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Objectives: SLE is an autoimmune disease characterized by aberrant autoantibody production and immune dysfunctions. Whether the anti-CMV immunity is impaired in SLE patients is poorly understood. We investigated the specific anti-viral T-cell response in SLE patients with CMV infection and its possible impacts on clinical manifestations in lupus., Methods: CD28 null T-cell percentages were measured by flow cytometry in 89 SLE patients and 58 healthy controls. A specific anti-CMV CD8 T-cell response was assessed ex vivo by the production of intracellular cytokines in response to CMV phosphoprotein 65 (pp65) by flow cytometry. Clinical manifestations and immune parameters were analysed in SLE patients according to their CMV serostatus., Results: CD28 null T cells were significantly expanded in SLE patients. When the anti-CMV pp65 CD8 polyfunctional T cell response was analysed, as defined by production of at least three of four functional cytokines or effectors (intracellular IFN-γ, IL-2, TNF-α and surface CD107a), the results demonstrated that it was not impaired in SLE patients. In contrast, when comparing clinical manifestations, there were lower anti-ds-DNA levels and decreased SLEDAI in SLE patients with CMV infection. Furthermore, the expansion of CD4+CD28 null T cells was negatively associated with anti-ds-DNA levels and SLEDAI in these lupus patients., Conclusion: In SLE patients with CMV infection, the specific anti-CMV CD8 T-cell response is preserved but is associated with decreased disease activity and lower anti-DNA levels among these patients, suggesting CMV infection may mitigate lupus activity., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

CD28H and B7-H5 have been identified as receptor-ligand pairs in the B7/CD28 family, and have co-stimulatory activity in immune cells. Here, we have systematically reviewed the research reports concerning the CD28H/B7-H5 pathway. It was found that CD28H is mainly expressed in T cells and natural killer (NK) cells with naive and poorly differentiated properties, and repeated antigen stimulation leads to permanent loss of CD28H. In tumors, CD28H is mainly expressed in tissue-resident memory (T RM ) lymphocyte T cells, which is associated with improved tumor prognosis. B7-H5 is a ligand for CD28H and is widely expressed in tumor cells. B7-H5 expression is closely related to the prognosis of the tumor. Studies have shown that high expression of B7-H5 in tumor is related to a worse prognosis for lung cancer, osteosarcoma, oral squamous cell carcinoma (OSCC), breast carcinoma, human clear cell renal cell carcinoma (ccRCC), intrahepatic cholangiocarcinoma (ICC), bladder urothelial carcinoma (BUC) and colorectal cancer (CRC), but is associated with a better prognosis for pancreatic ductal adenocarcinoma (PDAC) and glioma. Controversial views exist in studies on gastric cancer prognosis., (© 2020 British Society for Immunology.)

Background: Infection is the leading cause of death in antineutrophil cytoplasmic antibody-associated vasculitis (AAV). Expansion of CD4+CD28null T cells is associated with increased risk of infection and mortality, but is only present in cytomegalovirus (CMV)-seropositive individuals. We hypothesized that subclinical CMV reactivation drives CD4+CD28null T-cell expansion, that this is associated with impaired immune response to heterologous antigens, and that antiviral therapy may ameliorate this., Methods: In a proof-of-concept open-label clinical trial, 38 CMV-seropositive AAV patients were randomized to receive valacyclovir for 6 months or no intervention. CMV reactivation was measured monthly in plasma and urine. CD4+CD28null T cells were enumerated at baseline and at 6 months. At 6 months, 36 patients were vaccinated with a 13-valent pneumococcal vaccine. Serotype-specific immunoglobulin G was assayed before and 4 weeks postvaccination to calculate the antibody response ratio., Results: Valacyclovir treatment suppressed subclinical CMV reactivation and reduced CD4+CD28null T-cell proportion. CD4+CD28null T-cell reduction correlated with improved vaccine response, whereas CMV reactivation associated with reduced response to vaccination. Furthermore, expansion of CD4+CD28null T cells was associated with a reduction in the functional capacity of the CD4 compartment., Conclusions: Suppression of CMV may improve the immune response to a T-cell-dependent pneumococcal vaccination in patients with AAV, thus offering potential clinical benefit., Clinical Trials Registration: NCT01633476.

The in-depth understanding of skin resident memory CD8 + T lymphocytes (T RM ) may help to uncover strategies for their manipulation during disease. We investigated isolated T RM from healthy human skin, which expressed the residence marker CD69, and compared them to circulating CD8 + T cell populations from the same donors. There were significantly increased proportions of CD8 + CD45RA - CD27 - T cells in the skin that expressed low levels of killer cell lectin-like receptor G1 (KLRG1), CD57, perforin and granzyme B. The CD8 + T RM in skin were therefore phenotypically distinct from circulating CD8 + CD45RA - CD27 - T cells that expressed high levels of all these molecules. Nevertheless, the activation of CD8 + T RM with T cell receptor (TCR)/CD28 or interleukin (IL)-2 or IL-15 in vitro induced the expression of granzyme B. Blocking signalling through the inhibitory receptor programmed cell death 1 (PD)-1 further boosted granzyme B expression. A unique feature of some CD8 + T RM cells was their ability to secrete high levels of tumour necrosis factor (TNF)-α and IL-2, a cytokine combination that was not seen frequently in circulating CD8 + T cells. The cutaneous CD8 + T RM are therefore diverse, and appear to be phenotypically and functionally distinct from circulating cells. Indeed, the surface receptors used to distinguish differentiation stages of blood T cells cannot be applied to T cells in the skin. Furthermore, the function of cutaneous T RM appears to be stringently controlled by environmental signals in situ., (© 2018 British Society for Immunology.)

The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB). The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.

Type I IFN can exert pro- and anti-inflammatory activities in the immune system. Here, we have investigated the mechanism by which IFN-α enhances early expression of the anti-inflammatory cytokine IL-10 in human CD45RA + CD4 + T cells. With the use of transcriptomic and biochemical approaches, we found distinct and combined contributions of the IFN and the TCR signaling pathways to the induction of STAT1/2/3 and the basic leucine zipper activating transcription factor-like ( BATF ) family members. Moreover, IFN-induced STAT3 phosphorylation was prolonged by the TCR response, whereas IFN-induced STAT2 phosphorylation was of long duration. With the use of RNA interference (RNAi), we identified STAT3 as the major actor and STAT2 as a contributor of the IFN action on IL-10 Upon TCR/IFN costimulation, STAT3 directly bound at the IL-10 conserved noncoding sequence (CNS)- 9, an enhancer element known to recruit BATF in CD4 T cells. The cosilencing of the 3 BATFs resulted in an overall reduction of IL-10 expression, but the promoting activity of IFN-α was retained. These results support the notion that the IFN action is indexed on BATF function and provide evidence for a cooperation between BATFs and STAT3, the latter being activated via early IFN and delayed TCR effects., (© Society for Leukocyte Biology.)

Besides mobilizing stem cells into the periphery, granulocyte colony-stimulating factor (G-CSF) has been shown to influence various types of innate and adaptive immune cells. For example, it impairs the effector function of cytotoxic T lymphocytes (CTLs). It is assumed that this effect is mediated indirectly by monocytes, regulatory T cells and immunomodulatory cytokines influenced by G-CSF. In this study, isolated G-CSF-treated CD8(+) T cells were stimulated antigen-dependently with peptide-major histocompatibility complex (pMHC)-coupled artificial antigen-presenting cells (aAPCs) or stimulated antigen-independently with anti-CD3/CD28 stimulator beads. By measuring the changes in interferon (IFN)-γ and granzyme B expression at the mRNA and protein level, we showed for the first time that G-CSF has a direct effect on CD8(+) CTLs, which was confirmed based on the reduced production of IFN-γ and granzyme B by the cytotoxic T cell line TALL-104 after G-CSF treatment. By investigating further elements affected by G-CSF in CTLs from stem cell donors and untreated controls, we found a decreased phosphorylation of extracellular-regulated kinase (ERK)1/2, lymphocyte-specific protein tyrosine kinase (Lck) and CD3ζ after G-CSF treatment. Additionally, miRNA-155 and activation marker expression levels were reduced. In summary, our results show that G-CSF directly influences the effector function of cytotoxic CD8(+) T cells and affects various elements of T cell activation., (© 2016 British Society for Immunology.)

T cells have a central pathogenic role in the aetiopathogenesis of rheumatoid arthritis (RA), and are therefore a favoured target of immunotherapy aiming at physical or functional elimination. Here we report an efficacy test of FR104, a new co-stimulation inhibitor directly targeting CD28 on T cells, in a translationally relevant model, the rhesus monkey model of collagen-induced arthritis (CIA). As a relevant comparator we used abatacept [cytotoxic T lymphocyte antigen immunoglobulin (CTLA Ig)], an antagonist of CTLA-4 binding to CD80/86 clinically approved for treatment of RA. Treatment with either compound was started at the day of CIA induction. Although FR104 previously demonstrated a higher control of T cell responses in vitro than abatacept, both compounds were equally potent in the suppression of CIA symptoms and biomarkers, such as the production of C-reactive protein (CRP) and interleukin (IL)-6 and anti-collagen type II (CII) serum antibody (IgM/IgG). However, in contrast to abatacept, FR104 showed effective suppression of CII-induced peripheral blood mononuclear cell (PBMC) proliferation. The current study demonstrates a strong potential of the new selective CD28 antagonist FR104 for treatment of RA., (© 2015 British Society for Immunology.)

Crohn's disease (CD) is a chronic inflammatory disease associated with a dysregulated T cell response towards intestinal microflora. Vitamin D has immune modulatory effects on T cells through the nuclear vitamin D receptor (VDR) in vitro. It is unclear how oral vitamin D treatment affects VDR expression. The aim of this study was to establish a flow cytometry protocol, including nuclear and cytoplasmic VDR expression, and to investigate the effects of vitamin D treatment on T cell VDR expression in CD patients. The flow cytometry protocol for VDR staining was developed using the human acute monocytic leukaemia cell line (THP-1). The protocol was evaluated in anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) from vitamin D3- (n = 9) and placebo-treated (n = 9) CD patients. Anti-VDR-stained PBMCs were examined by flow cytometry, and their cytokine production was determined by cytokine bead array. VDR, CYP27B1 and RXRα mRNA expression levels in CD4(+) T cells were measured by quantitative reverse transcriptase polymerase chain reaction. The flow cytometry protocol enabled detection of cytoplasmic and nuclear VDR expression. The results were confirmed by confocal microscopy and supported by correlation with VDR mRNA expression. VDR expression in CD4(+) T cells increased following stimulation. This VDR up-regulation was inhibited with 30% by vitamin D treatment compared to placebo in CD patients (P = 0027). VDR expression was correlated with in-vitro interferon-γ production in stimulated PBMCs (P = 0.01). Flow cytometry is a useful method with which to measure intracellular VDR expression. Vitamin D treatment in CD patients reduces T cell receptor-mediated VDR up-regulation., (© 2015 British Society for Immunology.)

Background: Adoptive T cell transfer (ACT) is currently under investigation for the treatment of metastatic cancer. Recent evidence suggests that the coinhibitory PD-1-PD-L1 axis plays a major role in ACT failure. We hypothesized that a new fusion receptor reverting PD-1-mediated inhibition into CD28 costimulation may break peripheral tolerance., Methods: Different PD-1-CD28 fusion receptor constructs were created and retrovirally transduced into primary T cell receptor transgenic murine CD8(+) T cells specific for ovalbumin (OT-1). Cytokine release, proliferation, cytotoxicity, and tumor recognition were analyzed in vitro. Antitumor efficacy and mode of action were investigated in mice bearing subcutaneous tumors induced with the pancreatic carcinoma cell line Panc02 expressing the model antigen ovalbumin (Panc-OVA). For antitumoral efficacy, six to eight mice per group were used. All statistical tests are two-sided., Results: Transduction of the PD-1-CD28 receptor constructs mediated enhanced cytokine release, T cell proliferation, and T cell-induced lysis of target tumor cells. The PD-1-CD28 receptor function was dependent on two of the CD28-signaling motifs and IFN-γ release. Treatment of mice with established Panc-OVA tumors with fusion receptor-transduced OT-1 T cells mediated complete tumor regression. Mice rejecting the tumor were protected upon subsequent rechallenge with either ovalbumin-positive or -negative tumors, indicative of a memory response and epitope spreading in nine of 11 mice vs none of the six naïve mice (P < .001). Treatment efficacy was associated with accumulation of IFN-γ-producing T cells and an increased ratio of CD8(+) T cells to immunosuppressive myeloid-derived suppressor cells in the tumors., Conclusions: Transduction of T cells with this new PD-1-CD28 receptor has the potential of breaking the PD-1-PD-L1-immunosuppressive axis in ACT., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The biologically active form of vitamin D3 , 1, 25-dihydroxyvitamin D3 (calcitriol), is a potent modulator of the immune response. We have shown previously that calcitriol modulates the immunoglobulin response in vitro and in vivo in mice and humans. To analyse the underlying molecular mechanisms we studied whether calcitriol-primed B cells modulate T cell activation and function. Human B cells were stimulated with anti-CD40 and interleukin (IL)-4 in the presence of increasing concentrations of calcitriol. After removal of calcitriol, primed B cells were co-cultured with autologous CD4(+) T cells; the B cell phenotype T cell activation and their consecutive cytokine production were also assessed. Naive T cells co-cultured with calcitriol-primed naive B cells showed a reduced expansion, nuclear factor of activated T cells, cytoplasmic 2 (NFATc2) expression and cytokine production upon restimulation. CD86 expression on B cells after calcitriol priming was identified as an underlying mechanism, as T cell activation and expansion was rescued by activating anti-CD28 antibodies. Our data indicate that calcitriol-primed B cells display an impaired capacity to activate T cells. Taken together, we identified a novel B cell-dependent vitamin D immune regulatory mechanism, namely by decreased co-stimulation of calcitriol-primed B cells., (© 2014 British Society for Immunology.)

We hypothesized that the aberrant expression of microRNAs (miRNAs) in rheumatoid arthritis (RA) T cells was involved in the pathogenesis of RA. The expression profile of 270 human miRNAs in T cells from the first five RA patients and five controls were analysed by real-time polymerase chain reaction. Twelve miRNAs exhibited potentially aberrant expression in RA T cells compared to normal T cells. After validation with another 22 RA patients and 19 controls, miR-223 and miR-34b were over-expressed in RA T cells. The expression levels of miR-223 were correlated positively with the titre of rheumatoid factor (RF) in RA patients. Transfection of Jurkat cells with miR-223 mimic suppressed insulin-like growth factor-1 receptor (IGF-1R) and transfection with miR-34b mimic suppressed cAMP response element binding protein (CREB) protein expression by Western blotting. The protein expression of IGF-1R but not CREB was decreased in RA T cells. The addition of recombinant IGF-1-stimulated interleukin (IL)-10 production by activated normal T cells, but not RA T cells. The transfection of miR-223 mimic impaired IGF-1-mediated IL-10 production in activated normal T cells. The expression levels of SCD5, targeted by miR-34b, were decreased in RA T cells after microarray analysis. In conclusion, both miR-223 and miR-34b were over-expressed in RA T cells, but only the miR-223 expression levels were correlated positively with RF titre in RA patients. Functionally, the increased miR-223 expression could impair the IGF-1-mediated IL-10 production in activated RA T cells in vivo, which might contribute to the imbalance between proinflammatory and anti-inflammatory cytokines., (© 2014 British Society for Immunology.)

Chronic bone infection is associated with bone resorption. From animal studies, CD3/CD28-activated T cells are known to enhance osteoclastogenesis and bone resorption. Because CD28 is expressed constitutively on T cells and its expression is down-regulated by chronic exposure to the inflammatory environment, we characterized co-stimulatory molecule expression on T cells from chronically infected patients. We used cytofluorometric techniques to phenotypically characterize T cells, its co-stimulatory molecules and perforin secretion from infected and non-infected human bones. Chronic bone infection was defined as infection lasting for more than a month. We show a higher T cell activation [human leucocyte antigen D-related (HLA-DR⁺)] in infected compared to non-infected bones: median being 16 versus 7%, P = 0·009 for CD4 T cells, and 33 versus 15%, P = 0·038 for CD8 T cells, respectively. However, T cell proliferation (Ki67⁺) was lower for CD8 T cells in infected bones: 26 versus 34%, P = 0·045. In contrast, we detected no difference in apoptosis and regulatory T cells. In infected bone, we found higher CD28-negative CD4⁺ T cells compared to non-infected bone: 20 versus 8%, respectively (P = 0·005); this T cell subset had higher CD11b expression and perforin secretion. Chronically infected human bones are characterized by an increase of CD28-negative CD4⁺ T cells, indicating long-term activated cells with cytotoxic ability. Therefore, this alteration of co-stimulatory molecules may modify interactions with osteoclasts and impact bone resorption., (© 2013 British Society for Immunology.)

The activating receptor NKG2D is mainly expressed in NK cells and CD8(+) T cells. NKG2D and CD28 recruit the p85 subunit of PI3K to propagate their signals through the YXXM signaling motif. The function of CD28 as a costimulatory molecule is well-established in T cells. Ligation of NKG2D on T cells costimulates TCR signaling, although the intracellular signaling pathways triggered by the two receptors may not be identical. In this study, we analyzed the function of the NKG2D receptor in human CD8(+) T cell chemotaxis toward a CXCL12 gradient. We found that costimulation of the TCR together with CD28 or NKG2D impairs cell migration, although the signaling pathways responsible for this effect differ. Whereas the Rho GTPase Rac1 is activated upon TCR and costimulation via CD28 and NKG2D, the activity of Cdc42 is increased only upon CD3/NKG2D activation. Moreover, knockdown of N-WASp expression with siRNA rescues migration rates after NKG2D-mediated costimulation but not after CD3/CD28 activation. CD28- and NKG2D-mediated costimulation induces cofilin activation by dephosphorylation. Inhibition of N-WASp by wiskostatin further decreases phosphorylation levels of cofilin, although this effect is especially severe upon CD3/NKG2D activation. Thus, our findings reveal new differences in the signaling pathways between CD28- and NKG2D-mediated costimulation in the regulation of cell chemotaxis in human CD8(+) T cells.

Staphylococcus aureus and group A Streptococcus pyogenes (GAS) express superantigen (SAg) exotoxin proteins capable of inducing lethal shock. To induce toxicity, SAgs must bind not only to the major histocompatibility complex II molecule of antigen-presenting cells and the variable β chain of the T-cell receptor but also to the dimer interface of the T-cell costimulatory receptor CD28. Here, we show that the CD28-mimetic peptide AB103 (originally designated "p2TA") protects mice from lethal challenge with streptococcal exotoxin A, as well as from lethal GAS bacterial infection in a murine model of necrotizing soft-tissue infection. Administration of a single dose of AB103 increased survival when given up to 5 hours after infection, reduced inflammatory cytokine expression and bacterial burden at the site of infection, and improved muscle inflammation in a dose-dependent manner, without compromising cellular and humoral immunity. Thus, AB103 merits further investigation as a potential therapeutic in SAg-mediated necrotizing soft-tissue infection.

Allergen-specific immunotherapy (SIT) is the only treatment for allergic diseases that targets allergen-specific T helper type 2 (Th2) cells, which are the cause of the disease. There is an unmet requirement for adjuvants that increase the clinical efficacy of SIT allowing application of lower doses of the allergen, thereby reducing the risk of anaphylactic reactions. Cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA-4-Ig) has been shown to induce immunological tolerance in autoimmunity and allograft transplantation by blocking T cell co-stimulation and induction of the immunoregulatory enzyme indoleamine 2,3 dioxygenase (IDO). Previously, we showed that CTLA-4-Ig treatment at the time of allergen inhalation induced tolerance to subsequent allergen exposure in a mouse model of asthma. In this study, we test the hypothesis that CTLA-4-Ig acts as an adjuvant for experimental SIT. We evaluated the adjuvant effects of CTLA-4-Ig on SIT in a mouse model of ovalbumin-driven asthma. We used both wild-type and IDO-deficient mice to assess the role of IDO in the adjuvant effects of CTLA-4-Ig. Co-administration of CTLA-4-Ig strongly increased SIT-induced suppression of airway hyperreactivity (AHR), specific IgE in serum, airway eosinophilia and Th2 cytokine levels. Moreover, we found that CTLA-4-Ig, as an adjuvant for SIT, is equally effective in IDO-deficient and wild-type mice, demonstrating that the effect of CTLA-4-Ig is independent of IDO expression. We show that CTLA-4-Ig acts as a potent adjuvant to augment the therapeutic effects of SIT. As the adjuvant activity of CTLA-4-Ig is independent of IDO, we conclude that it acts by blocking CD28-mediated T cell co-stimulation., (© 2012 British Society for Immunology.)

Human lamina propria T lymphocytes (LPT) possess functional properties profoundly different from those of peripheral blood T lymphocytes (PBT). While they are characterized by a low proliferative response to T cell receptor (TCR)/CD3 stimulation in vitro their responsiveness to activation through the 'co-stimulatory' CD2-receptor is enhanced when compared to PBT. In this study, we demonstrate that engagement of another co-stimulatory receptor on both LPT and PBT, namely CD28, by a single monoclonal antibody (mAb), respectively, strongly activates the former but not the latter through a PI3-kinase dependent signalling pathway leading to the production of inflammatory cytokines such as interleukin (IL)-2, tumour necrosis factor (TNF)-α, interferon (IFN)-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition to the high sensitivity of LPT to CD2 stimulation, this finding supports the notion that 'non-specific/innate' mechanisms to activate T lymphocytes play a predominant role vis-à-vis'TCR driven/adaptive' responses in the intestinal mucosa. Furthermore, it suggests that results from preclinical tests for therapeutic antibodies performed with human blood derived T cells are probably insufficient to predict reactivities of tissue-resident immune cells, which--given their quantitative predominance--may critically determine the in-vivo response to such compounds., (© 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.)

CD7 is a cell-surface molecule, expressed on T lymphocytes and NK cells, which functions as a costimulatory receptor for T cell proliferation. SECTM1 has been proposed as a ligand for CD7. However, the expression pattern of this molecule in human immune cells and role in human T cell function remain unclear. In the present study, using human rSECTM1, we demonstrate that SECTM1 strongly costimulates CD4 and CD8 T cell proliferation and induces IFN-γ production, likely via a CD7-dependent mechanism. In addition, SECTM1 synergizes with suboptimal anti-CD28 to strongly augment T cell functions. We found a robust induction of IL-2 production when SECTM1 and anti-CD28 signals were present with TCR ligation. Furthermore, addition of SECTM1 into a MLR significantly enhanced proliferation of alloantigen-activated T cells, whereas blockade of SECTM1 inhibited T cell proliferation in a two-way MLR assay. Simultaneously blocking the effect of SECTM1, along with CTLA-4/Fc, diminishes two-way MLR. Finally, we demonstrated that expression of SECTM1 is not detected in monocytes and imMoDCs at the protein level. However, it is strongly induced by IFN-γ in monocytes and imMoDCs, and this induction is STAT1-dependent. These results indicate that SECTM1 is a broadly expressed, IFN-γ-inducible molecule, which functions as a potent costimulatory ligand for T cell activation and is synergistic with anti-CD28.

The non-catalytic region of tyrosine kinase (Nck) is proposed to play an essential role in T cell activation. However, evidence based on functional and biochemical studies has brought into question the critical function of Nck. Therefore, the aim of the present work was to investigate the role of Nck in T cell activation. To study this, the human Jurkat T cell line was used as a model for human T lymphocytes. The short interfering (si) RNA targeting Nck1 gene was used with electroporation to knock-down Nck1 protein expression in Jurkat T cells. Primary human CD4 T cells were also transfected with the siRNA of Nck1. The results showed that decreased Nck1 protein expression did not affect the apoptosis of the transfected Jurkat T cells compared with control siRNA-transfected cells and non-transfected cells. Upon CD3ε/CD28 stimulation, knock-down of Nck1 in Jurkat T cells caused a decrease in CD69 expression and in interleukin (IL)-2 secretion. Similarly, knock-down of Nck1 in primary CD4 T cells also caused decreased CD69 expression. However, no significant alterations of CD69 and IL-2 expression were found upon phytohaemagglutinin (PHA)/phorbol myristate acetate (PMA) stimulation. Knock-down of Nck1 had no effect on the proliferation of Jurkat T cells stimulated with either PHA or anti-T cell receptor (TCR) monoclonal antibody (C305). The reduced Nck1 expression in Jurkat cells was also associated with a reduced phosphorylation of extracellular regulated kinase (Erk)1 and Erk2 proteins upon CD3ε/CD28 stimulation. In conclusion, the decreased Nck1 protein in Jurkat T cells resulted in an impairment of TCR-CD3-mediated activation involving a defective Erk phosphorylation pathway., (© 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.)

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease; it is a leading cause of death and existing treatments have no proven disease-modifying effect. The mechanisms underlying this resistance are largely unknown, but suggest the presence of some self-maintaining pathogenic process, possibly initiated by cigarette smoking, that prevents the normal resolution of inflammation. We have previously reported increased production of proinflammatory cytokines and granzyme b by CD8(+) T cells in COPD; costimulatory receptor/ligand interactions required include CD80:86/CD28, B7-1/CTLA4, 4-1BB/1BBL and OX40/OX40L. We hypothesized that a dysregulated expression/function of these molecules may play a role in inflammatory/autoimmune components of COPD. We analysed T cell co-stimulatory molecules in blood from 34 controls, 15 smokers and 48 COPD subjects. We assessed the potential functional relevance of CD8/CD28(null) cells in COPD by measuring their production of proinflammatory cytokines, co-stimulatory molecules, granzyme and perforin. A smoke-exposed murine model was applied to investigate the relative expression of CD8/CD28(null) T cells in blood, lung tissue and airway. CD8/CD28(null) cells were increased in both current- and ex-smoker COPD groups; these cells expressed significantly more interferon (IFN)-γ, OX40, 4-1BB, CTLA4, granzyme and perforin when stimulated than CD8/CD28(+) T cells. There were no changes in CD4/CD28(null) T cells. In mice exposed to cigarette smoke for 12 weeks, CD8/CD28(null) T cells were significantly increased in the airway with a trend for an increase in lung tissue and blood. Increased production of proinflammatory cytokines and expression of alternative co-stimulatory molecules by CD8/CD28(null) T cells may play a role in inflammatory or autoimmune responses in COPD and identify therapeutic targets., (© 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.)

Background: CD28(-) T lymphocytes progressively increase during aging, autoimmunity, and HIV-1 infection. Expansion of these cells stands in contrast with their senescent phenotype described by several studies. Understanding the functional properties and phenotype of CD28(-) T cell during HIV-1 infection is important, because this subset incorporates T cells specific for HIV-1 and other chronic pathogens., Methods: Blood samples were obtained from 23 healthy and 43 HIV-1-infected individuals: 26 receiving antiretroviral therapy and 17 naive to treatment. The phenotype of CD28(-) and CD28(+) T cells was determined by flow cytometry. T cells were activated through T-cell receptor before apoptosis and proliferation measurements. Interleukin (IL)-2, tumor-necrosis factor, interferon-γ, and perforin production were analyzed using enzyme-linked immunosorbent assay., Results: CD28(-) T cells from patients receiving antiretroviral therapy exhibited a low sensitivity to apoptosis and enhanced proliferation after TCR stimulation, compared with T cells of uninfected individuals. On the contrary, CD28(-) T cells from viremic patients showed a decreased Bcl-2 expression, a high sensitivity to apoptosis, and poor proliferative ability, compared with treated patients and control subjects. T cells from untreated patients produced less IL-2, possibly underlying their decreased proliferative abilities., Conclusions: The level of HIV-1 replication and associated immunoactivation represent a critical factor in regulating survival and activation of CD28(-) T cells.

Aims: Diabetes mellitus (DM) is associated with high incidence of first and recurrent cardiovascular events, especially acute coronary syndromes (ACSs); however, the mechanisms involved are still unknown. We sought to investigate the role of CD4(+)CD28(null)T-lymphocytes, a rare long-lived subset of T-lymphocytes with proatherogenic and plaque-destabilizing properties, in the increased cardiovascular risk associated with DM., Methods and Results: CD4(+)CD28(null)T-cell frequency was analysed by flow-cytometry in 60 DM patients without overt cardiovascular disease (cDM), in 166 ACS patients with or without DM (ACS/DM+, n= 51 and ACS/DM-, n= 115), and in 60 healthy individuals. The incidence of cardiovascular events (death, myocardial infarction, unstable angina) was assessed at 36 months follow-up. CD4+CD28(null)T-cell frequency (median, range) was higher in ACS/DM+ (12.7%, 0.1-48) vs. ACS/DM- (3.9%, 0.2-35), cDM (3.1%, 0.3-22.4), and controls (1.5%, 0.1-9.1) (P< 0.001 for all comparisons). Notably, cDM patients had significantly higher CD4+CD28(null)T-cell frequency than controls (P= 0.001). Glycosylated haemoglobin A(1c) was the only parameter independently associated with CD4+CD28(null)T-cells in cDM. The 36-month event-free survival was significantly lower in cDM patients with CD4+CD28(null)T-cells ≥4% (90th percentile of normal distribution) than in those with CD4+CD28(null)T-cells <4% (P= 0.039). Among ACS patients, the 36-month event-free survival was the lowest in those with DM and CD4+CD28(null)T-cells ≥4% and highest in those without DM and CD4+CD28(null)T-cells <4% (P< 0.001), being intermediate in those with only one of these features., Conclusions: In DM patients, CD4+CD28(null)T-cells are expanded and are associated with poor glycaemic control; they also correlate with the occurrence of a first cardiovascular event and with a worse outcome after an ACS.

Background: Belatacept is thought to disrupt the interaction between CD80/86 and CD28, thus preventing T-cell activation by blocking the co-stimulatory second signal. However, the consequences on the T-cell profile in human renal transplant cases have not been determined., Methods: In this study, we analysed intra-graft levels of the mRNAs for Treg (FOXP3), cytotoxic CD8 T cells (Granzyme B), Th1 (INFγ, Tbet), Th2 (GATA3) and Th17 (RORγt and IL-17) in protocol biopsies obtained 12 months after renal transplantation in recipients treated with Belatacept or calcineurin inhibitor (CNI)., Results: Only the intra-graft abundance of FOXP3 mRNA was significantly lower (P < 0.001) in the Belatacept group than the CNI group. Conclusions. These results are in agreement with in vitro data suggesting that CD28 is a major co-stimulatory signal of both Tregs development and peripheral homeostasis but contrast with clinical trials showing a better 1-year graft function and a lower incidence of chronic allograft nephropathy in patients receiving Belatacept than patients treated with CNI. They suggest that immune benefits induced by Belatacept are not mediated by Treg expansion and that FOXP3 is not by itself a prognostic marker of long-term graft function in a non-inflammatory context. These results have to be, however, considered as preliminary since the size of our study population is limited.

Large scale T-cell expansion and efficient gene transduction are required for adoptive T-cell gene therapy. Based on our previous observations, human peripheral blood mononuclear cells (PBMCs) can be expanded efficiently while conserving a naïve phenotype by stimulating with both recombinant human fibronectin fragment (CH-296) and anti-CD3 monoclonal antibodies. In this article, we explored the possibility of using this co-stimulation method to generate engineered T cells using lentiviral vector. Human PBMCs were stimulated with anti-CD3 together with immobilized CH-296 or anti-CD28 antibody as well as anti-CD3/anti-CD28 conjugated beads and transduced with lentiviral vector simultaneously. Co-stimulation with CH-296 gave superior transduction efficiency than with anti-CD28. Next, PBMCs were stimulated and transduced with anti-CD3/CH-296 or with anti-CD3/CD28 beads. T-cell expansion, gene transfer efficiencies and immunophenotypes were analysed. Stimulation with anti-CD3/CH-296 resulted in more than 10-times higher cell expansion and higher gene transfer efficiency with conservation of the naïve phenotype compared with anti-CD3/CD28 stimulation method. Thus, lentiviral transduction with anti-CD3/CH-296 co-stimulation is an efficient way to generate large numbers of genetically modified T cells and may be suitable for many gene therapy protocols that use adoptive T-cell transfer therapy.

The precise biological mechanisms that caused the TGN1412 clinical trial tragedy (also known as 'The Elephant Man Clinical Trial') in March 2006 remain a mystery to this day. It is assumed widely that the drug used in this trial (TGN1412) bound to CD28 on T lymphocytes and following activation of these cells, a massive 'cytokine storm' ensued, leading ultimately to multi-organ failure in all recipients. The rapidity of this in vivo response (within 2 h), however, does not fit well with a classical T lymphocyte response, suggesting that other 'faster-acting' cell types may have been involved. In this study we have activated purified human peripheral blood leucocyte populations using various clones of mouse monoclonal anti-CD28 presented to cells in the form of a multimeric array. Cytokines were measured in cell-free supernatants at 2 h, and specific mRNA for tumour necrosis factor (TNF)-α, thought to be the initiator of the cytokine storm, was also measured in cell lysates by reverse transcription-polymerase chain reaction (RT-PCR). Monocytes were the only cell type found to show significant (P < 0·05) up-regulation of TNF-α at 2 h. Eleven other monocyte cytokines were also up-regulated by anti-CD28 within this time-frame. It therefore seems likely that monocytes and not T cells, as widely believed, were probably responsible, at least in part, for initiating the cytokine storm. Furthermore, we propose that a multimeric antibody array may have formed in vivo on the vascular endothelium via an interaction between TGN1412 and CD64 (FcγRI), and we provide some evidence in support of this hypothesis., (© 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.)

Background: Variability in human immunodeficiency virus (HIV) disease progression cannot be fully predicted by CD4(+) T cell counts or viral load (VL). Because central memory T (T(CM)) cells play a critical role in the pathogenesis of simian immunodeficiency virus disease, we hypothesized that quantifying these cells in early HIV infection could provide prognostic information., Methods: We measured expression of CD45RO, chemokine (C-C motif) receptor (CCR) 5, CCR7, CD27, and CD28 to enumerate naive and memory subsets in samples from recently infected individuals. We also quantified proliferation, CD127 expression, and cell-associated VL. Disease progression was compared across subgroups defined by these measurements, using Kaplan-Meier survival curves and multivariate Cox proportional hazards regression., Results: Four hundred sixty-six subjects contributed 101 events. The proportion or absolute count of T(CM) cells did not correlate with disease progression, defined as the time to AIDS or death. However, significant associations were observed for proliferation within CD4(+) or CD8(+) T cells, loss of naive or CD127(+) memory CD8(+) T cells, and CD4(+) T cell-associated VL., Conclusions: Our results demonstrate that the extent of the immunopathogenesis established early in HIV infection predicts the course of future disease. Because antiretroviral drug treatment reverses such defects in part, our study provides mechanistic clues to why early use of antiretrovirals may prove beneficial.

Objectives: The aim was to investigate the anti-inflammatory effects of Artemisia princeps extract on the activity of anti-CD3/CD28-stimulated CD4(+)CD25(-) T cells and antigen-expanded regulatory T cells., Methods: CD4(+)CD25(-) T cells were activated with coated anti-CD3 and anti-CD28 and cultured in the presence or absence of various concentrations of A. princeps extract. The cultures were pulsed on Day 6 with [(3)H]thymidine and, after harvesting the cells, [(3)H]thymidine incorporation was measured. For analysis of interleukin-2 and interferon-gamma secreted from CD4(+)CD25(-) T cells, culture supernatants were collected on Days 2 and 6. For the analysis of interleukin-10 secreted from the CD4(+)CD25(-) T cells and expanded regulatory T cells, supernatants were collected after 2 and 7 days, respectively. Cytokine levels were determined using an enzyme-linked immunosorbent assay. Potential medicinal components of the A. princeps extract were determined using gas chromatography-mass spectrometry., Key Findings: A. princeps (30 microg/ml) effectively suppressed proliferation of CD4(+)CD25(-) T cells that were stimulated with anti-CD3/CD28 without causing cytotoxicity in spleen cells incubated under conditions lacking antigen stimulation. A. princeps inhibited production of the pro-inflammatory cytokines interleukin-2 and interferon-gamma in anti-CD3/CD28-stimulated CD4(+)CD25(-) T cells. Also, the extract slightly increased production of the anti-inflammatory cytokine interleukin-10 in these cells. In regulatory T cells expanded by anti-CD3/CD28, A. princeps increased production of interleukin-10 and Foxp3., Conclusions: The results suggest that A. princeps may be useful in the treatment of autoimmune diseases and organ transplantation rejection by inhibiting proliferation of inflammatory T cells, suppressing inflammatory processes in antigen-stimulated CD4(+)CD25(-) T cells and increasing activity of expanded regulatory T cells.

IL-17-producing CD4+ T lymphocytes (Th17) are currently considered relevant participants in the pathogenesis of psoriasis skin lesions. However, little is known about the potential role of IL-17-producing CD8+ T cells, which are also present at the psoriatic plaque. We have addressed the functional characterization of this CD8+ subtype of T lymphocytes from psoriasis patients. Our results show that CD8+IL-17+ cells from psoriasis-inflamed skin tissue produce TNF-alpha and IFN-gamma (Th1-related cytokines) as well as IL-17, IL-21, and IL-22 (Th17-related cytokines) efficiently. A significant up-regulation of the RORC transcription factor is also observed. These cells are refractory to Tregs but show a proliferative response to anti-CD3/CD28 stimulation that is enhanced by IL-12 and IL-15. Blocking of TNF-alpha activity inhibits TCR-mediated activation and IL-17 production. CD8+IL-17+ T cells are cytotoxic cells that display TCR/CD3-mediated cytotoxic abilities to kill target cells. Thus, CD8+IL-17+ T cells share some key features with Th17 cells and exhibit remarkable differential abilities attributable to the CD8+ lineage of T lymphocytes, adding new insights into the functional resources of IL-17-producing cells from human epidermis that could be of potential interest to our understanding of the pathogenesis of psoriasis.

Treatment with gold in the form of aurothiomaleate, silver or mercury (Hg) in genetically susceptible mouse strains (H-2(s)) induces a systemic autoimmune condition characterized by anti-nuclear antibodies targeting the 34-kDa nucleolar protein fibrillarin, as well as lymphoproliferation and systemic immune-complex (IC) deposits. In this study we have examined the effect of single-gene deletions for interferon (IFN)-gamma, interleukin (IL)-4, IL-6 or CD28 in B10.S (H-2(s)) mice on heavy metal-induced autoimmunity. Targeting of the genes for IFN-gamma, IL-6 or CD28 abrogated the development of both anti-fibrillarin antibodies (AFA) and IC deposits using a modest dose of Hg (130 microg Hg/kg body weight/day). Deletion of IL-4 severely reduced the IgG1 AFA induced by all three metals, left the total IgG AFA response intact, but abrogated the Hg-induced systemic IC deposits. In conclusion, intact IFN-gamma and CD28 genes are necessary for induction of AFA with all three metals and systemic IC deposits using Hg, while lack of IL-4 distinctly skews the metal-induced AFA response towards T helper type 1. In a previous study using a higher dose of Hg (415 microg Hg/kg body weight/day), IC deposits were preserved in IL-4(-/-) and IL-6(-/-) mice, and also AFA in the latter mice. Therefore, the attenuated autoimmunity following loss of IL-4 and IL-6 is dose-dependent, as higher doses of Hg are able to override the attenuation observed using lower doses.

Most of our understanding of the development and phenotype of alternatively activated macrophages (AAMs) has been obtained from studies investigating the response of bone marrow- and peritoneal-derived cells to IL-4 or IL-13 stimulation. Comparatively little is known about the development of AAMs in the lungs, and how the complex signals associated with pulmonary inflammation influence the AAM phenotype. Here, we use Nippostrongylus brasiliensis to initiate AAM development and define the dynamics of surface molecules, gene expression, and cell function of macrophages isolated from lung tissue at different times postinfection (PI). Initially, lung macrophages take on a foamy phenotype, up-regulate MHC and costimulatory molecules, express reduced levels of TNF and IL-12, and undergo proliferation. Cells isolated between days 8 and 15 PI adopt a dense, granular phenotype and exhibit reduced levels of costimulatory molecules and elevated levels of programmed death ligand-1 (PDL-1) and PDL-2 and an increase in IL-10 expression. Functionally, AAMs isolated on days 13-15 PI demonstrate an enhanced capacity to take up and sequester antigen. However, these same cells did not mediate antigen-specific T cell proliferation and dampened the proliferation of CD3/CD28-activated CD4+ T cells. These data indicate that the alternative activation of macrophages in the lungs, although initiated by IL-4/IL-13, is a dynamic process that is likely to be influenced by other immune and nonimmune factors in the pulmonary environment.

Aging is commonly associated with immune deficiency and dysregulation. The aging of the immune system involves a progressive reduction in naïve T cell output associated with thymic involution and peripheral expansion of oligoclonal memory T cells. We have investigated frequency, phenotype, and function of CD3+CD8+CD28(-)CD25+ T cells in healthy volunteers over a wide age range. We demonstrate that the frequency of CD3+CD8+CD28(-)CD25+ T cells in healthy volunteers increases with age. Peripheral CD3+CD8+CD28(-)CD25+ T cells share phenotypic and functional features with CD3+CD4+CD25+ regulatory T cells (Tregs): In particular, they strongly express CTLA-4 and forkhead box P3. We observed that in vitro, functional titration assays of CD3+CD8+CD28(-)CD25+ T cells show equivalent regulatory function in young and elderly donors, with suppression of proliferation and cytokine production in response to polyclonal T cell stimulation. Finally, CD3+CD8+CD28(-)CD25+ T cells seem to specifically express the CD122 receptor. Altogether, these observations demonstrate an increase in peripheral blood CD8+ Tregs associated with aging.

Background: CX3CL1/Fractalkine (FKN) has been reported to play important roles in various inflammatory diseases. We examined the role of FKN and its receptor CX3CR1 in T-cell migration in the inflammatory bowel diseases (IBDs), ulcerative colitis (UC) and Crohn's disease (CD)., Methods: CX3CR1 expression on peripheral CD4(+) cells from normal controls (NL n = 24) and IBD patients (UC n = 28, CD n = 26) was examined using flow cytometry. CX3CR1(+)CD4(+) T cells were further characterized for surface antigens, cytokine production, and cytotoxic granule release by flow cytometry and ELISA. FKN expression in 53 colonic biopsy specimens (UC n = 20, CD n = 23, NL n = 10) was analyzed by quantitative PCR and immunohistochemistry. Isolated lamina propria and intraepithelial lymphocytes were also analyzed by flow cytometry (UC n = 10, CD n = 10, NL n = 6)., Results: CX3CR1(+)CD4(+) cells were increased in IBD while they were virtually absent in controls. Upregulation of CX3CR1 on CD4(+) T cells was positively correlated with disease activity. These unique T cells expressed markers for both effector memory and cytotoxic cells. Interestingly, CX3CR1 was expressed on CD4(+) T cells lacking CD28. CX3CR1(+)CD28(-)CD4(+) cells produced more IFN-gamma and TNF-alpha than CX3CR1(-) counterparts and released cytotoxic granules. FKN mRNA was upregulated in inflamed colonic tissues and robust expression of FKN was immunohistochemically observed on epithelial cells. Although CX3CR1(+) CD4(+) cells could not be detected in the gut, CD28(-)CD4(+) cells were found in IBD mainly as intraepithelial lymphocytes., Conclusions: FKN/CX3CR1 may contribute to the pathogenesis of IBD through the emergence of unique CX3CR1(+)CD28(-)CD4(+) T cells that can act both as proinflammatory and cytotoxic cells.

Beside the interaction of the antigen-presenting major histocompatibility complex with the T-cell receptor, a co-stimulatory signal is required for T-cell activation in an immune response. To reduce immune-mediated graft rejection in corneal transplantation, where topical application of drugs in ointments or eye-drops may be possible, we selected single-chain antibody fragments (scFv) with binding affinity to rat CD86 (B7.2) that inhibit the co-stimulatory signal. We produced the IgV-like domain of rat CD86 as a fusion protein in Escherichia coli by refolding from inclusion bodies. This protein was used as a target for phage display selection of scFv from HuCAL-1, a fully artificial human antibody library. Selected binding molecules were shown to specifically bind to rat CD86 and inhibit the interaction of CD86 with CD28 and CTLA4 (CD152) in flow cytometry experiments. In an assay for CD86-dependent co-stimulation, the selected scFv fragment successfully inhibited the proliferation of T-cells induced by CD86-expressing P815 cells.

The role of resveratrol and curcumin is well documented in cancer, inflammation, diabetes and various other diseases. However, their immunosuppressive action on T cells, B cells and macrophages is not well documented. In the present study, we have ascertained the effect of resveratrol and curcumin on T and B cells and macrophages. The most striking findings were that both resveratrol and curcumin suppressed the activity of T and B cells and macrophages, as evidenced by significant inhibition in proliferation, antibody production and lymphokine secretion. Interestingly, curcumin imparted immunosuppression by mainly down-regulating the expression of CD28 and CD80 and up-regulating CTLA-4. Resveratrol also functioned by decreasing the expression of CD28 and CD80, as well as by augmenting the production of interleukin (IL)-10.

Despite treatment, 10-30% of brucellosis patients develop chronic disease, characterized by atypical clinical picture and/or relapses. A defective T helper 1 (Th1) response and a low [corrected] percentage of CD4(+)/CD25(+) cells have been described in chronic brucellosis patients. CD80/CD28 co-stimulation is critical for an efficient Th1 response and has not been studied previously in human brucellosis. In order to investigate the role of CD80/CD28 co-stimulation, 13 acute brucellosis patients (AB), 22 chronic brucellosis patients (CB, 12/22 relapsing type-CB1 and 10/22 atypical type-CB2), 11 'cured' subjects and 15 healthy volunteers (controls) were studied. The percentage of CD4(+)/CD28(+) T lymphocytes and CD14(+)/CD80(+) monocytes were analysed by flow cytometry both ex vivo and after phytohaemagglutinin (PHA)-stimulation with or without heat-killed Brucella abortus (HkBA). Ex vivo analysis showed no differences between all groups studied. PHA stimulation up-regulated the percentage of CD80(+) monocytes in AB compared to 'cured' subjects and controls (P < 0.001), although the proportion of CD4(+)/CD28(+) cells did not alter. A higher percentage of CD80(+) monocytes was observed in the CB1 subgroup, compared to AB, 'cured' subjects and controls (P = 0.042, < 0.001 and < 0.001, respectively). CB2 was characterized by a lower percentage of CD80(+) monocytes in comparison to CB1 (P = 0.020). HkBA in PHA cultures down-regulated the percentage of CD80(+) monocytes compared to PHA alone in all groups, especially in AB and CB patients (P < 0.001 and P = 0.007, respectively). In conclusion, the diminished percentage of CD4(+)/CD25(+) T cells in CB is not associated with inadequate CD80/CD28 co-stimulation. We speculate that differential frequency of CD80(+) monocytes after PHA stimulation could serve as a qualitative parameter of disease status, related to the different clinical forms of chronic brucellosis.

The B7-1/B7-2-CD28/CTLA-4 pathway is crucial in regulating T cell activation and tolerance. Autoantibodies to surface molecules on lymphocytes have already been described in various immune conditions, such as autoimmune diseases, infections and blood transfusions. The objective of this study was to test sera from healthy individuals and from patients for association of CD28 autoantibodies with inflammatory and non-inflammatory diseases. First, CD28 was obtained by digestion of CD28-Ig fusion protein with trypsin. The cleavage products were separated by sodium dodecyl sulphate-page gel electrophoresis. Additionally, a CD28/GST fusion protein was expressed in Escherichia coli and was used to establish an enzyme-linked immunosorbent assay for detection of autoantibodies against CD28. Sera from healthy individuals (n = 72) and patients with different inflammatory and non-inflammatory skin diseases (n = 196) were tested for the presence of autoantibodies against CD28. Using mixed lymphocyte reaction (MLR), purified autoantibodies against CD28 were tested for their effects on CTLA-4-Ig-induced T cell anergy. In this study, for the first time, we describe the existence of autoantibodies against CD28 in humans which are associated with atopic diseases, e.g. allergic rhinitis and asthma. These antibodies stimulate T cells and overcome the CTLA-4-Ig-induced anergy of T cells in an MLR. The existence of autoantibodies against CD28, which may have a T cell-stimulating function, has been shown. The data indicate that autoantibodies against CD28 could be a new immunological mechanism in allergic inflammation. Additionally, autoantibodies against CD28 could be an important new marker to discriminate between atopic diseases and other inflammatory skin diseases.

Polychromatic flow-cytometric assays were used to analyze paired intrahepatic and peripheral lymphocyte samples from 37 patients with chronic hepatitis C. Compared with peripheral cells, intrahepatic T cells were selectively enriched with CD45RO+ memory T cells but had a lower percentage of CD4+ T cells expressing the differentiation markers CD27 and CD28. The percentage of intrahepatic CD45RO+ and CD28+ T cells correlated with the degree of liver inflammation, which suggests that memory T cells at relatively early stages of differentiation are directly involved in liver inflammation. Despite their memory phenotype, intrahepatic T cells were defective in proliferation capability, produced less interferon- gamma in response to stimulation by T cell receptor, and contained less perforin but expressed higher levels of Fas and Fas ligand, compared with their counterparts in peripheral blood. The distinct characteristics of intrahepatic T cells suggest that they play an important role in the immunopathogenesis of chronic hepatitis C.

Recruitment of naturally occurring CD4+ CD25+ regulatory T (T(reg)) cells is a highly promising approach for the treatment of experimental autoimmune encephalomyelitis (EAE), a widely used model of multiple sclerosis. Here, we studied the in vivo interaction of T(reg) cells, induced by the monoclonal anti-CD28 antibody JJ316, with encephalitogenic T cell lines established from eGFP-transgenic rats. By tracking these fluorescent cells using flow cytometry and confocal microscopy, we found that the activation and expansion of T(reg) cells inhibited infiltration of the CNS by pathogenic T cells. Interference with effector cell migration occured within the secondary lymphoid organs, since the early therapeutic effects were achieved despite the absence of T(reg) cells in the spinal cord. However, the delayed homing to the CNS seen after prophylactic JJ316 administration indicates that T(reg) cells may play an additional role within the target tissue. In addition, the blood-brain barrier remained largely intact after JJ316 treatment, the secretion of T(H)2 cytokines was augmented and the encephalitogenic T cells exhibited a reduced secretion of IFN-gamma. This in turn resulted in a reduced expression of the chemokine receptor CXCR-3 on effector T cells which may interfere with their capacity to infiltrate the CNS. Importantly, these effects were not achieved by direct action of JJ316 on the encephalitogenic cells. Our data rather suggest that polyclonal activation of T(reg) cells in the secondary lymphoid organs is instrumental in preventing the pathological transmigration of encephalitogenic T cells into the CNS. We anticipate that these results may help to better understand the role of T(reg) cells in controlling autoimmunity in the CNS.

Background: During human immunodeficiency virus (HIV) infection, reduced proportions of CD8 cells express CD28, the key costimulatory molecule for lymphocyte activation. However, it is unclear whether reduced CD28 expression affects immune responses to non-HIV antigens, potentially contributing to susceptibility to opportunistic infection., Methods: We measured CD4- and CD8-specific interferon- gamma responses to hepatitis C virus (HCV) peptide pools in subjects with chronic HCV monoinfection (n=14), in subjects with chronic HCV/HIV coinfection (n=15), and in healthy control subjects (n=10) by enzyme-linked immunospot assay in the presence and absence of CD28 costimulation., Results: Anti-CD28 agonist increased the cumulative frequency of HCV-specific CD4 cell responses in the subjects with HCV monoinfection and in those with HCV/HIV coinfection. In contrast, anti-CD28 agonist increased the breadth and cumulative frequency of HCV-specific CD8 cell responses only in the subjects with HCV monoinfection. Additionally, in the presence of anti-CD28 agonist, the proportion of subjects responding, the cumulative frequency, and the breadth of reactive CD8 cells were greater among the subjects with HCV monoinfection than among those with HCV/HIV coinfection. Finally, the HCV/HIV-coinfected subjects had lower proportions of CD8 cells that expressed CD28., Conclusions: These results indicate that, during HCV/HIV coinfection, memory-effector CD8 cells have reduced responsiveness to CD28 costimulation. This appears to reflect a global effect that HIV has on the activation or differentiation state of CD8 cells that are responsive to other microbial pathogens. This functional defect has implications for the pathogenesis of HCV/HIV coinfection.

Calcineurin antagonists are known as potent immunosuppressants working particularly on T cells by virtue of their capacity to block nuclear factor of activated T cell (NFAT) activation and translocation to the nucleus. In addition to interleukin (IL)-2 suppression, T helper cell type 1 (Th1) as well as Th2 cytokine transcription is blocked by calcineurin antagonists. Here, we show that calcineurin antagonists such as cyclosporin A (CsA) or tacrolimus can markedly enhance the production of interferon-gamma (IFN-gamma) by human T cells. This increased IFN-gamma production is dependent on T cell receptor (TCR) and CD28 signaling as well as on the presence of IL-12. IL-27, which could mimic the effect of IL-12, was however less potent in inducing IFN-gamma production in the presence of CsA and TCR stimulation. Other cytokines such as IL-23, IL-18, IL-2, or the Th2-related cytokine IL-4 are not able to support a calcineurin antagonist-dependent up-regulation of IFN-gamma. CsA-dependent IFN-gamma production is observable in therapeutic concentrations. The effect is independent of IL-10 or IL-4, as addition of these cytokines could not inhibit the CsA-induced IFN-gamma production. The effect of calcineurin antagonists is associated with an increased c-fos expression and DNA-binding activity of the transcription factor activated protein-1 but not with increased DNA-binding activity of T-bet. Our study further supports the relevance of known calcineurin activities other than NFAT activation. The presented data may help to explain why concomitant infections (resulting in increased IL-12 expression) under therapy with calcineurin antagonists often have a negative impact on the activity of the underlying disease (e.g., autoimmune disease).

Expression of tumour antigen-specific chimaeric receptors in T lymphocytes can redirect their effector functions towards tumour cells. Integration of the signalling domains of the co-stimulatory molecule CD28 into chRec enhances antigen-specific proliferation of polyclonal human T cell populations. While CD28 plays an essential role in the priming of naive CD4(+) T cells, its contribution to effector memory T cell responses is controversial. We compared the function of the chRec with and without the CD28 co-stimulatory domain, expressing it in peripheral blood T cells or Epstein-Barr virus (EBV)-specific T cell lines. The chimaeric T cell receptors contain an extracellular single-chain antibody domain, to give specificity against the tumour ganglioside antigen G(D2). The transduced cytotoxic T lymphocytes (CTL) maintained their specificity for autologous EBV targets and their capacity to proliferate after stimulation with EBV-infected B cells. Intracellular cytokine staining demonstrated efficient and comparable antigen-specific interferon (IFN)-gamma secretion by CTL following engagement of both the native and the chimaeric receptor, independent of chimaeric CD28 signalling. Furthermore, tumour targets were lysed in an antigen-specific manner by both chRec. However, while antigen engagement by CD28 zeta chRec efficiently induced expansion of polyclonal peripheral blood lymphocytes in an antigen-dependent manner, CD28 signalling did not induce proliferation of EBV-CTL in response to antigen-expressing tumour cells. Thus, the co-stimulatory requirement for the efficient activation response of antigen-specific memory cells cannot be mimicked simply by combining CD28 and zeta signalling. The full potential of this highly cytolytic T cell population for adoptive immunotherapy of cancer requires further exploration of their co-stimulatory requirements.

We have previously shown that jacalin, a CD4+ T cell lectin, induces phosphorylation of intracellular events, moderate levels of interleukin (IL)-2 secretion. We have also shown that in the presence of CD28 costimulation, jacalin induces IL-4 secretion. In the present study, we showed that stimulation of normal CD4+ T cells with jacalin plus CD28 cross-linking (CD28XL) resulted in phosphorylation of signal transducer and activator of transcription (STAT)-6 and expression of Bcl-2 and Bcl-xL, which were inhibited significantly when cells were cultured in the presence of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. We further generated jacalin-induced CD4+ T cell blasts, examined the effects of CD28XL, and observed enhanced up-regulation of p38 and activation of STAT-6, Bcl-2, and Bcl-xL. Engagement of CD28 alone induced a marked degree of phosphorylation of p38 MAPK and IL-4 secretion in memory T cells (jacalin blasts), whereas in naïve T cells, jacalin plus CD28XL was required to induce these molecules. Incubation of cells with p38 inhibitor prior to CD28XL resulted in down-modulation of all these molecules. Further treatment with IL-4 has not reversed this trend. Our studies imply that p38 MAPK may play an important role in induction of these molecules and a putative role in protecting cells from undergoing apoptosis.

Costimulatory signals play an important role in the development of T helper cell type 1 (Th1) or Th2 type. Little is known about jacalin plus CD28-mediated signaling and cytokine secretion. In the present study, we analyzed the intracellular signaling events following stimulation of CD4+ T cells with jacalin plus CD28 cross-linking (CD28XL) with anti-CD28 antibody. Our results indicate enhanced phosphorylation of Tec and linker for activation of T cells when compared with stimulation with jacalin alone or CD28XL alone. Stimulation with jacalin or CD28XL appears to be insufficient to induce interleukin (IL)-4 secretion; however, CD28XL followed by stimulation with jacalin resulted in enhanced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and increased secretion of IL-4. However, compared with stimulation with phorbol 12-myristate 13-acetate plus ionomycin, jacalin plus CD28XL resulted in decreased levels of tumor necrosis factor alpha secretion. Addition of p38 inhibitor, SB203580, inhibited p38 phosphorylation and IL-4 secretion. These data suggest that jacalin stimulation alone appears to be insufficient for Th2 development, and addition of CD28 costimulation induced Th2 generation. We propose that jacalin plus CD28XL induces Th2 differentiation via activation of p38 MAPK.

Costimulatory signals such as the ones elicited by CD28/B7 receptor ligation are essential for efficient T cell activation but their role in anti-tumour immune responses remains controversial. In the present study we compared the efficacy of DC vaccination-induced melanoma specific T cell responses to control the development of subcutaneous tumours and pulmonary metastases in CD28-deficient mice. Lack of CD28-mediated costimulatory signals accelerated tumour development in both model systems and also the load of pulmonary metastases was strongly increased by the end of the observation period. To scrutinize whether lack of CD28 signalling influences priming, homing or effector function of Trp-2(180-188)/K(b)-reactive T cells we investigated the characteristics of circulating and tumour infiltrating T cells. No difference in the frequency of Trp-2(180-188)/K(b)-reactive CD8+ T cells could be demonstrated among the cellular infiltrate of subcutaneous tumours after DC vaccination between both genotypes. However, the number of IFN-gamma-producing Trp-2-reactive cells was substantially lower in CD28-deficient mice and also their cytotoxicity was reduced. This suggests that CD28-mediated costimulatory signals are essential for differentiation of functional tumour-specific CD8+ T-effector cells despite having no impact on the homing of primed CD8+ T cells.

The mechanisms by which concanavalin A (Con A), a lectin, activates T cells are poorly studied. A low dose of Con A is stimulatory for T cells, whereas a high dose of Con A results in suppression of proliferation and enhanced T cell death. The expression and functional roles of costimulatory receptors, CD28 and cytotoxic T-lymphocyte antigen 4 (CTLA4), and their ligands, CD80 and CD86, on primary mouse CD4(+) T cells after activation with different doses of Con A were studied. CTLA4-CD80/CD86 interactions in this T:T cell activation model demonstrate distinct outcomes depending on the dose of Con A. CTLA4-CD80/CD86 interactions inhibit CD4(+) T cell cycling and survival after activation with a suppressive dose of Con A by increasing oxidative stress and decreasing levels of BclX(L). The enhanced CD4(+) T cell death with a suppressive dose of Con A is dependent on excess H(2)O(2) and nitric oxide but is independent of Fas and caspase activity. It is surprising that the increased proliferation of CD4(+) T cells with a suppressive dose of Con A on blocking CTLA4-CD80/CD86 interactions is largely interleukin (IL)-2-independent but is cyclosporine A-sensitive. On activation with a stimulatory dose of Con A, CTLA4-CD80/CD86 interactions enhance T cell activation and survival by reducing the production of reactive oxygen species, increasing IL-2 and BclX(L) levels. Here IL-10 but not transforming growth factor-beta plays a functional role. In summary, CTLA4-CD80/CD86 interactions on T cells integrate signal strength, based on the dose of Con A, to enhance or inhibit primary mouse CD4(+) T cell cycling and survival.

Our laboratory has demonstrated that down-regulation of proliferation and cytokine synthesis by CD4(+) T cells in mice fed diets rich in n-3 polyunsaturated fatty acids (PUFA) is highly dependent on the involvement of the co-stimulatory molecule, CD28. It has been reported that the inhibitory cytokine interleukin (IL)-10 acts directly on T cells which up-regulate IL-10 receptor (IL-10R) expression following stimulation via CD28 by efficiently blocking proliferation and cytokine production. Thus, it was hypothesized that dietary n-3 PUFA would suppress T cell function through the effects of IL-10. The proliferation of purified splenic CD4(+) T cells activated in vitro with anti-CD3 and anti-CD28 (alphaCD3/CD28) from conventional mice (C57BL/6) fed either a control corn oil (CO)-enriched diet devoid of n-3 PUFA, docosahexaenoic acid (DHA; 22 : 6) or eicosapentaenoic acid (EPA; 20 : 5) for 14 days was suppressed by dietary DHA and EPA. Surprisingly, a similar trend was seen in IL-10 gene knock-out (IL-10(-/-)) mice fed dietary n-3 PUFA. IL-10R cell surface expression was also significantly down-regulated on CD4(+) T cells from both the C57BL/6 and IL-10(-/-) mice fed dietary n-3 PUFA after 72 h of in vitro stimulation with alphaCD3/CD28. Enzyme-linked immunosorbent assay (ELISA) measurements revealed that C57BL/6 mice fed DHA had significantly reduced interferon (IFN)-gamma and IL-10 levels 48 h post-activation. However, CD4(+) T cells from IL-10(-/-) mice fed dietary n-3 PUFA produced significantly greater levels of IFN-gamma than the CO-fed group. Our data suggest that in the absence of IL-10, CD4(+) T cells from n-3 PUFA-fed mice may up-regulate IFN-gamma. Suppressed CD4(+) T cells from n-3 PUFA-fed C57BL/6 mice may use mechanisms other than IL-10 to down-regulate T cell function.

The objective of this study was to demonstrate the variable expression of cytokine receptors on naive versus memory human CD4+ T cell subpopulations in tonsillar tissue, cord blood and adult blood. We prove that the receptors for both interleukin (IL)-12 and IL-18 are expressed exclusively on memory T cells. This observation was seen not only on the CD45RO+ memory T cells but also on a significant percentage of the CD45RA+, CD62L-, CD27- and CCR7- populations. Furthermore, CD45RA+ CD62L+, CD27+ or CCR7+ CD4+ T cells that expressed IL-12Rbeta1 and IL-18Ralpha did not express CD31, a marker for recent thymic emigrants. We reveal that cord blood lymphocytes do not express IL-12Rbeta1 whereas IL-18Ralpha expression was detected at low levels. Importantly, the IL-12Rbeta2 signalling chain, which is absent in all resting T cells, was up-regulated in both CD45RA+ and CD45RO+ T cells as a result of stimulation with anti-CD3 and anti-CD28 in vitro. This observed up-regulation was, however, restricted to 80% of the total CD4+ population. Finally, a very small proportion of the CD4+ CD45RO+ tonsillar T cells expressed the IL-12 and IL-18 receptors, thereby establishing the differential expression of these receptors between peripheral and tonsillar memory T cell subpopulations.

Chemokines constitute a large family of structurally related proteins that play a role in leukocyte migration and differentiation. Indeed, the early expression of human CXC chemokine receptor 1 (hCXCR1) and hCXCR2 [homologous to mouse interleukin (IL)-8Rbeta] ligands by the epithelium is a hallmark of the mucosal host defense. Mice lack IL-8; however, granulocyte chemotactic protein-2 (GCP-2)/lipopolysaccharide-induced CXC chemokine, a murine homologue of human GCP-2, has 32% and 61% sequence identity to human IL-8 and GCP-2, respectively, and binds hCXCR1, hCXCR2, and mouse IL-8Rbeta. To better understand the role of GCP-2 in adaptive immunity and as a nasal adjuvant, we characterized the exogenous effects of this CXC chemokine on cellular and humoral mucosal immune responses. GCP-2 significantly enhanced serum immunoglobulin G (IgG) and mucosal IgA antibodies through increased cytokine secretion by CD4+ T cells. These alterations in humoral and cellular responses were preceded by an increase in the number of B cells in the nasal tract, a decrease in the number of CD4+ T cells in the nasal tract as well as cervical lymph nodes, and an increase in the number of neutrophils in the nasal tract 12 h after GCP-2 immunization. This chemokine also modulated CD28 expression by CD4+ T cells during CD3epsilon stimulation of wild-type mice. GCP-2 increased CD80 and CD86 expression on B cells during in vitro stimulation in a dose-dependent manner. In contrast, cytokine and costimulatory molecule enhancement by GCP-2 was not induced by lymphocytes from IL-8Rbeta-/- mice, suggesting that GCP-2 modulates cellular immunity in part through IL-8Rbeta interactions.

The interaction of T cell CD28/CTLA-4 receptors with B7 on antigen-presenting cells (APCs) represents an important co-stimulatory pathway in T cell activation or anergy. Our previous study indicated that recipients immunized with allogenic donor immature dendritic cells (DCs) or resting B cells could induce specific immune tolerance and prolong allograft survival. A possible mechanism for this observation is that the expression of B7 molecules is either at a low level or lacking on these cells. The present study investigates whether blockade of B7 molecules on donor splenocytes with a B7 antisense peptide (B7AP), i.e. a peptide analogue of the CD28-binding region, could induce specific immune tolerance and prolong allograft survival in the recipients. Both the lymphocyte proliferation reaction and the mice pinna cardiac allograft experiment were performed to evaluate the role of B7AP in inducing specific immune tolerance in recipients in vitro and in vivo. The results showed that 56.65% and 20.52% of C57BL/6 splenocytes expressed B7.1 and B7.2 molecules, respectively, on their cell surface. There were no significant changes of the B7 expression on such splenocytes after being treated by the B7AP (53.28% and 19.06%, respectively). B7AP inhibited the mixed lymphocyte reaction by up to 38.4% and a dose-response correlation was observed for inhibition. The recipients (BALB/c) immunized with B7AP-pretreated C57BL/6 splenocytes induced a specific immune hypo-response (43%versus control) and notably prolonged survival of the C57BL/6 cardiac allograft by up to 20.3 days. In contrast to the normal saline group (average: 8.6 days) and FTD(10) control peptide group (<4 days), the cardiac allograft survival of the test group was extended for an additional 11.7 days. These results strongly support the notion that immunization with donor splenocytes, which had been pretreated with B7AP, induced specific immune tolerance and prolonged allograft survival in the recipients.