Detection of donor-derived cells by polymerase chain reaction in neonatally tolerant mice. Microchimerism fails to predict tolerance.

Although it has long been appreciated that establishment of chimerism is important in the acquisition and maintenance of allograft tolerance, the importance of this relationship has been reemphasized recently. Using the exquisite sensitivity of the polymerase chain reaction we have studied qualitatively and quantitatively the presence of donor-derived chimeric cells in relation to the ability of neonatally injected mice to display skin graft tolerance or rejection. We have found that virtually all mice that receive neonatal injections of F1 hematopoietic cells acquire donor-derived chimerism that is detectable in blood, spleen, lymph nodes, and thymus. Surprisingly, neither the presence nor the quantity of chimeric cells predicts whether a particular neonatally injected mouse will accept or reject donor-specific skin allografts. Moreover, whether the test skin allograft is accepted (tolerance) or rejected by neonatally injected mice, chimerism typically remains detectable within recipient lymphoid tissues. In functionally tolerant mice, challenge with a test skin allograft actually leads to a remarkable expansion in donor-derived genetic sequences, implying that donor-derived cells have been induced by the graft to undergo proliferation. Since persistence of chimerism without proliferation after test grafting is characteristic of mice that fail to display tolerance, we believe that achievement of a threshold level of donor-derived alloantigen may be necessary to retain the tolerant state. We conclude that chimerism is essential for the induction of neonatally induced tolerance, and its expansion may play an important role in the maintenance of that tolerance, when challenged by an allograft.

Cellular mechanisms that maintain neonatally-induced tolerance of class II alloantigens. Evidence that factor-mediated suppression silences cytotoxic T cell activity.

Virtually all neonatal mice of the A strain background are rendered profoundly and permanently tolerant of test skin allografts if they receive an i.v. inoculation of semiallogeneic hematopoietic cells expressing class II disparate alloantigens. After neonatally injected mice reach immunologic maturity, their lymphoid organs have been found to contain 1) tolerogen-specific CD4+ T cells that proliferate and secrete IL-4 when stimulated in vitro with class II tolerogens and 2) tolerogen-specific CD8+ T cell that fail to differentiate into cytotoxic effector cells. In this study, experiments are described that investigate the possibility that tolerance is maintained by regulatory T cells of the Th2-type. When A.TH T cells were stimulated in vitro with A.TL alloantigens in the presence of lymphoid cells from tolerant mice, tolerogen-specific cytotoxic T cells responses were absent or greatly diminished. When regulatory cells from tolerant mice were fractionated and tested, the cell type responsible for suppression proved to be CD4+ class II tolerogen-specific and gamma irradiation sensitive. Moreover, suppression of tolerogen-specific cytotoxic T cell generation was achieved when regulatory cells and naive responder cells were separated by a transwell barrier and supernatants harvested from cultures in which tolerant cells were stimulated specifically with class II tolerogens also inhibited cytotoxic T cell generation. Thus, suppression appears to be mediated by a soluble factor(s) produced by regulatory T cells. We conclude that tolerance of class II alloantigens is maintained, at least in part, by regulatory cells of the Th2-type that secrete factors that suppress the generation of tolerogen-specific effector cells required for rejection of solid tissue allografts.

Cellular mechanisms that maintain neonatally-induced tolerance of class II alloantigens. Evidence that precursor cytotoxic T cells are present but silenced.

Clonal deletion of alloantigen-specific lymphocytes was the first explanation advanced to explain why neonatal mice that receive injections of alloantigenic hematopoietic cells mature into adults that accept donor-derived skin allografts indefinitely, i.e., they are immunologically tolerant. However, numerous other passive and active regulatory mechanisms have been invoked to explain neonatal transplantation tolerance. In A strain mice (A.TH, las, A.TL, lak) rendered tolerant of class II-only alloantigens, formal evidence exists demonstrating that tolerogen-reactive T cells are not eliminated. In fact, tolerogen-reactive T cells are present in peripheral lymphoid organs and can secrete lymphokines (IL-2/IL-4) when stimulated with tolerogen-bearing cells in vitro. Despite the presence of cytokines in these cultures, class II-specific T cells are usually not generated, raising the possibility that selective deletion of these cells may contribute to the tolerant state. To examine this issue, limiting dilution analysis was performed and revealed that tolerant mice possess significantly diminished precursor cytotoxic T cell frequencies. Virtually all cytotoxic T cells generated by normal A.TH mice in response to A.TL class II Ags are CD8+ cells. Moreover, the frequency of donor I-E reactive V beta 5 cells among CD4+ and CD8+ subpopulations among tolerant mice was comparable to naive mice. This suggests that the peripheral lymphoid organs of tolerant mice are functionally deleted of tolerogen-specific cytotoxic T cells and that tolerogen-specific CD8+ T cells are present in normal numbers of tolerant mice. Therefore, this circumstantial evidence implies that tolerogen-specific T cells have not been physically eliminated in class II tolerant mice. Instead, either tolerogen-specific CD8+ T cells have been rendered anergic or active suppression prevents their activation in vitro and presumably in vivo.

Ontogeny of tolerogen-responsive lymphocytes following neonatal inoculation of class II disparate semiallogeneic cells.

Spleens of adult mice of the A strain background that were rendered tolerant as neonates of class II alloantigens only (A.TH tolerant of A.TL, A.TL tolerant of A.TH) contain large numbers of tolerogen-responsive T cells, many of which secrete IL-4, but not IL-2. Since these spleens also contain suppressor cells that can adoptively transfer skin allograft acceptance in vivo and can prevent generation of class II-specific cytotoxic T cells in vitro, it is important to determine the origins during postnatal development of these cells. Class II disparate, semiallogeneic haematopoietic cells were injected into newborn A.TH and A.TL mice. Periodically thereafter (1 to 60 days post-injection, but prior to challenge with a tolerogen-bearing skin graft), thymocytes and splenocytes from these mice were examined in vitro for tolerogen-specific reactivity in mixed lymphocyte reactions during which proliferation and IL-2 and IL-4 production were assayed. Within 24 hours of neonatal injection, the thymus and spleens of injected mice were profoundly depleted of tolerogen-responsive T cells. However, there was no commensurate loss of I-E-related V beta 5+ cells in the thymus of A.TH mice that received neonatal inoculations of I-E-bearing A.TL cells. During the ensuing weeks, tolerogen-responsive proliferative and IL-4-secreting T cells were detected in thymus and spleen. However, not until after the mice were more than 60 days of age were tolerogen-responsive cells able to secrete IL-2. Since physical clonal deletion of tolerogen-related V beta 5+ cells is a characteristic of neither neonatal nor adult A.TH and A.TL mice that received injections of semiallogeneic cells at birth, and since tolerogen-responsive IL-4 producing cells exist in adult mice that have permanently accepted (A.TH x A.TL)F1 skin grafts, our results imply that the tolerogen-responsive T cells detected in adult tolerant mice are descendants of the novel IL-4-producing T cells that arise in the thymus almost immediately after the tolerance conferring inoculum of semiallogeneic cells. The possible mechanisms responsible for generation of IL-4-producing, tolerogen-responsive T cells and the role of these cells in maintenance of tolerance of class II alloantigens are discussed.

Peripheral tolerance induced in lymph nodes by syngeneic spleen cells inhibits generation of CTLs to hapten-altered self antigens but not to alloantigens.

The immunological tolerance that is induced in lymph nodes that have been exposed to syngeneic spleen cells has been examined. Development of cytotoxic T lymphocytes was used to assess the immunological status of the lymph node cells. The tolerance was studied from the viewpoint of its induction, its activation, and its specificity. We had already reported that injecting either T or B cells of splenic origin into a regional lymph node environment a week prior to immunization for CTL to hapten-altered self antigens prevents development of the CTL. Here, we confirm that syngeneic splenic cells but not lymph node cells will induce the suppression provided that spleen cells are not coupled with hapten. We now report that splenic cells that cannot replicate or synthesize and secrete protein are capable of inducing the suppression. The data suggest a preformed surface marker peculiar to spleen cells and perhaps on cells that traverse the thymus induces local tolerance that is mediated by suppressor cells. Triggering the induced suppressor T cells (previously identified as CD8-) was achieved by syngeneic spleen cells as well as by H-2-compatible, Mls-disparate spleen cells but not by syngeneic lymph node cells or apparently by allogeneic spleen cells. Furthermore, triggering suppression was achieved by hapten-coupled syngeneic spleen cells whereas such cells would not induce the suppression. Thus, activating the suppressor cells requires reexposure to splenic cells of the proper MHC haplotype, unaltered or coupled with either TNP or FITC. Once triggered, the suppression was manifested toward CTL generation against hapten-coupled syngeneic antigens on either spleen or lymph node cells but not against allogeneic antigens. Thus, the specificity of the tolerance was directed to altered self antigens despite its induction by unaltered spleen antigen. Furthermore, for suppression to be seen the spleen antigen was not required to be on the hapten-coupled syngeneic cells used for the CTL immunization. The relationship of the splenic cell "antigen" to hapten-altered self antigens and to other surface markers and its site of acquisition within the body and its significance for cell homing have become intriguing questions of importance. This information has been discussed from the viewpoint of its applicability to autoimmune diseases as well as to cessation of inflammatory reactions that may be mediated by lymph node cells.

L3T4 (CD4+) cells that mediate contact sensitivity to trinitrochlorobenzene express I-A determinants.

The present study has further characterized the T cell-mediated inflammatory response of contact sensitivity (CS) to the hapten trinitrochlorobenzene (TNCB) in mice. A discernible CS response was found to be induced as early as 2 days after epicutaneous application of TNCB. The response peaked on Days 4 to 5 and it then declined to a nearly undetectable level by Days 10 to 11. Examination of the draining lymph nodes demonstrated that development of CS coincided with an increase in cellular proliferation and in the total number of cells present. Despite a severalfold increase in the cellular contents of the draining lymph nodes of sensitized mice, the relative percentages of most subsets of T cells remained unchanged. Flow cytometric studies revealed that the subpopulation of T cells characterized as Thy 1.2+ L3T4+ I-A+ increased substantially in comparison to its presence in unsensitized mice. Whether the Thy 1.2+ L3T4+ I-A+ cells that increased following sensitization represented the effector population that mediates CS was then examined. Four-day immune lymph node T cells or L3T4 cells positively selected from them were capable of adoptively transferring CS to normal mice. However, these cells, after treatment with anti-Ia antibody or anti-I-A monoclonal antibody and complement, were unable to transfer CS. These findings imply that expression of I-A determinants may indicate antigen-induced T cell activation in vivo and that L3T4 cells that mediate CS are I-A positive.

Suppressor T cells induced by epidermally applied hapten are located in bone marrow.

Mice painted epidermally with trinitrochlorobenzene (TNCB) develop delayed type dermal hypersensitivity within five days, however, subsequent immunization with TNP-syngeneic antigens (TNP-H2k) does not generate CTLs to the same antigens. These studies were undertaken to determine what prevents development of the CTL. We report here, that a suppressor cell for CTL generation is found in the bone marrow (BM) of TNCB-painted mice. This suppressor cell is not present in spleens or lymph nodes, but is readily detected by adoptive transfer of BM cells. The cell responsible for suppression is an Lyt2+ (CD8+) T cell. Further studies with two monoclonal antibodies (one directed to a T cell suppressor factor [mAb 14-12], and the other directed to a suppressor T cell inducer factor [mAb 14-30]), demonstrated that the suppression could be reversed by either antibody when they were given prior to epidermal hapten painting. However, when each mAb was administered to recipients of BM cells from hapten painted donors, only mAb 14-12 reversed suppression of CTL generation. Examination of the number of resident BM cells revealed that TNCB-sensitized mice had 35% more cells than normal controls. When cultured in vitro with inactivated, syngeneic TNP-thymocytes, BM from normal mice readily developed TNP-self specific CTL, whereas whole BM from hapten-painted mice did not. The inability to generate CTLs was found to be attributable to suppressor cells, since BM cells from hapten-painted mice prevented CTL development by splenic T cells in culture. BM cells from normal mice did not suppress CTL generation. Suppression in vitro was not overcome by the presence of exogenous IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)