CD45RC isoforms define two types of CD4 memory T cells, one of which depends on persisting antigen.

The cellular basis of immunological memory remains a controversial area with respect to the identity of memory T cells and the role of persisting antigen. CD4 T cells are phenotypically divided by the expression of high and low molecular weight isoforms of CD45, surface markers that are frequently used to identify "naive" (CD45Rhigh) and "memory" (CD45Rlow) subsets. The latter subset responds rapidly in antigen recall assays but paradoxically has a short life span, a property that is difficult to reconcile with long-term memory. The present study examines these issues using a DTH (delayed-type hypersensitivity) model in which contact sensitivity to dinitrochlorobenzene (DNCB) was transferred to athymic nude rats by recirculating CD4 T cell subsets defined in the rat by the anti-CD45RC mAb OX22. As expected, CD45RC+ (but not RC-) CD4 T cells from normal unprimed rats transferred a DNCB-specific DTH response, whereas, 4 d after sensitization the CD45RC- (memory) subset alone contained the DNCB reactivity. However, when donor cells were collected from thymectomized rats sensitized two mo earlier, DNCB-specific responses were transferred by both CD45RC- and RC+ subsets suggesting that many of the latter had developed from cells with a memory phenotype. This was confirmed when CD45RC CD4 T cells from 4-d primed rats were parked in intermediate nude recipients and recovered 2 mo later. DNCB-specific activity was now found wholly within the CD45RC+ "revertant" subset; the CD45RC-CD4 T cell population was devoid of activity. Importantly, we found that the total switch-back from CD45RC- to RC+ could be prevented, apparently by persisting antigen. The results indicate that there are two functionally distinct categories of memory T cells: one, a short-lived CD45Rlow type which orchestrates the rapid kinetics, the other, a longer-lived CD45Rhigh revertant which ensures that immunological memory endures.

Fidelity of the repertoire in T cell reconstituted athymic nude rats. Preservation of a deficit in alloresponsiveness over one year.

A single intravenous injection of a relatively small number of T cells contained in the population of rat thoracic duct lymphocytes (TDL) is sufficient to restore to normal the peripheral T cell pool of athymic PVG.rnu/rnu nude rats. The donor T cells expand greater than 10-15-fold, self-renew, and restore immunocompetency to nude recipients permanently (greater than 2 yr). We asked whether the T cell repertoire was affected by the expansion and self-renewal process. Nude recipients were injected with syngeneic PVG TDL that had been allospecifically depleted (negatively selected) by consecutive passage from blood to thoracic duct lymph through two irradiated (DAxPVG)F1 intermediate rats. Negatively selected TDL were tested before transfer by the P----F1 popliteal LN GVH assay and showed a greater than 90% depletion of specific reactivity to DA alloantigens. Surviving cells or their progeny were recovered from LN or TDL of nude recipients 8 and 12 mo after transfer. The deficit in GVH reactivity to the DA haplotype persisted, but normal GVH activity was demonstrated against a third party (AOxPVG)F1 alloantigen. The "hole" in the repertoire could not be attributed to tolerance induced by the co-transfer of contaminating irradiated F1 TDL. PVG TDL passaged consecutively through (AOxPVG)F1 and (DAxPVG)F1 intermediates and devoid of (AOxPVG)F1 cells remained specifically depleted to both AO and DA haplotypes when recovered from nude recipients 4 and 13 mo later, but displayed GVH activity to a third-party (BNxPVG)F1 alloantigen. Thus the exact specificity of the T cell repertoire of the original inoculum was faithfully maintained in nude recipients throughout the initial phase of rapid expansion and the continued self-renewal of the mature peripheral T cell pool.

In vitro antibody synthesis in 20 microliters hanging drops: initiation of secondary responses and a simple method of cloning hybridomas.

An in vitro culture system is described in which antibody synthesis is initiated by 10(5) hapten-primed spleen cells in 20 microliters hanging drops. Plaque-forming cell (PFC) responses to thymus dependent and thymus independent antigens were obtained in drop cultures comparable to the micro Mishell--Dutton system. Antigens could be administered in soluble form or on antigen-pulsed peritoneal cells. The hanging drop method also provides a reliable yet simple procedure for cloning hybridoma cells while permitting direct visualization of the number of cells in each drop.

Lymphocyte traffic within the bone marrow and selective retention of alloreactive cells.

Earlier studies showed that large numbers of isotopically labelled thoracic duct lymphocytes (TDLs) enter the bone marrow (BM) within hours of injection but depart equally as rapidly by 12 to 24 hr. The significance of this rapid flux was investigated further. Early (1/2 to 2 hr) after the i.v. injection of TDLs, BM was shown to contain T cells capable of initiating a graft-versus-host (GVH) reaction in F1 hybrids and in other experiments memory cells against human serum albumin (HSA). Both GVH and memory cell activity had markedly declined in the BM by 12 hr. In contrast to the rapid departure of TDLs from syngeneic BM, F1 hybrid BM retained parental lymphocytes with GVH activity for alloantigens of the opposite parent. F1 hybrid BM under these circumstances supported the transformation and proliferation of lymphocytes activated in situ by alloantigens. These selectively retained T cells also reacted to third-party alloantigens. In addition, TDLs with memory for HSA were retained in the BM of F1 hybrids. The BM is a site in which alloreactive immune responses may be initiated or sustained.

CD45RC+ CD4 T cell subsets are maintained in an unresponsive state by the persistence of transfusion-derived alloantigen.

The ability of preoperative blood transfusion to extend the survival of organ allografts is well known but poorly understood. To study this phenomenon, adult PVG (RT1c) rats were rendered tolerant of DA (RT1a) cardiac allografts by prior donor-specific blood transfusion (DST). We investigated the cellular basis of the transfusion effect by adoptively transferring CD4 T cell subsets, obtained from thoracic duct lymph of tolerant rats, into cardiac allografted athymic PVG nude recipients. Surprisingly, CD4 T cells from DST rats evoked acute rejection on adoptive transfer. Evidence indicated that CD8 T cells played no role in DST-induced tolerance. Analysis of CD4 T cell subsets, defined in the rat by mAb OX22 (anti-CD45RC), revealed an unusual pattern of responsiveness. CD45RC+ CD4 T cells (normally capable of inducing prompt rejection), when obtained from rats given a specific blood transfusion, were depleted of alloreactive cells and deficient at inducing rejection. In contrast, the CD45RC- subset (normally slow at evoking graft destruction) was highly active and ten-fold-enriched in its ability to induce rejection. Destruction of cardiac allografts by this latter subset was, however, completely inhibited by giving nude recipients a specific (but not a third-party) blood transfusion two weeks before heart grafting and cell transfer. Apparently, tolerance was maintained by residual elements of the prior blood transfusion that prevented the specific CD45RC- subset from regaining an alloaggressive capacity.

Allograft rejection in athymic nude rats by transferred T cell subsets. II. The response of naive CD4+ and CD8+ thoracic duct lymphocytes to an isolated MHC class I disparity.

Athymic PVG-rnu/rnu (RT1c) rats were grafted with skin bearing isolated MHC disparities 7-14 days in advance of cell transfer. The ability of naive CD4+ or CD8+ thoracic duct lymphocytes to induce rejection was assessed by adoptive transfer of one or both T cell subsets into nude recipients bearing congenic PVG.r1 (MHC class I-only disparity, Aa) or PVG.r19 (class I and II-only disparity, Aa B/Da) skin grafts. Recipients of purified CD4+ TDL always rejected r19 allografts, whereas CD8+ TDL were ineffective against this class I + II difference. Neither the injection of CD4+ TDL nor CD8+ TDL alone resulted in destruction of r1 skin grafts. However, rejection of r1 tissue was observed in 63% of cases (19/30) when both CD4+ and CD8+ TDL were present in the nude recipients. Rejection of r1 skin was also induced in some recipients when CD8+ TDL were transferred 8 weeks in advance of CD4+ TDL. In contrast, sequential transfer in the reverse order apparently induced tolerance in the CD4+ population--i.e., surviving r1 skin grafts on 8 week CD4+ T cell-reconstituted nude recipients were not rejected following the subsequent transfer of CD8+ TDL. We conclude that CD4+ T cells were required for rejection of both class I and class II differences. In the presence of a class II difference, CD4+ T cells function autonomously to initiate both the inducer and effector stages of rejection. When the disparity is confined to class I, CD8+ T cells are required (probably at the effector stage) but are dependent on CD4+ T cells for help. There was no evidence of CD4+ effector T cells that could recognize class I directly within the graft.

Role of classical (RT1.A) and nonclassical (RT1.C) MHC class I regions in natural killer cell-mediated bone marrow allograft rejection in rats.

BACKGROUND: We have studied the role of the different MHC (RT1) subregions in acute natural killer (NK) cell-mediated bone marrow allograft rejection in lethally irradiated, bone marrow cell (BMC) reconstituted rats. METHODS: We employed a series of MHC congenic and intra-MHC recombinant rat strains so that effects of mismatches in defined RT1 subregions could be studied systematically. BMC allograft survival was measured as 125IUdR uptake in the spleen between day 5 and day 7 after irradiation and BMC reconstitution. RESULTS: We found that in certain RT1 haplotype combinations, nonclassical RT1.C disparities by themselves could determine graft rejection (i.e., in the u/av1 recombinant haplotypes), whereas in another combination (between the av1 and c haplotypes) a mismatch for an isolated classical RT1.A region was decisive for engraftment. Thus, PVG.R1 BMC failed to proliferate in PVG rats, differing in the RT1.A region only, whereas in PVG.1U rats rejection could be determined by isolated differences in the RT1.C region (LEW.1WR1). Also, RT1 homozygous rats (RT1.U) rejected semi-allogeneic F1 hybrid BMC. The acute rejection of BMC was mediated by NK cells, as athymic nude rats, lacking alloreactive T cells but with normal alloreactive NK cells, showed the same patterns of rejection as did normal rats. Nude rats also rejected allogeneic lymphocytes, a previously documented NK-mediated phenomenon, with identical requirements of MHC disparity. CONCLUSIONS: This investigation shows that rat effector NK cells are radioresistant, independent of the thymus, and capable of recognizing and rejecting MHC mismatched transplanted BMC on the basis of mismatches in both classical and nonclassical class I regions in vivo. The studies underline the importance also of NK cells in determining BMC allograft survival.

Contrasting outcomes of donor-specific blood transfusion: effectiveness against cell-mediated but not antibody-mediated rejection.

BACKGROUND: Giving recipients a prior donor-specific blood transfusion (DST) is effective in prolonging organ allograft survival in some inbred strains but not in others. The present investigation analyzed two such contrasting strains of rats in an attempt to define the basis for this variation. METHODS AND RESULTS: The survival of fully mismatched Dark Agouti (RT1a) cardiac allografts was significantly prolonged (from 7 to 44 days, median survival times) in PVG (RT1c) rats given a prior (-14 day) DST, whereas it shortened survival in the high-responder PVG-RT1u strain. Injecting PVG recipients with blood from strains bearing defined differences indicated that each disparity contributed to the increased survival time in an incremental way: blood and heart matched at the MHC class I (A) and/or class II (B/D) loci had a major influence on survival; class I-like (C) and non-MHC antigens made only minor contributions. MHC disparities had contrasting effects in RT1u rats. Blood transfusions from Dark Agouti or PVG-R8 (AaB/DuCu) rats induced accelerated rejection and anti-Aa alloantibody formation; transfusing PVG-R23 (AuB/DaCa) blood, a class II and class I-like difference, induced indefinite R23 heart allograft survival. Although produced in high titer, anti-class II antibody was not able to induce rejection in RT1u rats. Specific anti-Aa alloantibody was able, after passive transfer, to destroy class I-disparate allografts in both RT1u nude and PVG nude recipients. However, under normal circumstances, acute rejection in the PVG strain occurred in the absence of anti-Aa antibodies, presumably by a cell-mediated mechanism. CONCLUSION: Anti-class I alloantibody, when produced, seemed to override the unresponsiveness induced by DST. The results indicated that DST was effective only when rejection was induced by a cell-mediated response. The two contrasting response patterns in animals may reflect the experience of transplant patients who either benefit from DST or become sensitized instead.

Prevention of chronic rejection by donor-specific blood transfusion in a new model of chronic cardiac allograft rejection.

Chronic graft rejection is now a major barrier to the long-term survival of cardiac transplants. A major hallmark of chronic rejection is intimal thickening of arteries in the graft leading to vascular occlusion. Current animal models of chronic rejection generally utilize immunosuppression to prevent acute rejection of grafts disparate at major histocompatibility antigens or graft disparities involving minor histocompatibility antigens alone. In the present communication we describe a new model of chronic rejection involving grafting of PVG-R23 hearts into PVG-RT1(u) recipients. The R23 hearts, which differ from the RT1(u) recipients at class II MHC, are rejected with a chronic time course and demonstrate extensive severe vascular myointimal proliferation within the coronary arteries. Furthermore we demonstrate for the first time that donor-specific blood transfusion can prevent chronic rejection and the intimal thickening of the coronary arteries.

Allograft rejection in CD4+ T cell-reconstituted athymic nude rats--the nonessential role of host-derived CD8+ cells.

PVG-rnu/rnu nude rats reject fully allogenic renal (DA) and skin (BN, AO) allografts after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many nude-derived CD8+ leukocytes within the graft. In addition, mononuclear cells infiltrating the rejecting renal grafts in these animals display cytotoxic activity in vitro against specific and third-party alloantigens. In this investigation we have treated CD4+ T cell-restored nude rats bearing renal or skin allografts with the mAb MRC OX8 to deplete the host of CD8+ cells. In vivo treatment with OX8 completely eliminated CD8+ cells from rejecting grafts of both kidney and skin, but it did not prevent graft rejection, nor did OX8 treatment abolish the cytotoxic effector cells found in nude rat spleen or in graft-infiltrating cells (GIC) of rejecting renal allografts. The nature of the cytotoxic activity was examined with anti-CD3 mAb 1F4, which was shown to block conventional CD8+ Tc killing in vitro but did not inhibit allogeneic target cell lysis by spleen cells from nude rats. The cytotoxic activity found in GIC of rejecting allografts was not inhibited by anti-CD3 mAb, suggesting that these cytotoxic effector cells were CD3-CD8- and were of extrathymic origin. We conclude that non-thymus-derived CD8+ GIC are not essential for allograft rejection in CD4+ T cell-restored nude rats.

Renal allograft rejection in CD4+ T cell-reconstituted athymic nude rats. The origin of CD4+ and CD8+ graft-infiltrating cells.

PVG-rnu/rnu nude rats reject a fully allogeneic DA renal allograft after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many CD8+ leukocytes within the graft. In order to clearly establish the provenance of these CD8+ cells infiltrating rejecting kidney allografts, nude recipients (PVG-RT7a) were injected with CD4+ T cells from the PVG-RT7b congenic strain bearing an allotypic variant of the leukocyte-common antigen. Dual fluorescence and immunohistochemistry demonstrated that approximately 75% of the total infiltrate was host-derived; the donor-derived RT7b population was almost entirely (92-99%) CD4+, CD5+, CD3+, and alpha beta TCR+. At least 97% of the CD8+ cells were of nude origin. There was no evidence of donor-derived CD8+ cells or of a CD4+8+ double-staining population. Unexpectedly, nearly half of the alpha beta TCR+ cells from the grafts were of nude origin.

Prolongation of rat corneal graft survival by treatment with anti-CD4 monoclonal antibody.

A rat model of orthotopic corneal graft rejection was used to investigate the effect of depletion of subpopulations of immune cells by treatment with monoclonal antibodies. Though CD4+ cells were not eliminated completely by anti-CD4 monoclonal antibodies there was a profound delay in the rejection times of orthotopic corneal allografts. Furthermore a third of the CD4+ depleted animals failed to reject corneal allografts by 100 days post grafting. Despite an almost complete depletion of circulating CD8+ cells, the anti-CD8 antibody treated animals rejected corneal allografts in a similar time course to allografted controls treated with a non-reactive control antibody OX21. These results demonstrate that CD8+ T-cells are not required for rejection of corneal allografts whereas CD4+ T-cells play a critical role in the rejection response. Treatment with anti-CD4 antibodies may have a useful clinical application.

The toxicity, distribution and excretion of ricin holotoxin in rats.

The distribution and excretion of the plant toxin ricin were studied in rats after intravenous injection. 125I-labelled ricin was equal in toxicity to native ricin. Following injection, the liver was the major organ of localisation - 46% of injected dose at 0.5 h. The spleen and muscle were next with 9.9% and 13%, respectively, at 0.5 h. Ricin was relatively concentrated in the spleen (33% of injected dose/g of tissue) compared with the liver (7.4%/g) and the bone marrow (5.5%/g). The concentration in the lymph nodes was very low (1.2%/g). Ricin was quickly cleared from the animal; only 11% of the initial radioactivity remained 24 h later with 70% excreted in the urine. Excretion into the intestine via the bile duct was less than 5% by 24 h, 10-12% of the radioactivity was found in the intestinal contents or intestinal wall between 3 h and 12 h, and much of this was reabsorbed since less than 2% was recovered in faeces.

Comparative evaluation of the immune status of congenitally athymic and euthymic rat strains bred and maintained at different institutes: 1. Euthymic rats.

We performed a comparative evaluation of the immune status, focused on the T-cell system, of euthymic rat strains in which the nude mutation had been introduced. From 10 institutes, we sampled 12 groups of euthymic rats at ages of 1 1/2-2 months and 1/2 year. We analyzed weight of body, spleen and thymus; antibody response and delayed-type hypersensitivity response to ovalbumin immunization; and (immuno)histopathology of spleen, lymph nodes, and lymphoid tissue along the gastrointestinal tract. In the spleen morphometric analysis was done of the periarteriolar lymphocyte sheath (using the antibody R73 recognizing the alpha beta-T-cell receptor) and of the red pulp (using the antibody ED2 recognizing red pulp macrophages). For almost all parameters tested, statistically significant differences between the groups (origin of the animals) were observed. A cluster analysis on the basis of body weight, spleen weight, and morphometric data of spleen did not yield clusters with a different composition among animals from individual groups. Based on the antibody response to ovalbumin, clustering revealed groups of "fast-and-high", "slow-and-low", and "intermediate" responders. The various groups differed in location within these clusters, i.e. the speed and extent of the immune response depends on the background euthymic strain. Considering the microbiological status assessed by serology, a variation was found both in post-infection state at entrance in the study, and in primo-infection associated with a rise in antibody concentrations during the study. These states showed no negative effect on anti-ovalbumin reactivity. Rather, the response in primo-infection to Rat Corona Virus, Sendai Virus, and Pneumonia Virus of Mice was the highest in animals clustered as "fast-and high" responders to ovalbumin.

Comparative evaluation of the immune status of congenitally athymic and euthymic rat strains bred and maintained at different institutes: 2. Athymic rats.

We performed a comparative evaluation of the immune status, focused on the T-cell system, in congenitally athymic rat strains. From 11 institutes around the world, we sampled 15 groups of animals at ages of 1 1/2-2 months and 1/2 year. The analysis included weight of body and spleen; antibody response and delayed-type hypersensitivity response after immunization with ovalbumin; and (immuno)histology of spleen, lymph nodes and lymphoid tissue along the gastrointestinal tract. Morphometric analysis was done for alpha beta-T-cell receptor-bearing cells in spleen tissue as a measure of the periarteriolar lymphocyte sheath; it was also done for splenic red pulp using the antibody ED2 recognizing red pulp macrophages. For almost all variables analyzed, statistically significant differences between the groups were observed. The extent of alpha beta-T-cell receptor-bearing cells in the spleen increased with age. The functioning of these cells in immunological responses can be questioned, because an immune response to ovalbumin was invariably absent. But secondary follicles with germinal centers, reflecting T-cell-dependent B-cell reactivity, were observed in lymph nodes and Peyer's patches (up to 40% and 75%, respectively, depending on the group), with a higher prevalence in older animals. A cluster analysis on the basis of body and spleen weight and composition of spleen compartments did not yield clusters with a different profile in regard to the animals' group of origin. The data presented are useful when comparing studies performed with various athymic rat strains at different institutes.

Summary of a project comparing student performance forms.

This paper is a summary of a pilot project designed to provide data which would lead to more effective methods of evaluating field work performance of occupational therapy students in Canada. Three evaluation forms were selected for comparison. A quantitative-descriptive type of research was utilized and a questionnaire to occupational therapy clinicians and students was the method used for data collection. The three major findings were: i) that the American Occupational Therapy Association Field Work Performance Report Form was the most complete form in evaluating student performance in specific skill areas; ii) the Queen's University Occupational Therapy Clinical Training Report Form contained essential components concerning interaction skills; and iii) the Canadian Association of Occupational Therapy Report of Clinical Training was in need of major revisions if its use was to be continued. This paper includes an outline of the results and the major limitations of the pilot project; however, the conclusions and recommendations arising from the project are the main focus of discussion.

Muriel Driver Memorial Lecture 1980--Directions for the decade.

Following an introductory summary of role theory, this paper examines one element of professional development -- that of ROLE NEGOTIATION. Research findings as outlined which provide evidence of the need for developing role negotiation skills within the occupational therapy profession in Canada. The paper describes four role strategies which can be used to professional advantage, as therapists negotiate their roles. The final emphasis is on the positive professional implications of effective role negotiations in the 80's.