Persistent suppression of virus-specific cytotoxic T cell responses after transient depletion of CD4+ T cells in vivo

Co-administration of soluble Ag and anti-CD4 mAb has been successfully used to induce long term Ag-specific tolerance. The mechanisms underlying persistent immunologic unresponsiveness are unclear. We have now studied whether tolerance toward complex viral Ag expressed on Moloney sarcoma virus (MSV)-transformed tumor cells can be induced when given at the time of severe helper cell depletion. Although mice that had been injected with anti-CD4 mAb at the time of immunization regained the ability to recognize MSV Ag, their humoral and cytotoxic immunity to MSV were severely compromised. Ag-specific low responsiveness was maintained for more than 6 mo. To analyze the T cell repertoire of low responder mice we have estimated precursor frequencies of MSV-specific proliferative and cytotoxic T cells after the CD4+ T cell subset was fully reconstituted. There was no difference in the frequencies of control and low responder mice excluding clonal deletion as the mechanism maintaining low responsiveness. In co-culture experiments the defect in low responder mice could be localized to the regenerated CD4+ T cell subset, suggesting the induction of CD4+ suppressor-inducer cells. Alternatively, regenerated CD4+ cells in anti-CD4 conditioned mice had acquired a defect to provide help for MSV-specific responses. In spite of the potentials to induce low responsiveness to selected Ag by anti-CD4 conditioning, the risk to cause persistent virus-specific immunodeficiency might limit the clinical application of anti-CD4 therapy.     

Induction of foxP3+ regulatory T cells in the periphery of T cell receptor transgenic mice tolerized to transplants

Transplantation tolerance can be induced in mice by grafting under the cover of nondepleting CD4 plus CD8 or CD154 mAbs. This tolerance is donor Ag specific and depends on a population of CD4(+) regulatory T cells that, as yet, remain poorly defined in terms of their specificity, origin, and phenotype. Blocking of the Ag-specific response in vitro with an anti-CD4 mAb allowed T cells from monospecific female TCR-transgenic mice against the male Ag Dby, presented by H-2E(k), to express high levels of foxP3 mRNA. foxP3 induction was dependent on TGF-beta. The nondepleting anti-CD4 mAb was also able to induce tolerance in vivo in such monospecific TCR-transgenic mice, and this too was dependent on TGF-beta. As in conventional mice, acquired tolerance was dominant, such that naive monospecific T cells were not able to override tolerance. Splenic T cells from tolerant mice proliferated normally in response to Ag, and secreted IFN-gamma and some IL-4, similar to control mice undergoing primary or secondary graft rejection. High levels of foxP3 mRNA, and glucocorticoid-induced TNFR superfamily member 18 (GITR)(+) CD25(+) T cells were found within the tolerated skin grafts of long-term tolerant recipients. These data suggest that regulatory T cells maintaining transplantation tolerance after CD4 Ab blockade can be induced de novo through a TGF-beta-dependent mechanism, and come to accumulate in tolerated grafts.     

Peripheral deletion of mature CD8+ antigen-specific T cells after in vivo exposure to male antigen.

It has been well established that T cell tolerance to self Ag occurs primarily via clonal deletion of immature thymocytes in the thymus. Evidence also exists that there are additional mechanisms operative on mature T cells for establishing and maintaining tolerance in the periphery. To follow the fate of mature Ag-specific T cells in vivo, we used female transgenic mice, which contain a large population of male H-Y Ag-specific T cells that can be identified by immunostaining with mAb directed against CD8 and the transgenic TCR. H-Y Ag was introduced into these mice by injecting Ag-bearing male lymphocytes using conditions known to induce CTL precursor response reduction. The number of Ag-reactive CD8+ transgenic T cells in the periphery started to decrease after 2 days of in vivo exposure to male Ag. Decline was maximum (up to 80% of total) by 7 days, and stayed at this level for at least 6 wk. CD4+ cells and those CD8+ cells that did not carry the transgenic TCR were not affected. Most or all of the remaining Ag-reactive CD8+ cells in the periphery were fully responsive when stimulated by male Ag in vitro. Maturation of transgenic T cells in the thymus of injected mice remained the same as that of control animals. Our data provide direct evidence that mature Ag-reactive CD8+ cells are susceptible to clonal deletion in the periphery when exposed to the Ag in vivo. These findings suggest the presence of two types of APC in the periphery: stimulatory APC (e.g., macrophages and dendritic cells) required for initiating an active immune response; and functionally deleting APC (or veto cells) capable of deleting mature T lymphocytes that recognize Ag presented on their surface. Functionally deleting APC that present self Ag to peripheral T cells may provide a fail-safe mechanism against autoreactive cells that escaped deletion during differentiation in the thymus.     

CD40 ligand blockade induces CD4+ T cell tolerance and linked suppression.

The CD40-CD40 ligand (CD40L) interaction is a key event in the initiation of an adaptive immune response, and as such the therapeutic value of CD40L blockade has been studied in many experimental models of tissue transplantation and autoimmune disease. In rodents, transplantation of allogeneic tissues under the cover of anti-CD40L Abs has resulted in prolonged graft survival but not tolerance. In this report, we show that failure to induce tolerance probably results from the inability of anti-CD40L Abs to prevent graft rejection elicited by the CD8+ T cell subset. When the CD8+ T cell population is controlled independently, using anti-CD8 Abs, then tolerance is possible. Transplantation tolerance induced by anti-CD4 mAbs can often be associated with dominant regulation, manifested as infectious tolerance and linked suppression, both of which are mediated by CD4+ T cells. We show here that CD4+ T cells rendered tolerant using anti-CD40L therapy exhibit the same regulatory property of linked suppression, as demonstrated by their ability to accept grafts expressing third party Ags only if they are expressed in conjunction with the tolerated Ags. This observation of linked suppression reveals a hitherto undocumented consequence of CD40L blockade that suggests the tolerant state is maintained by a dominant regulatory mechanism. Our results suggest that, although anti-CD40L Abs are attractive clinical immunotherapeutic agents, additional therapies to control aggressive CD8+ T cell responses may be required.     

Treatment of allograft recipients with donor-specific transfusion and anti-CD154 antibody leads to deletion of alloreactive CD8+ T cells and prolonged graft survival in a CTLA4-dependent manner

A two-element protocol consisting of one donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb greatly prolongs the survival of murine islet, skin, and cardiac allografts. To study the mechanism of allograft survival, we determined the fate of tracer populations of alloreactive transgenic CD8+ T cells in a normal microenvironment. We observed that DST plus anti-CD154 mAb prolonged allograft survival and deleted alloreactive transgenic CD8+ T cells. Neither component alone did so. Skin allograft survival was also prolonged in normal recipients treated with anti-CD154 mAb plus a depleting anti-CD8 mAb and in C57BL/6-CD8 knockout mice treated with anti-CD154 mAb monotherapy. We conclude that, in the presence of anti-CD154 mAb, DST leads to an allotolerant state, in part by deleting alloreactive CD8+ T cells. Consistent with this conclusion, blockade of CTLA4, which is known to abrogate the effects of DST and anti-CD154 mAb, prevented the deletion of alloreactive transgenic CD8+ T cells. These results document for the first time that peripheral deletion of alloantigen-specific CD8+ T cells is an important mechanism through which allograft survival can be prolonged by costimulatory blockade. We propose a unifying mechanism to explain allograft prolongation by DST and blockade of costimulation.     

Identification of the anti-CD3-unresponsive subpopulation of CD4+, CD45RA+ peripheral T lymphocytes.

The majority of peripheral CD4+ T lymphocytes proliferate in vitro in response to anti-CD3 in presence of autologous APC. The present study describes a subpopulation of CD4+ T cells that cannot be activated and progress into cell cycle by stimulation with anti-CD3 plus APC or with mitogenic combinations of anti-CD2. The in vitro responses of these anti-CD3-unresponsive CD4+ T cells were investigated with a panel of mAb to CD2, CD3, and CD28, and found to be similar to those previously observed for mature thymocytes: only the combination of anti-CD2 plus anti-CD28 produced cell proliferation. Anti-CD3-unresponsive T cells were CD45RA+, but represented only 14 to 22% of the CD4+, CD45RA+ T cell population. Activation with anti-CD2 plus anti-CD28 mAb resulted in major changes in the cell surface phenotype and functional properties: a loss of CD45RA+ occurred and an increased expression of CD45RO, CD29, and CD58 (LFA3), as well as a gain in responsiveness to anti-CD3 and anti-CD2. This change in CD45 phenotype from CD45RA to CD45RO occurs in both the anti-CD3-responsive and in the anti-CD3-unresponsive subsets of the CD45RA+, CD4+ cells after cell proliferation. The anti-CD3-unresponsive subset may represent a pool of not yet fully differentiated peripheral T cells. The acquisition of anti-CD3 responsiveness could occur as a consequence of Ag priming or by an Ag-independent mechanism. Involvement of the CD28 Ag in this process is suggested from the present study.     

Analysis of the requirements for the induction of CD4+ T cell alloantigen hyporesponsiveness by ex vivo anti-CD40 ligand antibody

A major goal of the transplant field is to selectively tolerize only those donor T cells recognizing host alloantigen and mediating graft-vs-host disease (GVHD). Recently, we described an ex vivo approach in which the blockade of the CD40 ligand (CD40L):CD40 costimulatory pathway in bulk MLR cultures induces donor CD4+ T cells to become specifically tolerant to MHC class II-disparate alloantigenic-bearing stimulators, resulting in a profound reduction in GVHD generation in vivo. In studies presented in this work, we investigated the ex vivo requirements for tolerance induction. We found that CD4+ T cells become profoundly more hyporesponsive to alloantigen restimulation with prolonged culture duration such that 7 to 10 but not 4 days is needed to achieve maximum alloantigen hyporesponsiveness as assessed in secondary MLR cultures and GVHD generation. By day 7, both primed and tolerized cells had substantially increased blastogenesis and CD25 expression. Primed but not tolerized cells substantially down-regulated L-selectin expression, indicating that the tolerized cells do not become fully Ag experienced. Both Th1 and Th2 cytokine production is severely impaired by CD40L:CD40 blockade. Analysis of culture supernatants and results from IL-4 and IL-10 knockout mice indicated that GVHD prevention was not mediated by a skewing toward a Th2 phenotype. The addition of IL-4 to the cultures as a survival factor precluded the induction of tolerance in the anti-CD40L-cultured cells. These data provide further impetus for the ex vivo use of anti-CD40L mAb to block GVHD generation.     

Injection of soluble antigen into the anterior chamber of the eye induces expansion and functional unresponsiveness of antigen-specific CD8+ T cells

The injection of soluble Ag into the anterior chamber (a.c.) of the eye induces systemic tolerance, termed a.c.-associated immune deviation (ACAID), characterized by Ag-specific inhibition of delayed-type hypersensitivity responses and a reduction in complement-fixing Abs. Recently, we have shown that CD8(+) CTL responses are also inhibited in ACAID. In this study, we have used an adoptive transfer approach to follow the fate of Ag-specific CD8(+) TCR transgenic (OT-I) T cells in vivo during the induction and expression of ACAID. C57BL/6 (B6) recipients of OT-I splenocytes that were injected with chicken OVA in the a.c. displayed reduced OVA-specific delayed-type hypersensitivity and CTL responses, compared with those of mice given OVA in the subconjunctiva or an irrelevant Ag human IgG in the a.c. OT-I T cells increased 9-fold in the submandibular lymph nodes and 3-fold in the spleen following an a.c. injection with OVA, indicating that expansion rather than deletion of Ag-specific CD8(+) T cells was induced by this treatment. OT-I T cells expanded equivalently upon administration of OVA in CFA to mice previously given OVA in the a.c. or subconjunctiva. However, the lytic activity attributed to OT-I T cells was reduced on a per-cell basis in mice previously given OVA in the a.c. We conclude that tolerance of CTL responses in mice given Ag via the a.c. results from unresponsiveness of Ag-specific CD8(+) T cells.     

Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific

CD4+CD25+ T cells represent a unique population of "professional" suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+CD25- cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+CD25- Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+CD25- T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this T-T interaction remain to be characterized.     

Low-dose antigen promotes induction of FOXP3 in human CD4+ T cells

Low Ag dose promotes induction and persistence of regulatory T cells (Tregs) in mice, yet few studies have addressed the role of Ag dose in the induction of adaptive CD4(+)FOXP3(+) Tregs in humans. To this end, we examined the level of FOXP3 expression in human CD4(+)CD25(-) T cells upon activation with autologous APCs and varying doses of peptide. Ag-specific T cells expressing FOXP3 were identified by flow cytometry using MHC class II tetramer (Tmr). We found an inverse relationship between Ag dose and the frequency of FOXP3(+) cells for both foreign Ag-specific and self Ag-specific T cells. Through studies of FOXP3 locus demethylation and helios expression, we determined that variation in the frequency of Tmr(+)FOXP3(+) T cells was not due to expansion of natural Tregs, but instead, we found that induction, proliferation, and persistence of FOXP3(+) cells was similar in high- and low-dose cultures, whereas proliferation of FOXP3(-) T cells was favored in high Ag dose cultures. The frequency of FOXP3(+) cells positively correlated with suppressive function, indicative of adaptive Treg generation. The frequency of FOXP3(+) cells was maintained with IL-2, but not upon restimulation with Ag. Together, these data suggest that low Ag dose favors the transient generation of human Ag-specific adaptive Tregs over the proliferation of Ag-specific FOXP3(-) effector T cells. These adaptive Tregs could function to reduce ongoing inflammatory responses and promote low-dose tolerance in humans, especially when Ag exposure and tolerance is transient.     

CD4+ and CD8+ T cell priming for contact hypersensitivity occurs independently of CD40-CD154 interactions

The primary effector cells of contact hypersensitivity (CHS) responses to dintrofluorobenzene (DNFB) are IFN-gamma-producing CD8(+) T cells, whereas CD4(+) T cells regulate the magnitude and duration of the response. The requirement for CD40-CD154 engagement during CD8(+) and CD4(+) T cell priming by hapten-presenting Langerhans cells (hpLC) is undefined and was tested in the current study. Similar CHS responses to DNFB were elicited in wild-type and CD154(-/-) animals. DNFB sensitization of CD154(-/-) mice primed IFN-gamma-producing CD8(+) T cells and IL-4-producing CD4(+) T cells. However, anti-CD154 mAb MR1 given during hapten sensitization inhibited hapten-specific CD8(+), but not CD4(+), T cell development and the CHS response to challenge. F(ab')(2) of MR1 failed to inhibit CD8(+) T cell development and the CHS response suggesting that the mechanism of inhibition is distinct from that of CD40-CD154 blockade. Furthermore, anti-CD154 mAb did not inhibit CD8(+) T cell development and CHS responses in mice depleted of CD4(+) T cells or in CD4(-/-) mice. During in vitro proliferation assays, hpLC from mice treated with anti-CD154 mAb during DNFB sensitization were less stimulatory for hapten-primed T cells than hpLC from either control mice or mice depleted of CD4(+) T cells before anti-CD154 mAb administration. These results demonstrate that development of IFN-gamma-producing CD8(+) T cells and the CHS response are not dependent on CD40-CD154 interactions. This study proposes a novel mechanism of anti-CD154 mAb-mediated inhibition of CD8(+) T cell development where anti-CD154 mAb acts indirectly through CD4(+) T cells to impair the ability of hpLC to prime CD8(+) T cells.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

Induction of immune tolerance during administration of monoclonal antibody to L3T4 does not depend on depletion of L3T4+ cells

Treatment of mice with mAb to L3T4 profoundly depletes T helper cells. This treatment inhibits humoral and cellular immunity, retards autoimmunity, and permits the induction of Ag-specific tolerance. Treatment of BALB/c mice with F(ab')2 anti-L3T4 inhibits humoral immunity without depleting L3T4+ cells, which is evidence that mAb to L3T4 may inhibit T helper cell function in vivo. In this report, we demonstrate that F(ab')2 anti-L3T4 also permits the induction of immune tolerance in a manner that is independent of T cell depletion. C57BL/6 mice were treated with 1 mg of F(ab')2 anti-L3T4 every other day for 18 days and from the onset were challenged weekly with the immunogen 2C7, a rat mAb to chicken ovalbumin. These mice failed to respond to 2C7 not only during the treatment period but also for at least 5 mo thereafter. This immune tolerance was Ag-specific; the mice rapidly produced antibodies to subsequent challenge with another Ag, human gamma-globulin. Unlike intact anti-L3T4, which immediately depletes L3T4+ cells by greater than 90%, F(ab')2 anti-L3T4 did not initially deplete cells and caused only a partial reduction by the end of the 18-day treatment. This partial reduction of L3T4+ cells did not contribute to the induction of tolerance because mice that were first challenged with 2C7 3 days after stopping the F(ab')2 anti-L3T4 treatment, when L3T4+ cells were lowest, had a normal Ir to 2C7. These findings demonstrate that mAb to L3T4 permits induction of Ag-specific immune tolerance by a mechanism independent of its ability to deplete L3T4+ cells. They also show that F(ab')2 anti-L3T4 treatment does not impair humoral immunity when immunization is initiated after treatment is stopped. Because L3T4 is homologous to CD4 in humans, our findings suggest that F(ab')2 anti-CD4 may offer significant advantages over the use of intact anti-CD4 as an immunosuppressive agent in humans.     

Peripheral deletion of antigen-specific T cells leads to long-term tolerance mediated by CD8+ cytotoxic cells

Peripheral deletion is one mechanism by which potentially self-reactive clones are removed whether they escape thymic deletion. We have examined the consequences of deleting Ag-specific T cells by i.v. injection of soluble Ag. Deletion of DO11.10 T cells by peptide was mediated predominately via a Fas/FasL mechanism. Animals that underwent deletion were tolerant to subsequent immunization with Ag, even when tolerant mice were given fresh Ag-specific DO11.10 T cells before immunization. Tolerance was mediated by CD8(+) T cells that killed the DO11.10-transgenic T cells in vivo. These data demonstrate that the programmed cell death of large numbers of T cells leads to peripheral tolerance mediated by CD8(+) CTLs.     

CD8+ T cell tolerance in nonobese diabetic mice is restored by insulin-dependent diabetes resistance alleles

Although candidate genes controlling autoimmune disease can now be identified, a major challenge that remains is defining the resulting cellular events mediated by each locus. In the current study we have used NOD-InsHA transgenic mice that express the influenza hemagglutinin (HA) as an islet Ag to compare the fate of HA-specific CD8+ T cells in diabetes susceptible NOD-InsHA mice with that observed in diabetes-resistant congenic mice having protective alleles at insulin-dependent diabetes (Idd) 3, Idd5.1, and Idd5.2 (Idd3/5 strain) or at Idd9.1, Idd9.2, and Idd9.3 (Idd9 strain). We demonstrate that protection from diabetes in each case is correlated with functional tolerance of endogenous islet-specific CD8+ T cells. However, by following the fate of naive, CFSE-labeled, islet Ag-specific CD8+ (HA-specific clone-4) or CD4+ (BDC2.5) T cells, we observed that tolerance is achieved differently in each protected strain. In Idd3/5 mice, tolerance occurs during the initial activation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes where CD25+ regulatory T cells (Tregs) effectively prevent their accumulation. In contrast, resistance alleles in Idd9 mice do not prevent the accumulation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes, indicating that tolerance occurs at a later checkpoint. These results underscore the variety of ways that autoimmunity can be prevented and identify the elimination of islet-specific CD8+ T cells as a common indicator of high-level protection.     

CTLA-4 engagement and regulatory CD4+CD25+ T cells independently control CD8+-mediated responses under costimulation blockade

Blockade of costimulatory signals is a promising therapeutic target to prevent allograft rejection. In this study, we sought to characterize to what extent CTLA-4 engagement contributes to the development of transplantation tolerance under the cover of CD40/CD40L and CD28/CD86 blockade. In vitro, we found that inhibition of the primary alloresponse and induction of alloantigen hyporesponsiveness by costimulation blockade was abrogated by anti-CTLA-4 mAb. In addition, regulatory CD4(+)CD25(+) T cells (T(REG)) were confirmed to play a critical role in the induction of hyporesponsiveness by anti-CD40L and anti-CD86 mAb. Our data indicated that CTLA-4 engagement is not required for activation or suppressor function of T(REG). Instead, in the absence of either CTLA-4 signaling or T(REG), CD8(+) T cell division was enhanced, whereas the inhibition of CD4(+) T cell division by costimulation blockade remained largely unaffected. In vivo, the administration of additional anti-CTLA-4 mAb abrogated anti-CD40L- and anti-CD86 mAb-induced cardiac allograft survival. Correspondingly, rejection was accompanied by enhanced allograft infiltration of CD8(+) cells. We conclude that CTLA-4 signaling and T(REG) independently cooperate in the inhibition of CD8(+) T cell expansion under costimulation blockade.     

Manipulation of CD98 resolves type 1 diabetes in nonobese diabetic mice

The interplay of CD4(+) and CD8(+) T cells targeting autoantigens is responsible for the progression of a number of autoimmune diseases, including type 1 diabetes mellitus (T1D). Understanding the molecular mechanisms that regulate T cell activation is crucial for designing effective therapies for autoimmune diseases. We probed a panel of Abs with T cell-modulating activity and identified a mAb specific for the H chain of CD98 (CD98hc) that was able to suppress T cell proliferation. The anti-CD98hc mAb also inhibited Ag-specific proliferation and the acquisition of effector function by CD4(+) and CD8(+) T cells in vitro and in vivo. Injection of the anti-CD98hc mAb completely prevented the onset of cyclophosphamide-induced diabetes in NOD mice. Treatment of diabetic NOD mice with anti-CD98hc reversed the diabetic state to normal levels, coincident with decreased proliferation of CD4(+) T cells. Furthermore, treatment of diabetic NOD mice with CD98hc small interfering RNA resolved T1D. These data indicate that strategies targeting CD98hc might have clinical application for treating T1D and other T cell-mediated autoimmune diseases.     

Anti-human CD4 induces peripheral tolerance in a human CD4+, murine CD4-, HLA-DR+ advanced transgenic mouse model

Selection in vivo of potent mAbs to human CD4 useful for immunotherapy, e.g., for the induction of immunological tolerance, is restricted for ethical reasons. We therefore used multiple transgenic mice that lack murine CD4, but express human CD4 specifically on Th cells, and HLA-DR3 as its natural counterligand (CD4/DR3 mice). The injection of CD4/DR3 mice with anti-human CD4 (mAb Max.16H5) before immunization with tetanus toxoid (TT, day 0) totally blocked the formation of specific Abs. This state of unresponsiveness persisted a subsequent boost again performed in the presence of anti-human CD4. When these mice were left untreated for at least 40 days, and were then re-exposed with TT, but in the absence of anti-human CD4, they consistently failed to induce specific Abs (long-term unresponsiveness). Exposure to second party Ags (hen egg lysozyme, human acetylcholine receptor) induced specific Abs comparable with control mice, demonstrating that the anti-CD4-induced unresponsiveness was Ag specific (immunological tolerance). Importantly, the concurrent injection of TT and anti-human CD4 at day 0, followed by another two anti-CD4 treatments, also led to tolerant animals, indicating that tolerance was inducible at the same day as the Ag exposure is provided. We finally demonstrate a limited ability of spleen cells to respond to TT in vitro, indicating that T cells are essentially involved in the maintenance of TT-specific tolerance. These data show for the first time that the human CD4 coreceptor mediates tolerance-inducing signals when triggered by an appropriate ligand in vivo.     

Cytotoxic T lymphocyte triggering via CD16 is regulated by CD3 and CD8 antigens. Studies with T cell receptor (TCR)-alpha beta+/CD3+16+ and TCR-gamma delta+/CD3+16+ granular lymphocytes

The role of CD3 and CD8 Ag in CD16-mediated CTL triggering was studied in TCR-alpha beta+ and TCR-gamma delta+ granular lymphocytes (GL). In TCR-alpha beta+/CD3+4-8+16+ GL obtained from patients with GL-proliferative disorders, antibody-dependent cellular cytotoxicity was inhibited by anti-CD3 and anti-CD8 mAb. Anti-CD3 mAb also inhibited antibody-dependent cellular cytotoxicity activity of TCR-gamma delta+/CD3+4-8-16+ GL from a patient and that of TCR-gamma delta+/CD3+4-8+/-16+ T cell clones established from patients with proliferating TCR-gamma delta+ GL. In TCR-gamma delta+ T cell clones, cytotoxicity against Fc gamma R+ targets was induced by stimulation of CD16 Ag with anti-CD16 mAb, and such cytotoxicity was also inhibited by anti-CD3 mAb. These results indicate that CD3 and CD8 molecules play a regulatory role in CD16-mediated CTL triggering.