T-cell Receptor Transgenic Mice Research Articles

Maintenance of TCR clonality in T cells expressing genes for two TCR heterodimers.

T cell receptor (TCR) allelic exclusion is believed to be primarily mediated by suppression of further recombination at the TCR locus after the expression of a functional TCR protein. Genetic allelic exclusion has been shown to be leaky for the beta chain and, more commonly, for the alpha chain. Here, we demonstrate an additional mechanism by which T cells can maintain monoclonality. T cells from double TCR transgenic mice express only one or the other of the two available TCRs at the cell surface. This "functional allelic exclusion" is apparently due to control of the TCR assembly process because these T cells express RNA and protein for all four transgenic TCR proteins. Lack of cell surface expression of the second TCR may be controlled by a failure to assemble the TCR heterodimer.

T–cell anergy and peripheral T–cell tolerance

The discovery that T-cell recognition of antigen can have distinct outcomes has advanced understanding of peripheral T-cell tolerance, and opened up new possibilities in immunotherapy. Anergy is one such outcome, and results from partial T-cell activation. This can arise either due to subtle alteration of the antigen, leading to a lower-affinity cognate interaction, or due to a lack of adequate co-stimulation. The signalling defects in anergic T cells are partially defined, and suggest that T-cell receptor (TCR) proximal, as well as downstream defects negatively regulate the anergic T cell's ability to be activated. Most importantly, the use of TCR-transgenic mice has provided compelling evidence that anergy is an in vivo phenomenon, and not merely an in vitro artefact. These findings raise the question as to whether anergic T cells have any biological function. Studies in rodents and in man suggest that anergic T cells acquire regulatory properties; the regulatory effects of anergic T cells require cell to cell contact, and appear to be mediated by inhibition of antigen-presenting cell immunogenicity. Close similarities exist between anergic T cells, and the recently defined CD4+ CD25+ population of spontaneously arising regulatory cells that serve to inhibit autoimmunity in mice. Taken together, these findings suggest that a spectrum of regulatory T cells exists. At one end of the spectrum are cells, such as anergic and CD4+ CD25+ T cells, which regulate via cell-to-cell contact. At the other end of the spectrum are cells which secrete antiinflammatory cytokines such as interleukin 10 and transforming growth factor-beta. The challenge is to devise strategies that reliably induce T-cell anergy in vivo, as a means of inhibiting immunity to allo- and autoantigens.

Phagocytic dendritic cells from myelomas activate tumor-specific T cells at a single cell level

AbstractAntigen-presenting cells (APCs) from subcutaneous mouse MOPC315 plasmacytoma phagocytosed immunoglobulin G–coated magnetic beads, enabling efficient isolation within 2 hours by magnetic separation (APC-MB). Cell morphology was heterogeneous, with some of the cells having dendrites. The surface phenotype of purified tumor APCs-MB was CD11b+, CD11c+, CD40+, CD80+, CD86+, and MHC class II+. Tumor APCs-MB expressed messenger RNA for fractalkine and ABCD-1 chemokines, and for CC-type chemokine receptors CCR5 and CCR7, indicating the presence of mature dendritic cells (DCs). Visualized at a single cell level within 4 hours after disruption of the tumor, APCs-MB induced rapid Ca++ mobilization in MHC class II–restricted tumor idiotype (Id)–specific cloned CD4+ T cells. In long-term assays, tumor APCs-MB induced proliferation of naive T cells from Id-specific T-cell receptor transgenic mice. The results suggest that tumor APCs-MB represent a heterogeneous cell population that includes myeloid-derived DCs of various stages of maturation. A considerable fraction (≥ 15%) of DCs is spontaneously primed with tumor-specific antigen.

TH2 Lymphocytes Secrete Functional VIP upon Antigen Stimulation

In addition to the peptidergic innervation, immune cells may also represent a source for VIP in the lymphoid organs. Previous studies reported increased VIP mRNA and protein expression in mitogen-stimulated B and T lymphocytes. To determine whether specific T cell subsets are responsible for VIP production, we derived TH1 and TH2 effector cell lines from T-cell receptor transgenic mice. TH1 and TH2 cells were stimulated with the specific (pigeon cytochrome C peptide) or nonspecific (ovalbumin) antigen presented by MHC class II compatible antigen-presenting cells. Upon stimulation with the specific antigen, TH2, but not TH1 cells express VIP mRNA and intracelllular VIP protein, as determined by Northern blots and FACS analysis. Supernatants harvested from antigen-stimulated TH2 cells contain secreted VIP, as determined by Elisa, and induce cAMP in HEK293 cells transfected with the specific VIP/PACAP receptor VPAC1. These results confirm that TH2, but not TH1 cells, express and secrete functional VIP following specific antigen stimulation. The release of VIP within the lymphoid microenvironment following antigenic stimulation provides a physiological basis for the immunoregulatory effects of VIP on neighboring immune cells, such as downregulation of macrophage activation, effects on lymphocyte migration, on antigen-induced T cell apoptosis, and on T cell differentiation.

A murine model of allogeneic adrenocortical cell transplantation: Perspectives for the treatment of Addison's disease in humans

Background. Hormone substitution for the treatment of adrenocortical insufficiency (Addison's disease) does not adequately substitute the hormone peaks required in stress situations. Therefore, allogeneic transplantation of adrenal cortex could offer an intriguing alternative. Methods. Major histocompatibility complex (MHC) class I transgenic mice were used for the implementation of an animal model of adrenocortical cell transplantation in adrenalectomized mice. Kb-transgenic cells and allogeneic adrenocortical cells were cocultured in mixed lymphocyte cultures to examine the alloimmune response. Lymphocytes from T-cell receptor transgenic mice and normal allogeneic mice served as responder cells. The effect of corticosteroids secreted by adrenocortical cells was antagonized by the steroid receptor antagonist mifepristone (RU486). Results. In vitro coculture experiments showed that MHC class I disparate adrenocortical cells failed to activate B10.BR and T-cell receptor transgenic lymph node cells. In the presence of mifepristone this inhibitory effect was antagonized, resulting in strong lymphocyte proliferation. Activation of B10.BR lymphocytes by Kb-disparate spleen cells was also abolished in the presence of adrenocortical cells. This effect, however, could not be reversed by mifepristone. Conclusions. In vitro, the presence of adrenocortical cells potently suppressed allogeneic immune responses. This effect was only in part due to the secretion of corticosteroids, pointing to an additional immunomodulatory property of adrenocortical cells. (Surgery 2000;128:999-1006.)

T-cell activation and receptor downmodulation precede deletion induced by mucosally administered antigen.

The fate of antigen-specific T cells was characterized in myelin basic protein (MBP) T-cell receptor (TCR) transgenic (Tg) mice after oral administration of MBP. Peripheral Th cells are immediately activated in vivo, as indicated by upregulation of CD69 and increased cytokine responses (Th1 and Th2). Concurrently, surface TCR expression diminishes and internal TCR levels increase. When challenged for experimental autoimmune encephalomyelitis during TCR downmodulation, Tg mice are protected from disease. To characterize Th cells at later times after antigen feeding, it was necessary to prevent thymic release of naive Tg cells. Therefore, adult Tg mice were thymectomized before treatment. TCR expression returns in thymectomized Tg mice 3 days after MBP feeding and then ultimately declines in conjunction with MBP-specific proliferation and cytokine responses (Th1-type and Th2-type). The decline correlates with an increase in apoptosis. Collectively, these results demonstrate that a high dose of fed antigen induces early T-cell activation and TCR downmodulation, followed by an intermediate stage of anergy and subsequent deletion.

T-cell receptor antagonists induce Vav phosphorylation by selective activation of Fyn kinase.

T cell receptor (TCR) antagonists inhibit antigen-induced T cell activation and by themselves fail to induce phenotypic changes associated with T cell activation. However, we have recently shown that TCR antagonists are inducers of antigen-presenting cell (APC)-T cell conjugates. The signaling pathway associated with this cytoskeleton-dependent event appears to involve tyrosine phosphorylation and activation of Vav. In this study, we investigated the role played by the protein tyrosine kinases Fyn, Lck, and ZAP-70 in antagonist-induced signaling pathway. Antagonist stimulation increased tyrosine phosphorylation and kinase activity of Fyn severalfold, whereas little or no increase in Lck and ZAP-70 activity was observed. Second, TCR stimulation of Lck(-), Fyn(hi) Jurkat cells induced strong tyrosine phosphorylation of Vav. In contrast, minimal increase in tyrosine phosphorylation of Vav was observed in Lck(hi), Fyn(lo) Jurkat cells. Finally, study of T cells from a Fyn-deficient TCR transgenic mouse also showed that Fyn was required for tyrosine phosphorylation and activation of Vav induced by both antagonist and agonist peptides. The deficiency in Vav phosphorylation in Fyn-deficient T cells was associated with a defect in the formation of APC-T cell conjugates when T cells were stimulated with either agonist or antagonist peptide. We conclude from these results that Vav is a selective substrate for Fyn, especially under conditions of low-affinity TCR-mediated signaling, and that this signaling pathway involving Fyn, Vav, and Rac-1 is required for the cytoskeletal reorganization that leads to T cell-APC conjugates and the formation of the immunologic synapse.

The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte-antigen-presenting cell interface.

During the interaction of a T cell with an antigen-presenting cell (APC), several receptor ligand pairs, including the T cell receptor (TCR)/major histocompatibility complex (MHC), accumulate at the T cell/APC interface in defined geometrical patterns. This accumulation depends on a movement of the T cell cortical actin cytoskeleton toward the interface. Here we study the involvement of the guanine nucleotide exchange factor vav in this process. We crossed 129 vav(-/-) mice with B10/BR 5C.C7 TCR transgenic mice and used peptide-loaded APCs to stimulate T cells from the offspring. We found that the accumulation of TCR/MHC at the T cell/APC interface and the T cell actin cytoskeleton rearrangement were clearly defective in these vav(+/-) mice. A comparable defect in superantigen-mediated T cell activation of T cells from non-TCR transgenic 129 mice was also observed, although in this case it was more apparent in vav(-/-) mice. These data indicate that vav is an essential regulator of cytoskeletal rearrangements during T cell activation.

Differential requirement of perforin and IFN-gamma in CD8 T cell-mediated immune responses against B16.F10 melanoma cells expressing a viral antigen.

Perforin-mediated lysis and secretion of IFN-gamma belong to the key effector functions of CD8 T cells. To compare the anti-tumor activity of these two mechanisms, we used B16.F10 melanoma cells (B16GP33) expressing the cytotoxic T cell epitope GP33 and T cell receptor transgenic (TCR-tg) mice specific for GP33 and deficient in perforin or IFN-gamma. B16GP33 tumor cells, injected either i.v. to induce experimental metastases or s.c., were similarly controlled in both wild-type and perforin-deficient, but not in IFN-gamma-deficient TCR-tg mice. A similar result was obtained when the therapeutic efficacy of adoptively transferred TCR-tg effector cells from these mice was examined in the corresponding perforin- or IFN-gamma-deficient C57BL/6 hosts. Criss-cross experiments further revealed that IFN-gamma production by the host strongly influenced the efficiency of the adoptively transferred effector cells. In contrast to the potent ability of GP33-specific effector cells to mediate B16GP33 tumor regression without perforin, GP33-specific memory cells, induced with recombinant vaccinia virus expressing GP33, failed to control B16GP33 tumor growth in the absence of perforin. In conclusion, our data demonstrate a crucial role for IFN-gamma in B16GP33 tumor cell elimination in vivo and indicate a differential requirement of perforin by effector versus memory CD8 T cells in anti-tumor immunity.

Nondeletional T-cell receptor transgenic mice: model for the CD4(+) T-cell repertoire in chronic hepatitis B virus infection.

Chronicity after infection with the hepatitis B virus (HBV) can occur for a variety of reasons. However, once established, chronicity may be maintained by high levels of viral proteins circulating in the serum. To examine the characteristics of T cells capable of coexisting with the secreted hepatitis B e antigen (HBeAg), T-cell receptor (TCR) transgenic (Tg) mice were produced. To ensure that HBeAg-specific T cells would not be deleted in the presence of serum HBeAg, the TCR alpha- and beta-chain genes used to produce the TCR-Tg mice were derived from T-cell hybridomas produced from immunizing HBeAg-Tg mice. A TCR-Tg lineage (11/4-12) was produced that possessed a high frequency ( approximately 67%) of CD4(+) T cells that expressed a Tg TCR specific for the HBeAg. As predicted, when 11/4-12 TCR-Tg mice were bred with HBeAg-Tg mice no deletion of the HBeAg-specific CD4(+) T cells occurred in the thymus or the spleen. Functional analysis of the TCR-Tg T cells revealed that the HBeAg-specific CD4(+) T cells escaped deletion in the thymus and periphery by virtue of low avidity. Regardless of their low avidity, HBeAg-specific TCR-Tg T cells could be activated by exogenous HBeAg, as measured by cytokine production in vitro and T-helper-cell function for anti-HBe antibody production in vitro and in vivo. Furthermore, activated TCR-Tg HBeAg-specific T cells polarized to the Th1 subset were able to elicit liver injury when transferred into HBeAg or HBcAg-Tg recipients. Therefore, HBeAg-specific CD4(+) T cells that can survive deletion or anergy in the presence of circulating HBeAg nonetheless are capable of being activated and of mediating liver injury in vivo. The 11/4-12 TCR-Tg lineage may serve as a monoclonal model for the HBe/HBcAg-specific CD4(+) T-cell repertoire present in chronically infected HBV patients.

The Fas antigen is involved in thymic T-cell development as a costimulatory molecule, but not in the deletion of neglected thymocytes

To determine whether the Fas antigen (Fas) is involved in thymic T-cell development, we introduced the lymphoproliferation (lpr) mutation into a T-cell receptor-αβ transgenic mouse (DO10 mouse) and generated 4 genotypes of T-cell receptor transgenic mice homozygous or heterozygous for the lpr mutation with selecting or nonselecting H-2 haplotype. Unexpectedly, we found that the homozygous Fas mutation (lpr/lpr) induced a marked reduction in CD4+CD8+ double-positive (DP) thymocytes in mice with nonselecting background and that the thymus showed severe cortical atrophy. We also found that the homozygous Fas mutation inhibited the activation of DP thymocytes in the process of positive selection, as indicated by the lower levels of CD5 and CD69 expressions on DP thymocytes in lpr/lpr mice with both selecting and nonselecting background than those of lpr/+ mice. Furthermore, we found a significant skewing from CD4+ to CD8+ single-positive thymocytes in lpr/lpr mice with nonselecting background compared with that in the corresponding lpr/+ mice. Taken together, these results indicate that Fas is involved in thymic T-cell development, DP thymocyte generation and positive selection, as a costimulatory molecule but is not involved in the deletion of neglected thymocytes. (J Allergy Clin Immunol 2000;106:S19-31.)

Eradication of Cryptosporidium parvum infection by mice with ovalbumin-specific T cells.

CD154 is necessary for mice to clear a Cryptosporidium parvum infection, but whether this ligand has to be expressed on T cells with specificity for C. parvum has not been determined. We infected DO11.10 (ovalbumin specific) T-cell receptor transgenic mice that had been bred to a RAG(-/-) background with C. parvum and found that the infection was cleared within 6 weeks, while RAG(-/-) controls were unable to clear C. parvum infection. Recovery was accompanied by an increase in the number of splenic T cells with the CD44(high) phenotype that characterizes memory cells. To determine whether a C. parvum-infected environment sufficed to activate transgenic T cells, we reconstituted C. parvum-infected BALB/c SCID mice with DO11.10 RAG(-/-) splenocytes. Fecal excretion of C. parvum antigen ceased in the 12 weeks following the adoptive transfer, unless the mice were also injected with tolerizing doses of ovalbumin. DO11.10 T cells were found in the submucosa of C. parvum-infected, but not uninfected, BALB/c SCID hosts within 48 h of injection. The transferred DO11.10 T cells divided and acquired a CD44(high) memory phenotype in C. parvum-infected, but not uninfected, recipients. DO11.10 splenocytes from CD154 knockout donors failed to clear a C. parvum infection, confirming a requirement for CD154 in recovery. In vitro, the DO11.10 cells did not proliferate in response to C. parvum antigen, and a tBlast GenBank search revealed no matches between the ovalbumin peptide and C. parvum DNA sequences. C. parvum-infected SCID mice given RAG(-/-) CD8(+) T cells with a Listeria-specific transgene did not recover from C. parvum infection. Our data suggest that antigen-nonspecific CD4(+) T-cell effector mechanisms in combination with the innate arm of the immune system are sufficient for the eradication of C. parvum infection.

Host resistance and immune deviation in pigeon cytochrome c T-cell receptor transgenic mice infected with Toxoplasma gondii.

Resistance to Toxoplasma gondii has been shown to be mediated by gamma interferon (IFN-gamma) produced by NK, CD4(+), and CD8(+) T cells. While studies of SCID mice have implicated NK cells as the source of the cytokine in acute infection, several lines of evidence suggest that IFN-gamma production by CD4(+) T lymphocytes also plays an important role in controlling early parasite growth. To evaluate whether this function is due to nonspecific as opposed to T-cell receptor (TCR)-dependent stimulation by the parasite, we have examined the resistance to T. gondii infection of pigeon cytochrome c transgenic (PCC-Tg) Rag-2(-/-) mice in which all CD4(+) T lymphocytes are unreactive with the protozoan. When inoculated with the ME49 strain, PCC-Tg animals exhibited only temporary control of acute infection and succumbed by day 17. Intracellular cytokine staining by flow cytometry revealed that, in contrast to infected nontransgenic controls, infected PCC-Tg animals failed to develop IFN-gamma-producing CD4(+) T cells. Moreover, the CD4(+) lymphocytes from these mice showed no evidence of activation as judged by lack of upregulated expression of CD44 or CD69. Nevertheless, when acutely infected transgenic mice were primed by PCC injection, the lymphokine responses measured after in vitro antigen restimulation displayed a strong Th1 bias which was shown to be dependent on endogenous interleukin 12 (IL-12). The above findings argue that, while T. gondii-induced IL-12 cannot trigger IFN-gamma production by CD4(+) T cells in the absence of TCR ligation, the pathogen is able to nonspecifically promote Th1 responses against nonparasite antigens, an effect that may explain the immunostimulatory properties of T. gondii infection.

Serum IgE response to orally ingested antigen: A novel IgE response model with allergen-specific T-cell receptor transgenic mice

Background: The mechanism by which orally ingested allergens elicit an IgE response remains unclear because there are few animal models available for investigation of this response. Objective: We tried to develop a murine model suitable for investigation of the IgE response to orally ingested allergens, which would allow us to identify T cells that could promote IgE production. Methods: Ovalbumin (OVA)-specific T-cell receptor transgenic mice were fed a diet containing OVA, and both the serum antibody response and cytokine production by splenocytes were examined. Results: Oral administration of OVA to transgenic mice led to an increase in the levels of both antigen-specific IgE and total IgE in the sera. Subsequent intravenous challenge of OVA-fed transgenic mice with OVA resulted in anaphylactic shock. Analysis of cytokine production by splenocytes revealed that high IL-4–producing T cells appeared in the spleen 1 week after the start of feeding the OVA diet. T cells from these mice were found to promote IgE secretion by BALB/c B cells in vitro. This helper activity and the levels of IL-4 secretion were diminished after long-term feeding. These findings suggest the possibility that the orally ingested antigen elicited a response by a subpopulation of T cells that produce high levels of TH2-type cytokines and that promote IgE secretion, and these same T cells were tolerized by the orally ingested antigen. Conclusion: This experimental model with transgenic mice may be a useful tool for further studies of the cellular and molecular mechanisms of the T-cell and IgE responses to orally ingested antigens. (J Allergy Clin Immunol 2000;105:788-95.)

Fulminant spontaneous autoimmunity of the central nervous system in mice transgenic for the myelin proteolipid protein-specific T cell receptor.

Proteolipid protein (PLP)-139-151 is the dominant encephalitogenic peptide that induces experimental autoimmune encephalomyelitis (EAE) in SJL (H-2(s)) mice. To examine the contribution of T cell receptor (TCR) specificity in the induction of EAE, we generated transgenic mice expressing the rearranged TCR genes from an encephalitogenic or a nonencephalitogenic PLP-139-151/I-A(s)-specific T cell clone. Both types of transgenic lines developed spontaneous EAE, but, remarkably, the lines expressing the TCR from the nonencephalitogenic clone showed increasingly higher frequencies of disease (60-83%) in progressive SJL backcrosses and could not be propagated on the susceptible background. The T cells from the transgenic mice were not tolerized, because they responded vigorously to the antigen in vitro and mediated EAE when the mice were immunized with antigen. Besides being the only description of a TCR transgenic mice for the PLP-139-151/I-A(s) epitope, the results demonstrate that the TCR from a nonencephalitogenic PLP-specific T cell clone can induce autoimmune disease when expressed appropriately in vivo.

Dendritic cells purified from myeloma are primed with tumor-specific antigen (idiotype) and activate CD4+ T cells.

Multiple myelomas produce tumor-specific antigen (TSA) in the form of idiotype (Id) on monoclonal Ig. CD4(+) T cells can recognize Id-peptide on MHC class II molecules and protect against challenges with MOPC315 cells, which are, as common for myelomas, class II-negative. The present study explains these previous results by demonstrating that Id can be transferred from myeloma cells to antigen-presenting cells (APC), which present processed Id-peptide on their class II molecules to Id-specific T cell receptor-transgenic (TCR-TG) CD4(+) T cells. Id-primed tumor APC were heterogeneous, the majority being dendritic cells with class II(+), CD11b(+) CD11c(+) CD40(+) CD80(+) CD86(+) markers. The APC were localized beneath CD31(+) endothelial cells of tumor microvessels, and their frequency declined with tumor progression. The APC could stimulate Id-specific naive TCR-TG, short-term polarized TCR-TG, and cloned CD4(+) T cells to proliferate and produce cytokines in vitro. Furthermore, small MOPC315 tumors established in Id-specific TCR-TG mice contained clusters of activated (CD69(+)CD25(+)) and proliferating (BrdUrd(+)) Id-specific transgenic CD4(+) blasts. The activated Id-specific T cells were located adjacent to Id-primed dendritic cells in the tumor. Thus, a TSA can be transferred in vivo from myeloma, and possibly other types of cancer cells to APC for MHC class II presentation to CD4(+) T cells.

Injury primes the immune system for an enhanced and lethal T-cell response against bacterial superantigen.

We previously reported the high lethality of CD4+ T-cell activation in burn-injured T-cell receptor (TCR) transgenic mice. This suggested to us that T-cells may play a role in the development of the systemic inflammatory response syndrome (SIRS) which can occur after severe injury. In this study, we sought a more clinically relevant model to test the hypothesis that naturally produced bacterial toxins that are known to act as potent polyclonal T-cell activating agents may induce a similar lethal shock-like response in injured, non-TCR transgenic mice. Accordingly, sham- or burn-injured mice were treated with various doses of staphylococcal enterotoxin A (SEA), then observed for 48-hour mortality. We observed 94% and 56% 48-h mortality when burn-injured mice were given 15 microg and 10 microg of SEA, respectively, while neither SEA dose caused mortality in sham-injured mice. The assessment of serum cytokine levels demonstrated significantly elevated interleukin 2 (IL-2) and tumor necrosis factor alpha (TNFalpha) levels when compared to sham mice (P < 0.01). In vitro studies confirmed our in vivo results and also demonstrated elevated levels of interferon gamma (IFNgamma) (P < 0.01). We also observed a novel injury-dependent switch from CD4+ to CD8+ T-cells as the dominant T-cell type producing TNFalpha and IFNgamma in response to SEA stimulation in vitro. Taken together, our findings indicate that injury primes the immune system for an augmented early T-cell response that can result in a lethal shock-like syndrome.

Mechanisms of tolerance induction after intrathymic islet injection: determination of the fate of alloreactive thymocytes.

Intrathymic (IT) administration of antigen when combined with peripheral T-cell depletion has been shown to induce operational tolerance in a wide range of experimental protocols. IT injection of pancreatic islets has been demonstrated not only to induce tolerance to alloantigen but also to prevent the development of autoimmune beta-cell destruction in models of type I diabetes. However, little is known about the mechanisms involved in tolerance induction after IT islet injection. A protocol for the induction of tolerance to fully allogeneic (C57BL/10; H2b) peripheral islet allografts was developed in CBA/Ca (H2k) recipients by the IT injection of allogeneic islets combined with depletion of peripheral CD4+ T cells. This protocol was based upon our own data and those of others showing that CD4+ T cells play a critical role in islet allograft rejection. Using this regimen, donor-type peripheral islet allografts survived indefinitely whereas third-party grafts were rejected. To determine the fate of alloreactive thymocytes that recognize donor major histocompatibility complex antigens via the direct pathway, T-cell receptor transgenic mice specific for the major histocompatibility complex class I molecule Kb (BM3 and DES) were used as recipients. IT injection of islets expressing the specific alloantigen Kb resulted in clonal deletion of alloreactive thymocytes in T-cell receptor transgenic recipients. No evidence of clonal inactivation in the residual peripheral alloreactive population was observed in this system. IT injection of allogeneic islets and concomitant CD4+ T-cell depletion is able to induce donor-specific unresponsiveness. One mechanism responsible for this unresponsiveness is the clonal deletion of thymocytes that recognize alloantigen via the direct pathway.

Tolerance and autoimmunity in TCR transgenic mice specific for myelin basic protein.

Summary: T‐cell receptor (TCR) transgenic mice provide the ability to follow the maturation and fate of T cells specific for self‐antigens in vivo. This technology represents a major breakthrough in the study of autoimmune diseases in which specific antigens have been implicated. Proteins expressed within the central nervous system are believed to be important autoantigens in multiple sclerosis, TCR transgenic models specific for myelin basic protein (MBP) allowed us to assess the role of tolerance in providing protection from T cells with this specificity Our studies demonstrate that T cells specific for the immunodominant epitope of MBP do not undergo tolerance in vivo and that TCR transgenic mice are susceptible to spontaneous autoimmune disease. The susceptibility to spontaneous disease is dependent on exposure to microbial antigens, MBP TCR transgenic models expressing TCRs specific for the same epitope of MBP but utilizing different Vα genes exhibit differing susceptibilities to, spontaneous disease. These data support the idea that genetic and environmental differences play a role in susceptibility to autoimmunity MBP TCR transgenic models are playing an important role in defining mechanisms by which infectious agents trigger autoimmune disease as well as defining mechanisms by which tolerance is induced to distinct epitopes within self‐antigens.

Induction of cytokine production in naive CD4 + T cells by antigen-presenting murine liver sinusoidal endothelial cells but failure to induce differentiation toward T h1 cells

Background & Aims: Murine liver sinusoidal endothelial cells (LSECs) constitutively express accessory molecules and can present antigen to memory T h1 CD4 + T cells. Using a T-cell receptor transgenic mouse line, we addressed the question whether LSECs can prime naive CD4 + T cells. Methods: Purified LSECs were investigated for their ability to induce activation and differentiation of naive CD4 + T cells in comparison with bone marrow–derived antigen-presenting cells and macrovascular endothelial cells. Activation of T cells was determined by cytokine production. LSECs were further studied for expression of interleukin (IL)-12 by reverse-transcription polymerase chain reaction, and the unique phenotype of LSECs was determined by flow cytometry. Results: We provide evidence that antigen-presenting LSECs can activate naive CD62L high CD4 + T cells. Activation of naive CD4 + T cells by LSECs occurred in the absence of IL-12. In contrast, macrovascular endothelial cells from aorta could not activate naive CD4 + T cells. The unique functional characteristics of microvascular LSECs together with a unique phenotype (CD4 +, CD11b +, CD11c +, CD80 +, CD86 +) make these cells different from macrovascular endothelial cells. Furthermore, LSECs did not require in vitro maturation to activate naive CD4 + T cells. Most importantly, LSECs failed to induce differentiation toward T h1 cells, whereas conventional antigen-presenting cell populations induced a T h1 phenotype in activated CD4 + T cells. Upon restimulation, CD4 + T cells, which were primed by antigen-presenting LSECs, expressed interferon gamma, IL-4, and IL-10, which is consistent with a T h0 phenotype. Exogenous cytokines (IL-1β, IL-12, or IL-18) present during T-cell priming by antigen-presenting LSECs could not induce a T h1 phenotype, but neutralization of endogenously produced IL-4 during T-cell priming led to a reduced expression of IL-4 and IL-10 by CD4 + T cells upon restimulation. The addition of spleen cells to cocultures of LSECs and naive CD4 + T cells during T-cell priming led to differentiation of T cells toward a T h1 phenotype. Conclusions: The ability of antigen-presenting LSECs to induce cytokine expression in naive CD4 + T cells and their failure to induce differentiation toward a T h1 phenotype may contribute to the unique hepatic microenvironment that is known to promote tolerance. GASTROENTEROLOGY 1999;116:1428-1440