Complement C3 and marginal zone B cells promote IgG-mediated enhancement of RBC alloimmunization in mice.

Administration of anti-RhD immunoglobulin (Ig) to decrease maternal alloimmunization (antibody-mediated immune suppression [AMIS]) was a landmark clinical development. However, IgG has potent immune-stimulatory effects in other settings (antibody-mediated immune enhancement [AMIE]). The dominant thinking has been that IgG causes AMIS for antigens on RBCs but AMIE for soluble antigens. However, we have recently reported that IgG against RBC antigens can cause either AMIS or AMIE as a function of an IgG subclass. Recent advances in mechanistic understanding have demonstrated that RBC alloimmunization requires the IFN-α/-ß receptor (IFNAR) and is inhibited by the complement C3 protein. Here, we demonstrate the opposite for AMIE of an RBC alloantigen (IFNAR is not required and C3 enhances). RBC clearance, C3 deposition, and antigen modulation all preceded AMIE, and both CD4+ T cells and marginal zone B cells were required. We detected no significant increase in antigen-specific germinal center B cells, consistent with other studies of RBC alloimmunization that show extrafollicular-like responses. To the best of our knowledge, these findings provide the first evidence of an RBC alloimmunization pathway which is IFNAR independent and C3 dependent, thus further advancing our understanding of RBCs as an immunogen and AMIE as a phenomenon.

The role of RBC antigen transgene integration sites on RBC biology in mice.

BACKGROUND: Transgenic mice expressing RBC specific antigens are widely used in mechanistic studies of RBC alloimmunization. Existing RBC donor strains have random transgene integration, potentially disrupting host elements that can confound biological interpretation. STUDY DESIGN AND METHODS: Integration site and genomic alterations were characterized by both targeted locus amplification and congenic backcrossing in the five most commonly used RBC alloantigen donor strains (KEL-K2hi , KEL-K2med , and KEL-K2lo , and KEL-K1). A targeted transgenic approach was developed to allow RBC specific transgene expression from a safe harbor locus (ROSA26). Alloimmune responses were assessed by transfusing alloantigen expressing RBCs into wild-type recipients and measuring alloantibodies by flow cytometry. RESULTS/FINDINGS: Four of the five analyzed strains had at least one gene disrupted by the transgene integration but none of the disrupted genes are known to be involved in RBC biology. The integration of KEL-K2med potentially altered the immunological properties of RBCs, although the biological significance of the observed changes is unclear. The ROSA26 targeted approach resulted in a single copy of the transgene that maintains RBC specific expression without random disruption of genomic elements. CONCLUSION: These findings provide a detailed characterization of genomic disruption by transgene integration found in commonly used RBC donor strains that is relevant to numerous previous publications as well as future studies. With the possible exception of KEL-K2med , transgene integration is not predicted to affect RBC biology in existing models, and new models can avoid this concern using the described targeted transgenic approach.

Long-Acting Recombinant Human Interleukin-7, NT-I7, Increases Cytotoxic CD8 T Cells and Enhances Survival in Mouse Glioma Models.

PURPOSE: Patients with glioblastoma (GBM) are treated with radiotherapy (RT) and temozolomide (TMZ). These treatments may cause prolonged systemic lymphopenia, which itself is associated with poor outcomes. NT-I7 is a long-acting IL7 that expands CD4 and CD8 T-cell numbers in humans and mice. We tested whether NT-I7 prevents systemic lymphopenia and improves survival in mouse models of GBM. EXPERIMENTAL DESIGN: C57BL/6 mice bearing intracranial tumors (GL261 or CT2A) were treated with RT (1.8 Gy/day × 5 days), TMZ (33 mg/kg/day × 5 days), and/or NT-I7 (10 mg/kg on the final day of RT). We followed the mice for survival while serially analyzing levels of circulating T lymphocytes. We assessed regulatory T cells (Treg) and cytotoxic T lymphocytes in the tumor microenvironment, cervical lymph nodes, spleen, and thymus, and hematopoietic stem and progenitor cells in the bone marrow. RESULTS: GBM tumor-bearing mice treated with RT+NT-I7 increased T lymphocytes in the lymph nodes, thymus, and spleen, enhanced IFNγ production, and decreased Tregs in the tumor which was associated with a significant increase in survival. NT-I7 also enhanced central memory and effector memory CD8 T cells in lymphoid organs and tumor. Depleting CD8 T cells abrogated the effects of NT-I7. Furthermore, NT-I7 treatment decreased progenitor cells in the bone marrow. CONCLUSIONS: In orthotopic glioma-bearing mice, NT-I7 mitigates RT-related lymphopenia, increases cytotoxic CD8 T lymphocytes systemically and in the tumor, and improves survival. A phase I/II trial to evaluate NT-I7 in patients with high-grade gliomas is ongoing (NCT03687957).

Antibodies to Low-Copy Number RBC Alloantigen Convert a Tolerogenic Stimulus to an Immunogenic Stimulus in Mice.

Red blood cells expressing alloantigens are well known to be capable of inducing robust humoral alloantibody responses both in transfusion and pregnancy. However, the majority of transfusion recipients and pregnant women never make alloantibodies, even after repeat exposure to foreign RBCs. More recently, RBCs have been used as a cellular therapeutic-very much like transfusion, engineered RBCs are highly immunogenic in some cases but not others. In animal models of both transfusion and RBC based therapeutics, RBCs that do not induce an immune response also cause tolerance. Despite a robust phenomenology, the mechanisms of what regulates immunity vs. tolerance to RBCs remains unclear. However, it has been reported that copy number of alloantigens on the RBCs is a critical factor, with a very low copy number causing non-responsiveness (in both humans and mice) and also leading to tolerance in mice. Recently, we reported that an IgG2c specific for an RBC antigen can substantially enhance the humoral immune response upon transfusion of RBCs expressing that antigen. Herein, we report that an IgG2c converts RBCs with low antigen copy number from a tolerogenic to an immunogenic stimulus. These findings report the first known stimulus that induces humoral alloimmunization to a low copy number RBC alloantigen and identify a previously undescribed molecular switch that has the ability to affect responder vs. non-responder phenotypes of transfusion recipients.

Defining phenotypic and functional heterogeneity of glioblastoma stem cells by mass cytometry.

Most patients with glioblastoma (GBM) die within 2 years. A major therapeutic goal is to target GBM stem cells (GSCs), a subpopulation of cells that contribute to treatment resistance and recurrence. Since their discovery in 2003, GSCs have been isolated using single-surface markers, such as CD15, CD44, CD133, and α6 integrin. It remains unknown how these single-surface marker-defined GSC populations compare with each other in terms of signaling and function and whether expression of different combinations of these markers is associated with different functional capacity. Using mass cytometry and fresh operating room specimens, we found 15 distinct GSC subpopulations in patients, and they differed in their MEK/ERK, WNT, and AKT pathway activation status. Once in culture, some subpopulations were lost and previously undetectable ones materialized. GSCs that highly expressed all 4 surface markers had the greatest self-renewal capacity, WNT inhibitor sensitivity, and in vivo tumorigenicity. This work highlights the potential signaling and phenotypic diversity of GSCs. Larger patient sample sizes and antibody panels are required to confirm these findings.

In utero exposure to alloantigens primes alloimmunization to platelet transfusion in mice.

BACKGROUND: Platelet transfusions remain a mainstay of treatment for many patients with thrombocytopenia, but can lead to alloantibodies to Human Leukocyte Antigens (anti-HLA) resulting in inadequate responses to subsequent platelet transfusions (refractoriness), as well as complicate transplantation. Despite substantial decreases in alloimmunization with the implementation of leukoreduction, a significant percentage of patients still become alloimmunized following platelet transfusions. It remains unclear why some patients make anti-HLA antibodies, but others do not make anti-HLA antibodies even with chronic transfusion. Antecedent pregnancy correlates with risk of alloimmunization due to platelet transfusion in humans - however, isolation of pregnancy as a single variable is not possible in human populations. STUDY DESIGN AND METHODS: A tractable murine model of pregnancy and transfusion was engineered by breeding C57BL/6 (H-2b ) dames with BALB/c (H-2d ) sires. After pregnancy, female mice were transfused with leukoreduced platelets from F1 (H-2b/d ) donors that expressed the same paternal major histocompatibility complex (MHC) H-2d alloantigens as the sires. Control groups allowed isolation of pregnancy or transfusion alone as independent variables. Alloimmunization was determined by testing serum for antibodies to H-2d MHC alloantigens. RESULTS: No alloantibodies were detected after pregnancy alone, or in response to transfusion of platelets alone; however, significant levels of alloantibodies were detected when pregnancy was followed by transfusion. CONCLUSIONS: These findings isolate antecedent pregnancy as a causal contribution to increased frequencies of alloimmunization by subsequent platelet transfusion in mice and provide a platform for ongoing mechanistic investigation.

Zika virus oncolytic activity requires CD8+ T cells and is boosted by immune checkpoint blockade.

Glioblastoma multiforme (GBM) is a fatal human cancer in part because GBM stem cells are resistant to therapy and recurrence is inevitable. Previously, we demonstrated Zika virus (ZIKV) targets GBM stem cells and prevents death of mice with gliomas. Here, we evaluated the immunological basis of ZIKV-mediated protection against GBM. Introduction of ZIKV into the brain tumor increased recruitment of CD8+ T and myeloid cells to the tumor microenvironment. CD8+ T cells were required for ZIKV-dependent tumor clearance because survival benefits were lost with CD8+ T cell depletion. Moreover, while anti-PD-1 antibody monotherapy moderately improved tumor survival, when coadministered with ZIKV, survival increased. ZIKV-mediated tumor clearance also resulted in durable protection against syngeneic tumor rechallenge, which also depended on CD8+ T cells. To address safety concerns, we generated an immune-sensitized ZIKV strain, which was effective alone or in combination with immunotherapy. Thus, oncolytic ZIKV treatment can be leveraged by immunotherapies, which may prompt combination treatment paradigms for adult patients with GBM.

ZBTB32 restrains antibody responses to murine cytomegalovirus infections, but not other repetitive challenges.

ZBTB32 is a transcription factor that is highly expressed by a subset of memory B cells and restrains the magnitude and duration of recall responses against hapten-protein conjugates. To define physiological contexts in which ZBTB32 acts, we assessed responses by Zbtb32-/- mice or bone marrow chimeras against a panel of chronic and acute challenges. Mixed bone marrow chimeras were established in which all B cells were derived from either Zbtb32-/- mice or control littermates. Chronic infection of Zbtb32-/- chimeras with murine cytomegalovirus led to nearly 20-fold higher antigen-specific IgG2b levels relative to controls by week 9 post-infection, despite similar viral loads. In contrast, IgA responses and specificities in the intestine, where memory B cells are repeatedly stimulated by commensal bacteria, were similar between Zbtb32-/- mice and control littermates. Finally, an infection and heterologous booster vaccination model revealed no role for ZBTB32 in restraining primary or recall antibody responses against influenza viruses. Thus, ZBTB32 does not limit recall responses to a number of physiological acute challenges, but does restrict antibody levels during chronic viral infections that periodically engage memory B cells. This restriction might selectively prevent recall responses against chronic infections from progressively overwhelming other antibody specificities.

Metabolic and Transcriptional Modules Independently Diversify Plasma Cell Lifespan and Function.

Plasma cell survival and the consequent duration of immunity vary widely with infection or vaccination. Using fluorescent glucose analog uptake, we defined multiple developmentally independent mouse plasma cell populations with varying lifespans. Long-lived plasma cells imported more fluorescent glucose analog, expressed higher surface levels of the amino acid transporter CD98, and had more autophagosome mass than did short-lived cells. Low amino acid concentrations triggered reductions in both antibody secretion and mitochondrial respiration, especially by short-lived plasma cells. To explain these observations, we found that glutamine was used for both mitochondrial respiration and anaplerotic reactions, yielding glutamate and aspartate for antibody synthesis. Endoplasmic reticulum (ER) stress responses, which link metabolism to transcriptional outcomes, were similar between long- and short-lived subsets. Accordingly, population and single-cell transcriptional comparisons across mouse and human plasma cell subsets revealed few consistent and conserved differences. Thus, plasma cell antibody secretion and lifespan are primarily defined by non-transcriptional metabolic traits.

ZBTB32 Restricts the Duration of Memory B Cell Recall Responses.

Memory B cell responses are more rapid and of greater magnitude than are primary Ab responses. The mechanisms by which these secondary responses are eventually attenuated remain unknown. We demonstrate that the transcription factor ZBTB32 limits the rapidity and duration of Ab recall responses. ZBTB32 is highly expressed by mouse and human memory B cells but not by their naive counterparts. Zbtb32(-/-) mice mount normal primary Ab responses to T-dependent Ags. However, Zbtb32(-/-) memory B cell-mediated recall responses occur more rapidly and persist longer than do control responses. Microarray analyses demonstrate that Zbtb32(-/-) secondary bone marrow plasma cells display elevated expression of genes that promote cell cycle progression and mitochondrial function relative to wild-type controls. BrdU labeling and adoptive transfer experiments confirm more rapid production and a cell-intrinsic survival advantage of Zbtb32(-/-) secondary plasma cells relative to wild-type counterparts. ZBTB32 is therefore a novel negative regulator of Ab recall responses.

Amelioration of experimental autoimmune encephalomyelitis by probiotic mixture is mediated by a shift in T helper cell immune response.

The immunomodulatory effect of probiotics has been shown mainly in gastro-intestinal immune disorders and little information is available on the inflammation of central nervous system. Recently we reported that IRT5 probiotics, a mixture of 5 probiotics, could suppress diverse experimental inflammatory disorders. In this study, we evaluated the prophylactic and therapeutic effects of IRT5 probiotics in experimental autoimmune encephalomyelitis (EAE), a T cell mediated inflammatory autoimmune disease of the central nervous system. Pretreatment of IRT5 probiotics before disease induction significantly suppressed EAE development. In addition, treatment with IRT5 probiotics to the ongoing EAE delayed the disease onset. Administration of IRT5 probiotics inhibited the pro-inflammatory Th1/Th17 polarization, while inducing IL10(+) producing or/and Foxp3(+) regulatory T cells, both in the peripheral immune system and at the site of inflammation. Collectively, our data suggest that IRT5 probiotics could be applicable to modulate T cell mediated neuronal autoimmune diseases, including multiple sclerosis.

Looping mediated interaction between the promoter and 3' UTR regulates type II collagen expression in chondrocytes.

Type II collagen is the major component of articular cartilage and is mainly synthesized by chondrocytes. Repeated sub-culturing of primary chondrocytes leads to reduction of type II collagen gene (Col2a1) expression, which mimics the process of chondrocyte dedifferentiation. Although the functional importance of Col2a1 expression has been extensively investigated, mechanism of transcriptional regulation during chondrocyte dedifferentiation is still unclear. In this study, we have investigated the crosstalk between cis-acting DNA element and transcription factor on Col2a1 expression in primary chondrocytes. Bioinformatic analysis revealed the potential regulatory regions in the Col2a1 genomic locus. Among them, promoter and 3' untranslated region (UTR) showed highly accessible chromatin architecture with enriched recruitment of active chromatin markers in primary chondrocytes. 3' UTR has a potent enhancer function which recruits Lef1 (Lymphoid enhancer binding factor 1) transcription factor, leading to juxtaposition of the 3' UTR with the promoter through gene looping resulting in up-regulation of Col2a1 gene transcription. Knock-down of endogenous Lef1 level significantly reduced the gene looping and subsequently down-regulated Col2a1 expression. However, these regulatory loci become inaccessible due to condensed chromatin architecture as chondrocytes dedifferentiate which was accompanied by a reduction of gene looping and down-regulation of Col2a1 expression. Our results indicate that Lef1 mediated looping between promoter and 3' UTR under the permissive chromatin architecture upregulates Col2a1 expression in primary chondrocytes.

Nuclear factor of activated T cells 1 (NFAT1)-induced permissive chromatin modification facilitates nuclear factor-κB (NF-κB)-mediated interleukin-9 (IL-9) transactivation.

IL-9 regulates diverse inflammatory immune responses. Although the functional importance of IL-9 has been investigated in various pathophysiological conditions, molecular mechanisms by which TCR stimulation induced IL-9 gene expression are still unclear. In this study, we investigated the functional importance of the NFAT1 and NF-κB (p65) in IL-9 gene transcription in CD4(+) T cells. In vivo binding of NFAT1 and NF-κB (p65) to the IL-9 promoter was observed. NFAT1 binding induced a transcriptionally active chromatin configuration at the IL-9 promoter locus, whereas NF-κB (p65) binding transactivated the IL-9 promoter. Mouse deficient in NFAT1 shows a significant down-regulation of IL-9 expression that resulted from an inaccessible chromatin configuration at the IL-9 promoter. In parallel, knockdown of NF-κB (p65) also resulted in reduced IL-9 expression. In this process, NFAT1 plays a pivotal role as a core protein that creates an accessible platform for the assembly of transcription activators. The presence of NFAT1 correlates with recruitment of NF-κB (p65), p300, and active histone markers on the IL-9 promoter, resulting in a transcriptionally competent promoter. NFAT1 deficiency significantly reduced the recruitment of the above activation complex to the IL-9 promoter. In summary, our data suggest that functional cooperation of NFAT1 and NF-κB synergistically enhances IL-9 transcription in CD4(+) T cells.

Stat6 and c-Jun mediate Th2 cell-specific IL-24 gene expression.

TCR signaling regulates multiple aspects of T cell function by controlling expression of various cytokine genes. IL-24 is a multifunctional cytokine belonging to the IL-10 family. It displays anticancer effects in diverse cancer cells and regulates immunopathology of psoriasis and rheumatoid arthritis. IL-24 also plays an important role in B cell differentiation. Mouse IL-24 gene is selectively expressed in activated Th2 cells upon TCR stimulation. However, the molecular mechanisms by which TCR stimulation induces IL-24 gene expression are still unclear. In this study, to elucidate the mechanism of Th2 cell-specific expression of IL-24, we identified a proximal promoter region (-157/+95 bp) that plays critical role in activating the IL-24 gene in Th2 cells. This region has a Th2 cell-specific open chromatin structure along with permissive histone modifications. In vivo binding of Stat6 and AP-1 (c-Jun) to the IL-24 promoter locus in Th2 cells synergistically transactivated the IL-24 promoter. Stat6 and c-Jun proteins were found to physically cooperate with each other and upregulated IL-24 gene transcription. Knockdown of either Stat6 or c-Jun suppressed endogenous IL-24 gene expression in Th2 cells. In summary, TCR stimulation induces IL-24 expression in Th2 cells by the coordinate action of Stat6 and c-Jun transcription factors at the transcriptional level.

Topical application of porcine placenta extract inhibits the progression of experimental contact hypersensitivity.

AIM OF STUDY: Placenta extract features as a composition of ointments used for skin beautification, dermatological diseases and skin dryness. However, little evidence has been cited about its underlying mechanisms of action by which it exerts a beneficial role in dermatological diseases in vivo. In this study, we intended to test the effect of topical application of porcine placenta extract in mouse model of contact hypersensitivity and elucidate its mechanism of action. MATERIALS AND METHODS: To test the in vitro effect of porcine placenta extract, RAW 264.7 cells were cocultured with porcine placenta extract and stimulated with LPS (1 µg/ml) and the expression of inflammatory mediator TNF-α was estimated by RT-PCR at the mRNA level and by intracellular staining at the protein level. To further test in vivo efficacy, porcine placenta extract was topically applied to the mice with experimental skin hypersensitivity. For in vivo studies placenta extract in gel form was topically applied to ear of DNCB-induced contact hypersensitivity mouse model everyday for 2 weeks and progression of the disease was estimated by following criteria: (a) ear thickness, (b) serum IgE level by ELISA, (c) histological examination of ear tissue by H&E staining and (d) cytokine profile of total cells and CD4(+) T cells by real time PCR. RESULTS: Topical application of porcine placenta extract on mouse ears with contact hypersensitivity decreased the severity and progression of the disease manifested by reducing ear swelling, inflammation and edema. Histological evaluation showed that placenta extract treatment reduced lymphocyte infiltration in the ear tissues. Protective effect of placenta extract is also associated with down-regulation of serum IgE level and inflammatory cytokine production (IL-1ß, IFN-γ, TNF-α, IL-4, IL-12 and IL-17) in total lymph node cells and CD4(+) T cells. CONCLUSIONS: Our data indicate that protective effect of porcine placenta extract in contact hypersensitivity is mediated by inhibition of the inflammatory responses and IgE production, suggesting a potential therapeutic application of porcine placenta extract to modulate skin inflammation.

Lactobacillus casei enhances type II collagen/glucosamine-mediated suppression of inflammatory responses in experimental osteoarthritis.

AIMS: We previously reported that Lactobacillus casei (L. casei) has beneficial effects in experimental rheumatoid arthritis (RA) by suppressing inflammatory immune responses. The major purpose of this study was to evaluate therapeutic effects of L. casei on pathological responses in experimental rodent model of osteoarthritis (OA). MAIN METHODS: Experimental OA was induced by intra-articular injection of monosodium iodoacetate (MIA) in Wistar rats. L. casei alone or together with type II collagen (CII) and glucosamine (Gln) was orally administered into OA rats. The pathophysiological aspects of OA were investigated by analyzing mechanical hyperalgesia and histology of articular tissues. Expression of inflammatory molecules was analyzed in CD4(+) T cells, synovial fibroblasts, and chondrocytes by quantitative real-time PCR. KEY FINDINGS: Oral administration of L. casei together with CII and Gln more effectively reduced pain, cartilage destruction, and lymphocyte infiltration than the treatment of Gln or L. casei alone. This co-administration also decreased expression of various pro-inflammatory cytokines (interleukin-1ß (IL-1ß), IL-2, IL-6, IL-12, IL-17, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)) and matrix metalloproteinases (MMP1, MMP3, and MMP13), while up-regulating anti-inflammatory cytokines (IL-4 and IL-10). These results are concomitant with reduced translocation of NF-κB into the nucleus and increased expression of the tissue inhibitor of MMP1 (TIMP1) and CII in chondrocytes. SIGNIFICANCE: Our study provides evidence that L. casei could act as a potent nutraceutical modulator for OA treatment by reducing pain, inflammatory responses, and articular cartilage degradation.

Lymphoid enhancer binding factor 1 regulates transcription through gene looping.

Efficient transcription depends upon efficient physical and functional interactions between transcriptosome complexes and DNA. We have previously shown that IL-1beta-induced lymphoid enhancer binding factor 1 (Lef1) regulates the transcription of its target genes COX2 and MMP13 in mouse chondrocytes by binding to the Lef1 binding sites located in the 3' region. In this study, we investigated how the 3' region-bound Lef1 regulates expression of target genes. IL-1beta stimulation induced gene looping in COX2 and MMP13 genomic loci, which is mediated by the physical interaction of Lef1 with its binding partners, including beta-catenin, AP-1, and NF-kappaB. As shown by chromosome conformation capture (3C) assay, the 5' and 3' genomic regions of these genes were juxtaposed in an IL-1beta-stimulation dependent manner. Lef1 played a pivotal role in this gene looping; Lef1 knockdown decreased the incidence of gene looping, while Lef1 overexpression induced it. Physical interactions between the 3' region-bound Lef1 and promoter-bound transcription factors AP-1 or NF-kappaB in COX2 and MMP13, respectively, were increased upon stimulation, leading to synergistic up-regulation of gene expression. Knockdown of RelA or c-Jun decreased the formation of gene loop and down-regulated cyclooxygenase 2 (COX2) or matrix metalloproteinase 13 (MMP13) transcription levels. However, overexpression of RelA or c-Jun along with Lef1 increased the looping and their expression levels. Our results indicate a novel function of Lef1, as a mediator of gene looping between 5' and 3' regions. Gene looping may serve to delineate the transcription unit in the inducible gene transcription of mammalian cells.

Defect in TCR-CD3zeta signaling mediates T cell hypo-responsiveness in mesenteric lymph node.

Mesenteric lymph node (MLN) in gut-associated lymphoid tissue plays obligatory roles in the induction of oral tolerance and ignorance to commensals. However, little is known about its immunological characteristics. In this study, we investigated the hypo-responsiveness of MLN CD4(+) T cells, comparing them with spleen CD4(+) T cells. MLN CD4(+) T cells were hypo-proliferative and expressed low levels of Th1-type cytokines in response to antigen or CD3/T cell receptor (TCR) stimulation. The hypo-responsiveness of MLN CD4(+) T cells is linked neither with changes in the regulatory T cell population (CD4(+)CD25(+), CD4(+)Foxp3(+)) nor the apoptotic population. Rather, MLN CD4(+) T cells showed deformity of T cell:APC conjugation and reduced expression of TCR signaling molecules such as CD3zeta, PLC-gamma1, PKC-theta, Zap70, with reduced phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs). Among the alterations in TCR signaling molecules, defective CD3zeta expression is the most evident, and reversal of the anergic state by CD3/CD28 costimulation restored CD3zeta expression levels. Collectively, we suggest that reduced CD3zeta expression and defects in TCR signaling mediate the anergy state of MLN CD4(+) T cells, which play a critical role in maintenance of mucosal tolerance in gut-associated lymphoid tissue.