL ADENOMA IN RENAL ALLOGRAFT

Autopsy findings of a 34-year-old Japanese male with stomach cancer in a renal allograft are presented. The renal graft was given by his 53-year-old mother, who had the same HLA type as the patient and showed negative response in the Mixed Leukocyte Culture Reaction test. Stomach cancer occurred 4 years and 5 months after the renal graft, and infiltrated widely into the abdominal cavity, causing rectal stricture. In his original left kidney, a small renal adenoma was found. The tumor cells of the adenoma were composed of clear cells. In the adenoma, infiltration of stomach cancer was found. The renal allograft functioned very well until his death in spite of several rejection episodes. This is the seventh reported case in Japan in which a renal allotransplant was accompanied by malignancies.

DLA-D-specific suppressor cells characterized by monoclonal antibodies.

Canine lymphocytes alloantigen-primed in mixed leukocyte culture (MLC) were tested for their ability to suppress MLC reactivity. These cells were added to fresh responder cells in MLC at ratios of 1:50 and 1:20 and suppressed reactivity 90 +/- 7% (mean +/- 1 SD) and 91 +/- 3% respectively. At lower ratios no suppression was observed. Monoclonal antibody E11 (IgG3) recognized a subset of canine Thy1+ T cells, and with complement treatment was capable of depleting suppressor cells from bulk MLC (11 +/- 6% of control suppression), but DT-2 (IgG2a), another canine T-cell antibody, had no such effect (84 +/- 11%) (P = 0.028). Antibody A5 (IgG2b), reactive with all T and most non-T cells, also eliminated suppression. Positively selected E11+ bulk-MLC cells suppressed (91%) and E11- bulk-MLC cells did not (4%). At the ratios described, suppression was specific for the DLA-D phenotype of the stimulator cells. In tests with the original stimulators, suppression was 91 +/- 7%; in tests with DLA-D identical stimulators it was 92 +/- 5%; and for DLA-D nonidentical stimulators it was 38 +/- 25% (P = 0.005). Nonspecific suppression increased with increasing numbers of suppressor cells. At ratios of 1:10 and 1:5 nonspecific suppression was 72 +/- 13% and 92 +/- 8%, respectively. These studies show that under appropriate conditions, Thy1+, E11+, A5+, DT-2- alloantigen-activated canine T lymphocytes function as DLA-D-specific suppressor cells and DT-2+ cells do not.

Preliminary characterization of a soluble immunosuppressive molecule from DBA/2 spleen cells using monoclonal antibody immunoadsorbence

In a previous publication a monoclonal antibody (B16G) which appeared to recognize T suppressor cells and a T-suppressor factor (TsF) in the spleens of DBA/2 mice was described. B16G appears to be directed to a public specificity of DBA/2 TsF and therefore has been shown to inhibit a variety of immunological reactions. The present study involves preliminary characterization of the material with which B16G reacts. It was found that the B16G-reactive protein (putative TsF) could be absorbed and eluted specifically from a B16G immunoadsorbent column. Material eluting from the B16G column reacted with B16G in an ELISA and appeared to run as two or more bands of 40–45 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The eluted material was biologically active (i.e., suppressive) in the standard assay (mixed leukocyte reaction of DBA/2 splenocytes with B10.BR targets), and its suppressive activity was abrogated by the addition of B16G to the mixed leukocyte reaction cultures. Sephadex G-150 chromatography of the B16G-reactive material showed that under these conditions, its native molecular mass was between 80–90 kDa, indicating that it might occur as a dimer under natural conditions.

Electrophoretic variation between class II molecules expressed on HLA-DRw8 homozygous typing cells reveals multiple distinct haplotypes.

Two-dimensional (2D) gel electrophoresis of immunoprecipitated HLA-DR antigens from eight homozygous typing cells (HTC) expressing the HLA-DRw8 specificity revealed a clustering of polymorphic beta chain patterns into distinct electrophoretic variants. The variant patterns correlate with three discrete HLA-D clusters that are defined in the mixed leukocyte culture reaction (MLR) using DRw8-positive HTC. These HLA-D clusters have been provisionally designated Dw"8.1", detected primarily in Caucasoids, Dw"8.2", detected primarily in American Indians, and Dw"8.3", detected predominantly in Orientals. All three HLA-Dw"8.1" cell lines express a single DR-locus product as defined by immunoprecipitation with a DR-specific monoclonal antibody, P4.1. This DR beta chain is identical among the Dw"8.1" cell lines and different from the DR beta chains of the Dw"8.2" and Dw"8.3" cell lines. Two separate Dw"8.2" HTC express a shared DR beta chain that is slightly more basic than the 8.1 DR molecule; interestingly, one of these lines also expresses an additional DR-like beta chain not found in the other cells. Thus, the two lines defining the Dw"8.2" cluster share one distinct class II molecule, but differ in another and therefore are not biochemically HLA-identical. Cells from the Dw"8.3" cluster are likewise distinct from all other Dw8 clusters. One additional DRw8-positive HTC has been analyzed and found to be distinct from the Dw"8.1", "8.2" and "8.3" clusters by both MLR and 2D gels. Immunoprecipitates using monoclonal antibody 1B5 [anti-DR and anti-DQ(DS)] identify additional polymorphic class II variants among the cell lines tested. These data indicate that HLA-DRw8 is a public serologic specificity present on class II molecules expressed on multiple distinct haplotypes. These haplotypes differ from each other in expression of polymorphic class II molecules encoded by at least two HLA loci. They also differ in HLA-D, even though they all type as HLA-DRw8 homozygous. In Dw"8.2", variation in expressed beta chains is not reflected in variation in HLA-D, indicating that MLR, as well as serologic typing, does not detect the full degree of allelic polymorphism within HLA.

Early recognition of transplantation antigens by T lymphocytes. II. Specificity and characterization of the recognition process.

In crude supernates of interactions of parental strain T spleen cells with H-2 antigen-bearing F1 hybrid spleen cells, a product (soluble early product of immune recognition, SEPIR) can be found which enhances mixed leukocyte culture (MLC) reactions set up with suboptimal cell concentrations (SMLC). Supernates of parental T cells reacting with F1 spleen cell also produce this factor. The product ameliorates SMLC proliferative responses specifically since identical antigenic specificities have to be recognized in both SEPIR formation and in test SMLC. Further evidence for SEPIR being an immunologically specific product is documented by the finding that spleen cells from mice made specifically tolerant at birth cannot respond to the tolerogen with SEPIR formation, but are fully reactive towards unrelated H-2 antigens. No responses to syngeneic or autologous antigens could be discerned. Characterization of allorecognition resulting in SEPIR formation has revealed that spleen cells of Lyt 23 phenotype recognize H-2K/D-encoded antigens. Lyt 1 cells appear to be incapable of forming the early product and H-2I-determined antigens fail to incite a response. Reactivity to class I antigens was amenable to specific inhibition by monoclonal alloantibodies.

Mixed Leukocyte Reaction, Graft-Versus-Host Reaction, and Skin Allograft Rejection in the Lizard, Chalcides ocellatus

In optimal seasonal conditions, splenocytes derived from adult lizards, Chalcides ocellatus, responded to vigorous proliferation in two- and one-way mixed leukocyte reaction cultures (MLRC). Data based on a large number of reciprocal MLRC provided evidence for the presence of strong and diverse lymphocyte activating determinants. These findings were fully confirmed in studies of systemic graft-versus-host reaction as intraperitoneal injection of splenocytes into newborn allogeneic recipients consistently induced splenomegaly, retarded growth and mortality. In favourable ambient conditions, adult lizards were also able to reject skin allografts in a subacute manner (mean survival time ± S.E. = 28.8 ± 0.88). The results clearly indicate that the lizard, Chalcides ocellatus, is endowed with an advanced type of cellmediated immunity, and possesses strong and polymorphic histocompatibility antigens. Cellular alloreactivity in MLRC and towards skin grafts was, however, abrogated in winter and significantly diminished during spring through mid-summer as compared to mid-summer till autumn. The present study, thus, suggests that immunological defects attributed to reptiles might be more apparent than real, and essentially ascribable to the fact that the immune capacity of these ectothermic vertebrates is profoundly modulated by environmental conditions.

Surgery, trauma and immune suppression. Evolving the mechanism.

A major surgical procedure can impair the delayed hypersensitivity response. This impairment is associated with suppressor cell activity that can alter either afferent or efferent responses. Using the third party mixed leukocyte culture to define cell types involved, major immune impairment was seen with the combination of both nonadherent and plastic adherent cells, suggesting that a T cell-macrophage interaction is required. A serum factor(s) is present in operated mice that can impair mixed leukocyte culture reactivity. A serum factor(s) in an adoptive transfer experiment is also capable of enhancing primary tumor growth. A unifying hypothesis, based predominantly on data from the current literature, is presented in an attempt to elucidate the mechanism by which all forms of major trauma are associated in some patients with "paradoxical" immune suppression.

Heterogeneity of cytolytic and suppressor clones of alloactivated cells generated from soft agar colonies

Long-term cell cultures (or clones) were developed from soft agar colonies of lymphocytes alloactivated in mixed leukocyte culture reaction (MLR). Two types of colonies were identified: upper colonies that grew on the agar surface, and lower colonies found within the agar layer. Virtually all cytolytic clones originated exclusively from the upper colonies. Two groups of cytolytic clones could be distinguished, one with strong and the other with weak proliferation upon restimulation. Upper clones were capable of inhibiting primary MLR proliferation and this appeared to be related to their cytolytic effect on the stimulator. Many noncytolytic lower clones were found to suppress primary MLR cultures. Considerable heterogeneity was apparent from differences in the magnitude of suppression and the ability of the clones themselves to undergo stimulator-induced proliferation. Kinetic studies of MLR suppression were conducted to further analyze this heterogeneity. Two major kinetic patterns were observed. One showed a biphasic proliferation pattern of the MLR + clone culture. The first peak appeared to reflect an enhanced proliferation of the clone. The second phase seemed to represent diminished proliferation of the MLR responder. This type of suppression may be related to T cell growth factor depletion from MLR by the proliferating clone. The other kinetic pattern showed a consistently low proliferation of the MLR + clone culture throughout the 8-day assay period. Subsequent testing of these suppressor clones in third-party MLR cultures suggested that the specificity of suppression was unrelated to HLA-DR, MB, MT, and SB.