Regulation of the immune response to alloantigens: suppressor and helper T cells generated in the primary MLR of the rat

The primary MLR of the rat was used to generate suppressor, cytotoxic, and helper T cells from lymph node cells of the WF (RT1 mu) inbred strain. They were assayed in 51Cr-release cytotoxic assays and by their effect on proliferation of fresh unprimed responder cells. Suppression by MLR cellular products was antigen-specific and generation and functional expression were directed to class II (RT1.B,D) antigens of stimulator cells in the strains tested. In contrast, help was not antigen-specific. The monoclonal antibodies OX8 and W3/25 were used to separate the primed products of the MLR into the constitutive subsets, suppressor/cytotoxic (OX8+) and helper/inducer (W3/25+). Gamma irradiation of OX8+ MLR-primed cells caused modest reductions in suppressive activity, but had no effect on the helper activity of W3/25+ cells. MLR-derived suppressor cells are effective only when added in the early stages of the test primary MLR, whereas helper cells can augment proliferation even when added late. Feedback suppression is not mediated by classical cytotoxic T cells, because of differences in kinetics of development, cell numbers required, susceptibility to freezing, and expression of the RT6 differentiation antigen.     

Cellular basis of allograft rejection in vivo. V. Examination of the mechanisms responsible for the differing efficacy of monoclonal antibody to CD4+ T cell subsets in low- and high-responder rat strains

MRC OX35, an anti-CD4 mAb, was used to treat high responder Wistar Furth (W/F) (RT1u) and low responder DA (RT1a) rats which had been grafted with directly vascularized hearts from PVG (RT1c) rats across a full MHC plus non-MHC incompatibility. Four doses of mAb at 7 mg/kg given in the first 2 wk postgrafting induced indefinite graft survival (greater than 150 days) in DA hosts, but only delayed rejection to 18 to 42 days in W/F as compared to rejection times of 6 to 8 days in untreated rats. The extension of MRC OX35 treatment to 6 wk in W/F rats induced indefinite graft survival in three of six rats. During treatment MRC OX35 therapy only partially depleted CD4+ cells, and all circulating CD4+ cells were coated with MRC OX35. The capacity of naive CD4+ and CD8+ cells from W/F and DA to be activated to PVG alloantigen was compared both in vitro in an MLC assay and in vivo by an adoptive transfer assay of their capacity to restore rejection of PVG heart grafts in irradiated syngeneic hosts. CD4+ cells from both W/F and DA proliferated in MLC and restored graft rejection. W/F CD8+ cells both proliferated in MLC and restored rejection, but DA CD8+ cells neither proliferated nor reconstituted rejection. Examination of lymphocytes from MRC OX35 treated hosts with long-surviving grafts showed that they were neither depleted of CD4+ T cells nor did they lack the capacity to proliferate to PVG Ag in MLC, this response being similar to that to third-party Ag or by naive lymphocytes. Compared to first-set rejection, PVG skin graft rejection was delayed 2 to 3 days in W/F and 10 to 12 days in DA rats with long-surviving grafts after MRC OX35 therapy, whereas they rejected third-party skin grafts in first-set tempo. These studies show that differences in graft survival in anti-CD4 treated low and high responder strains may be due to the inherent capacity of CD8+ cells to be activated to effect rejection independent of CD4+ cells in W/F but not in DA. In those hosts that accept grafts, there is no evidence of clonal deletion, but there appears to be a form of unresponsiveness akin to that induced in adult rats by other immunosuppressive therapies that protects the graft from rejection.     

T cell subsets mediating lethal graft versus host disease: demonstration that synergy between CD4+ and CD8+ T cells is the predominant mechanism in low responder rat strains

T cell subsets from rat strains that have been characterized as high and low responders to alloantigen were examined for their capacity to mediate lethal graft versus host disease (GVHD) across strain combinations incompatible for class I, class II, and non-MHC antigens. Inocula of 5 X 10(7) lymph node and spleen cells (LC) from low responder DA (RT1a) and high responder W/F (RT1u) strains caused lethal GVHD in (W/F X DA)F1 hybrids given 6 Gy whole body irradiation. W/F CD4+ (W3/25+) cells (2 X 10(7], equal to the number in 5 X 10(7) LC mediated lethal GVHD but 10(8) DA CD4+ cells were required to cause lethal GVHD. CD8+ (MRC OX8+) cells (5 X 10(7] from W/F rats alone caused lethal GVHD but those from DA rats could not. Mixtures of CD4+ and CD8+ DA T cells, equivalent to the number in 5 X 10(7) LC, did mediate lethal GVHD, demonstrating that synergy between the subsets was the predominant mechanism with DA cells. These results suggest that differences in alloreactivity between the strains tested may be due to alternate requirements for the alloactivation of T cell subsets; the high responder subsets being self-sufficient and the low responder subsets being dependent upon each other.     

Xenogeneic monoclonal antibody to an Ly-6-linked murine cell surface antigen: differential reactivity with T cell subpopulations and bone marrow cells

The patterns of cellular and strain reactivity of a monoclonal antibody (6C3 MAb) derived from the fusion of SP2/0 cells with splenocytes from rats immunized against MRL/MpJ-lpr/lpr T cells were characterized by using flow cytofluorometry (FCF) analysis. This MAb was found to stain 70 to 90% of T cells of mice with the lpr/lpr genotype and 20 to 60% of T cells of congenic +/+ strains. Dual-parameter FCF analysis of Lyt-2 vs 6C3 expression revealed the existence of several Lyt-2- and Lyt-2+ T cell subsets, one of which (Lyt-2- bright 6C3+) was expanded in lpr/lpr-bearing mice. The 6C3 MAb stained only 2 to 5% normal thymocytes but reacted with 40 to 50% bone marrow (BM) cells. A strain survey demonstrated the expression of the 6C3 antigen on peripheral T cells (and BM cells) of all strains examined, with the exception of NOD, NZB/B1NJ, and ST/bJ. Interestingly, in the positive strains, two types of 6C3 staining patterns of T cells were observed: bimodal or trimodal. Study of BXH and CXB recombinant inbred (RI) strains demonstrated that the bimodal and trimodal 6C3 patterns are associated with the Ly-6.1 and Ly-6.2 phenotypes, respectively. Linkage of 6C3 expression with the Ly-6 locus was confirmed by using the congenic C3H.B6-Ly-6b strain. Moreover, the 6C3 staining of T cells in Ly-6.2 strains was reduced by preincubation with the H9/25 and SK-142-446 MAb, which are known to recognize Ly-6.2-associated antigens. Therefore, the 6C3 MAb appears to detect a frame-work determinant on an Ly-6-linked antigen that is absent from T cells of NOD, NZB, and ST/bJ mice. Analysis of (NZB x C58) NX8 RI strains demonstrated a correlation between the lack of 6C3 expression on T cells and unresponsiveness in autologous mixed lymphocyte reaction (a property of NZB/B1NJ mice). The 6C3 MAb should prove useful for further genetic and biochemical analysis of the Ly-6 locus and its product(s), and for the delineation of functional subsets of T cells and BM cells in normal and lpr/lpr-bearing mice.     

Autoimmune effector cells. IX. Inhibition of adoptive transfer of autoimmune encephalomyelitis with a monoclonal antibody specific for interleukin 2 receptors

This study was conducted to determine whether a monoclonal antibody (MAb) specific for rat interleukin 2 receptors (IL 2R) inhibits the activation of effector T cells that adoptively transfer experimental allergic encephalomyelitis (EAE). MAb OX 39 appears to be specific for IL 2R because it binds to concanavalin A-activated, but not resting, rat lymphocytes and inhibits mitogen- and IL 2-induced proliferation of rat spleen cells. Moreover, this MAb inhibits the in vitro activation of effector cells of EAE by myelin basic protein when added to immune donor spleen cell at the start of 72-hr culture or after 24 hr, but not when added after 48 hr of culture. Other studies employed MAb W3/25, which reacts with the rat helper T cell subset and appears to define the rat homolog of the human CD4 marker present on T4-positive cells. MAb W3/25 also blocks in vitro activation of EAE effector cells, and this blocking effect can be abrogated by adding rat T cell growth factor or partially purified IL 2 to the donor spleen cell cultures. T cell growth factor alone is incapable of activating EAE effector cells. These findings are discussed with respect to the role of lymphokines in the generation of autoreactive T cells.     

T cell-mediated cytotoxicity induced by Listeria monocytogenes. III. Phenotypic characteristics of mediator T cells

Cytotoxic thymus-derived lymphocytes (CTL) generated in vitro by restimulating rat cells with Listeria antigen- (LMA) pulsed syngeneic accessory cells were characterized in respect to their surface membrane markers. LM-dependent CTL were devoid of detectable surface immunoglobulin (Ig) and receptors for the Fc region of rabbit IgG. Experiments with monoclonal antibodies to rat T cell markers revealed that these cytotoxic cells have the phenotypic profile W3/13+, W3/25-, MRC OX 8+. LM-dependent CTL also bind the monoclonal antibody, MRC OX 3, which recognizes an Ia-antigen-like determinant on rat cells. Although LM-dependent CTL lack the W3/25 marker, their generation depends on the cooperative interplay of W3/25+ and W3/25- T cells.     

Characterization of RT6 bearing rat lymphocytes. I. Ontogeny of the RT6+ subset

The RT6 alloantigen is present on approximately 70% of peripheral T cells in the rat, but is absent from thymocytes and bone marrow lymphocytes. The results of further phenotypic analysis in the present study demonstrated that the RT6 alloantigen is expressed on approximately 45% of the helper/inducer (CD4; W3/25+) and 80% of the cytotoxic/suppressor (CD8; OX8+) peripheral T-cell subsets. Ontogenetic and thymus ablation studies indicated that the RT6+ T-cell subset is thymus-dependent and normally develops after the appearance of RT6-T cells in neonatal rats, and that the expression of RT6 is a post-thymic maturational event. Furthermore, intrathymic adoptive transfer of bone marrow cells demonstrated that RT6+ T cells are thymus-derived cells. These results show that most if not all RT6+ T cells are the progeny of RT6- T cells. However, they do not exclude the possibility that a separate lineage of RT6- T cells exists, which also has OX8+ and W3/25+ subsets. The possible developmental and functional relationships of RT6- and RT6+ T cells in the rat are discussed.     

The membrane phenotype of in vivo induced tumor selective cytolytic lymphocytes of the rat: a distinction from NK cells

The phenotype of tumor selective cytolytic lymphocytes of the rat is defined by staining of peritoneal cells of tumor-immunized donors with the monoclonal antibodies OX19, OX8, and W3/25, sorting in a cytofluorometer and evaluating cytolytic capacity in a 51Cr release assay. It is shown that the tumor selective cytolytic lymphocytes bear the OX19 and OX8 markers but are lacking the W3/25 marker. It is also shown that the OX19+ lymphocytes do not contain any NK-like activity. The OX19 marker can therefore distinguish cells that execute selective cytolysis from cells with NK-like activities.     

An investigation of genetic variation within a series of congenic strains of mice

2 congenic strains of mice, B6N.AKN-Ahk and D2N.B6N-Ahb, imported from the USA, were found to be either segregating or fixed for an incorrect allele at a number of biochemical loci. B6N.AKN-Ahk, supposedly congenic with C57BL/6N, had the wrong genotype at 6 out of 12 biochemical loci; D2N.B6N-Ahb, supposedly congenic with DBA/2N, was segregating at 3 out of 9 loci. There was genetic variation in mandible shape within the 2 strains but no abnormal coat colours were found and no hybrid vigour in breeding performance was detected. Analyses in the USA confirmed these results and showed that 2 other congenic strains, C3N.D2N-Ahd and AKN.B6J-Ahb, were also segregating at a number of loci. Some of the alleles found in the C3N.D2N-Ahd mice must be the result of a genetic contamination. The simplest explanation for this breakdown in the backcrossing programme is genetic contamination with other congenic strains or recombinant inbred lines under development in the same laboratory. These findings emphasize the importance of continual genetic monitoring of all genetic stocks at regular intervals and in particular during the development of congenic and recombinant lines.     

Experimental autoallergic sialadenitis in the LEW rat. III. Role of CD4+ T cells in EAS induction

Experimental autoallergic sialadenitis (EAS) in the LEW rat is an induced autoimmune disease of the salivary tissues. EAS is characterized by a lymphocytic infiltration that consists of both CD4+ (helper/inducer T-cell subset) and CD8+ (cytotoxic/suppressor T-cell subset) T cells and results in the immune-mediated destruction of the exocrine salivary glands. To investigate the role that each of the T-cell subsets may have in the pathogenesis of EAS, LEW rats sensitized with WF SMG homogenate were injected with monoclonal antibodies to deplete or inactivate, in vivo, the CD4, CD5 (OX19; pan T lymphocyte), CD8, or RT6 (70% of peripheral T cells) T-cell populations. Treatment with the OX8 (CD8), OX19 (CD5), or W3/25 (CD4) only partially reduced in vivo the respective splenic or lymph node T-cell subsets when analyzed on Day 14, while treatment with DS4.23 (anti-RT6) resulted in greater than 95% depletion of RT6+ spleen and lymph node T cells. EAS incidence and severity was significantly reduced in the W3/25 (CD4) treatment group (11% incidence rate; histologic score 1.0) as compared to medium-injected controls (88% incidence rate; histologic score 2.9). Although the incidence and severity of EAS in the OX19 (71%; histologic score 1.7), OX8 (55%; histologic score 1.7), and RT6 (67%; histologic score 1.6) treatment groups appeared decreased, the reduction was not statistically significant. These results provide evidence that CD4+ T cells have an important role in EAS induction and demonstrate that in vivo treatment with anti-CD4 can ameliorate and/or prevent EAS in the LEW rat.     

The regulatory role of CD45 on rat NK cells in target cell lysis.

To investigate the role of CD45 in rat NK cell function, we developed new mAbs directed against rat CD45. mAb ANK12 binds to a high molecular isoform of CD45 and mAb ANK74 binds to the common part on all known CD45 isoforms, as has been described for the anti-rat CD45 mAb OX1. The ability of these mAbs to affect NK cell-mediated lysis was tested using the Fc receptor-positive target cell line P815. mAb ANK12 was found to significantly enhance the lysis of P815, whereas ANK74 and the anti-CD45 mAb OX1 did not. In addition, cross-linking of the CD45 isoform by ANK12 induced tyrosine phosphorylation of specific proteins in NK cells. Subsequently, the involvement of CD45 in the negative signaling after "self" MHC class I recognition by rat NK cells was investigated. The anti-CD45 mAbs were found to affect NK cell-mediated lysis of syngeneic tumor cell lines, depending upon the expression level of MHC class I on target cells. mAbs ANK74 and OX1 only inhibited lysis of the syngeneic tumor cell lines that expressed low levels of MHC class I. Furthermore, both mAbs caused an inhibition of NK cell-mediated lysis of these tumor cell lines when MHC class I molecules on the tumor cell lines were masked by an Ab. These results suggest that CD45 regulates the inhibitory signal pathway after self MHC class I recognition, supposedly by dephosphorylation of proteins.     

Genes of SHR rats protect spontaneously diabetic BB/OK rats from diabetes: lessons from congenic BB.SHR rat strains

Diabetes in BB rats share many common features with human type 1 diabetes. One of them is the complex and polygenic nature of disease. Analysis of cross hybrids of diabetic BB/OK rats and rats of different diabetes-resistant strains has demonstrated that beside the MHC genes, Iddm1 and the lymphopenia, Iddm2, additional non-MHC genes are involved in diabetes development. To study the importance of the non-MHC genes, Iddm4 and Iddm3, two congenic BB.SHR rat strains were generated by recombining a segment of the SHR chromosome 6 (Iddm4; termed BB.6S; 15cM) or chromosome 18 (Iddm3; termed BB.18S; 24cM) into the BB/OK background by serial backcrossing and marker-aided selection. The characterization of both congenic strains demonstrates a drastic reduction of diabetes frequency in comparison to the BB/OK strain (86% vs 14% and 34%). It is supposed that diabetes protective genes of SHR must be located on both chromosomal segments and that these suppress the action of the essential and most important genes of diabetes development in the BB/OK rat, Iddm1, and Iddm2.     

Natural killer cell number and function in the spontaneously diabetic BB/W rat

The BB/W rat provides a good model of spontaneous autoimmune diabetes. Diabetes-prone (DP) rats have a virtual lack of OX 8+ OX 19+ T cytotoxic/suppressor cells in peripheral blood lymphocytes (PBL) and spleen, suggesting that the OX 8+ OX 9- natural killer (NK) cells are the predominant cytotoxic cell in this animal. In this study, we have shown that rat NK cells belong to the OX 8+ OX 19- asialo GM1 bright population, and that rat NK cell function may be depleted in vivo by administration of OX 8 antibody. Furthermore, evidence is provided to indicate that NK cell number and activity are enhanced on a per cell basis in DP rats as compared to the diabetes-resistant W line rat. DP rats had about threefold more NK cells than did W-line rats. The cytotoxic activity mediated by spleen and PBL against the YAC-1 target generally correlated with the relative number of cells having the OX 8+ OX 19- phenotype. DP lymphocytes mediated low levels of cytolytic activity against the relatively resistant NK target cell K562. To more directly compare the activity of W-line and DP NK cells, spleen NK cells were isolated by flow sorting of the OX 8+ OX 19- population. At a 5:1 E:T ratio, DP OX 8+ OX 19- cells elicited 21% +/- 3 specific lysis and W-line cells elicited 7% +/- 2 specific lysis. To determine whether the elevated levels of NK cells and NK cell activity in DP rats were a consequence of NK cell proliferation, spleen cells were size-separated by centrifugal elutriation. The NK cell activity was predominantly mediated by small to medium-size lymphocytes and not blast-size enriched populations. Moreover, when the DNA content of splenic OX 8+ cells was measured, 98% of the cells were in the G0-G1 phase of the cell cycle. These data indicate that NK cell number and activity are elevated in DP rats, and support a role for NK cells in the pathogenesis of BB/W diabetes.     

Escherichia coli-specific T lymphocytes in experimental pyelonephritis

We have assessed the phenotype and specificity of infiltrating mononuclear cells in a model of unilateral ascending acute pyelonephritis induced in rats with nephritogenic Escherichia coli or Pseudomonas aeruginosa. Histologic examination showed a predominance of mononuclear cells in the interstitium at all periods examined (4, 8, 15, 21, and 25 days), although at 4 and 8 days neutrophils were also abundant. Most of the mononuclear cells had the morphologic appearance of large lymphocytes. Immunoperoxidase studies with mAb showed that most of the mononuclear cells were W3/25+; many were W3/13+ and a small proportion were OX8+. Many of the mononuclear cells were Ia+. T cells were propagated in IL-2-containing media from small fragments of renal tissue with pyelonephritic lesions. Most of the propagated cells were W3/25+; fewer than (10%) were OX8+ or Ia+. T cells propagated from kidneys infected with E. coli responded, in proliferation assays, to the infecting strain or other E. coli strains, but not to P. aeruginosa or enterococci. The response to non-p-pilus-bearing E. coli was as great or greater than to E. coli with adhesins. T cells derived from lesions induced by P. aeruginosa responded to the infecting organisms, but not to E. coli. The response to the infecting organism (E. coli or P. aeruginosa) was MHC restricted, as indicated by the requirement for syngeneic APC. The results show that large numbers of T lymphocytes, especially with the "helper/inducer" phenotype, accumulate in the lesions of acute pyelonephritis in rats. Among the infiltrating T lymphocytes are activated cells and cells with specific reactivity to the infecting bacteria (or related strains). The findings indicate that T lymphocytes play a role within the kidney in response to the invading bacteria.     

Phenotypic heterogeneity of Gross virus-induced thymic lymphomas in the rat: cellular origins and migratory properties

Neoplastic thymocytes from rat thymic lymphoma-leukemias induced by the rat-adapted Gross leukemia virus (RAGV) were analyzed for a variety of differentiation markers. The neoplasms from individual rats all expressed the antigenic phenotype MP+, W3/13+, Thy-1+, RT-1+, RT-7+, W3/25-. However, approximately two-thirds of the neoplasms were positive for the OX 8 antigen, and one-third were negative. The OX 8- neoplasms only involved the thymus, whereas approximately 40% of the OX 8+ neoplasms involved the spleen as well as the thymus. Virtually all OX 8+ and OX 8- neoplastic cells contained terminal deoxynucleotidyl transferase (TdT), and both OX 8+ and OX 8- lymphomas expressed the lactate dehydrogenase (LDH)-5' isozyme and the primary, but not the secondary, ADA isozyme. This enzymatic phenotype is characteristic of thymocyte precursors, but not thymocytes. Our results therefore indicate that RAGV-induced lymphomas arise from transformed prethymic TdT+ cells which contain the LDH-5' and the primary ADA isozymes. These preleukemic cells presumably migrate to the thymus where they express the RT-7 pan-T-cell antigen and, in some instances, the OX 8 antigen during the development of overt leukemia. The OX 8+ neoplasms, being more differentiated than their OX 8- counterparts, then migrate to peripheral lymphoid tissues.     

Selective induction of OX19+ (CD5+) or OX19- (CD5-) alloreactive cytolytic lymphocytes in the rat

The phenotypes of alloselective cytolytic lymphocytes of the rat are defined by staining of peritoneal cells of alloimmunized donors with monoclonal antibodies, sorting in a cytofluorometer and evaluating cytolytic capacity in a 51Cr-release assay. We demonstrate that alloimmunization of BN rats can result in either OX19+ (CD5+) or OX19- (CD5-) cytolytic alloselective lymphocytes and show that the OX19- (CD5-) cytolytic cells are OX34+ W3/25- (CD4-) OX8+ (CD8+) lymphocytes not exposing surface Ig. It is further demonstrated that the appearance of CD5+ and CD5- cytolytic alloselective lymphocytes are mutually exclusive; immunization with (WF X BN) F1 cells leading exclusively to appearance of OX19+ effector cells while immunization with WF cells leads to OX19- effector cells. Alloimmunization of WF rats only results in appearance of OX19+ cytolytic lymphocytes.     

Altered levels of mononuclear leukocytes in tumor-bearing rats: decrease of helper T lymphocytes and increase of suppressor cells

In the spleen and peripheral blood of BN rats with progressive tumors, W3/25+ T helper cells were significantly reduced and OX8+ T suppressor/cytotoxic cells were significantly increased. The ratio of helper to suppressor elements was decreased to 1.6 from a ratio of 3 in normal BN rats without tumors, and this decreased ratio correlated with tumor growth. When tumors were eliminated in vivo by infusion of effector cells (W3/25+ T lymphocytes), the levels of W3/25+ and OX8+ T cells returned to normal and the ratio of helper to suppressor/cytotoxic cells in the spleen and peripheral blood reverted to 3.0 or higher. Macrophages and null cells, T-sIg-, were also elevated in the spleen and peripheral blood of rats bearing expanding tumors and returned to normal levels after cure. Assays of spleen cells for cell-mediated cytotoxicity in rats with large tumors revealed little or no specific cytotoxicity. Cytotoxic activity was high in spleen of rats cured of their neoplasms by infusion of helper cells.     

Genetic analysis of theT-H-2 region in non-t Chromosomes

A general breeding protocol useful in the construction of congenic lines of mice disparate in the 15 cMT-H-2 region of chromosome 17 in non-t chromosomes is described. Two such congenic lines, B6.TC2/Rn and B6.TC3/Rn, were derived from the C57BL/6J and B6.C-H-2 ^d/By strains using this protocol. Both B6.TC2 and B6.TC3 dissociate the quantitative activity locus for glyoxalase I (Qglo-1) from theH-2 complex, and hence possess BALB/cBy DNA centromeric toH-2. However, neither new strain is able to map anyH-2-associated restriction fragment length polymorphism with anH-2 cDNA probe even though both strains are recombinant in the 2 cMQglo-1-H-2K interval.     

Absence of the RT-6 T cell subset in diabetes-prone BB/W rats

Diabetes-prone Bio-breeding/Worcester (DP) rats exhibit a severe T cell lymphopenia and autoimmune pancreatic insulitis. The present results indicate that the T cell lymphopenia is due in large part, if not entirely, to the absence of the RT-6+ peripheral T cell subset, which includes members of both the helper/inducer (W3/25) and suppressor/cytotoxic (OX 8) antigenic phenotypes. Delineation of the causal mechanism(s) for the selective absence of RT-6+ T cells in DP rats may provide important insights into the cellular basis of the insulin-dependent diabetes mellitus syndrome in these animals.