The phenotype and survival of antigen-stimulated transgenic CD4 T cells in vivo: the influence of persisting antigen.

Naive and primed/memory CD4 T cells are distinguished by changes in the expression of activation/adhesion molecules that correspond with an altered function. Adoptively transferred TCR transgenic (tg) CD4 T cells specific for ovalbumin peptide (OVA-pep) were analysed for changing phenotype and the speed of change in vivo following antigen challenge with alum-precipitated (ap) OVA-pep, a conjugate that stimulated a Th2-type cytokine response. The change of CD45RB in relation to number of divisions showed that the transition from CD45RB(hi) (naive) to CD45RB(low) (primed/memory) was incremental; with each cell cycle the number of CD45RB(hi) molecules on the cell surface was diluted by approximately half and replaced by the low-weight isoform. Similarly, the change to CD44(hi) expression increased gradually during four rounds of proliferation. The loss of CD62L expression occurred early and was independent of cell division. CD69 was up-regulated quickly within 1-2 cycles, but down-regulated after about seven divisions. The expression of CD49d was not altered during the early rounds of division, although it was up-regulated on 30-60% of tg T cells dividing repeatedly (>or=8 cycles). When analysed on day 3 following stimulation, CD25 was no longer up-regulated. The intra-peritoneal injection of ap-OVA-pep stimulated tg T cells in the spleen and mesenteric lymph node one day in advance of those in more distant peripheral lymph nodes. Evidence indicated that residual antigen persisted for at least 4 weeks and was able to stimulate naive tg T cells. However, residual antigen had no net effect on extending or reducing survival of the transferred population.

Naive and memory T cells migrate in comparable numbers through the normal rat lung: only effector T cells accumulate and proliferate in the lamina propria of the bronchi.

T cells reach the lung via the pulmonary and bronchial arteries that supply the alveolar and bronchial regions. Although these regions are differentially affected by T cell-mediated diseases, the migration of T-cell subsets in these two regions has not been studied. Naive, memory, and effector T cells were injected into congenic rats and traced in sections of normal lung. All three T-cell subsets were found in large numbers in the alveolar region and exited again quickly. Only effector T cells accumulated in the lamina propria of the bronchi. Further, 72 h after injection 6% of the effector T cells still proliferated in the lung, whereas apoptotic effector T cells were only observed 1 h after injection (0.2%). Thus, not only effector and memory but also naive T cells continuously migrated through the lung. The preferential accumulation of effector T cells in the bronchial lamina propria may explain why some diseases preferentially affect the bronchial region.

Both CD45R(low) and CD45R(high) "revertant" CD4 memory T cells provide help for memory B cells.

During a primary response to thymus dependent antigens, B cells undergo a number of qualitative changes to become memory B cells - processes that require co-stimulatory signals and cytokine help from CD4 T cells. The question of whether distinct, antigen-experienced memory CD4 T cells are subsequently needed to program memory B cells into antibody synthesis has not been clearly resolved. Using an adoptive transfer model in which memory but not naive B cells were stimulated, we evaluated CD4 T cell help using lymphocytes obtained from primed or unprimed thymectomized donors and expressing a naive (CD45R(high)) or a memory (CD45R(low)) phenotype. Memory B cells, most of which were committed to the IgG1 (Th2) subclass, could be stimulated to produce antibody using help transferred by the CD45R(high) naive subset of unprimed donors (slow onset of response), the CD45R(low) subset of 7 day primed donors (large, rapid antibody response) or by both the CD45R(low) and the CD45R(high) "revertant" subsets of 6 month primed donors. We found that antigen primed CD45R(low) CD4 T cells reverted (defaulted) with time to a CD45R(high) resting state, a change that was prevented by persisting antigen. The evidence suggests that CD4 memory T cells are partitioned into two different functional states (CD45R(high) and CD45R(low)) and that these determine the characteristics of the memory B cell response in terms of speed, size and longevity.

Specific B cell tolerance is induced by cyclosporin A plus donor-specific blood transfusion pretreatment: prolonged survival of MHC class I disparate cardiac allografts.

Donor-specific blood transfusion (DST), designed to prolong allograft survival, sensitized recipients of the high-responder PVG-RT1u strain, resulting in accelerated rejection of MHC-class I mismatched (PVG-R8) allografts. Rejection was found to be mediated by anti-MHC class I (Aa) alloantibody. By pretreating recipients 4 wk before grafting with cyclosporin A (CsA) daily (x7), combined with once weekly (x4) DST, rejection was prevented. The investigation explores the mechanism for this induced unresponsiveness. CD4 T cells purified from the thoracic duct of CsA/DST-pretreated RT1u rats induced rejection when transferred to R8 heart-grafted RT1u athymic nude recipients, indicating that CD4 T cells were not tolerized by the pretreatment. To determine whether B cells were affected, nude recipients were pretreated, in the absence of T cells, with CsA/DST (or CsA/third party blood) 4 wk before grafting. The subsequent transfer of normal CD4 T cells induced acute rejection of R8 cardiac allografts in third party- but not DST-pretreated recipients; prolonged allograft survival was reversed by the cotransfer of B cells with the CD4 T cells. Graft survival correlated with reduced production of anti-MHC class I (Aa) cytotoxic alloantibody. The results indicated that the combined pretransplant treatment of CsA and DST induced tolerance in allospecific B cells independently of T cells. The resulting suppression of allospecific cytotoxic Ab correlated with the survival of MHC class I mismatched allografts. The induction of B cell tolerance by CsA has important implications for clinical transplantation.

Naive and memory T lymphocytes migrate in comparable numbers through normal rat liver: activated T cells accumulate in the periportal field.

Although the liver is known to contain a significant number of lymphocytes, migration of these through the compartments of the liver, parenchyma and periportal field, has not been studied. The periportal field, in particular, is affected in several immunological disorders of the liver. Populations of labeled naive, activated, and memory T cells were injected into congenic rats. The recipient livers and draining lymph nodes were removed at various time points, and cryostat sections were analyzed for the presence of donor cells using quantitative immunohistology. Donor cell proliferation and apoptosis were examined in vivo by BrdU (5 microM 5-bromo-2-deoxyuridine) incorporation and the TUNEL technique, respectively. Early after injection (0.5-1 h), naive, activated, and memory T cells were localized to the parenchyma and periportal field in comparable numbers. With time, all T cell subsets left the parenchyma but remained or, in the case of activated T cells, significantly accumulated in the periportal field. Furthermore, 12% of activated donor T cells proliferated in vivo within the periportal field, and 0.5% showed evidence of apoptosis. Taken together, not only activated and memory, but also naive T cells continuously migrate through the liver, showing a preference for the periportal field, and activated T cells mainly proliferate there. This may explain why many immunological liver diseases predominantly affect the periportal field.

Persisting alloantigen prevents primed CD45RC- CD4 T cells from inducing allograft rejection: implications for immunological memory.

Antigen stimulation induces specific CD4 T cells to change from a resting phenotype (CD45RC+) to a "memory" phenotype (CD45RC-), an isoform switch that is reversible and regulated by persisting antigen. We show here that CD4 T cells responsible for mediating allograft rejection undergo a similar CD45RC+ to CD45RC- switch irrespective of whether antigen priming results in sensitization or tolerance in vivo. Thus, skin allograft priming, designed to induce second set rejection, and a donor-specific blood transfusion (DST), designed to prolong cardiac allograft survival, will generate CD45RC- CD4 T cells that induce acute rejection when adoptively transferred to T cell-deficient athymic nude recipients. The ability of CD45RC- T cells, obtained from DST donors, to induce graft rejection was prevented by giving nude recipients a DST 14, 28 or even 56 days before grafting and T cell transfer. Thus, prolonged allograft survival in rats after DST was found to be strongly linked with persisting alloantigen from the blood transfusion but was not associated with detectable microchimerism. Importantly, CD45RC- T cells from skin graft-primed animals were similarly prevented from inducing rejection by residual DST-derived alloantigen. The investigation shows (1) that an allogeneic blood transfusion primes (not tolerizes) alloreactive CD4 T cells and (2) that residual DST-derived alloantigen can block the action of specifically primed "memory" CD4 T cells. These findings have implications for understanding immunological memory.

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.

Lymphocyte trafficking: CD4 T cells with a 'memory' phenotype (CD45RC-) freely cross lymph node high endothelial venules in vivo.

Antigen encounter not only induces a change in surface expression of CD45RC isoforms in the rat from a high (CD45RC+) to a low molecular weight molecule (CD45RC-), but also stimulates changes in expression of adhesion molecules that regulate CD4 T-cell migration. T cells with an activated or 'memory' phenotype (CD45RC-) are thought to enter lymph nodes almost exclusively via afferent lymphatics whereas T cells in a resting state (CD45RC+) migrate across high endothelial venules (HEV). The present study monitored the rapid recirculation from blood to lymph of allotype-marked CD45RC T-cell subsets. Surprisingly, we found that CD45RC- CD4 T cells entered the thoracic duct slightly faster and reached peak numbers 3 hr earlier (18 hr) than did the CD45RC+ subset. To determine whether the entrance of CD45RC+ and RC- subsets was restricted to HEV and afferent lymphatics, respectively, recirculation of CD4 T cells was monitored in mesenteric lymphadenectomized (MLNx) rats (on healing the intestinal afferent lymphatics are joined directly to the thoracic duct), or in recipients that had had the mesenteric lymph node (MLN) acutely (2-3 hr) deafferentized (entry would be restricted to HEV). In these studies CD45RC- CD4 T cells entered the MLN across HEV on an equal basis with T cells expressing a CD45RC+ phenotype. Contrary to currently held dogma the results showed that, in vivo, CD4 T cells with a memory phenotype freely enter lymph nodes (LN) across HEV as well as via afferent lymphatics.

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.

Identical expression of CD45R isoforms by CD45RC+ 'revertant' memory and CD45RC+ naive CD4 T cells.

Naive and memory CD4 T cells are frequently defined by exon-specific monoclonal antibodies (mAb) which stain (or not) high- or low-molecular-weight (MW) isoforms of the leucocyte common antigen CD45. The link between isoform and the naive/memory designation is complicated by the fact that CD4 T cells with a 'memory' phenotype (CD45RA-, RB-, RC-, or CD45RO+) may revert ('revertants') and re-express the high mw isoform (CD45RA+, RB+, RC+). Isoform expression also changes during normal T-cell development. Furthermore, the picture may be incomplete since an exon-specific mAb will not detect all possible isoforms on a cell. We have used molecular techniques to determine whether revertant CD4 memory T cells were different from naive T cells with respect to CD45R isoform expression. Using the anti-CD45RC mAb OX22 to purify rat lymphocyte subsets, CD45R isoform expression was examined at the mRNA level in CD4 T cells at different stages of development and compared with that of B cells and unseparated lymphocytes. B cells contained abundant message for the highest MW 3-exon isoform ABC, the 2-exon isoforms AB and BC, and the null isoform O. Both immature CD45RC- (i.e. CD4+8- 'single positive' thymocytes, and peripheral Thy-1+ recent thymic emigrants) and mature CD45RC- 'antigen-experienced' CD4 T cells had message for single-exons B, possibly C and for the O exon. In contrast, CD45RC+ CD4 T cells contained mRNA coding for ABC (low level), AB, BC, B, C (low level) and O (low level). Importantly, there was no difference between CD45RC+ T cells that had not seen antigen ('truly native') and CD45RC+ antigen-experienced revertant memory T cells. This observation has implications for understanding long-term immunological memory.

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.

CD4+ T cells of both the naive and the memory phenotype enter rat lymph nodes and Peyer's patches via high endothelial venules: within the tissue their migratory behavior differs.

It is thought that naive T cells predominantly enter lymphoid organs such as lymph nodes (LN) and Peyer's patches (PP) via high endothelial venules (HEV), whereas memory T cells migrate mainly into non-lymphoid organs. However, direct evidence for the existence of these distinct migration pathways in vivo is incomplete, and nothing is known about their migration through the different compartments of lymphoid organs. Such knowledge would be of considerable interest for understanding T cell memory in vivo. In the present study we separated naive and memory CD4+ T cells from the rat thoracic duct according to the expression of the high and low molecular weight isoforms of CD45R, respectively. At various time points after injection into congenic animals, these cells were identified by quantitative immunohistology in HEV, and T and B cell areas of different LN and PP. Three major findings emerged. First, both naive and memory CD4+ T cells enter lymphoid organs via the HEV in comparable numbers. Second, naive and memory CD4+ T cells migrate into the B cell area, although in small numbers and continuously enter established germinal centers (GC) with a bias for memory CD4+ T cells. Third, memory CD4+ T cells migrate faster through the T cell area of lymphoid organs than naive CD4+ T cells. Thus, our study shows that memory CD4+ T cells are not excluded from the HEV route. In addition, "memory" might depend in part on the ability of T cells to specifically enter the B cell area and GC and to screen large quantities of lymphoid tissues in a short time.

The peripheral T-cell pool: regulation by non-antigen induced proliferation?

Despite continuous perturbations, the recirculating lymphocyte pool remains relatively constant. Expansion of the pool is compensated for by cell loss. When the T-cell pool is in deficit, either from a congenital defect or an acquired immunodeficiency, T-cell numbers are restored by extrathymic division-a response that occurs without deliberate provocation. We have considered how the recirculating pool may be stably maintained and how a T-cell deficit might be restored to equilibrium. Recent evidence suggests that depleted T-cell compartments are replenished by CD4 T-cell proliferation in the absence of specific antigen, a response that occurs without engaging the T-cell receptor.

Both activated and nonactivated leukocytes from the periphery continuously enter the thymic medulla of adult rats: phenotypes, sources and magnitude of traffic.

Although the thymus is primarily noted for the export of T cells to the periphery, a small influx of cells has also been observed. It is still a matter of debate whether entry into the thymus depends on prior activation. The phenotypes, sources and degree of immigration are largely unknown. We monitored by quantitative immunohistochemistry the entry of cells from the periphery into the rat thymus in three experimental models. We injected i.v. recirculating, small, nonactivated CD4+ T cell subsets, often referred to as naive (CD45RC+) and memory or antigen-experienced (CD45RC-) cells, purified from thoracic duct lymph of allotype-marked donors, allotype-marked leukocytes released from spleen or lung transplants, or leukocytes labeled in the periphery for 12 weeks during the S-phase of the cell cycle by oral application of 5-bromo-2-deoxyuridine (BrdUrd). Early after i.v. injection (0.5 h), significantly more antigen-experienced (CD45RC-) CD4+ T cells entered the thymus, and by 24 h four times as many cells from the CD45RC- subset as from the CD45RC+ subset had entered the thymus and localized to the medulla. None of the thymic entrants expressed the interleukin (IL)-2 receptor. Following spleen transplantation approximately 40% of donor cells entering the thymic medulla were T cells and approximately 55% were B cells. In contrast, from a lung transplant, approximately 85% of peripheral immigrants were T cells and approximately 10% were B cells. After both procedures, a small number of NK cells and monocytes/macrophages were found among the immigrants (< 5%). Rats were fed BrdUrd continuously for 12 weeks, a procedure which labeled approximately 30% of peripheral lymphocytes but not cortical thymocytes. BrdUrd-labeled cells were localized almost exclusively to the thymic medulla and represented approximately 10% of medullary cells. Of the thymic immigrants approximately 50% were T cells, approximately 30% were B cells (including approximately 15% IgD+ cells), approximately 15% were NK cells and the remainder (approximately 5%) were monocytes/macrophages. Only a quarter of BrdUrd-labeled cells expressed the IL-2 receptor. The thymus is continuously infiltrated by both activated and nonactivated leukocytes from the periphery, including T cells, B cells, NK cells and monocytes. These immigrants are supplied by lymphoid and nonlymphoid organs in a characteristic subset composition. Their entry is facilitated by prior antigen experience or activation. Thus, the participation of the thymic medulla in general leukocyte traffic suggests a mechanism by which the T cell repertoire could potentially be modulated by the peripheral tissues.

The contrasting effects of CD8+ T cells on primary, established and Nippostrongylus brasiliensis-induced IgE responses.

Recent data have indicated that CD8+ T cells suppress rodent IgE responses. In this study we investigated the effect of CD8+ T cells on primary and established IgE responses in euthymic and athymic nude rats. Euthymic PVG rats were depleted of CD8+ T cells by intraperitoneal injection of a CD8-specific monoclonal antibody (OX8), which resulted in an apparent loss of 92% of splenic and 98% of peripheral blood CD8+ T cells. The CD8+ T-cell depleted animals failed to mount a significant IgE response compared with control animals given an irrelevant monoclonal antibody (OX21). Furthermore, PVG nude rats reconstituted with purified CD4+ thoracic duct lymphocytes (TDL) alone failed to mount a significant IgE response, while animals given unfractionated TDL (containing CD4+ and CD8+ T cells) did. Depletion of CD8+ T cells 7 days prior to immunization and subsequent reconstitution at the time of immunization restored the IgE response. In contrast, removal of CD8+ T cells 1 month after induction of IgE by immunization with ovalbumin (OVA) and ricin prolonged the IgE response. In all cases IgG antibody responses were unaffected by the presence or absence of CD8+ T cells. This study shows that some CD8+ T cells are required for IgE, but not IgG, production to soluble antigen in a primary immune response. However, later in the immune response CD8+ T cells were shown to inhibit IgE production. These effects were apparently restricted to the immune response to soluble antigen, as Hooded Lister rats infected with 9000 larvae of the nematode Nippostrongylus brasiliensis produced high sustained levels of circulating IgE, in excess of 10 micrograms/ml, regardless of whether CD8+ T cells were depleted before or 1 month after infection.

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.

Neonatally tolerant rats actively eliminate donor-specific lymphocytes despite persistent chimerism.

Rats from the allotype-marked PVG-RT7b and PVG-RT1u-RT7b strains were injected at birth with semi-allogenic F1 bone marrow (BM) cells from athymic nude rats (PVG-rnu/rnu x PVG-RT1u-rnu/rnu) to induce neonatal tolerance. As adults, 97% of the animals accepted donor-specific allogeneic skin grafts and a majority (65%) of rats were chimeric, expressing the major histocompatibility complex class I and allotype marker of the donor strain. Similar results were obtained when PVG-RT1u-RT7b rats were injected at birth with fully allogeneic PVG-rnu/rnu nude BM cells: as adults, 94% accepted donor-specific skin allografts and 76% of recipients were chimeric. Donor derived CD4 T cells, CD8 T cells and B cells were found in low numbers (less than 2%) in peripheral blood of rats made tolerant by F1 BM cells. A large proportion of T cells bore the phenotype of recent thymic emigrants, suggesting that they were newly produced. All the evidence was consistent with clonal deletion tolerance, induced centrally within the thymus. The thymus was chimeric and thymocytes failed to respond in vitro to alloantigens of the donor-specific haplotype; donor-specific skin allografts survived indefinitely on athymic nude recipients reconstituted with CD4+CD8- thymocytes or peripheral CD4 T cells from tolerant animals. The chimeric state was interesting, since the PVG and PVG-RT1u rat strains contain a natural killer (NK) cell system that rapidly eliminates (within 24 h) intravenously injected allogeneic or semi-allogeneic lymphocytes--a phenomenon known as allogeneic lymphocyte cytotoxicity or ALC. When neonatal tolerant rats were tested, the ALC index (a measure of cell killing) was unchanged in nonchimeric tolerant rats and significantly altered (reduced killing), but not abolished in chimeric animals. Hence, the injection of allogeneic BM cells which induced specific tolerance in the T cell population failed to tolerize the NK cell system, allowing the constant killing of newly produced donor-derived lymphocytes and putting at risk the very survival of the allogenic BM cells. This has interesting implications for clinical transplantation.

Migration pathways of CD4 T cell subsets in vivo: the CD45RC- subset enters the thymus via alpha 4 integrin-VCAM-1 interaction.

The present investigation examines the localization and migration of purified T cell subsets in comparison with B cells, CD8 T cells and CD4+ CD8- single-positive thymocytes. CD4 T cell subsets in the rat are defined by mAb MRC OX22 (anti-CD45RC), which distinguishes resting CD4 T cells (CD45RC+) from those (CD45RC-) which have encountered antigen in the recent past--subpopulations often referred to as 'naive' and 'memory'. Purified, 51Cr-labelled CD45RC+ CD4 T cells broadly reflected the migration pattern of CD8 T cells and B cells. Early localization to the spleen was followed by a redistribution to mesenteric lymph nodes (MLN) and cervical lymph nodes (CLN), B cells migrating at a slightly slower tempo. There was almost no localization of these subpopulations to the small or large intestine [Peyer's patches (PP) excluded]. In contrast, CD45RC- CD4 T cells (indistinguishable in size from the CD45RC+ subset) localized in large numbers to the intestine; they were present here at the earliest time point (0.5 h), persisted for at least 48 h but did not accumulate, indicating a rapid exit. Numerically, localization of CD45RC- CD4 T cells in the MLN could be accounted for entirely by afferent drainage from the intestine. Unexpectedly, CD45RC- CD4 T cells (but not other subsets) localized and accumulated in the thymus. In vivo treatment with mAb HP2/1 against the integrin alpha 4 subunit inhibited almost entirely CD45RC- CD4 T cell migration into the PP (98.1%), intestine (87.1%), MLN (89.1%) and thymus (93.5%); migration into the CLN was only reduced by half. To distinguish between recognition of MAdCAM-1 and VCAM-1 by alpha 4-containing integrins, recipients were treated with mAb 5F10 against rat VCAM-1. Except for the thymus and a small reduction in CLN, localization of CD45RC- CD4 T cells was unaffected; entry to the thymus was almost completely blocked (92.3%) by anti-VCAM-1. The results indicated (i) that CD45RC- CD4 T cells alone showed enhanced localization to the gut and PP, probably via alpha 4 beta 7-MAdCAM-1 interaction; (ii) that many CD45RC- cells entered non-mucosal LN independently of alpha 4 integrin or VCAM-1; and (iii) that entry of mature recirculating CD45RC- CD4 T cells into the thymus across thymic endothelium was apparently regulated by alpha 4 integrin-VCAM-1 interaction.

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.