Little is known regarding the functional effects of common autoimmune susceptibility variants on human immune cells. The SNP CT60 (rs3087243; A/G) located in the 3' UTR of the CTLA4 gene has been associated with autoimmune diseases. We examined a cohort of healthy individuals stratified by genotypes at CTLA4 to gain insight into the functional effects of allelic variation on T cell signaling. Using phospho-site-specific mAbs, we tested the hypothesis that the CT60 genotype at CTLA4 is associated with altered T cell antigen receptor (TCR) signaling in naive and/or memory T cells. By normalizing for the extent of the initial TCR signaling event at CD3zeta, we observed that the relative responsiveness to TCR stimulation as assessed by phosphorylation levels of downstream signaling molecules was altered in naive (CD4(+)CD45RA(high)) and memory (CD4(+)CD45RA(low)) T cells obtained from individuals with the disease-susceptibility allele at CTLA4. Thus, allelic variation associated with autoimmune disease can alter the signaling threshold of CD4(+) T cells. These experiments provide a rational approach for the dissection of T cell-susceptibility genes in autoimmune diseases.

Graves' disease (GD) and Hashimoto's thyroiditis (HT) make up the autoimmune thyroid diseases (AITD), with classical clinical characteristics arising as a result of environmental factors in people who are genetically predisposed. Three gene regions consistently associated with AITD include the Human Leucocyte Antigen (HLA) region, CTLA4 and PTPN22, which represent general autoimmune risk loci and encode molecules vital for correct immune system function. AITD patients in addition are likely to carry at least one thyroid specific susceptibility locus. Recent genetic studies have refined association of the TSHR with GD to within a 40kb region including intron 1, of the TSHR itself, with preliminary evidence to suggest altered mRNA isoform expression. These findings, combined with previous functional data demonstrating that the TSHR A-subunit is the primary autoantigenic region, suggests novel mechanisms for the onset of GD and a potential therapeutic target.

BACKGROUND

More than 200 articles have been published in the past 20 years on associations between genetic variants and risk of cervical cancer but the results have generally been inconsistent.

OBJECTIVE

To provide a synopsis of the current understanding of the genetic architecture of the risk of cervical cancer by conducting a systematic review and meta-analysis.

METHODS

We conducted a systematic literature search by a two-stage strategy using PubMed and other databases on or before 31 March 2012.

METHODS

Cross-sectional, case-control or cohort studies about the relationship between genetic variants and cervical cancer were included.

METHODS

Study outcomes were presented as odds ratios (ORs) with a 95% confidence interval.We did the meta-analysis for genetic variants which had at least three data sources and for which the significant associations were assessed using the Venice criteria.

RESULTS

A total of 5605 publications were screened, of which 286 were eligible. Meta-analysis was conducted for 58 variants in 25 genes or loci. Fourteen variants in 11 genes or loci could increase the risk of cervical cancer and five variants in three genes or loci could decrease the risk. The epidemiological evidence of the association was graded as strong for four variants in CTLA4 and HLA DQB1, moderate for five variants in IL-1B, IL-10, XRCC3 and HLA DQA1, and weak for 10 variants.

CONCLUSIONS

Many genetic variants were associated with the risk of cervical cancer as supported by the epidemiological evidence in this meta-analysis.

BACKGROUND

Systemic inhibition of the inflammatory enzyme cyclooxygenase (COX) 2 decreases the risk of breast cancer and its recurrence. However, the biology of COX-2 in the multicellular tumor microenvironment is poorly defined.

METHODS

Mammary tumor onset and multiplicity were examined in ErbB2 transgenic mice that were deficient in mammary epithelial cell COX-2 (COX-2(MEC)KO) compared to wild type (WT) mice. Tumors were analyzed, by real time PCR, immune-staining and flow cytometry, for proliferation, apoptosis, angiogenesis and immune microenvironment. Lentiviral shRNA delivery was used to knock down (KD) COX-2 in ErbB2-transformed mouse breast cancer cells (COX-2KD), and growth as orthotopic tumors was examined in syngenic recipient mice, with or without depletion of CD8+ immune cells.

RESULTS

Mammary tumor onset was delayed, and multiplicity halved, in COX-2(MEC)KO mice compared to WT. COX-2(MEC)KO tumors showed decreased expression of Ki67, a proliferation marker, as well as reduced VEGFA, its receptor VEGFR2, endothelial NOS and the vascular endothelial marker CD31, indicating reduced tumor vascularization. COX-2(MEC)KO tumors contained more CD4+ T helper (Th) cells and CD8+ cytotoxic immune cells (CTL) consistent with increased immune surveillance. The ratio of Th markers Tbet (Th1) to GATA3 (Th2) was higher, and levels of Retnla, a M2 macrophage marker, lower, in COX-2(MEC)KO tumor infiltrating leukocytes compared to WT, suggesting a prevalence of pro-immune Th1 over immune suppressive Th2 lymphocytes, and reduced macrophage polarization to the immune suppressive M2 phenotype. Enhanced immune surveillance in COX-2(MEC)KO tumors was coincident with increased intratumoral CXCL9, a T cell chemoattractant, and decreased expression of T lymphocyte co-inhibitory receptors CTLA4 and PD-1, as well as PD-L1, the ligand for PD-1. PD-L1 was also decreased in IFNγ-treated COX-2KD mouse mammary cancer cells in vitro and, compared to control cells, growth of COX-2KD cells as orthotopic tumors in immune competent mice was markedly suppressed. However, robust growth of COX-2KD tumor cells was evident when recipients were depleted of CD8+ cells.

CONCLUSIONS

The data strongly support that, in addition to its angiogenic function, tumor cell COX-2 suppresses intratumoral cytotoxic CD8+ immune cell function, possibly through upregulation of immune checkpoints, thereby contributing to tumor immune escape. COX-2 inhibition may be clinically useful to augment breast cancer immunotherapy.

OBJECTIVE

Genome-wide association studies have revealed several susceptibility genes among patients with autoimmune thyroid disease (AITD), including CTLA4, PTPN22, FCRL3, and ZFAT. However, any possible association between these genes and AITD prognosis remains unknown. The objective of this study was to identify associations between polymorphisms of these genes and AITD prognosis.

METHODS

We genotyped functional polymorphisms, including CTLA4 CT60, CTLA4 +49A/G, CTLA4 -1147C/T, CTLA4 -318C/T, PTPN22 -1123C/G, PTPN22 SNP37, CD40 -1C/T, FCRL3 -169C/T, ZFAT Ex9b-SNP10, and ZFAT Ex9b-SNP2, in 197 AITD patients carefully selected from 456 registered AITD patients, and 86 control subjects. The restriction fragment length polymorphism method was used for genotyping.

RESULTS

The CD40 -1CC genotype and C allele were significantly more frequent in patients with Graves' disease (GD) in remission than in those with intractable GD (P = 0.041 and P = 0.031, respectively). The FCRL3 -169TT genotype was significantly less frequent in patients with intractable GD than in those with GD in remission (P = 0.0324). For a ZFAT Ex9b-SNP10 polymorphism, the TT genotype and T allele were significantly more frequent in patients with severe Hashimoto's disease (HD) than in those with mild HD (P = 0.0029 and P = 0.0049, respectively). For a CTLA4 CT60 polymorphism, the antithyrotropin receptor antibody levels at the onset of GD were significantly higher in those with the GG genotype than in those with other genotypes (P = 0.0117).

CONCLUSIONS

CD40 and FCRL3 gene polymorphisms were associated with GD intractability, and ZFAT polymorphism was associated with HD severity but not its development.

CTLA4 is currently viewed as a late-appearing T cell surface receptor which is able to inhibit the proliferation of activated T cells. We sought to identify how CTLA4 ligation exerts these anti-proliferative effects by studying its influence on the activities of the relevant nuclear transcription factors AP-1, NFAT and NF-kappaB. We found that cross-linking CTLA4 on activated T cells completely blocks AP-1 and NFAT transcription factor activity before any effects on T cell proliferation can be observed, with NF-kappaB activity affected to a lesser degree. The suppression of AP-1 and NFAT transcriptional activity correlates with reduced levels of AP-1 and NFAT DNA binding as early as 10 h after T cell activation, prior to detectable up-regulation of CTLA4 on the T cell surface. Additionally, inhibitory effects on T cell proliferation only occurred when CTLA4 molecules were ligated in proximity to signaling TCR complexes, and inhibition of transcription factor DNA binding and activity was observed in the absence of CD28 stimulation. CTLA4 can thus act early during T cell activation to reduce the activity of several key nuclear transcription factors important for continued T cell proliferation and differentiation.

Mesenchymal stem cells (MSCs) have fueled ample translation for the treatment of immune-mediated diseases. They exert immunoregulatory and tissue-restoring effects. MSC-mediated transfer of mitochondria (MitoT) has been demonstrated to rescue target organs from tissue damage, yet the mechanism remains to be fully resolved. Therefore, we explored the effect of MitoT on lymphoid cells. Here, we describe dose-dependent MitoT from mitochondria-labeled MSCs mainly to CD4⁺ T cells, rather than CD8⁺ T cells or CD19⁺ B cells. Artificial transfer of isolated MSC-derived mitochondria increases the expression of mRNA transcripts involved in T-cell activation and T regulatory cell differentiation including FOXP3, IL2RA, CTLA4, and TGFβ1, leading to an increase in a highly suppressive CD25⁺ FoxP3⁺ population. In a GVHD mouse model, transplantation of MitoT-induced human T cells leads to significant improvement in survival and reduction in tissue damage and organ T CD4⁺ , CD8⁺ , and IFN-γ⁺ expressing cell infiltration. These findings point to a unique CD4⁺ T-cell reprogramming mechanism with pre-clinical proof-of-concept data that pave the way for the exploration of organelle-based therapies in immune diseases.

The immune system ensures optimum T-effector (Teff) immune responses against invading microbes and tumor antigens while preventing inappropriate autoimmune responses against self-antigens with the help of T-regulatory (Treg) cells. Thus, Treg and Teff cells help maintain immune homeostasis through mutual regulation. While Tregs can contribute to tumor immune evasion by suppressing anti-tumor Teff response, loss of Treg function can result in Teff responses against self-antigens leading to autoimmune disease. Thus, loss of homeostatic balance between Teff/Treg cells is often associated with both cancer and autoimmunity. Co-stimulatory and co-inhibitory receptors, collectively known as co-signaling receptors, play an indispensable role in the regulation of Teff and Treg cell expansion and function and thus play critical roles in modulating autoimmune and anti-tumor immune responses. Over the past three decades, considerable efforts have been made to understand the biology of co-signaling receptors and their role in immune homeostasis. Mutations in co-inhibitory receptors such as CTLA4 and PD1 are associated with Treg dysfunction, and autoimmune diseases in mice and humans. On the other hand, growing tumors evade immune surveillance by exploiting co-inhibitory signaling through expression of CTLA4, PD1 and PDL-1. Immune checkpoint blockade (ICB) using anti-CTLA4 and anti-PD1 has drawn considerable attention towards co-signaling receptors in tumor immunology and created renewed interest in studying other co-signaling receptors, which until recently have not been as well studied. In addition to co-inhibitory receptors, co-stimulatory receptors like OX40, GITR and 4-1BB have also been widely implicated in immune homeostasis and T-cell stimulation, and use of agonistic antibodies against OX40, GITR and 4-1BB has been effective in causing tumor regression. Although ICB has seen unprecedented success in cancer treatment, autoimmune adverse events arising from ICB due to loss of Treg homeostasis poses a major obstacle. Herein, we comprehensively review the role of various co-stimulatory and co-inhibitory receptors in Treg biology and immune homeostasis, autoimmunity, and anti-tumor immunity. Furthermore, we discuss the autoimmune adverse events arising upon targeting these co-signaling receptors to augment anti-tumor immune responses.

Preoperative immunotherapy with anti-PD1 plus anti-CTLA4 antibodies has shown remarkable pathological responses in melanoma¹ and colorectal cancer². In NABUCCO (ClinicalTrials.gov: NCT03387761), a single-arm feasibility trial, 24 patients with stage III urothelial cancer (UC) received two doses of ipilimumab and two doses of nivolumab, followed by resection. The primary endpoint was feasibility to resect within 12 weeks from treatment start. All patients were evaluable for the study endpoints and underwent resection, 23 (96%) within 12 weeks. Grade 3-4 immune-related adverse events occurred in 55% of patients and in 41% of patients when excluding clinically insignificant laboratory abnormalities. Eleven patients (46%) had a pathological complete response (pCR), meeting the secondary efficacy endpoint. Fourteen patients (58%) had no remaining invasive disease (pCR or pTisN0/pTaN0). In contrast to studies with anti-PD1/PD-L1 monotherapy, complete response to ipilimumab plus nivolumab was independent of baseline CD8⁺ presence or T-effector signatures. Induction of tertiary lymphoid structures upon treatment was observed in responding patients. Our data indicate that combined CTLA-4 plus PD-1 blockade might provide an effective preoperative treatment strategy in locoregionally advanced UC, irrespective of pre-existing CD8⁺ T cell activity.

Potent immunosuppressive mechanisms within the tumor microenvironment contribute to the resistance of aggressive human cancers to immune checkpoint blockade (ICB) therapy. One of the main mechanisms for myeloid-derived suppressor cells (MDSCs) to induce T cell tolerance is through secretion of reactive nitrogen species (RNS), which nitrates tyrosine residues in proteins involved in T cell function. However, so far very few nitrated proteins have been identified. Here, using a transgenic mouse model of prostate cancer and a syngeneic cell line model of lung cancer, we applied a nitroproteomic approach based on chemical derivation of 3-nitrotyrosine and identified that lymphocyte-specific protein tyrosine kinase (LCK), an initiating tyrosine kinase in the T cell receptor signaling cascade, is nitrated at Tyr394 by MDSCs. LCK nitration inhibits T cell activation, leading to reduced interleukin 2 (IL2) production and proliferation. In human T cells with defective endogenous LCK, wild type, but not nitrated LCK, rescues IL2 production. In the mouse model of castration-resistant prostate cancer (CRPC) by prostate-specific deletion of Pten, p53, and Smad4, CRPC is resistant to an ICB therapy composed of antiprogrammed cell death 1 (PD1) and anticytotoxic-T lymphocyte-associated protein 4 (CTLA4) antibodies. However, we showed that ICB elicits strong anti-CRPC efficacy when combined with an RNS neutralizing agent. Together, these data identify a previously unknown mechanism of T cell inactivation by MDSC-induced protein nitration and illuminate a clinical path hypothesis for combining ICB with RNS-reducing agents in the treatment of CRPC.

We used expression microarrays to characterize the changes most specific for pure T cell-mediated rejection (TCMR) compared to other diseases including antibody-mediated rejection in 703 human kidney transplant biopsies, using a Discovery Set-Validation Set approach. The expression of thousands of transcripts--fold change and association strength--changed in a pattern that was highly conserved between the Discovery and Validation sets, reflecting a hierarchy of T cell signaling, costimulation, antigen-presenting cell (APC) activation and interferon-gamma (IFNG) expression and effects, with weaker associations for inflammasome activation, innate immunity, cytotoxic molecules and parenchymal injury. In cell lines, the transcripts most specific for TCMR were expressed most strongly in effector T cells (e.g. CTLA4, CD28, IFNG), macrophages (e.g. PDL1, CD86, SLAMF8, ADAMDEC1), B cells (e.g. CD72, BTLA) and IFNG-treated macrophages (e.g. ANKRD22, AIM2). In pathway analysis, the top pathways included T cell receptor signaling and CTLA4 costimulation. These results suggest a model in which TCMR creates an inflammatory compartment with a rigorous hierarchy dominated by the proximal aspects of cognate engagement of effector T cell receptor and costimulator triggering by APCs. The prominence of inhibitors like CTLA4 and PDL1 raises the possibility of active negative controls within the rejecting tissue.

Ipilimumab is a monoclonal antibody that inhibits the CTLA4 receptor on cytotoxic T lymphocytes, resulting in immune-mediated tumor cell death. Ipilimumab is most often used in the treatment of metastatic melanoma, and rarely liver toxicity necessitating cessation of treatment occurs. The aim of this study was to characterize the histologic features and clinical course of ipilimumab-associated hepatitis. Eleven patients with clinical suspicion of ipilimumab-induced hepatitis, due to the development of abnormal liver function tests (LFTs) while receiving treatment, and who underwent liver biopsy, were identified over a 6-year period. Ten patients were male and 1 female (median age 58 y), and all received 1 to 4 doses of ipilimumab. None had known preexisting liver disease. Two patients were obese, and another had a history of alcohol abuse. Viral and autoimmune serologies were negative in all patients except 1 who had a mildly elevated ANA titer. Nine biopsies showed active hepatitis with 2 distinct histologic patterns: panlobular hepatitis in 6 cases and zone 3 hepatitis in 3. The inflammatory infiltrate was similar in composition in both patterns, composed predominantly of CD8+ T lymphocytes, admixed histiocytes, scattered plasma cells, and eosinophils. Prominent histiocytic sinusoidal infiltrates were present in 7 cases and frequently formed loose histiocytic aggregates. Central vein endothelialitis was present in 8 cases. Patients in this group tended to have markedly elevated ALT, AST, and total bilirubin. Two cases did not fit into the above 2 histologic groups: 1 showed portal inflammation with cholangitis, and the other showed morphologic features indistinguishable from nonalcoholic steatohepatitis. Discontinuation of ipilimumab and administration of immunosuppressives resulted in resolution or marked improvement of LFTs in all patients within 3 months of presentation. Ipilimumab may potentially unmask previously subclinical liver disease, for example, fatty liver disease, and the diagnosis of ipilimumab-induced liver injury may only be recognized with certainty after cessation of the drug leads to normalization of LFTs. Overall, ipilimumab-associated hepatitis most often presents with a panlobular active hepatitis that resembles autoimmune hepatitis. Prominent sinusoidal histiocytic infiltrates and central vein damage with endothelialitis may be helpful histologic clues to the diagnosis of ipilimumab-associated hepatitis.

Preclinical models predict that blockade of the coinhibitory molecule cytotoxic T lymphocyte-associated antigen 4 (CTLA4) on lymphocytes results in the release of a cell cycle inhibitory checkpoint, allowing lymphocyte proliferation, tumor targeting, and regression. However, there is a paucity of data demonstrating that lymphocyte proliferation does occur in humans treated with CTLA4-blocking antibodies.

METHODS

We tested the role of whole-body molecular imaging in patients with advanced melanoma receiving the CTLA4-blocking antibody tremelimumab, allowing the analysis of changes in glucose metabolism using the PET probe (18)F-FDG and cell replication with the PET probe 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT).

RESULTS

PET/CT scans obtained at a median of 2 mo after initial dosing did not demonstrate significant changes in lesion size or (18)F-FDG or (18)F-FLT uptake when focusing on metastatic lesions. Similarly, there was no difference in (18)F-FDG uptake in the non-melanoma-involved spleen. However, there were significant increases in standardized uptake values for (18)F-FLT in the spleen using post- and pretremelimumab treatment scans.

CONCLUSIONS

Molecular imaging with the PET probe (18)F-FLT allows mapping and noninvasive imaging of cell proliferation in secondary lymphoid organs after CTLA4 blockade in patients with metastatic melanoma.

The major goal of this study was to perform an in depth characterization of the "gene signature" of human FoxP3(+) T regulatory cells (Tregs). Highly purified Tregs and T conventional cells (Tconvs) from multiple healthy donors (HD), either freshly explanted or activated in vitro, were analyzed via RNA sequencing (RNA-seq) and gene expression changes validated using the nCounter system. Additionally, we analyzed microRNA (miRNA) expression using TaqMan low-density arrays. Our results confirm previous studies demonstrating selective gene expression of FoxP3, IKZF2, and CTLA4 in Tregs. Notably, a number of yet uncharacterized genes (RTKN2, LAYN, UTS2, CSF2RB, TRIB1, F5, CECAM4, CD70, ENC1 and NKG7) were identified and validated as being differentially expressed in human Tregs. We further characterize the functional roles of RTKN2 and LAYN by analyzing their roles in vitro human Treg suppression assays by knocking them down in Tregs and overexpressing them in Tconvs. In order to facilitate a better understanding of the human Treg gene expression signature, we have generated from our results a hypothetical interactome of genes and miRNAs in Tregs and Tconvs.

The tumor development and metastasis are closely related to the structure and function of the tumor microenvironment (TME). Recently, TME modulation strategies have attracted much attention in cancer immunotherapy. Despite the preliminary success of immunotherapeutic agents, their therapeutic effects have been restricted by the limited retention time of drugs in TME. Compared with traditional delivery systems, nanoparticles with unique physical properties and elaborate design can efficiently penetrate TME and specifically deliver to the major components in TME. In this review, we briefly introduce the substitutes of TME including dendritic cells, macrophages, fibroblasts, tumor vasculature, tumor-draining lymph nodes and hypoxic state, then review various nanoparticles targeting these components and their applications in tumor therapy. In addition, nanoparticles could be combined with other therapies, including chemotherapy, radiotherapy, and photodynamic therapy, however, the nanoplatform delivery system may not be effective in all types of tumors due to the heterogeneity of different tumors and individuals. The changes of TME at various stages during tumor development are required to be further elucidated so that more individualized nanoplatforms could be designed.

Keywords: AC-NPs, antigen-capturing nanoparticles; ANG2, angiopoietin-2; APCs, antigen-presenting cells; Ab, antibodies; Ag, antigen; AuNCs, gold nanocages; AuNPs, gold nanoparticles; BBB, blood-brain barrier; BTK, Bruton's tyrosine kinase; Bcl-2, B-cell lymphoma 2; CAFs, cancer associated fibroblasts; CAP, cleavable amphiphilic peptide; CAR-T, Chimeric antigen receptor-modified T-cell therapy; CCL, chemoattractant chemokines ligand; CTL, cytotoxic T lymphocytes; CTLA4, cytotoxic lymphocyte antigen 4; CaCO3, calcium carbonate; Cancer immunotherapy; DCs, dendritic cells; DMMA, 2,3-dimethylmaleic anhydrid; DMXAA, 5,6-dimethylxanthenone-4-acetic acid; DSF/Cu, disulfiram/copper; ECM, extracellular matrix; EGFR, epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; EPG, egg phosphatidylglycerol; EPR, enhanced permeability and retention; FAP, fibroblast activation protein; FDA, the Food and Drug Administration; HA, hyaluronic acid; HB-GFs, heparin-binding growth factors; HIF, hypoxia-inducible factor; HPMA, N-(2-hydroxypropyl) methacrylamide; HSA, human serum albumin; Hypoxia; IBR, Ibrutinib; IFN-γ, interferon-γ; IFP, interstitial fluid pressure; IL, interleukin; LMWH, low molecular weight heparin; LPS, lipopolysaccharide; M2NP, M2-like TAM dual-targeting nanoparticle; MCMC, mannosylated carboxymethyl chitosan; MDSCs, myeloid-derived suppressor cells; MPs, microparticles; MnO2, manganese dioxide; NF-κB, nuclear factor κB; NK, nature killer; NO, nitric oxide; NPs, nanoparticles; Nanoparticles; ODN, oligodeoxynucleotides; PD-1, programmed cell death protein 1; PDT, photodynamic therapy; PFC, perfluorocarbon; PHDs, prolyl hydroxylases; PLGA, poly(lactic-co-glycolic acid); PS, photosensitizer; PSCs, pancreatic stellate cells; PTX, paclitaxel; RBC, red-blood-cell; RLX, relaxin-2; ROS, reactive oxygen species; SA, sialic acid; SPARC, secreted protein acidic and rich in cysteine; TAAs, tumor-associated antigens; TAMs, tumor-associated macrophages; TDPA, tumor-derived protein antigens; TGF-β, transforming growth factor β; TIE2, tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2; TIM-3, T cell immunoglobulin domain and mucin domain-3; TLR, Toll-like receptor; TME, tumor microenvironment; TNF-α, tumor necrosis factor alpha; TfR, transferrin receptor; Tregs, regulatory T cells; Tumor microenvironment; UPS-NP, ultra-pH-sensitive nanoparticle; VDA, vasculature disrupting agent; VEGF, vascular endothelial growth factor; cDCs, conventional dendritic cells; melittin-NP, melittin-lipid nanoparticle; nMOFs, nanoscale metal-organic frameworks; scFv, single-chain variable fragment; siRNA, small interfering RNA; tdLNs, tumor-draining lymph nodes; α-SMA, alpha-smooth muscle actin.

T cell-dependent regulation of B cell growth and differentiation involves an interaction between CD40, a B cell surface molecule, and the CD40 ligand (CD40L) which is expressed on activated CD4+ T cells. In the current study, we show that recombinant membrane-bound murine CD40L induces B cells to express costimulatory function for the proliferation of CD4+ T cells. CD40L- or lipopolysaccharide (LPS)-activated, but not control-cultured B cells were strong costimulators of anti-CD3 or alloantigen-dependent T cell responses. The molecular interactions responsible for the increased costimulatory functions were examined by analyzing the activated B cells for changes in the expression of two costimulatory molecules, B7 and heat-stable antigen (HSA), as well as by the use of antagonists of B7 and HSA (CTLA4.Fc and 20C9, respectively). The expression of both B7 and HSA was enhanced on B cells activated with LPS. As observed in previous studies, the costimulatory activity of the LPS-activated B cells was dependent on both B7 and HSA and was completely inhibited in the presence of a combination of CTLA4.Fc and 20C9. In contrast, activation of B cells with CD40L induced the expression of B7 but did not enhance the expression of HSA. In addition the costimulatory activity of the CD40L-activated B cells was partially, but not completely, inhibited by the combination of CTLA4.Fc and 20C9. These results demonstrate that CD40L regulates costimulatory function of B cells in part by inducing the expression of B7 and suggest that CD40L-activated B cells express an additional costimulatory activity that is not associated with LPS-activated B cells.

Antigen-specific T cells, which express CD154 rapidly, but remain untested in alloimmunity, were measured with flow cytometry in 16-h MLR of 58 identically-immunosuppressed children with liver transplantation (LTx), to identify Rejectors (who had experienced biopsy-proven rejection within 60 days posttransplantation). Thirty-one children were sampled once, cross-sectionally. Twenty-seven children were sampled longitudinally, pre-LTx, and at 1-60 and 61-200 days after LTx. Results were correlated with proliferative alloresponses measured by CFSE-dye dilution (n = 23), and CTLA4, a negative T-cell costimulator, which antagonizes CD154-mediated effects (n = 31). In cross-sectional observations, logistic regression and leave-one-out cross-validation identified donor-specific, CD154 + T-cytotoxic (Tc)-memory cells as best associated with rejection outcomes. In the longitudinal cohort, (1) the association between CD154 + Tc-memory cells and rejection outcomes was replicated with sensitivity/specificity 92.3%/84.6% for observations at 1-60 days, and (2) elevated pre-LTx CD154 + Tc-memory cell responses were associated with significantly increased incidence (p = 0.02) and hazard (HR = 7.355) of rejection in survival/proportional hazard analysis. CD154 expression correlated with proliferative alloresponses (r = 0.835, p = 7.1e-07), and inversely with CTLA4 expression of allospecific CD154 + Tc-memory cells (r =-0.706, p = 3.0e-05). Allospecific CD154 + T-helper-memory cells, not CD154 + Tc-memory, were inhibited by increasing Tacrolimus concentrations (p = 0.026). Collectively, allospecific CD154 + T cells provide an estimate of rejection risk in children with LTx.

To determine whether genetic heterogeneity exists in patients with Graves' disease (GD), the cytotoxic T-lymphocyte associated 4 (CTLA-4) gene, which is implicated a susceptibility gene for GD by considerable genetic and immunological evidence, was used for association analysis in a Chinese Han cohort recruited from various geographic regions. Our association study for the SNPs in the CTLA4 gene in 2640 GD patients and 2204 control subjects confirmed that CTLA4 is the susceptibility gene for GD in the Chinese Han population. Moreover, the logistic regression analysis in the combined Chinese Han cohort revealed that SNP rs231779 (allele frequencies p = 2.81x10(-9), OR = 1.35, and genotype distributions p = 2.75x10(-9), OR = 1.42) is likely the susceptibility variant for GD. Interestingly, the logistic regression analysis revealed that SNP rs35219727 may be the susceptibility variant to GD in the Shandong population; however, SNP, rs231779 in the CTLA4 gene probably independently confers GD susceptibility in the Xuzhou and southern China populations. These data suggest that the susceptibility variants of the CTLA4 gene varied between the different geographic populations with GD.

Abatacept (CTLA4-Ig) is a novel fusion protein designed to modulate the T cell co-stimulatory signal mediated through the CD28-CD80/86 pathway. Clinical trials have provided preliminary evidence of the efficacy of this compound in the treatment of rheumatoid arthritis. This review describes the molecular and biologic bases for the use of abatacept in rheumatoid arthritis and summarizes the current clinical data on its safety and effectiveness in this disease.

OBJECTIVE

To assess salivary gland tissues obtained from patients with primary Sjögren's syndrome (SS) for the gene expression profile of the candidate genes TNFRSF6 (Fas), TNFSF6 (FasL), SSA1 (Ro52 alpha and the splice variant Ro52 beta), SSB (La), CTLA4, PDCD1 (PD-1), and ORM2, which were selected on the basis of their putative participation in salivary gland inflammation.

METHODS

Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the expression of messenger RNA (mRNA). Tissue localization of the expressed proteins was detected by immunohistochemistry.

RESULTS

Expression of mRNA was increased for Fas (5.1-fold; P < 0.001), FasL (8.8-fold; P < 0.05), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (11.2-fold; P < 0.01), programmed cell death 1 (PD-1) (15.2-fold; P < 0.05), Ro52 alpha (3.0-fold; P < 0.01), La (2.3-fold; P < 0.05), and orosomucoid 2 (ORM2) (4.4-fold; P < 0.05) in patients compared with controls when GAPDH was used as endogenous standard in duplex runs. In single runs using 2 other endogenous standards (18S and beta-actin), statistically significant differences between patients and controls were confirmed for expression of Fas, FasL, CTLA-4, and PD-1, but this difference was not observed for Ro52 alpha, La, and ORM2. Expression of Ro52 beta mRNA was similar in patients and controls.

CONCLUSIONS

The present study demonstrates a substantial increase in expression of the negative regulator molecules PD-1 and CTLA-4 and the apoptotic signal molecules Fas and FasL in patients with primary SS compared with controls, which corresponded to the immunomorphologic pattern. The results strongly indicate that these molecules have central roles in the inflammatory process in the salivary glands of patients with primary SS.

The role of costimulation in Ag-induced responses of naive and differentiated IL-2 and IL-4-producing CD4+ T cells has been examined using cells from mice expressing a transgenic TCR specific for cytochrome c (81-104) + I-Ek. IL-2 secretion by naive and pre-activated T cells is dependent on costimulation, because it is not induced with chemically fixed APCs and is significantly inhibited by the B7 antagonist, CTLA4-Ig. In contrast, IL-4 secretion by in vitro differentiated Th2-like cells is relatively independent of costimulators. Thus, the requirement for costimulation is related more to lymphokine secretion profiles than to the previous activation status of CD4+ T lymphocytes.

Tolerogenic dendritic cells (Tol-DCs) and regulatory T cells (Treg) are key factors in the induction and maintenance of transplantation tolerance. We previously demonstrated that ex vivo-isolated Tol-DCs promote Treg generation, and vice versa, in an in vitro co-culture system. Here we demonstrate the occurrence of such an immune regulatory feedback loop in vivo. Tol-DC generated in vitro by treatment with LF 15-0195 exhibited features of immature DC and express low levels of MHC class II, CD86 and CD40. These Tol-DCs were capable of augmenting CD4(+)CD25(+)CTLA4(+) and FoxP3(+) Treg cell numbers and activity in cardiac allograft recipients. On the other hand, Tol-DCs possessed an ability to generate Treg cells in vitro. The adoptive transfer of these in vitro-generated Treg cells resulted in an increase of Tol-DC in vivo, suggesting that an immune regulatory feedback loop, between Tol-DC and Treg, exists in vivo. Furthermore, the administration of in vitro-generated Tol-DCs or Treg cells prevented rejection of allografts. Co-administration of Tol-DC and Treg synergized efficacy of promoting allograft survival heart transplantation. The present study highlights the therapeutic potential of preventing allograft rejection using in vitro-generated Tol-DCs and Treg.

OBJECTIVE

Defining an optimal costimulatory blockade-based immune suppression protocol enabling engraftment and functional development of E42 pig embryonic pancreatic tissue in mice.

METHODS

Considering that anti-CD40L was found to be thrombotic in humans, we sought to test alternative costimulatory blockade agents already in clinical use, including CTLA4-Ig, anti-LFA1, and anti-CD48. These agents were tested in conjunction with T-cell debulking by anti-CD4 and anti-CD8 antibodies or with conventional immunosuppressive drugs. Engraftment and functional development of E42 pig pancreatic tissue was monitored by immunohistology and by measuring pig insulin blood levels.

RESULTS

Fetal pig pancreatic tissue harvested at E42, or even as early as at E28, was fiercely rejected in C57BL/6 mice and in Lewis rats. A novel immune suppression comprising anti-LFA1, anti-CD48, and FTY720 afforded optimal growth and functional development. Cessation of treatment with anti-LFA1 and anti-CD48 at 3 months posttransplant did not lead to graft rejection, and graft maintenance could be achieved for >8 months with twice-weekly low-dose FTY720 treatment. These grafts exhibited normal morphology and were functional, as revealed by the high pig insulin blood levels in the transplanted mice and by the ability of the recipients to resist alloxan induced diabetes.

CONCLUSIONS

This novel protocol, comprising agents that simulate those approved for clinical use, offer an attractive approach for embryonic xenogeneic transplantation. Further studies in nonhuman primates are warranted.

It is well established that B7-CD28/CTLA4 interactions play an important role in the induction of T helper cells for T-dependent antibody responses. However, targeted mutation of CD28 does not significantly affect production of IgG and activation of CD4 T helper cells in response to infections by some viruses and nematode parasites. To test whether the CD28-independent induction of Ig class switches requires costimulation by the heat-stable antigen (HSA), we compared T helper cell induction and antibody response in mice deficient for either HSA, CD28, or both genes. We found that after immunization with KLH-DNP, mice deficient for both CD28 and HSA lack DNP-specific IgA and all subtypes of IgG. This deficiency corresponds to a reduced number of effector helper T cells that rapidly produce IL-2, IL-4, and IFN-gamma after in vitro stimulation with carrier antigen KLH. In contrast, priming of T helper cells and Ig class switch are normal in mice deficient with either HSA or CD28 alone. IgM responses are not affected by any of these targeted mutations. These results demonstrate that CD28-independent induction of T helper cells and Ig class-switches requires costimulation by the HSA.