Transplantation tolerance from a historical perspective.

Although transplantation immunology as a distinctive field began with the development of experimental models that showed the feasibility of bone marrow transplantation, organ engraftment was accomplished first in humans, and was thought for many years to occur by drastically different mechanisms. Here, we present our view of the concepts of allograft acceptance and acquired tolerance from a historical perspective, and attempt to amalgamate them into simple and unifying rules that might guide improvements in clinical therapy.

CD4+ T-cell-epitope escape mutant virus selected in vivo.

Mutations in viral genomes that affect T-cell-receptor recognition by CD8+ cytotoxic T lymphocytes have been shown to allow viral evasion from immune surveillance during persistent viral infections. Although CD4+ T-helper cells are crucially involved in the maintenance of effective cytotoxic T-lymphocyte and neutralizing-antibody responses, their role in viral clearance and therefore in imposing similar selective pressures on the virus is unclear. We show here that transgenic virus-specific CD4+ Tcells, transferred into mice persistently infected with lymphocytic choriomeningitis virus, select for T-helper epitope mutant viruses that are not recognized. Together with the observed antigenic variation of the same T-helper epitope during polyclonal CD4+ T-cell responses in infected pore-forming protein-deficient C57BL/6 mice, this finding indicates that viral escape from CD4+ T lymphocytes is a possible mechanism of virus persistence.

PrP expression in B lymphocytes is not required for prion neuroinvasion.

Prion diseases are typically initiated by infection of peripheral sites, as in the case of bovine spongiform encephalopathy, new variant Creutzfeldt-Jakob disease, kuru and most cases of iatrogenic Creutzfeldt-Jakob disease. In mouse scrapie, prion infectivity accumulates in lymphoid organs, and the absence of mature B lymphocytes prevents peripherally administered prions from inducing central nervous system disease. We have now assessed whether expression of the cellular prion protein, PrPc, is required for B lymphocytes to mediate neuroinvasion. We found that repopulation of SCID and Rag-1(-/-) mice with fetal liver cells from either PrP-expressing or PrP-deficient mice and from T-cell deficient mice, but not from B-cell deficient mice, is equally efficient in restoring neuroinvasion after intraperitoneal inoculation of scrapie prions. These results indicate that cells whose maturation depends on B cells or their products, such as follicular dendritic cells, may enhance neuroinvasion. Alternatively, B cells may transport prions to the nervous system by a PrP-independent mechanism.

Antigen persistence and time of T-cell tolerization determine the efficacy of tolerization protocols for prevention of skin graft rejection.

We studied antigen-specific T-cell tolerization therapy using skin transplantation across a defined minor histocompatibility antigen difference. Specific tolerization protocols using short-lived peptide or long-lived spleen cells presenting the peptide as antigen prevented graft rejection without immunosuppression when started before or as long as 10 days after transplantation. Peptide-induced T-cell tolerance was transient, and antigen presentation by the graft was not sufficient to maintain tolerance. In contrast, transfer of antigen-expressing lymphoid cells induced long-lasting tolerance correlating with donor cell chimerism. These findings show that antigen-specific tolerization can induce graft acceptance even when begun after transplantation and that long-term graft survival depends on persistence of the tolerizing antigen.

T-cell independent IgM and enduring protective IgG antibodies induced by chimeric measles viruses.

B-cell activation depends on the intensity of B-cell receptor cross-linking. Studies of haptenated antigens and vesicular stomatitis virus (VSV) have demonstrated a correlation between antigen repetitiveness and the degree to which B-cell activation is independent of T cells. Here, we compare neutralizing antibody responses to inactivated VSV with those to two inactivated human pathogenic viruses: highly cytopathic poliovirus (PV) and poorly cytopathic measles virus (MV). The rigidly structured PV efficiently induced neutralizing IgM antibodies independent of T cells. In contrast, neutralizing antibodies to the pleomorphic MV were dependent on helper T cells. To test whether this resulted from the differences in virus structure or the capacity of MV to induce cell fusion and/or immunosuppression, we analyzed antibody responses to chimeric MV expressing VSV glycoprotein instead of MV fusion protein and hemagglutinin. IgM antibodies were independent of T cells; in addition, we found IgG responses dependent on T-cell help that were enduring and protective against lethal VSV infection. Because chimeric MV viruses look like MV ultrastructurally, we conclude that not only structural differences in the envelope but also the ability of MV to induce immunosuppression may limit its capacity to directly activate B cells. These findings are relevant for our understanding of B-cell activation by two prototypic human pathogenic viruses and for the design of new recombinant vaccines.

Complement facilitates early prion pathogenesis.

New-variant Creutzfeldt-Jakob disease and scrapie are typically initiated by extracerebral exposure to the causative agent, and exhibit early prion replication in lymphoid organs. In mouse scrapie, depletion of B-lymphocytes prevents neuropathogenesis after intraperitoneal inoculation, probably due to impaired lymphotoxin-dependent maturation of follicular dendritic cells (FDCs), which are a major extracerebral prion reservoir. FDCs trap immune complexes with Fc-gamma receptors and C3d/C4b-opsonized antigens with CD21/CD35 complement receptors. We examined whether these mechanisms participate in peripheral prion pathogenesis. Depletion of circulating immunoglobulins or of individual Fc-gamma receptors had no effect on scrapie pathogenesis if B-cell maturation was unaffected. However, mice deficient in C3, C1q, Bf/C2, combinations thereof or complement receptors were partially or fully protected against spongiform encephalopathy upon intraperitoneal exposure to limiting amounts of prions. Splenic accumulation of prion infectivity and PrPSc was delayed, indicating that activation of specific complement components is involved in the initial trapping of prions in lymphoreticular organs early after infection.

Selection of restriction specificities of virus-specific cytotoxic T cells in the thymus: no evidence for a crucial role of antigen-presenting cells.

The proposal was tested that (P1 X P2) F1 leads to P1 irradiation bone marrow chimeras expressed predominantly P1-restricted T cells because donor derived stem cells were exposed to recipient derived antigen-presenting cells in the thymus. Because P1 recipient-derived antigen-presenting cells are replaced only slowly after 6-8 wk by (P1 X P2) donor-derived antigen-presenting cells in the thymus and because replenished pools of mature T cells may by then prevent substantial numbers of P2-restricted T cells to be generated, a large portion of thymus cells and mature T cells were eliminated using the following treatments of 12-20-wk-old (P1 X P2) F1 leads to P1 irradiation bone marrow chimeras: (a) cortisone plus antilymphocyte serum, (b) Cytoxan, (c) three doses of sublethal irradiation (300 rad) 2d apart, and (d) lethal irradiation (850 rad) and reconstitution with T cell-depleted (P1 X P2) F1 stem cells. 12-20 wk after this second treatment, (P1 X P2) leads to P1 chimeras were infected with vaccinia-virus. Virus-specific cytotoxic T cell reactivity was expressed by chimeric T cells of (P1 X P[2) F1 origin and was restricted predominantly to P1. Virus-specific cytotoxic T cells, therefore, do not seem to be selected to measurable extent by the immigrating donor-derived antigen-presenting cells in the thymus; their selection depends apparently from the recipient-derived radioresistant thymus cells.

H-2 restriction of virus-specific T-cell-mediated effector functions in vivo. II. Adoptive transfer of delayed-type hypersensitivity to murine lymphocytic choriomeningits virus is restriced by the K and D region of H-2.

In mice, primary footpad swelling after local infection with lymphocytic choriomeningitis virus (LCMV) and delayed-type hypersensitivity (DTH) adoptively transferred by LCMV immune lymphocytes are T-cell dependent. Nude mice do not develop primary footpad swelling, and T-cell depletion abrogates the capacity to transfer LCMV-specific DTH. Effector T cells involved in eliciting dose-dependent DTH are virus specific in that vaccinia virus-immune lymphocytes could not elicit DTH in LCMV-infected mice. The adoptive transfer of DTH is restricted to H-2K or H-2D compatible donor-recipient combinations. Distinct from the fowl-gamma-globulin DTH model, I-region compatibility is neither necessary nor alone sufficient. Whatever the mechanisms involved in this K- or D-region associated restriction in vivo, it most likely operates at the level of T-cell recognition of "altered self" coded in K or D. T cells associated with the I region (helper T cells and DTH-T cells to fowl-gamma-globulin) are specific for soluble, defined, and inert antigens. T cells associated with the K and D region (T cells cytotoxic in vitro and in vivo for acute LCMV-infected cells, DTH effector T cells, and anti-viral T cells) are specific for infectious, multiplying virus. The fact that T-cell specificity is differentially linked with the I region or with the K and D regions of H-2 may reflect the fundamental biological differences of these antigens. Although it cannot be excluded that separate functional subclasses of T-effector cells could have self-recognizers for different cell surface structures coded in I or K and D, it is more likely that the antigen parameters determine whether T cells are specific for "altered" I or "altered" K- or D-coded structures.

H-2 compatibility requirement for virus-specific T-cell-mediated cytolysis. The H-2K structure involved is coded by a single cistron defined by H-2Kb mutant mice.

Lymphocytic choriomeningitis or vaccinia virus-immune spleen cells of H-2 mutant mice carrying a point mutation in the K region (B6 H-2ba, B6 H-2bf) cannot lyse infected wild-type H-2Kb targets and vice versa. Yet, cytotoxic T cells specific for infected H-2Kba or H-2Kbf targets are generated during virus infections as shown by cold target competition experiments. The critical structure for the apparent restriction by the K or D regions of the H-2 gene complex of cytolytic interactions between T cells and virus-infected target cells are therefore each coded, at least as shown for the K region, by a single cistron. This finding is most readily accommodated within the altered self concept (postulating that T cells are specific for virus-modified self structures) but cannot exclude the possibility of a physiological interaction mechanism being responsible for the apparent H-2 restriction of virus-specific cytotoxic T cells.

H-2 restriction of virus-specific cytotoxicity across the H-2 barrier. Separate effector T-cell specificities are associated with self-H-2 and with the tolerated allogeneic H-2 in chimeras.

During infection with lymphocytic choriomeningitis or vaccinia virus, F1 irradiation chimeras reconstituted with bone marrow cells from or both parents generate cytotoxic T cells which can lyse targets across the H-2 barrier. However, activity of chimera T cells is H-2 restricted as shown by cold target competition experiments and selective restimulation of a secondary response in vitro; T cells of H-2k specificity which lyse tolerated infected H-2d target cells do not lyse infected H-2k or unrelated target cells and vice versa. Therefore, H-2 restriction of virus-specific cytotoxic T cells probably does not reflect need for like-like self-interactions for lysis to occur. The specificity of virus immune T cells is thus determined by the H-2K and H-2D specificities present in the infected animal and which are probably recognized unidirectionally by T cells. The results are compatible with the idea the T cells are specific for "altered alloantigen", i.e., a complex of cell surface marker and viral antigen. Alternatively, explained with a dual recognition model, T cells may possess two independently, clonally expressed receptors, a self-recognizer which is expressed for one of the syngeneic or tolerated allogeneic K or D "self" markers, and an immunologically specific receptor for viral antigen.

Antiviral protection by virus-immune cytotoxic T cells: infected target cells are lysed before infectious virus progeny is assembled.

Virus-immune cytotoxic T cells can inhibit effectively growth of vaccinia virus in acutely infected target cells in vitro by destroying infected target cells before infectious virus progeny is assembled. Together with the fact that virus-specific T cells are demonstrable after 3 days, very early during infection, and with strong circumstantial evidence from adoptive transfer models in vivo, these data suggest that in some virus infections T cells may in fact act cytolytically in vivo to prevent virus growth and spread and be an important early antiviral effector mechanism.

Virus-triggered immune suppression in mice caused by virus-specific cytotoxic T cells.

Normal mice infected with 10(5) infectious doses of lymphocytic choriomeningitis virus (LCMV, WE isolate) generated a reduced or no T cell-independent IgM and/or T cell-dependent IgG response to a subsequent vesicular stomatitis virus Indiana (VSV-IND) injection; this transient immune suppression lasted for weeks to months. Connatally infected LCMV-carrier mice or acutely infected T cell-deficient nude mice had normal anti-VSV IgM and IgG or IgM responses respectively. LCMV-infected nude mice transfused with helper cell-depleted LCMV-specific immune spleen cells were immunosuppressed. Normal mice infected with LCMV but treated with a rat anti-CD8 mAb (that had been shown previously to eliminate cytotoxic T cells in vivo) and then infected with VSV exhibited a normal anti-VSV IgM and IgG response. Since no IFN-alpha or -beta was detected on, or after, day 6 of LCMV infection, neither LCMV alone, nor IFN induced by it caused the observed immune suppression; the presented evidence suggests that LCMV-immune CD8+ T cells were responsible for it. It is conceivable that a similar pathogenesis where virus-specific cytotoxic T cells may destroy virus-infected cells essentially involved in an immune response (APC, T helper cells, etc.) may be involved in other virally triggered immune suppression or in AIDS.

Monoclonal antibodies against viral determinants are not restricted to the K/D end of the major histocompatibility complex.

Monoclonal antibodies against lymphocytic choriomeningitis virus (LCMV), a natural, high-replicating, noncytolytic pathogen in mice, were obtained from fusions between myeloma cells and lymphoid cells of mice of different H-2 haplotypes at various times (4-24 d) after infection. Supernatants from growing hybridomas were tested in a RIA, and approximately 15% of all supernatants were positive when tested for specificity on infected vs. uninfected cells of different haplotypes. Upon retesting for specific fluorescence, only some RIA+ supernatants exhibited specific surface staining of acetone-fixed infected cells or unfixed infected cells. In all these experiments and using various detection methods we could not find antibodies with any preference of recognition of viral antigen in conjunction with the H-2 haplotype of the responder mouse. The absence of H-2 restricted antibodies after a primary virus infection in vivo, whether assayed by RIA or surface immunofluorescence, suggests that antibodies obtained in other experiments using infected tumor cells for induction and in the RIA may not represent the general case.

Thymic reconstitution of nude F1 mice with one or both parental thymus grafts.

Thymus-derived lymphocytes (T cells) have two outstanding characteristics that distinguish them from other lymphocytes: (a) they express two specificities, one for self-antigens, the major transplantation antigens (H) coded by the major histocompatibility gene complex (MHC), and a second specificity for foreign antigenic determinants. (b) T cells must undergo differentiation or maturation in the thymus (1, 2). Apparently, an important step in T-cell differentiation in the thymus is the selection of T-cells' restriction specificity for self-H. This interpretation stems from experiments with chimeras formed by lethally irradiating parental type mice and reconstituting them with F(1) stem cells: the maturing F(1) T cells expressed predominantly the restriction specificities for the recipient parental MHC type (3-8). Alternatively, adult F(1) mice that were thymectomized, lethally irradiated, reconstituted with bone marrow, and then engrafted with a parental thymus had T cells that were restricted predominantly to the thymus donors' H-2 (4-8). The present study first extends these observations to nude mice that are born without a thymus and therefore do not develop functional T cells and second, attempts to study the possibility that suppression may be responsible for the apparent influence of the radioresistant portion of the thymus on T- cell restriction specificities. We tested the immunocompetence and restriction specificities expressed by lymphocytes from F(1) nude mice reconstituted with both parental thymus grafts; our expectation was that suppression of the expression of T-cell restriction specificity should result either in complete immunoincompetence or emergence of only one of the two possible sets of restriction specificities. Nude F(1)mice that simultaneously received thymus gratis from both parents developed spleen cells restricted to both parental H-2 types. These results are compatible with the idea that the thymus' influence on T- cell restriction is via positive selection rather than by suppression.

H-2 compatability requirement for T-cell-mediated lysis of target cells infected with lymphocytic choriomeningitis virus. Different cytotoxic T-cell specificities are associated with structures coded for in H-2K or H-2D;.

Use of syngeneic, allogeneic, F1, AND H-2 recombinatn mice has shown that animals injected with lymphocytic choriomeningitis (LCM) virus generate T cells which are cytotoxic for H-2K or H-2D compatible, but not H-2 different, virus-infected target cells. Three separate lines of evidence are presented which indicate that these immune T cells are sensitized to "altered-self," the self antigens involved being coded for in the H-2K or H-2d regions. Firstly, cytotoxic activity associated with mutuality at H-2D iy, lysis mediated by immune T cells from F1 or H-2 recombinant mice is specifically inhibited only by presence of unlabeled, virus-infected cells that are H-2 compatible with the targets. Thirdly, LCM-immune F1 and H-2 recombinant T cells inoculated into irradiated, virus-infected recipients proliferate only to kill target cells that are H-2 compatible with both the donor and the recipient. All of these experiments establish that there is a dissociation of T-cell activities between parental haplotypes in F1 mice, and between H-2K and H-2D in recombinants. It would thus seem that there are at least two specificities of tlcm-immune T cells in homozygotes, associated with either H-2K or H-2D, and four specificities in F1 hybrids. The significance of these findings, with respect both to gene duplication and to the marked polymorphism in the H-2 system, is discussed.

H-2 compatibility is required for T-cell-mediated lysis of target cells infected with lymphocytic choriomeningitis virus.

Maximal cell-mediated lysis of targets infected with lymphocytic choriomeningitis virus occurs only within a H-2 compatible system. Syngeneic immune spleen cells are at least 100 times as effective as are allogeneic lymphocytes. Reciprocal restriction of cytotoxic T-cell activity has been shown to operative between H-2k, H-2d, and H-2b. Experiments with cogenic mice have localized the effect to the H-2 gene complex. Furthermore, the observation that lymphocytes from H-2a mice cause high specific 51Cr release from either H-2d virus-infected cells, indicates that identity at either the K or the D end of the H-2 gene complex is sufficient for this lytic interaction.

Thymic selection of H-2-incompatible bone marrow cells in SCID mice. Differences in T help for induction of B cell IgG responses versus cytotoxic T cells.

Mice with congenital severe combined immunodeficiency disease (SCID) failed to mount either a T cell-independent IgM or T cell-dependent IgG anti-vesicular stomatitis virus (VSV) Indiana (IND) response. They did not generate cytotoxic T cells against lymphocytic choriomeningitis virus (LCMV) or vaccinia virus, but exhibited NK cell-like activities. When SCID mice were given bone marrow from syngeneic BALB/c (H-2d) nu/nu mice, all immune responses were expressed at control levels. If SCID mice were reconstituted with allogeneic H-2b C57BL/6 nu/nu bone marrow, the following primary anti-viral immune responses were measured. T-independent IgM anti-VSV-IND were normal, but T-dependent IgG anti-VSV-IND responses were absent. Cytotoxic T cell responses to LCMV and vaccinia virus were within normal ranges, were donor cell mediated, and were specific exclusively for the recipient SCID H-2d type. Since antigen presentation by spleen cells was functional in these chimaeras, the presented results indicate that (a) thymic selection of T cell restriction is strict; and (b) the type of T help necessary for B cells depends upon H-2-restricted contact between T and B cells, whereas, such contact-dependent help is not mandatory for the induction of virus-specific cytotoxic T cells.

Virus-induced transient bone marrow aplasia: major role of interferon-alpha/beta during acute infection with the noncytopathic lymphocytic choriomeningitis virus.

The hematologic consequences of infection with the noncytopathic lymphocytic choriomeningitis virus (LCMV) were studied in wild-type mice with inherent variations in their interferon (IFN)-alpha/beta responder ability and in mutant mice lacking alpha/beta (IFN-alpha/beta R0/0) or gamma IFN (IFN-gamma R0/0) receptors. During the first week of infection, wild type mice demonstrated a transient pancytopenia. Within a given genetic background, the extent of the blood cell abnormalities did not correlate with the virulence of the LCMV isolate but variations were detected between different mouse strains: they were found to depend on their IFN-alpha/beta responder phenotype. Whereas IFN-gamma R0/0 mice were comparable to wild-type mice, IFN-alpha/beta R0/0 mice exhibited unchanged peripheral blood values during acute LCMV infection. In parallel, the bone marrow (BM) cellularity, the pluripotential and committed progenitor compartments were up to 30-fold reduced in wild type and IFN-gamma R0/0, but remained unchanged in IFN-alpha/beta R0/0 mice. Viral titers in BM 3 d after LCMV infection were similar in these mice, but antigen localization was different. Viral antigen was predominantly confined to stromal BM in normal mice and IFN-gamma R0/0 knockouts, whereas, in IFN-alpha/beta R0/0 mice, LCMV was detected in > 90% of megakaryocytes and 10-15% of myeloid precursors, but not in erythroblasts Although IFN-alpha/beta efficiently prevented viral replication in potentially susceptible hematopoietic cells, even in overwhelming LCMV infection, unlimited virus multiplication in platelet and myeloid precursors in IFN-alpha/beta R0/0 mice did not interfere with the number of circulating blood cells. Natural killer (NK) cell expansion and activity in the BM was comparable on day 3 after infection in mutant and control mice. Adaptive immune responses did not play a major role because comparable kinetics of LCMV-induced pancytopenia and transient depletion of the pluripotential and committed progenitor compartments were observed in CD8(0/0) and CD4(0/0) mice, in mice depleted of NK cells, in lpr mice, and in perforin-deficient (P0/0) mice lacking lytic NK cells. Thus, the reversible depression of hematopoiesis during early LCMV infection was not mediated by LCMV-WE-specific cytotoxic T lymphocyte, cytolysis, or secreted IFN-gamma from virally induced NK cells but was a direct effect of IFN-alpha/beta.

Distinct fates of self-specific T cells developing in irradiation bone marrow chimeras: clonal deletion, clonal anergy, or in vitro responsiveness to self-Mls-1a controlled by hemopoietic cells in the thymus.

Elimination of potentially self-reactive T lymphocytes during their maturation in the thymus has been shown to be a major mechanism in accomplishing self-tolerance. Previous reports demonstrated that clonal deletion of self-Mls-1a-specific V beta 6+ T lymphocyte is controlled by a radiosensitive I-E+ thymic component. Irradiation chimeras reconstituted with I-E- bone marrow showed substantial numbers of mature V beta 6+ T cells despite host Mls-1a expression. Analysis of the functional properties of such chimeric T cells revealed a surprising variability in their in vitro reactivity to host Mls-1a, depending on the H-2 haplotype of stem cells used for reconstitution. In chimeras reconstituted with B10.S (H-2s) stem cells, mature V beta 6+ lymphocytes were present but functionally anergic to host-type Mls-1a in vitro. In contrast, in chimeras reconstituted with B10.G (H-2q) bone marrow, nondeleted V beta 6+ cells were highly responsive to Mls-1a in vitro. These findings suggest that clonal anergy of V beta 6+ cells to self-Mls-1a may be controlled by the affinity/avidity of T cell receptor interactions with bone marrow-derived cells in the thymus depending on the major histocompatibility complex class II molecules involved. Furthermore, chimeras bearing host (Mls-1a)-reactive V beta 6+ cells did not differ clinically from those with anergic or deleted V beta 6+ cells and survived more than one year without signs of autoimmune disease. Interestingly, their spleen cells had no Mls-1a stimulatory capacity in vitro. Therefore, regulation at the level of antigen presentation may be an alternative mechanism for maintenance of tolerance to certain self-antigens such as Mls-1a.

Bystander activation of cytotoxic T cells: studies on the mechanism and evaluation of in vivo significance in a transgenic mouse model.

Bystander activation, i.e., activation of T cells specific for an antigen X during an immune response against antigen Y may occur during viral infections. However, the low frequency of bystander-activated T cells has rendered it difficult to define the mechanisms and possible in vivo relevance of this nonspecific activation. This study uses transgenic mice expressing a major histocompatibility complex class I-restricted TCR specific for glycoprotein peptide 33-41 of lymphocytic choriomeningitis virus (LCMV) to overcome this limitation. CD8+ T cells from specific pathogen-free maintained, unimmunized "naive" TCR transgenic mice can differentiate into LCMV-specific cytolytic effector CTL during infections with vaccinia virus or Listeria monocytogenes in vivo or mixed lymphocyte culture in vitro. We show that in these model situations (a) nonspecifically activated CTL are able to confer antiviral protection in vivo, (b) bystander activation is largely independent of the expression of a second T cell receptor of different specificity, (c) bystander activation is not mediated by a broadly cross-reactive TCR, but rather by cytokines, (d) bystander activation can be mediated by cytokines such as IL-2, but not alpha/beta-IFN in vitro; (e) bystander activation is, overall, a rare event, occuring in vivo in roughly 1 in 200 of the LCMV-specific CTL during infection of TCR transgenic mice with vaccinia virus; (f) bystander activation does not have a significant functional impact on nontransgenic CTL memory under the conditions tested; and (g) even in the TCR transgenic situation, where unphysiologically high numbers of T cells of a single specificity are present, bystander activation is not sufficient to cause clinically manifest autoimmune disease in a transgenic mouse model of diabetes. We conclude that although bystander activation via cytokines may generate cytolytically active CTL from naive precursors, quantitative considerations suggest that this is usually not of major biological consequence.