CD4+ but not CD8+ cells are essential for allorejection

The generation of knockout mice with targeted gene disruption has provided a valuable tool for studying the immune response. Here we describe the use of CD4 and CD8 knockout mice to examine the role of CD4+ and CD8+ cells in initiating allotransplantation rejection. Pretreatment with a brief course of depletive anti-CD4 monoclonal antibody therapy allowed permanent survival of heart, but not skin, allografts transplanted across a major histocompatibility barrier. However, skin as well as heart grafts were permanently accepted in the CD4 knockout mice. Transfer of CD4+ cells into CD4 knockout recipient mice 1 d before skin engraftment reconstituted rejection, demonstrating that CD4+ cells are necessary for initiating rejection of allogeneic transplants. Major histocompatibility complex disparate heart and skin allografts transplanted into CD8 knockout recipients were rejected within 10 d. This study demonstrates that CD4+ but not CD8+ T cells are absolutely required to initiate allograft rejection.     

In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes

The low number of CD4+ CD25+ regulatory T cells (Tregs), their anergic phenotype, and diverse antigen specificity present major challenges to harnessing this potent tolerogenic population to treat autoimmunity and transplant rejection. In this study, we describe a robust method to expand antigen-specific Tregs from autoimmune-prone nonobese diabetic mice. Purified CD4+ CD25+ Tregs were expanded up to 200-fold in less than 2 wk in vitro using a combination of anti-CD3, anti-CD28, and interleukin 2. The expanded Tregs express a classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions. Most significantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity.     

Tumor immunity and autoimmunity induced by immunization with homologous DNA.

The immune system can recognize self antigens expressed by cancer cells. Differentiation antigens are prototypes of these self antigens, being expressed by cancer cells and their normal cell counterparts. The tyrosinase family proteins are well characterized differentiation antigens recognized by antibodies and T cells of patients with melanoma. However, immune tolerance may prevent immunity directed against these antigens. Immunity to the brown locus protein, gp75/ tyrosinase-related protein-1, was investigated in a syngeneic mouse model. C57BL/6 mice, which are tolerant to gp75, generated autoantibodies against gp75 after immunization with DNA encoding human gp75 but not syngeneic mouse gp75. Priming with human gp75 DNA broke tolerance to mouse gp75. Immunity against mouse gp75 provided significant tumor protection. Manifestations of autoimmunity were observed, characterized by coat depigmentation. Rejection of tumor challenge required CD4(+) and NK1.1(+) cells and Fc receptor gamma-chain, but depigmentation did not require these components. Thus, immunization with homologous DNA broke tolerance against mouse gp75, possibly by providing help from CD4(+) T cells. Mechanisms required for tumor protection were not necessary for autoimmunity, demonstrating that tumor immunity can be uncoupled from autoimmune manifestations.     

TCR stimulation with modified anti-CD3 mAb expands CD8+ T cell population and induces CD8+CD25+ Tregs

Modified anti-CD3 mAbs are emerging as a possible means of inducing immunologic tolerance in settings including transplantation and autoimmunity such as in type 1 diabetes. In a trial of a modified anti-CD3 mAb [hOKT3gamma1(Ala-Ala)] in patients with type 1 diabetes, we identified clinical responders by an increase in the number of peripheral blood CD8+ cells following treatment with the mAb. Here we show that the anti-CD3 mAb caused activation of CD8+ T cells that was similar in vitro and in vivo and induced regulatory CD8+CD25+ T cells. These cells inhibited the responses of CD4+ cells to the mAb itself and to antigen. The regulatory CD8+CD25+ cells were CTLA4 and Foxp3 and required contact for inhibition. Foxp3 was also induced on CD8+ T cells in patients during mAb treatment, which suggests a potential mechanism of the anti-CD3 mAb immune modulatory effects involving induction of a subset of regulatory CD8+ T cells.     

CD4+CD25+ regulatory T cell therapy for the induction of donor-specific clinical transplantation tolerance

Naturally occurring CD4+CD25+ regulatory T cells (Tregs) have been shown to play a key role in the control of autoimmunity. Interestingly, they are also capable of mediating transplantation tolerance and they can have indirect allospecificity for donor antigens. An increasing body of evidence in experimental studies has indicated that adoptive transfer of in vitro expanded CD4+CD25+ Tregs with indirect antidonor allospecificity can induce long-term donor-specific transplantation tolerance. Thus, adoptive cell therapy using patient-specific CD4+CD25+ Tregs as individualised medicine to promote clinical transplantation tolerance is promising.     

Cellular basis of skin allograft rejection across a class I major histocompatibility barrier in mice depleted of CD8+ T cells in vivo.

The present study was undertaken to define the cellular mechanisms involved in the rejection of major histocompatibility complex (MHC) class I disparate skin grafts by mice depleted of CD8+ T cells in vivo. Mice were effectively depleted of CD8+ T cells by adult thymectomy followed by in vivo administration of anti-CD8 monoclonal antibody (mAb) and then engrafted with allogeneic skin. We found that CD8 depleted mice did reject MHC class I disparate skin grafts, but only when the grafts also expressed additional alloantigens. Despite the marked depletion of CD8+ T cells in these mice, we found that their rejection of MHC class I disparate grafts was mediated by CD8+ cytolytic T lymphocyte (CTL) effectors that had escaped depletion. These CD8+ CTL effectors were unique in that: (a) their generation was dependent upon the injected anti-CD8 mAb and upon exposure to class I MHC alloantigens expressed on the engrafted skin, and (b) their effector function was resistant to blockade by anti-CD8 mAb. We observed that the additional alloantigens coexpressed on MHC class I disparate grafts that triggered graft rejection in CD8-depleted mice could be MHC-linked or not and that they functioned in these rejection responses to activate third party specific CD4+ T helper (Th) cells to provide helper signals for the generation of CD8+ anti-CD8 resistant CTL effector cells. Thus, mice depleted of CD8+ T cells by thymectomy and in vivo administration of anti-CD8 mAb harbor a unique population of anti-CD8 resistant, CD8+ effector cells that mediate anti-MHC class I responses in vivo and in vitro, but require help from third party specific Th cells to do so.     

DNA vaccination with CD25 protects rats from adjuvant arthritis and induces an antiergotypic response

Ab's to the alpha-chain of the IL-2 receptor (anti-CD25) are used clinically to achieve immunosuppression. Here we investigated the effects of DNA vaccination with the whole CD25 gene on the induction of rat adjuvant arthritis. The DNA vaccine protected the rats and led to a shift in the cytokine profile of T cells responding to disease target antigens from Th1 to Th2. The mechanism of protection was found to involve the induction of an antiergotypic response, rather than the induction of anti-CD25 Ab's. Antiergotypic T cells respond to activation molecules, ergotopes, expressed on syngeneic activated, but not resting, T cells. CD25-derived peptides function as ergotopes that can be recognized by the antiergotypic T cells. Antiergotypic T cells taken from control sick rats did not proliferate against activated T cells and secreted mainly IFN-gamma. In contrast, antiergotypic cells from CD25-DNA-protected rats proliferated against activated T cells and secreted mainly IL-10. Protective antiergotypic T cells were found in both the CD4+ and CD8+ populations and expressed alpha/beta or gamma/delta T cell receptors. Antiergotypic alpha/beta T cells were MHC restricted, while gamma/delta T cells were MHC independent. Thus, CD25 DNA vaccination may induce protection from autoimmunity by inducing a cytokine shift in both the antiergotypic response and the response to the antigens targeted in the disease.     

CD8(+) T cell tolerance to a tumor-associated antigen is maintained at the level of expansion rather than effector function

CD8+ T cell tolerance to self-proteins prevents autoimmunity but represents an obstacle to generating T cell responses to tumor-associated antigens. We have made a T cell receptor (TCR) transgenic mouse specific for a tumor antigen and crossed TCR-TG mice to transgenic mice expressing the tumor antigen in hepatocytes (gag-TG). TCRxgag mice showed no signs of autoimmunity despite persistence of high avidity transgenic CD8+ T cells in the periphery. Peripheral CD8+ T cells expressed phenotypic markers consistent with antigen encounter in vivo and had upregulated the antiapoptotic molecule Bcl-2. TCRxgag cells failed to proliferate in response to antigen but demonstrated cytolytic activity and the ability to produce interferon gamma. This split tolerance was accompanied by inhibition of Ca(2+) flux, ERK1/2, and Jun kinase phosphorylation, and a block in both interleukin 2 production and response to exogenous interleukin 2. The data suggest that proliferation and expression of specific effector functions characteristic of reactive cells are not necessarily linked in CD8+ T cell tolerance.     

Induction of donor-specific tolerance in sublethally irradiated recipients by gene therapy.

Donor-specific transplantation tolerance can be established through the induction of molecular chimerism following reconstitution of lethally irradiated mice with autologous bone marrow expressing retrovirally transduced allogeneic MHC antigens. Here, we set out to define nonmyeloablative host conditioning regimens that would allow for establishment of molecular chimerism and the induction of donor-specific tolerance. Recipient mice received various doses of whole-body irradiation, together with costimulatory blockade using anti-CD154 monoclonal antibody prior to reconstitution with syngeneic bone marrow cells transduced with retroviruses carrying the gene encoding H-2K(b). Conditioning consisting of 3 Gy whole-body irradiation and treatment with anti-CD154 was sufficient to induce molecular chimerism resulting in stable multilineage expression of K(b) on hematopoietic cells. T cells from molecular chimeras were unable to lyse allogeneic targets expressing K(b) and contained substantially fewer K(b)-reactive IL-2- and IFN-gamma-producing CD4 T cells than controls receiving mock-transduced bone marrow. Induction of molecular chimerism using nonmyeloablative host conditioning allowed for permanent survival of K(b)-disparate allogeneic skin grafts. These data suggest that nonmyeloablative host conditioning can be used effectively to induce molecular chimerism resulting in transplantation tolerance.     

Activation of diverse repertoires of autoreactive T cells enhances the loss of anti-dsDNA B cell tolerance

CD4+ helper T cells play a critical role in the production of the antinuclear autoantibodies that characterize systemic lupus erythematosus in mice and humans. A key issue is whether this help is derived from a diverse repertoire of autoreactive CD4+ T cells or from a select number of T cells of limited specificity. We used the chronic graft-versus-host disease model to define the diversity of the CD4+ T cell repertoire required to induce the autoantibody response. By transferring clonally restricted versus clonally diverse populations of MHC class II-reactive CD4+ T cells, we show that the loss of B cell tolerance to nuclear antigens has two distinct components with different CD4+ cell requirements. Activation of limited repertoires of CD4+ T cells was sufficient for the expansion of anergized anti-double-stranded DNA B cells and production of IgM autoantibodies. Unexpectedly, we found that CD4+ T cell diversity was necessary for CD4+ T cell trafficking into the follicle and for the generation of isotype-switched IgG autoantibodies. Importantly, combining two limited repertoires of T cells provides sufficient CD4+ T cell diversity to drive antinuclear Ab production. These data demonstrate that a diverse CD4+ T cell repertoire is required to generate a sustained effector B cell response capable of mediating systemic autoimmunity.     

CD25+ CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes

In the nonobese diabetic (NOD) mouse model of type 1 diabetes, the immune system recognizes many autoantigens expressed in pancreatic islet beta cells. To silence autoimmunity, we used dendritic cells (DCs) from NOD mice to expand CD25+ CD4+ suppressor T cells from BDC2.5 mice, which are specific for a single islet autoantigen. The expanded T cells were more suppressive in vitro than their freshly isolated counterparts, indicating that DCs from autoimmune mice can increase the number and function of antigen-specific, CD25+ CD4+ regulatory T cells. Importantly, only 5,000 expanded CD25+ CD4+ BDC2.5 T cells could block autoimmunity caused by diabetogenic T cells in NOD mice, whereas 10(5) polyclonal, CD25+ CD4+ T cells from NOD mice were inactive. When islets were examined in treated mice, insulitis development was blocked at early (3 wk) but not later (11 wk) time points. The expanded CD25+ CD4+ BDC2.5 T cells were effective even if administered 14 d after the diabetogenic T cells. Our data indicate that DCs can generate CD25+ CD4+ T cells that suppress autoimmune disease in vivo. This might be harnessed as a new avenue for immunotherapy, especially because CD25+ CD4+ regulatory cells responsive to a single autoantigen can inhibit diabetes mediated by reactivity to multiple antigens.     

Immune tolerance after delivery of dying cells to dendritic cells in situ

Peripheral immune tolerance is believed to be induced by the processing and presentation of self-tissues that die during physiologic tissue turnover. To examine the mechanism that mediates tolerance, we injected mice with dying syngeneic TAP(-/-) splenocytes loaded with small amounts of the protein antigen, ovalbumin (OVA). After ingestion and presentation of cell-associated OVA by the CD8(+) subset of dendritic cells in situ, large numbers of antigen-reactive, CD8(+) T cell receptor (TCR) transgenic T lymphocytes were driven into cell cycle, but then the T cells were deleted. The animals were also tolerant to challenge with OVA in complete Freund's adjuvant. An agonistic anti-CD40 monoclonal antibody was then administered together with the OVA-loaded splenocytes, so that the dendritic cells in the recipient mice would mature. In contrast to observations made in the steady state, the antigen-reactive T cells expanded in numbers for 1-2 wk and produced large amounts of interleukin 2 and interferon gamma, while the animals retained responsiveness to antigen rechallenge. The specific tolerance that develops when dendritic cells process self tissues in the steady state should prevent or reduce the development of autoimmunity when dying cells are subsequently processed during infection.     

FK506 augments activation-induced programmed cell death of T lymphocytes in vivo.

FK506 is an immunosuppressive drug that inhibits T cell receptor-mediated signal transduction. This drug can induce immunological tolerance in allograft recipients. In this study, we investigated the in vivo effects of FK506 on T cell receptor-mediated apoptosis induction. Injection of anti-CD3 antibody (Ab) in mice resulted in the elimination of CD4+ CD8+ thymocytes by DNA fragmentation. FK506 treatment significantly augmented thymic apoptosis induced by in vivo anti-CD3 Ab administration. Increased thymic apoptosis resulted in the disappearance of CD4+ CD8+ thymocytes after anti-CD3 Ab/FK506 treatment. DNA fragmentation triggered by FK506 was induced exclusively in antigen-stimulated T cells, since enhanced DNA fragmentation induced by in vivo staphylococcal enterotoxin B (SEB) injection was confirmed in SEB-reactive V beta 8+ thymocytes but not in SEB-nonreactive V beta 6+ thymocytes. In addition to thymocytes, mature peripheral T cells also die by activation-induced programmed cell death. A similar effect of FK506 on activation-induced programmed cell death was observed in SEB-activated peripheral spleen T cells. In contrast, cyclosporin A treatment did not enhance activation-induced programmed cell death of thymocytes and peripheral T cells. Apoptosis is required for the generation and maintenance of self-tolerance in the immune system. Our findings suggest that FK506-triggered apoptosis after elimination of antigen-activated T cells may represent a potential mechanism of the immunological tolerance achieved by FK506 treatment.     

Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood

It has been known for years that rodents harbor a unique population of CD4(+)CD25(+) "professional" regulatory/suppressor T cells that is crucial for the prevention of spontaneous autoimmune diseases. Here we demonstrate that CD4(+)CD25(+)CD45RO(+) T cells (mean 6% of CD4(+) T cells) are present in the blood of adult healthy volunteers. In contrast to previous reports, these CD4(+)CD25(+) T cells do not constitute conventional memory cells but rather regulatory cells exhibiting properties identical to their rodent counterparts. Cytotoxic T lymphocyte-associated antigen (CTLA)-4 (CD152), for example, which is essential for the in vivo suppressive activity of CD4(+)CD25(+) T cells, was constitutively expressed, and remained strongly upregulated after stimulation. The cells were nonproliferative to stimulation via their T cell receptor for antigen, but the anergic state was partially reversed by interleukin (IL)-2 and IL-15. Upon stimulation with allogeneic (but not syngeneic) mature dendritic cells or platebound anti-CD3 plus anti-CD28 the CD4(+)CD25(+) T cells released IL-10, and in coculture experiments suppressed the activation and proliferation of CD4(+) and CD8(+) T cells. Suppression proved IL-10 independent, yet contact dependent as in the mouse. The identification of regulatory CD4(+)CD25(+) T cells has important implications for the study of tolerance in man, notably in the context of autoimmunity, transplantation, and cancer.     

Induction of anti-allo-class I H-2 tolerance by inactivation of CD8+ helper T cells, and reversal of tolerance through introduction of third-party helper T cells

The intravenous sensitization of C57BL/6 (B6) mice with class I H-2-disparate B6-C-H-2bm1 (bm1) spleen cells resulted in the abrogation of CD8+ T cell-mediated anti-bm1 (proliferative and interleukin 2-producing) T helper (Th) cell activities. In vitro stimulation of lymphoid cells from these mice with bm1 cells, however, generated a reduced, but appreciable, anti-bm1 cytotoxic T lymphocyte (CTL) response. Moreover, the anti-bm1 CTL response, upon stimulation with [bm1 x B6-C-H-2bm12 (bm12)]F1 spleen cells, was enhanced when compared with the response induced upon stimulation with bm1 cells. These in vitro results were reflected on in vivo graft rejection responses; bm1 skin grafts engrafted in the bm1-presensitized B6 mice exhibited prolonged survival, whereas (bm1 x bm12)F1 grafts placed collateral to bm1 grafts (dual engrafted mice) inhibited the tolerance to bm1. In the B6 mice 1-2 d after rejecting the bm1 grafts, anti-bm1 Th activities remained marginal, whereas potent anti-bm1 CTL responses were found to be generated from their spleen cells. Administration in vivo of anti-CD4 antibody into bm1-presensitized, dual graft-engrafted mice prolonged bm1 graft survival and interfered with enhanced induction of anti-bm1 CTL activity. These results indicate that anti-class I alloantigen (bm1) tolerance as induced by intravenous presensitization with the relevant antigens is not ascribed to the elimination of CD8+ CTL precursors, but to the specific inactivation of CD8+ Th cells, whose function can be bypassed by activating third-party Th cells.     

The CD8+ dendritic cell subset selectively endocytoses dying cells in culture and in vivo

Dendritic cells (DCs) are able in tissue culture to phagocytose and present antigens derived from infected, malignant, and allogeneic cells. Here we show directly that DCs in situ take up these types of cells after fluorescent labeling with carboxyfluorescein succinimidyl ester (CFSE) and injection into mice. The injected cells include syngeneic splenocytes and tumor cell lines, induced to undergo apoptosis ex vivo by exposure to osmotic shock, and allogeneic B cells killed by NK cells in situ. The CFSE-labeled cells in each case are actively endocytosed by DCs in vivo, but only the CD8+ subset. After uptake, all of the phagocytic CD8+ DCs can form major histocompatibility complex class II-peptide complexes, as detected with a monoclonal antibody specific for these complexes. The CD8+ DCs also selectively present cell-associated antigens to both CD4+ and CD8+ T cells. Similar events take place with cultured DCs; CD8+ DCs again selectively take up and present dying cells. In contrast, both CD8+ and CD8- DCs phagocytose latex particles in culture, and both DC subsets present soluble ovalbumin captured in vivo. Therefore CD8+ DCs are specialized to capture dying cells, and this helps to explain their selective ability to cross present cellular antigens to both CD4+ and CD8+ T cells.     

The induction of experimental autoimmune myocarditis in mice lacking CD4 or CD8 molecules [corrected] [published erratum appears in J Exp Med 1994 Jan 1;179(1):371]

Experimental induction of most autoimmune diseases appears to depend on the activation of CD4+ T helper cells, while CD8+ lymphocytes may have a role in disease progression. To study the role of CD4+ and CD8+ T cell subsets in T cell-dependent autoimmunity, mice lacking CD4 or CD8 molecules after gene targeting were injected with cardiac myosin to induce organ specific autoimmune myocarditis. Mice homozygous for the CD8 mutation (CD8-/-) developed significantly more severe disease as compared to CD4+/-CD8+/- controls. Surprisingly, CD4-/- mice developed autoimmune myocarditis with infiltration of TCR alpha beta +CD4-CD8- T cells in the heart tissue and appearance of autoantibodies. These data demonstrate that the lack of CD4+ or CD8+ T cells has no significant influence on the initiation of autoimmune myocarditis. CD4+ and CD8+ cells regulate disease severity and these results may explain the occurrence of autoimmunity in CD4 immunodeficiencies.     

Costimulation controls diabetes by altering the balance of pathogenic and regulatory T cells

The development of autoimmune diabetes in the nonobese diabetic (NOD) mouse results from a breakdown in tolerance to pancreatic islet antigens. CD28-B7 and CD40 ligand-CD40 (CD40L-CD40) costimulatory pathways affect the development of disease and are promising therapeutic targets. Indeed, it was shown previously that diabetes fails to develop in NOD-B7-2-/- and NOD-CD40L-/- mice. In this study, we examined the relative role of these 2 costimulatory pathways in the balance of autoimmunity versus regulation in NOD mice. We demonstrate that initiation but not effector function of autoreactive T cells was defective in NOD-B7-2-/- mice. Moreover, the residual proliferation of the autoreactive cells was effectively controlled by CD28-dependent CD4+CD25+ regulatory T cells (Treg's), as depletion of Treg's partially restored proliferation of autoreactive T cells and resulted in diabetes in an adoptive-transfer model. Similarly, disruption of the CD28-B7 pathway and subsequent Treg deletion restored autoimmunity in NOD-CD40L-/- mice. These results demonstrate that development of diabetes is dependent on a balance of pathogenic and regulatory T cells that is controlled by costimulatory signals. Thus, elimination of Treg's results in diabetes even in the absence of costimulation, which suggests a need for alternative strategies for immunotherapeutic approaches.