Search

This study highlights the key role of polycytotoxic CD8+ T cells in the severity of toxic epidermal necrolysis., Toxic epidermal necrolysis (TEN) is a life-threatening cutaneous adverse drug reaction. To better understand why skin symptoms are so severe, we conducted a prospective immunophenotyping study on skin and blood. Mass cytometry results confirmed that effector memory polycytotoxic CD8+ T cells (CTLs) are the main leucocytes in TEN blisters at the acute phase. Deep T cell receptor (TCR) repertoire sequencing identified massive expansion of unique CDR3 clonotypes in blister cells. The same clones were highly expanded in patient’s blood, and the degree of their expansion showed significant correlation with disease severity. By transducing α and β chains of the expanded clonotypes into a TCR-defective cell line, we confirmed that those cells were drug specific. Collectively, these results suggest that the relative clonal expansion and phenotype of skin-recruited CTLs condition the clinical presentation of cutaneous adverse drug reactions.

Several populations of memory T cells have been described that differ in their migration and function. In this study, we have identified a unique subset of memory T cells, which we have named recirculating memory T cells (TRCM). By exposing Kaede transgenic mouse skin to violet light, we tracked the fate of cutaneous T cells. One population of memory CD4+ T cells remained in the skin. A second population migrated from the skin into draining lymph nodes (LNs) in a CCR7-dependent manner. These migrating CD4+ T cells expressed a novel cell surface phenotype (CCR7int/+CD62LintCD69−CD103+/− E-selectin ligands+) that is distinct from memory T cell subsets described to date. Unlike memory T cell subsets that remain resident within tissues long-term, or that migrate either exclusively between lymphoid tissues or into peripheral nonlymphoid sites, CD4+ TRCM migrate from the skin into draining LNs. From the draining LNs, CD4+ TRCM reenter into the circulation, distal LNs, and sites of non-specific cutaneous inflammation. In addition, CD4+ TRCM upregulated CD40L and secreted IL-2 following polyclonal stimulation. Our results identify a novel subset of recirculating memory CD4+ T cells equipped to deliver help to both distal lymphoid and cutaneous tissues.

Tregs play an important role in protecting the skin from autoimmune attack. However, the extent of Treg trafficking between the skin and draining lymph nodes (DLNs) is unknown. We set out to investigate this using mice engineered to express the photoconvertible fluorescence protein Kaede, which changes from green to red when exposed to violet light. By exposing the skin of Kaede-transgenic mice to violet light, we were able to label T cells in the periphery under physiological conditions with Kaede-red and demonstrated that both memory phenotype CD4+Foxp3- non-Tregs and CD4+Foxp3+ Tregs migrated from the skin to DLNs in the steady state. During cutaneous immune responses, Tregs constituted the major emigrants and inhibited immune responses more robustly than did LN-resident Tregs. We consistently observed that cutaneous immune responses were prolonged by depletion of endogenous Tregs in vivo. In addition, the circulating Tregs specifically included activated CD25hi Tregs that demonstrated a strong inhibitory function. Together, our results suggest that Tregs in circulation infiltrate the periphery, traffic to DLNs, and then recirculate back to the skin, contributing to the downregulation of cutaneous immune responses.

The transfer of nuclei of fully differentiated cells into enucleated oocytes is a well-recognized method for the generation of embryonic stem (ES) cells. Here, we demonstrate that nuclear transferred ES (NT-ES) cells can be established with high efficiency using innate-like B lymphocytes as donor cells. We established two mouse lines carrying rearranged immunoglobulin heavy and light chains using NT-ES cells containing nuclei from peritoneal cavity B1 cells. Analysis of B1 clone lines revealed that the B1-cell generation critically depends on the interaction between antigen (possibly self-antigen) and surface immunoglobulin, while the B1-cell maintenance requires the peritoneal environment. The B1-cell expansion takes place in spleen, and is held in check by competitor B2 cells. The results indicate that the NT-ES method could replace the transgenic or knock-in mouse approaches currently used to study the biology of cells that undergo somatic rearrangements of their antigen receptor genes.

The trans presentation of IL-15 by cells expressing the specific high-affinity receptor α-chain (IL-15Rα) to cells expressing the signaling receptor β-chain and γ-chain is essential for the generation and maintenance of CD8 memory T cells, NK cells, and NKT cells in an in vivo mouse system. We have also demonstrated in vitro that cell-surface IL-15Rα on cells expressing all the receptor components present IL-15 to receptor β-chain/γ-chain coexpressed on the same cell surface (cis presentation). However, although mouse CD8 T cells express all the IL-15R components, they show no evidence of cis presentation. In this study, we demonstrate that increased expression of mouse IL-15Rα in mouse CD8 T cells by retrovirus-mediated gene transfer changes the ability of the T cell to use cis presentation on the cell surface, indicating that cis presentation requires high expression of mouse IL-15Rα on the cell surface. Using cell lines expressing human or mouse receptors, we demonstrate that cis presentation occurs more efficiently in the human receptor–ligand combination than in that of the mouse system. Moreover, we found that primary human CD8 T cells do not require trans presentation of human IL-15 in vitro. These findings raise the possibility that the maintenance and generation of memory CD8 T cells are achieved via distinct mechanisms in humans and mice. Therefore, careful study of the human immune system, rather than extrapolation from the murine model, is necessary to achieve more complete understanding of memory CD8 T cell development in humans.

Hyperplasia associated with a loss of tissue homeostasis can induce DNA replication stress, leading to precancerous dysregulation. Epidermal {gamma}{delta} T cells reside in the primary barrier that protects against diverse environmental insults; however, the functions of these T cells in tissue surveillance are not completely understood. In mice with inducible Notch1 inactivation in keratinocytes that causes epidermal hyperplasia, epidermal {gamma}{delta} T cells sensed stressed keratinocytes and migrated into the cutaneous draining lymph nodes. Simultaneous induction of β-galactosidase (β-Gal) as a putative antigen expressed in the process of precancerous dysregulation and Notch1 ablation in the epidermis resulted in elevated β-Gal-specific IgG2a production. Epidermal {gamma}{delta} T cells were found to have the capacity to express chemokine (C-C motif) receptor 7 and migrate into the lymph nodes. Cutaneous draining lymph node cells in Notch1-inactivated mice expressed high levels of IFN-{gamma} upon anti-CD3 plus anti-CD28 stimulation. Furthermore, induced expression of β-Gal in mice that lacked epidermal {gamma}{delta} T cells failed to induce anti-β-Gal IgG. These results suggest that epidermal {gamma}{delta} T cells play an essential role in the initiation process of epidermal antigen-specific humoral immune responses and demonstrate the importance of epidermal {gamma}{delta} T cells in sensing precancerous dysregulation and activating adaptive immunity.

IFN-gamma signaling-deficient non-obese diabetic (NOD) mice develop diabetes with similar kinetics to those of wild-type NOD mice. However, the immunization of IFN-gamma signaling-deficient NOD mice with CFA failed to induce long-term protection, whereas wild-type NOD mice receiving CFA remained diabetes-free. CFA also failed to protect IFN-gamma receptor-deficient (IFN-gammaR(-/-)) NOD mice from the autoimmune rejection of transplanted islets, as it does in diabetic NOD mice, and from disease transfer by spleen cells from diabetic NOD mice. These data clearly show that the pro-inflammatory cytokine IFN-gamma is necessary for the CFA-mediated protection of NOD mice from diabetes. There is no difference in the T(h)1/T(h)17 balance between IFN-gammaR(-/-) NOD and wild-type NOD mice. There is also no difference in the total numbers and percentages of regulatory T (Treg) cells in the lymph node CD4(+) T-cell populations between IFN-gammaR(-/-) NOD and wild-type NOD mice. However, pathogenic T cells lacking IFN-gammaR are resistant to the suppressive effect of Treg cells, both in vivo and in vitro. Therefore, it is likely that CFA-mediated protection against diabetes development depends on a change in the balance between Treg cells and pathogenic T cells, and IFN-gamma signaling seems to control the susceptibility of pathogenic T cells to the inhibitory activity of Treg cells.

Most of the immunoglobulin A (IgA) in the gut is generated by B cells in the germinal centers of Peyer's patches through a process that requires the presence of CD4 + follicular B helper T(T FH ) cells. The nature of these T FH cells in Peyer's patches has been elusive. Here, we demonstrate that suppressive Foxp3 + CD4 + T cells can differentiate into T FH cells in mouse Peyer's patches. The conversion of Foxp3 + T cells into T FH cells requires the loss of Foxp3 expression and subsequent interaction with B cells. Thus, environmental cues present in gut Peyer's patches promote the selective differentiation of distinct helper T cell subsets, such as T FH cells.

Gads is a Grb2-like adaptor protein expressed in hematopoietic cells. We demonstrated that mast cells from Gads(-/-) mice have selective functional defects. Bone marrow-derived mast cells from Gads(-/-) mice failed to induce Ca(2+) mobilization, degranulation and cytokine production upon cross-linking of FcepsilonRI. In vivo passive cutaneous anaphylaxis was also greatly impaired in Gads(-/-) mice. In contrast, Gads was dispensable for Toll-like receptor-mediated cytokine production in mast cells. Accordingly, mast cell-dependent resistance to acute peritoneal bacterial infection is not reduced in Gads(-/-) mice in vivo. Moreover, mature T and B cell responses and antibody production upon immunization were apparently normal in Gads(-/-) mice. Thus, inhibition of Gads in vivo would suppress the IgE-mediated allergic reaction with minimum adverse effects on both innate and acquired immune responses, and Gads could be an ideal target for the control of allergic responses.

Non-obese diabetic (NOD) mice that are genetically deficient in either IFN-gamma or beta chain of the IFN-gammaR develop diabetes with similar kinetics to wild-type NOD mice. In the current study, we demonstrated that treatment of IFN-gamma signaling-deficient NOD mice with cyclophosphamide (CY) not only fails to induce acute diabetes but also confers permanent protection from diabetes. Protection was mediated by the preferential generation of regulatory T cells (Treg cells) that are capable of suppressing the diabetogenic process, with no change in the total number and function of Treg cells. Moreover, CY treatment of IFN-gamma signaling-deficient NOD mice reversed the ongoing pathogenic process and eliminated cellular infiltrates of pancreatic islets. While these results have been derived using a genetically modified mouse model of diabetes, they indicate that knowledge of host genetic factors and environmental factors influencing the development of Type I diabetes mellitus may provide a rational approach to develop a means to reverse the development of Type I diabetes in human.

NF-(kappa)B is an important component of both autoimmunity and bone destruction in RA. NF-(kappa)B-inducing kinase (NIK) is a key mediator of the alternative arm of the NF-(kappa)B pathway, which is characterized by the nuclear translocation of RelB/p52 complexes. Mice lacking functional NIK have no peripheral lymph nodes, defective B and T cells, and impaired receptor activator of NF-kappaB ligand-stimulated osteoclastogenesis. We investigated the role of NIK in murine models of inflammatory arthritis using Nik-/- mice. The serum transfer arthritis model is initiated by preformed antibodies and required only intact neutrophil and complement systems in recipients. While Nik-/- mice had inflammation equivalent to that of Nik+/+ controls, they showed significantly less periarticular osteoclastogenesis and less bone erosion. In contrast, Nik-/- mice were completely resistant to antigen-induced arthritis (AIA), which requires intact antigen presentation and lymphocyte function but not lymph nodes. Additionally, transfer of Nik+/+ splenocytes or T cells to Rag2-/- mice conferred susceptibility to AIA, while transfer of Nik-/- cells did not. Nik-/- mice were also resistant to a genetic, spontaneous form of arthritis, generated in mice expressing both the KRN T cell receptor and H-2. Thus, NIK is important in the immune and bone-destructive components of inflammatory arthritis and represents a possible therapeutic target for these diseases.

CD4(-)CD8(-) thymocytes expressing a transgenic T cell receptor (TCR) alpha chain have decreased capacity to give rise to CD4(+)CD8(+) thymocytes when compared with wild-type thymocytes. This inefficient CD4(-)CD8(-) to CD4(+)CD8(+) maturation is mediated by the transgenic TCR alpha chain pairing with endogenous TCR beta chain but not with endogenous TCR gamma chain. Comparison between TCR alpha chain-transgenic mice with or without a functional pre-TCR alpha (pT alpha ) chain reveals that the formation of transgenic alpha/endogenous beta TCR on CD4(-)CD8(-) thymocytes inhibits the formation of pre-TCR, but at the same time mediates CD4(-)CD8(-) to CD4(+)CD8(+) maturation in the absence of pre-TCR, albeit inefficiently. These results indicate that alpha beta TCR and pre-TCR provide different signals for thymocyte development. They also suggest that the precise regulation of the sequential rearrangements of TCR beta and alpha loci and the cellular expansion induced by the pre-TCR may both be evolved to ensure the efficient generation of mature alpha beta T cells.

Diabetogenic BDC2.5 CD4 T cells induce diabetes when injected into NOD.scid mice. However, when co-transferred with the OVA-specific DO11.10 CD4 T cells, BDC2.5 T cells failed to cause diabetes. This inhibition depended upon the stimulation of DO11.10 T cells only with soluble OVA, which skewed their differentiation to a Th2-type pattern of cytokine secretion in vivo. However, in vivo neutralization of IL-4, IL-10 or TGF-beta using monoclonal antibodies did not prevent the inhibition whereas treatment with an antibody against the glucocorticoid-induced TNF receptor abrogated the protection from disease. In the protected mice, the diabetogenic T cells could be isolated from their spleens and shown to transfer diabetes when injected into new NOD.scid recipients. Thus, the inhibition took place without the physical or functional elimination of the diabetogenic T cells.

Costimulatory signals received by diabetogenic T cells during priming by or upon secondary encounter with autoantigen are decisive in determining the outcome of autoimmune attack. The OX40-OX40 ligand (OX40L) costimulatory pathway is known to influence T cell responses, prompting us to examine its role in autoimmune diabetes. A null allele at OX40L completely prevented diabetes development in nonobese diabetic mice and strongly reduced its incidence in a TCR transgenic model (BDC2.5). However, somewhat paradoxically, the initial activation of T cells responsive to islet β cell Ag was slightly faster and more efficient in the absence of OX40L, with an increased degree of cell proliferation and survival in the deficient hosts. Activated T cell migration into and retention within the islets was also slightly accelerated. When challenged in vitro, splenocytes from BDC2.5.OX40Lo/o mice showed no altered reactivity to exogenously added peptide, no bias to the Th1 or Th2 phenotype, and no alteration in T cell survival. Thus, the OX40/OX40L axis has the paradoxical effect of dampening the early activation and migration of autoimmune T cells, but sustains the long-term progression to autoimmune destruction.

Interferon-producing cells (IPCs) secrete high levels of type I interferon in response to certain viruses. The lack of lineage markers, the expression of major histocompatibility complex (MHC) class II and the capacity to stimulate allogeneic T cells have led these cells to be classified as a subset of dendritic cells (DCs), called plasmacytoid DCs (PDCs). However, the role of IPCs/PDCs in initiating primary immune responses remains elusive. Here we examined the antigen presenting capacity of murine IPCs in antigen specific systems. While CD8α+ and CD11b+ DCs induced logarithmic expansion of naive CD4 and CD8 T cells, without conferring T helper commitment at a first encounter, primary IPCs lacked the ability to stimulate naive T cells. However, when antigen-experienced, nonpolarized T cells expanded by classical DC subsets, were restimulated by IPCs, they proliferated and produced high amounts of IFN-γ. These data indicate that IPCs can effectively stimulate preactivated or memory-type T cells and exert an immune-regulatory role. They also suggest that expansion of naive T cells and acquisition of effector function during antigen-specific T cell responses may involve different antigen-presenting cell (APC) types. Independent and coordinated control of T cell proliferation and differentiation would provide the immune system with greater flexibility in regulating immune responses.

Little is known concerning the role of T cells in regulating an anti-polysaccharide Ig response to an intact pathogen. We previously reported that the in vivo Ig responses to Streptococcus pneumoniae (strain R36A), specific for pneumococcal surface protein A (PspA) and for the phosphorylcholine (PC) determinant of C-polysaccharide, were both dependent on TCR-αβ+ T cells and B7-dependent costimulation, although only PspA-specific memory was generated. In this report, we show that the T cell help underlying these two Ag-specific Ig responses is distinct. Using H-Y-specific T cell transgenic mice made “nonleaky” by crossing with mice genetically deficient for TCR-α, we demonstrate that the T cell help for the anti-PC, in contrast to the anti-PspA, response is TCR-nonspecific and occurs normally in the absence of germinal center formation, although it is still dependent on B7-dependent costimulation. Consistent with these data, we demonstrate, using cathepsin S−/− mice, that although the anti-PC response is largely dependent on CD4+ T cells, there is a reduced (or lack of) dependence, relative to the anti-PspA response, on the generation of new peptide-MHC class II complexes. In this regard, the T cell help for an optimal anti-PC response is delivered more rapidly than that required for an optimal anti-PspA response. Collectively, these data demonstrate a novel accelerated TCR-nonspecific B7-dependent form of T cell help for augmenting a polysaccharide-specific Ig response to an intact bacterium without the generation of memory.

Immunization with superantigen in vivo induces transient activation of superantigen-specific T cells, followed by a superantigen-nonresponsive state. In this study, using a TCR α knock-in mouse in which the knock-in α-chain can be replaced with endogenous α-chain through secondary rearrangement, we show that immunization of superantigen changes the TCR α-chain expression on peripheral superantigen-specific T cells, induces expression of recombination-activating genes, and generates DNA double-strand breaks at the TCR α-chain locus. These results suggest that viral superantigens are capable of inducing peripheral TCR revision. Our findings thus provide a new perspective on pathogen-immune system interaction.

We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-Ag7 molecule, the P9 pocket of I-Ag7 plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-Ag7 molecules or a mutant I-Ag7 molecule in which the β57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-Ag7-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-Ag7 were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-Ag7 molecule. These peptides did not favor acidic residues toward the carboxyl terminus.

Skin-derived dendritic cells (DCs) play a crucial role in the maintenance of immune homeostasis due to their role in antigen trafficking from the skin to the draining lymph nodes (dLNs). To quantify the spatiotemporal regulation of skin-derived DCs in vivo, we generated knock-in mice expressing the photoconvertible fluorescent protein KikGR. By exposing the skin or dLN of these mice to violet light, we were able to label and track the migration and turnover of endogenous skin-derived DCs. Langerhans cells and CD103(+)DCs, including Langerin(+)CD103(+)dermal DCs (DDCs), remained in the dLN for 4-4.5 days after migration from the skin, while CD103(-)DDCs persisted for only two days. Application of a skin irritant (chemical stress) induced a transient >10-fold increase in CD103(-)DDC migration from the skin to the dLN. Tape stripping (mechanical injury) induced a long-lasting four-fold increase in CD103(-)DDC migration to the dLN and accelerated the trafficking of exogenous protein antigens by these cells. Both stresses increased the turnover of CD103(-)DDCs within the dLN, causing these cells to die within one day of arrival. Therefore, CD103(-)DDCs act as sentinels against skin invasion that respond with increased cellular migration and antigen trafficking from the skin to the dLNs.

Using a TCRα chain knock-in mouse, we demonstrate that V-gene replacement can operate in the T cell receptor α locus. Functional TCRα chain transcripts generated by Vα-gene replacement at the site of the Vα-embedded heptamer were identified in splenic T cells. This finding shows that Vα-gene replacement can likely be used to shape the peripheral T cell repertoire.The conservation of the embedded heptamer in most Vα segments adds support to the notion that V-gene replacement is a mechanism maintained to diversify the immune system and that argues that itis common to B and T cells.

We generated transgenic mice that expressed hen egg-white lysozyme (HEL) under a class II MHC promoter. The A7 line expressed HEL with a point mutation in the Asp52 residue, the main anchor amino acid responsible for the selection of the chemically dominant family of peptides (52–60) by I-Ak molecules. Mice expressing HEL with Ala52 were completely unresponsive when immunized with the same protein, i.e., HEL A52. However, the same mice immunized with wild-type HEL elicited T cells that recognized a conformation of the 52–61 core sequence uniquely different between Asp52 and Ala52 containing peptides. Importantly, some T cells also recognized the HEL A52 peptide given exogenously but not the same peptide processed from HEL A52 protein. Thus, a core MHC anchor residue influences markedly the specificity of the T cells. We discuss the relevance of these findings to autoimmunity and vaccination with altered peptides.

Although interleukin (IL)-12 and IL-4 polarize naive CD4+ T cells toward T helper cell type 1 (Th1) or Th2 phenotypes, it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate. To distinguish these instructive and selective models, we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral-based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels. We observe IL-4–dependent redirection of phenotype in cells that have already committed to a non–IL-4–producing fate, inconsistent with predictions of the selective model. Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate. These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.

Mice expressing hen egg-white lysozyme (HEL) as a transgene are unresponsive to immunization with the HEL protein. Profound tolerance was found even in situations where the amounts of I-A k -peptide complexes was 100 or less per APC. Among the few T cells that escaped tolerance, we did not observe differential responses to the different HEL epitopes, perhaps because of the very high sensitivity of the negative selection process. The same HEL transgenic mice that did not respond to HEL responded to immunization with the 46–61 peptide of HEL. These peptide-specific T cells that escaped negative selection belonged to a set that reacted with a particular conformer of the HEL peptide-I-A k (type B). The presence of type B reactive T cells should be considered in autoimmunity.

We quantitated the number of peptide-class II MHC complexes required to affect the deletion or activation of 3A9 TCR transgenic thymocytes. Deletion of immature double positive thymocytes was very sensitive, taking place with approximately three peptide-MHC complexes per APC. However, the activation of mature CD4+ thymocytes required 100-fold more complexes per APC. Therefore, a “biochemical margin of safety” exists at the level of the APC. To be activated, autoreactive T cells in peripheral lymphoid tissues require a relatively high level of peptide-MHC complexes.

We examined the role of the peptide/MHC ligand in CD4+ T cell differentiation into Th1 or Th2 cells using a TCR αβ transgenic mouse specific for hemoglobin (Hb)(64-76)/I-Ek. We identified two altered peptide ligands of Hb(64-76) that retain strong agonist activity but, at a given dose, induce cytokine patterns distinct from the Hb(64-76) peptide. The ability of these peptides to produce distinct cytokine patterns at identical doses is not due to an intrinsic qualitative property. Each peptide can induce Th2 cytokines at low concentrations and Th1 cytokines at high concentrations and has a unique range of concentrations at which these distinct effects occur. The pattern of cytokines produced from limiting dilution of naive T cells demonstrated that the potential to develop an individual Th1 or Th2 cell is stochastic, independent of Ag dose. We propose that the basis for the observed effects on the Th1/Th2 balance shown by the altered peptide ligands and the amount of Ag dose involves the modification of soluble factors in bulk cultures that are the driving force that polarize the population to either a Th1 or Th2 phenotype.

The diabetes-susceptible class II MHC genes (in human and mouse) share unique nonaspartic acid residues at position 57 of the class II β-chain. Transgenic expression of a mutant I-Ag7, substituting histidine and serine at position 56 and 57 of β-chain with proline and aspartic acid (I-Ag7PD), respectively, inhibits diabetes development in the nonobese diabetic mouse model. Here, we demonstrate that immature thymocytes expressing a diabetogenic islet Ag-specific transgenic TCR are positively selected by I-Ag7PD class II MHC to give rise to mature CD4+ T cells. However, splenic APCs expressing the same I-Ag7PD fail to present pancreatic islet Ag to mature T cells bearing this diabetogenic TCR. These results indicate that nonaspartic acid residues at position 57 of class II MHC β-chain is important for diabetogenic CD4+ T cell activation in the periphery but is not essential for the formation of a diabetogenic T cell repertoire in the thymus.

Mice infected with murine acquired immunodeficiency syndrome (MAIDS) virus developed lymphoadenopathy and profound immunodeficiency. Concomitantly the expression of endogenous mammary tumor virus (MTV) mRNA increased significantly, especially for the 1.7-kb 3' open reading frame (ORF) mRNA encoding MTV superantigen. B cell lines that are established from MAIDS mice and exhibit superantigen activity also express a high level of 1.7-kb endogenous MTV and mRNA. Infection of a B cell tumor line in vitro with retrovirus containing the cloned MAIDS virus gene induced superantigen activity and this cell line also expressed the 1.7-kb superantigen coding MTV 3' ORF mRNA. These results strongly suggest a link between MAIDS virus infection and the induction of endogenous superantigen activity. This may play an important role in the pathogenesis of the MAIDS virus.

Mice bearing the I-Ag7class II major histocompatibility complex molecules contain a high number of spontaneous autoreactive T cells, as estimated by limiting-dilution assays. We found this autoreactivity in various strains that bear the I-Ag7molecule, such as the nonobese diabetic (NOD) mouse strain, which spontaneously develops autoimmune diabetes. However, NOD mice strains that do not express the I-Ag7molecule, but instead express I-Ab, do not have a high incidence of autoreactive T cells. About 15% of the autoreactive T cells also recognize the I-Ag7molecule expressed in the T2 line, which is defective in the processing of protein antigens. We interpret this to mean that some of the T cells may interact with class II molecules that are either devoid of peptides or contain a limited peptide content. We also find a high component of autoreactivity among antigen-specific T cell clones. These T cell clones proliferate specifically to protein antigens but also have a high level of reactivity to antigen-presenting cells not pulsed with antigen. Thus, the library of T cell receptors in NOD mice is skewed to autoreactivity, which we speculate is based on the weak peptide-binding properties of I-Ag7molecules.

T cell unresponsiveness to Ag stimulation can be induced by several means. The precise mechanism by which this process occurs remains poorly understood. Preincubating T cells with either EDCI-fixed APC or ionomycin is a proven means of inducing T cell anergy with reduced IL-2 production in response to Ag stimulation. Using T cells from mice expressing the TCR transgene DO11.10, which is specific for a peptide (323–339) derived from hen egg OVA, we demonstrate that naive cells obtained directly from the host are resistant to the anergy induction by either fixed APC or ionomycin. TCR transgenic mice also deficient in the recombination-activating gene-2 (RAG-2−/−), preventing the formation of T cells with endogenous TCRs, were immunized with OVA, and in vivo activated T cells with low expression of CD62 were isolated. These primed cells possess the same sensitivity to ionomycin-induced anergy as in vitro activated cell lines. This unresponsive state most profoundly affects Ag-induced IL-2 production, with IFN-γ and IL-3 affected to a lesser degree and no effect observed on IL-4 production. Thus, T cells in vivo can be distinguished phenotypically by their susceptibility to anergic stimuli. Anergy so induced affects selected T cell functions.

Listeriosis in mice with the SCID mutation results in a chronic infection. The chronic infection is characterized by abundant granulomas and neutrophil infiltrates. Both lesions were particularly noticeable in the liver. In the liver, about 95% are granulomas with 5% microabscesses involving intrahepatic infection. The majority of Listeria resided in membrane-bound vacuolar structures of the macrophages and not in the cytosol. Three manipulations resulted in alterations in the equilibrium between granulomas and liver microabscesses, with massive transfer of the infection to the hepatocyte and dissolution of the granulomas: depletion of neutrophils and neutralization of IFN-γ and TNF-α. We did not find a role for IL-12, IL-10, or nitric oxide. Adoptive transfer studies showed a decisive role for both CD4+ and CD8+ T cells for an effective immune response, i.e., clearance of bacteria, granuloma formation with lymphocytes, and disappearance of microabscess. Clearance of Listeria was induced by transfer of CD8+ T cells from mice with targeted disruption of the IFN-γ structural gene (IfgTM1KO), even in the presence of neutralizing mAb to IFN-γ. In marked contrast, transfer of CD4+ T cells from IfgTM1KO mice exacerbated the infection in the chronically infected SCID mice, resulting in increased mortality with dissolution of the granulomas and severe hepatic infection with neutrophil infiltration. Thus, these data indicate that both IFN-γ-dependent and -independent mechanisms are operative in the context of a chronic listerial infection.

We examine here how the beta chain of the class II MHC molecule I-Ag7 influences T cell recognition. Three sets of T cell clones were identified. The first set recognizes peptides bound to I-Ag7, I-Ad and I-Ag7 mutant in which the allele-specific residues His and Ser at position 56 and 57 were changed to the Pro at residue 56 and to non-polymorphic Asp at residue 57. The second set responds to the antigen presented only by I-Ag7 and does not recognize the peptides bound to the other class II molecules. The third set is also specific for I-Ag7 as a result of the poor binding of the peptide to I-Ad and the mutant I-Ag7. These results indicate that positions 56 and 57 of the I-Ag7 class II MHC beta chain play a role in both T cell recognition of the MHC-peptide complex and peptide binding to MHC. These two different functions may be involved in I-Ag7-restricted beta cell antigen recognition by diabetogenic T cell clones.

Osteoclasts are bone-resorbing polykaryons differentiated from monocyte/macrophage-lineage hematopoietic precursors. It remains unclear whether osteoclasts originate from circulating blood monocytes or from bone tissue–resident precursors. To address this question, we combined two different experimental procedures: 1) shared blood circulation “parabiosis” with fluorescently labeled osteoclast precursors, and 2) photoconversion-based cell tracking with a Kikume Green-Red protein (KikGR). In parabiosis, CX3CR1-EGFP knock-in mice in which osteoclast precursors were labeled with EGFP were surgically connected with wild-type mice to establish a shared circulation. Mature EGFP+ osteoclasts were found in the bones of the wild-type mice, indicating the mobilization of EGFP+ osteoclast precursors into bones from systemic circulation. Receptor activator for NF-κB ligand stimulation increased the number of EGFP+ osteoclasts in wild-type mice, suggesting that this mobilization depends on the bone resorption state. Additionally, KikGR+ monocytes (including osteoclast precursors) in the spleen were exposed to violet light, and 2 d later we detected photoconverted “red” KikGR+ osteoclasts along the bone surfaces. These results indicate that circulating monocytes from the spleen entered the bone spaces and differentiated into mature osteoclasts during a certain period. The current study used fluorescence-based methods clearly to demonstrate that osteoclasts can be generated from circulating monocytes once they home to bone tissues.

Murine acquired immunodeficiency syndrome (MAIDS) is caused by a defective murine leukemia virus. The disease is characterized by abnormal lymphoproliferation, impaired T and B cell function and aberrant regulation of cytokines. Both T and B lymphocytes show activated phenotypes, but undergo apoptotic death with characteristic DNA fragmentation. These results indicate the presence of a continuous activation death pathway of the lymphocytes in MAIDS. Overexpression of the bcl-2 transgene in lymphocytes showed no effect on the apoptotic cell death or on the development of the disease. In contrast, mice carrying mutations in either Fas or Fas ligand exhibited accelerated progression of the disease upon infection with MAIDS virus. These results suggest the involvement of Fas-Fas ligand system in the pathogenesis of MAIDS.

T cell hybridomas require the immediate-early gene NGFI-B (nur77) for T cell receptor (TCR)-mediated apoptosis, a model for negative selection of self-reactive T cells. TCR-mediated death was examined in mice bearing an NGFI-B loss-of-function mutation, either by administration of antibodies to CD3 (anti-CD3) or in two well-characterized transgenic models expressing self-reactive TCRs. Both the extent and the rate of thymocyte death were unimpaired. Anti-CD3-induced death was normal in CD4+ peripheral T cells, in which death is mediated predominantly by the Fas signaling pathway. Thus, no unique requirement for NGFI-B is observed for thymic or peripheral T cell death.

A defective murine leukemia virus is the causative agent of murine acquired immunodeficiency syndrome (MAIDS). We have cloned cDNAs from both virus infected and non-infected cells using the PCR methods with primers corresponding to the franking sequence of the unique p12 gag gene. Sequence analysis of these cDNA clones revealed: (i) the presence of endogenous virus related to MAIDS virus in C57BL/6 mice, (ii) B cell lineage specific expression of endogenous virus and (iii) extensive heterogeneity of MAIDS virus recovered from virus infected cells due to the recombination of the related viruses (defective pathogenic virus, ecotropic virus and endogenous virus). These findings suggest that the creation of virus variants in infected cells may play an important role in virus pathogenesis and escape from immune attack during the development of MAIDS.

Ly-49 can be expressed in interspecies hybridoma cells resulting from the fusion of Ly-49 negative lymphoid cells from normal mice and Ly-49 negative T cells from the rat thymoma W/ Fu(C58NT)D (C58). Several hundred hybridoma clones of this type were studied. Hybridoma clones derived from the fusion between activated C57BL/6 mouse spleen cells and C58 expressed Ly-49 on their surface although Ly-49 was undetectable on both parental cells. No Ly-49 antigen was expressed on hybrids when BW5147, a T lymphoma derived from AKR mouse, replaced C58 as parental cell. Thymocytes from C57BL/6 mice also produced Ly-49 positive interspecies hybridomas with C58. Subsequent manipulation of these thymocytes with a mixture of anti-CD4 and anti-CD8 antibodies plus complement to obtain double negative cells (CD4-/CD8-) prior to the fusion abolished the expression of Ly-49 on hybridomas. On the other hand, if the thymocytes were treated with either anti-CD4 or anti-CD8 antibody plus complement, the resultant thymocytes which were a mixture of cells with a single positive (CD4+/CD8- or CD4-/CD8+) and a double negative phenotype (CD4-/CD8-) retained the ability to produce Ly-49 positive hybridomas. Interspecies hybridomas between either CD4+/CD8- or CD4-/CD8+ functional T cell clones and C58 expressed Ly-49 on their surface, though these parental T cells lacked Ly-49 on their surfaces. Northern hybridization analysis using Ly-49 cDNA revealed that C58 cells do not express Ly-49 mRNA, indicating that a trans-acting factor(s) from C58 plays an important role in the induction of cell surface Ly-49 expression on interspecies hybridomas. Our data indicate the complex nature of the regulatory mechanisms for expression of the Ly-49 molecule.

Collagen type II-induced arthritis (CIA) is generated in susceptible rodent strains by intradermal injections of homologous or heterologous native type II collagen in complete Freund's adjuvant. Symptoms of CIA are analogous to those of the human autoimmune disease, rheumatoid arthritis. CIA is a model system for T cell-mediated autoimmune disease. To study the T cell receptor (TCR) repertoire of bovine type II-specific T cells that may be involved in the pathogenesis of CIA in DBA/1Lac.J (H-2q) mice, 13 clonally distinct T cell hybridomas specific for bovine type II collagen have been established and the alpha and beta chains of their TCRs have been analyzed. These T cell hybridomas recognize epitopes that are shared by type II collagens from distinct species and not by type I collagens, and exhibit a highly restricted TCR-alpha/beta repertoire. The alpha chains of the TCRs employ three V alpha gene subfamilies (V alpha 11, V alpha 8, and V alpha 22) and four J alpha gene segments (J alpha 42, J alpha 24, J alpha 37, and J alpha 32). The V alpha 22 is a newly identified subfamily consisting of approximately four to six members, and exhibits a high degree of polymorphism among four mouse strains of distinct V alpha haplotypes. In addition, the beta chains of the TCRs employ three V beta gene subfamilies (V beta 8, V beta 1, and V beta 6), however the V beta 8.2 gene segment is preferentially utilized (58.3%). In contrast, the J beta gene segment usage is more heterogeneous. On the basis of the highly limited TCR-alpha/beta repertoire of the TCRs of the panel of bovine type II-specific T cell hybrid clones, a significant reduction (60%) of the incidence of arthritis in DBA/1Lac.J mice is accomplished by the use of anti-V beta 8.2 antibody therapy.

A murine acquired immunodeficiency (MAIDS) virus transformed B cell line, B6-1710, was shown to express superantigen activity and stimulate T cell hybridomas to produce IL-2. However, T cell clones and lines from which B6-1710 reactive hybridomas were established failed to proliferate upon stimulation with B6-1710, while B6-1710 cells were capable of presenting bacterial superantigen to stimulate both T cell hybridomas and clones. Proliferative response of T cells to MAIDS virus superantigen could be detected by the addition of the pharmacological agent phorbol myristate acetate (PMA). Thus, B6-1710 seems to lack a stimulatory activity necessary for the proliferative response of T cells to the MAIDS viral superantigen. In the analysis of the response of naive splenic T cells to the MAIDS superantigen, T cells recovered from a culture stimulated with B6-1710 cells in the presence of PMA showed no dominant TCR V beta chain usage, while cells recovered from a culture stimulated with B6-1710 alone were dominated by T cells expressing V beta 5, 11, and 12 TCR chains. These results suggest that T cells bearing a majority of TCR V beta chains are capable of responding to B6-1710 superantigen. The avidity of interaction between T cells and viral superantigen may differ significantly among T cells and those T cells exhibiting weak interaction with MAIDS virus superantigen may require additional signals, such as PMA, for an in vitro proliferative response to B6-1710 cells.

Naive T lymphocytes recirculate through the lymph-vascular system and enter and exit lymphoid organs. Using mice expressing the photoconvertible fluorescence protein Kaede, we demonstrated that naive T cells seek to interact with endogenous Ags after migrating to the lymphoid organs. The interaction with endogenous Ags transiently induces CD69 expression on T cells, which prolongs retention in the lymphoid organs. Cells that fail to express CD69 or lose CD69 expression migrate to other lymphoid organs. Functionally, CD69+-naive CD4+ T cells exhibit faster and greater cytokine production than do CD69− naive CD4+ T cells. These results indicate that CD4+ T cells continuously migrate to interact with endogenous Ags, and such an interaction plays an important role in the Ag reactivity of naive CD4+ T cells.

In mammals, the gastrointestinal tract harbors an extraordinarily dense and complex community of microorganisms. The gut microbiota provide strong selective pressure to the host to evolve adaptive immune responses required for the maintenance of local and systemic homeostasis. The continuous antigenic presence in the gut imposes a dynamic remodeling of gut-associated lymphoid tissues (GALT) and the selection of multiple layered strategies for immunoglobulin (Ig) A production. The composite and dynamic gut environment also necessitates heterogeneous, versatile, and convertible T cells, capable of inhibiting (Foxp3+T cells) or helping (TFHcells) local immune responses. In this review, we describe recent advances in our understanding of dynamic pathways that lead to IgA synthesis, in gut follicular structures and in extrafollicular sites, by T cell–dependent and T cell–independent mechanisms. We discuss the finely tuned regulatory mechanisms for IgA production and emphasize the role of mucosal IgA in the selection and maintenance of the appropriate microbial composition that is necessary for immune homeostasis.

Recognition of conventional foreign antigen by T cells is determined by the expression of multiple variable regions of both alpha and beta chains of the T cell receptor (TCR) alpha/beta heterodimer. In contrast, there exists a class of antigens that appears to interact with the TCR alpha/beta heterodimer through the variable region on the beta chain (V beta), independent of other TCR components, a property that has led to their designation as superantigens. The goal of the present study was to analyze V alpha use in V beta 6+ T cells responsive to the superantigen, Mlaa. Results indicate that while deletion of T cells expressing V beta 6 in Mlsa-expressing mice is essentially complete and therefore appears to occur regardless of V alpha usage, in vitro Mlsa stimulation of T cells from Mlsa-negative mice results in significant skewing of V alpha use among responding V beta 6+ T cells. This indicates that V alpha expression influences recognition of the superantigen, Mlsa by mature peripheral T cells.

We investigated a mechanism leading to activation of murine T cells by staphylococcal enterotoxin E (SEE). L cells transfected with I-Ab genes but not control L cells supported IL-2 production by SEE-induced C57BL/6 T lymphoblasts upon restimulation with SEE. mAb to I-Ab markedly inhibited the above response. Flow cytometric analyses showed that SEE-induced C57BL/6 T lymphoblasts are composed of both CD4+ T cells and CD8+ T cells, and that larger parts of them bore V beta 11 (40-75%). mAb to V beta 11 markedly inhibited the SEE-induced proliferative response and IL-2 production by T cells. Analysis of SEE-induced IL-2 production in spleen cells from various mouse strains showed that C57BL/6 and B10.A(4R) mice (I-E, not expressed; V beta 11+ T cells, normally generated) are highly responsive to SEE. In contrast, BALB/c, C3H/HeN, (C57BL/6 x BALB/c or C3H/HeN) F1 mice (I-E, normally expressed and V beta 11+ T cells, deleted), and SJL and C57L mice (V beta 11 genes, deleted) are weakly responsive to SEE. The results indicate that SEE activates mainly T cells bearing V beta 11 in physical association with MHC class II molecules expressed on AC. In addition, the results indicate that SEE activates both CD4+ T cells and CD8+ T cells.

The monoclonal antibody (mAb) MR10-2 is specific for mouse T cells expressing the V beta 9 chain of T cell antigen receptor. This mAb reacts with 2% of peripheral T cells in a number of different strains of mice, but in mice expressing the minor lymphocyte stimulating (Mls)-1a antigen, V beta 9+ T cells recognized by MR10-2 are deleted. However, we failed to demonstrate the Mls-1 a antigen-specific response of naive, non-activated V beta 9 T cells in vitro. On the other hand, T cells activated initially with anti-V beta 9 (MR10-2) mAb in vitro are capable of responding to the Mls-1a antigen. These results demonstrate that Mls-1a reactivity of V beta 9 T cells depends on the activation state of the T cells, suggesting a differential activation requirement between naive, non-activated T cells and previously activated T cells.

Experimental allergic encephalomyelitis (EAE) is a model system for T cell-mediated autoimmune disease. Symptoms of EAE are similar to those of multiple sclerosis (MS) in humans. EAE is induced in susceptible animal strains by immunization with myelin basic protein (MBP) and potent adjuvant. The major T cell response to MBP in B10.PL mice is directed towards an NH2-terminal epitope and involves T cells expressing either V beta 8.2 or V beta 13 gene segments. Animals treated with a TCR V beta 8-specific mAb have a reduced incidence of EAE. We report here that the in vivo administration of a combination of anti-V beta 8.2 and anti-V beta 13 mAbs results in a long-term elimination of T cells involved in the response to MBP. When given before MBP immunization, anti-TCR antibody treatment leads to nearly complete protection against EAE. Antibody treatment also results in a dramatic reversal of paralysis in diseased animals. Thus, treatment with a combination of V beta-specific antibodies is a very effective therapy for the prevention and treatment of EAE. It is hoped that the future characterization of TCR V gene usage in human autoimmune diseases may lead to similar strategies of immune intervention.

Incomplete T cell antigen receptor-mediated signaling induces an unresponsive state known as anergy. Previously, we had shown that anergy can be induced in antigen-primed but not naive T cells. In this report, we found that in vitro primed T cells from IL-2R alpha-deficient mice were resistant to anergy induction in contrast to comparably treated wild-type T cells. This resistance persisted even after proliferation of IL-2R alpha chain-deficient CD4 T cells with high-dose IL-2-IL-2R beta gamma chains interaction. Thus, antigen activation, and/or progression through cell cycle are not sufficient to induce anergy susceptibility in T cells. The high-affinity IL-2-IL-2R interaction appears to play a critical role in this process.

We determined that, over a biologic time interval, from 4 to 8 weeks of age, female non-obese diabetic (NOD) mice develop antibodies against pancreatic beta-cell-surface antigens depending upon the presence of both the MHC class II susceptibility allele, I-A(g7), and other NOD background genes. We generated a mAb from a pre-diabetic NOD mouse that binds to the surface of insulinoma cells and isolated mouse beta cells, and identified the target as a retroviral envelope glycoprotein expressed on pancreatic beta cells. The cloned and expressed sequence for this protein was recognized by the mAb. The antibody as well as sera from pre-diabetic NOD mice recognized the recombinant protein. Spontaneous T cell reactivity against a peptide from the cloned protein was found in NOD mice. In conclusion, a beta cell retroviral envelope protein is a target antigen that is selected by the NOD mouse immune system early in the pathogenesis of autoimmune diabetes.

The third complementarity-determining region (CDR) of the TCR alpha and beta chains forms loops that engage amino acid residues of peptides complexed with MHC. This interaction is central to the specific discrimination of antigenic-peptide-MHC complexes by the TCR. The TCRbeta chain CDR3 loop is encoded by the Dbeta gene segment and flanking portions of the Vbeta and Jbeta gene segments. The joining of these gene segments is imprecise, leading to significant variability in the TCRbeta chain CDR3 loop length and amino acid composition. In marked contrast to other pairing antigen-receptor chains, the TCR beta and alpha chain CDR3 loop size distributions are relatively narrow and closely matched. Thus, pairing of TCR alpha and beta chains with relatively similar CDR3 loop sizes may be important for generating a functional repertoire of alpha beta TCR. Here we show that the TCRbeta chain CDR3 loop size distribution is minimally impacted by TCRbeta chain or alpha beta TCR selection during thymocyte development. Rather, this distribution is determined primarily at the level of variable-region gene assembly, and is critically dependent on unique features of the V(D)J recombination reaction that ensure Dbeta gene segment utilization.

CD8 is either an alpha alpha homodimer or an alpha beta heterodimer, although most peripheral CD8-lineage T cells express only the CD8 alpha beta heterodimer. The physiological function of CD8 beta was elucidated with mice that were chimeric for the homozygous disruption of the CD8 beta gene. The CD8 beta-1- T cells developed normally to CD4+CD8+ stage, but did not efficiently differentiate further, which resulted in few peripheral CD8+ T cells. The number of peripheral CD8+ T cells was restored by transfer of an exogenous CD8 beta gene into CD8 beta-deficient T cells. Thus, CD8 beta is necessary for the maturation of CD8+ T cells.

Glucocorticoids, administered in pharmacological doses, potently modulate immune system function and are a mainstay therapy for many common human diseases. Physiologic production of glucocorticoids may play a role in optimization of the immune repertoire both centrally and peripherally. Possible effects include alteration of lymphocyte development and down-regulation of cytokine responses, but essential roles remain unclear. To determine the part that endogenous glucocorticoids play in thymocyte development, we used fetal liver from mice lacking the glucocorticoid receptor GRko for immunological reconstitution of lethally irradiated wild-type (WT) mice. We find normal numbers and subset distribution of GRko thymocytes. GRko thymocytes also exhibit similar sensitivity to apoptosis induced by activating anti-CD3ε Ab as WT thymocytes in vitro. Surprisingly, GRko thymocytes are significantly more resistant than WT thymocytes to anti-CD3ε-mediated thymocyte apoptosis in vivo. Consistent with this finding, in vivo TCR complex activation induces sustained high levels of glucocorticoids that correlate strongly with thymocyte apoptosis in WT mice. We find that while direct engagement of the TCR complex may cause death of a subset of thymocytes, glucocorticoids are required for deletion of the majority of thymocytes. Thus, TCR stimulation by Ab administration may more accurately reflect polyclonal T cell activation than negative selection in vivo.