In all tissues that have been studied to date, dendritic leucocytes constitute only a small proportion of total cells and are difficult both to isolate and purify. This study reports on a method for the propagation of large numbers of dendritic cells (DC) from mouse spleen using granulocyte-macrophage colony-stimulating factor (GM-CSF) and their characteristics. Within a few days of liquid culture in GM-CSF, B10 BR (H-2k, I-E+) mouse splenocytes formed loosely adherent myeloid cell clusters. Mononuclear progeny released from these clusters at and beyond 4 days exhibited distinct dendritic morphology and strongly expressed leucocyte common antigen (CD45), CD11b, heat-stable antigen, Pgp-1 (CD44) and intercellular adhesion molecule-1 (ICAM-1; CD54). The intensity of expression of the DC-restricted markers NLDC 145 and 33D1, the macrophage marker F4/80, and Fc gamma RII (CDw32) was low to moderate, whereas the cells were negative for CD3, CD45RA and NK1.1. High and moderate levels, respectively, of cell surface staining for major histocompatibility complex (MHC) class II (I-Ek) and the B7 antigens (counter-receptors of CTLA4, a structural homologue of CD28) were associated with potent stimulation of unprimed, allogeneic T cells (B10; H-2b, I-E-). DC propagated in a similar fashion from DBA/2 mouse spleen proved to be strong antigen-presenting cells (APC) for MHC-restricted, syngeneic T-helper type 2 (Th2) cell clones specifically responsive to sperm whale myoglobin. Footpad or intravenous injection of GM-CSF-stimulated B10.BR spleen-derived DC into B10 (H-2b, I-E-) recipients resulted in homing of the allogeneic cells to T-cell-dependent areas of lymph nodes and spleen, where they strongly expressed donor MHC class II antigen 1-2 days later. These findings indicate that cells can be propagated from fresh splenocyte suspensions that exhibit distinctive features of DC, namely morphology, motility, cell-surface phenotype, potent allogeneic and syngeneic APC function and in vivo homing ability. Propagation of DC in this manner from progenitors present in lymphoid tissue provides an alternative and relatively convenient source of high numbers of these otherwise difficult to isolate but functionally important APC.

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.

A T cell-dependent immune response to group C meningococcal capsular polysaccharide (CPS) can be elicited when CPS is conjugated to the class 3 neisserial porin (CPS-porin). Treatment of CPS-porin-immunized mice with B7-2 blocking monoclonal antibody (MAb) caused a dramatic reduction in the CPS-specific IgG response, treatment with anti-B7-1 MAb had no effect, and concurrent blockade of B7-1 and B7-2 resulted in a synergistic abrogation of the CPS-specific IgG response while the CPS IgM response was unaffected. Anti-CD40L MAb treatment caused a significant reduction of both CPS-specific IgG and IgM levels. In contrast, blockade of CTLA4 interactions resulted in increases in both CPS IgG and IgM responses in CPS-porin-immunized mice. These data support the hypothesis that the ability of neisserial porins to improve the immune response to poorly immunogenic antigens (e.g., polysaccharides) is related to porin-induced increases in B7-2 expression on antigen-presenting cells and enhanced B/T cell interactions.

The proinflammatory cytokine IL-6 plays an important role in controlling T-cell differentiation, especially the development of Th17 and regulatory T cells. To determine the function of IL-6 in regulating allograft rejection and tolerance, BALB/c cardiac grafts were transplanted into wild-type or IL-6-deficient C57BL/6 mice. We observed that production of IL-6 and IFN-γ was upregulated during allograft rejection in untreated wild-type mice. In IL-6-deficient mice, IFN-γ production was greater than that observed in wild-type controls, suggesting that IL-6 production affects Th1/Th2 balance during allograft rejection. CD28-B7 blockade by CTLA4-Ig inhibited IFN-γ production in C57BL/6 recipients, but had no effect on the production of IL-6. Although wild-type C57BL/6 recipients treated with CTLA4-Ig rejected fully MHC-mismatched BALB/c heart transplants, treatment of IL-6-deficient mice with CTLA4-Ig resulted in graft acceptance. Allograft acceptance appeared to result from the combined effect of costimulatory molecule blockade and IL-6-deficiency, which limited the differentiation of effector cells and promoted the migration of regulatory T cells into the grafts. These data suggest that the blockade of IL-6, or its signaling pathway, when combined with strategies that inhibit Th1 responses, has a synergistic effect on the promotion of allograft acceptance. Thus, targeting the effects of IL-6 production may represent an important part of costimulation blockade-based strategies to promote allograft acceptance and tolerance.

The gut environment has been found to significantly influence autoimmune diseases such as multiple sclerosis; however, immune cell mechanisms are unclear. Here we show that the gut epithelium of myelin oligodendrocyte glycoprotein(35-55)-specific T-cell receptor transgenic mice contains environmental stimuli-induced intraepithelial lymphocytes (IELs) that inhibit experimental autoimmune encephalomyelitis on transfer. These cells express surface markers phenotypical of 'induced' IELs, have a TH17-like profile and infiltrate the central nervous system (CNS). They constitutively express Ctla4 and Tgfb1 and markedly upregulate Lag3 expression in the CNS, thereby inhibiting inflammation. We also demonstrate the suppressive capability of CD4(+) IELs with alternative antigen specificities, their proliferation in response to gut-derived antigens and contribution of the microbiota and dietary aryl hydrocarbon receptor ligands to their induction. Thus, the gut environment favours the generation of autoreactive CD4(+) T cells with unique regulatory functions, potentially important for preventing CNS autoimmunity.

OBJECTIVE

Islet autoantibody-positive children progress to type 1 diabetes at variable rates. In our study, we asked whether characteristic autoantibody and/or gene profiles could be defined for phenotypes showing extreme progression.

METHODS

Autoantibodies to insulin (IAA), GAD (GADA), insulinoma-associated antigen-2 (IA-2A) and zinc transporter 8 (ZnT8A) were measured in follow-up sera, and genotyping for type 1 diabetes susceptibility genes (HLA-DR/HLA-DQ, INS variable number of tandem repeats [VNTR] and single nucleotide polymorphisms at PTPN22, PTPN2, ERBB3, IL2, SH2B3, CTLA4, IFIH1, KIAA0350 [also known as CLEC16A], CD25, IL18RAP, IL10, COBL) was performed on the DNA samples of children born to a parent with type 1 diabetes and prospectively followed from birth for up to 22 years.

RESULTS

Of the 1,650 children followed, 23 developed multiple autoantibodies and progressed to diabetes within 3 years (rapid progressors), while 24 children developed multiple autoantibodies and remained non-diabetic for more than 10 years from seroconversion (slow progressors). Rapid and slow progressors were similar with respect to HLA-DR/HLA-DQ genotypes, development of IAA, GADA and ZnT8A, and progression to multiple autoantibodies. In contrast, IA-2A development was considerably delayed in the slow progressors. Furthermore, both groups were effectively distinguished by the combined presence or absence of type 1 diabetes susceptibility alleles of non-HLA genes, most notably IL2, CD25, INS VNTR, IL18RAP, IL10, IFIH1 and PTPN22, and discrimination was improved among children carrying high-risk HLA-DR/HLA-DQ genotypes.

CONCLUSIONS

Our data suggest that genotypes of non-HLA type 1 diabetes susceptibility genes influence the likelihood or rate of diabetes progression among children with multiple islet autoantibodies.

The aim of this study was to investigate the allelic association of a single nucleotide polymorphism in exon 1 of the cytotoxic T-lymphocyte antigen-4 (CTLA4) gene with rheumatoid arthritis (RA) in Chinese people. One hundred and eighty-six unrelated adults with RA and 203 randomly selected normal adults were studied. All were ethnic Chinese living in Taiwan. The CTLA4 A-G polymorphism was genotyped with a polymerase chain reaction (PCR) and digestion with the restriction enzyme BstEII. The genotype and allele frequencies of CTLA4 in patients with rheumatoid arthritis differed significantly from those of adult controls (P=0.022 and P=0.037, respectively). Genotype CTLA4 49 G/G and allele G were associated with an increased risk of RA (RR=1.72, 95% CI=1.15-2.57, P=0.008; RR=1.39, 95% CI=1.02-1.89, P=0.037, respectively), whereas genotype A/G and allele A were associated with protection against RA (RR=0.58, 95% CI=0.39-0.87, P=0.008 and RR=0.72, 95% CI=0.53-0.98, P=0.037, respectively). We concluded that, the CTLA4 49 A-G polymorphism is associated with RA in Chinese patients from Taiwan.

Like in many tumor types, immunotherapy is currently under investigation to assess its potential efficacy in glioblastoma patients. Trials are under way to assess the efficacy of new immune checkpoint inhibitors including anti-PD-1 or CTLA4. We here investigate the expression and efficacy of a novel immune-checkpoint inhibitor, called LAG-3. We show that LAG-3 is expressed in human glioblastoma samples and in a mouse glioblastoma model we show that knock out or LAG-3 inhibition with a blocking antibody is efficacious against glioblastoma and can be used in combination with other immune checkpoint inhibitors toward complete eradication of the model glioblastoma tumors. From a mechanistic standpoint we show that LAG-3 expression is an early marker of T cell exhaustion and therefore early treatment with LAG-3 blocking antibody is more efficacious than later treatment. These data provide insight and support the design of trials that incorporate LAG-3 in the treatment of glioblastoma.

Regulatory T (Treg) cells are fundamental in the control of immunity and excessive tissue pathology. In paracoccidioidomycosis, an endemic mycosis of Latin America, the immunoregulatory mechanisms that control the progressive and regressive forms of this infection are poorly known. Due to its modulatory activity on Treg cells, we investigated the effects of anti-CD25 treatment over the course of pulmonary infection in resistant (A/J) and susceptible (B10.A) mice infected with Paracoccidioides brasiliensis. We verified that the resistant A/J mice developed higher numbers and more potent Treg cells than susceptible B10.A mice. Compared to B10.A cells, the CD4(+)CD25(+)Foxp3(+) Treg cells of A/J mice expressed higher levels of CD25, CTLA4, GITR, Foxp3, LAP and intracellular IL-10 and TGF-β. In both resistant and susceptible mice, anti-CD25 treatment decreased the CD4(+)CD25(+)Foxp3(+) Treg cell number, impaired indoleamine 2,3-dioxygenase expression and resulted in decreased fungal loads in the lungs, liver and spleen. In A/J mice, anti-CD25 treatment led to an early increase in T cell immunity, demonstrated by the augmented influx of activated CD4(+) and CD8(+) T cells, macrophages and dendritic cells to the lungs. At a later phase, the mild infection was associated with decreased inflammatory reactions and increased Th1/Th2/Th17 cytokine production. In B10.A mice, anti-CD25 treatment did not alter the inflammatory reactions but increased the fungicidal mechanisms and late secretion of Th1/Th2/Th17 cytokines. Importantly, in both mouse strains, the early depletion of CD25(+) cells resulted in less severe tissue pathology and abolished the enhanced mortality observed in susceptible mice. In conclusion, this study is the first to demonstrate that anti-CD25 treatment is beneficial to the progressive and regressive forms of paracoccidioidomycosis, potentially due to the anti-CD25-mediated reduction of Treg cells, as these cells have suppressive effects on the early T cell response in resistant mice and the clearance mechanisms of fungal cells in susceptible mice.

IL-4 has been shown to be involved in the early stages of B cell maturation. Changes induced by IL-4 include cell enlargement, increased viability, and increased MHC class II expression. However IL-4 alone does not induce B cell activation as defined by proliferation, lymphokine production, or Ig class switching. In this study, we demonstrate that incubation with IL-4 enhances the ability of small splenic murine B cells, normally poor stimulators of murine Th1 clones, to stimulate lymphokine production and proliferation by Th1 clones. Moreover, small resting B cells induce anergy, whereas IL-4-treated B cells do not. IL-4-treated B cells were found to express both B7 (B7-1) and a second ligand for CTLA4Ig (B7-2). Although IL-4 induces both B7-1 and B7-2, the kinetics of expression of these molecules are different: B7-2 is detected by 6 h, whereas B7-1 is not detectable until 48 h. In addition, only CTLA4Ig fully blocks IL-4 induced costimulatory activity; a mAb to B7-1 does not. Thus, these results suggest that IL-4 may function indirectly as a costimulatory factor by inducing costimulatory molecules on resting B cells. Additionally, these findings support our previous findings that an alternative ligand for CD28 and CTLA4 is important in providing costimulation.

NK cells recognize and kill tumor cells and normal cells, and these play an important role in immune defense in cancer, infectious disease, and autoimmunity. NK killing is regulated by positive or negative signals derived from the interaction of surface receptors with ligands on the target cells. However, the mechanisms controlling the proliferation and maintenance of NK cells in normal human individuals are less clearly defined. In this study, using an entirely autologous system, we demonstrate that human peripheral blood CD3-CD56+, killer cell-inhibitory receptor (KIR)-expressing cells proliferate and expand in response to LPS. These responses are enhanced in the presence of anti-IL-10 receptor-blocking Abs or on the removal of CD14+ cells from the cultures. This enhancement is also reflected in substantial increases in cytolytic activity and IFN-gamma production. The negative effect of CD14+ cells may also be IL-10 mediated, IL-10 being lost from the culture supernatants of CD14-depleted PBMC and rIL-10 reversing the effect of this depletion. On the other hand, mRNA for the p35 and p40 subunits of IL-12 is still induced in CD14-depleted cultures. The expansion of CD3-CD56+ cells was also inhibited by CTLA4-Ig, indicating a role for CD80/86. B lymphocytes were not required for the expansion of CD3-CD56+ cells, whereas removal of MHC class II+ cells from CD14-depleted cultures resulted in a complete abrogation of these responses. Expansion of CD3-CD56+ cells was reconstituted in MHC class II-depleted cell cultures by adding back monocyte-derived dendritic cells. These results indicate that the responses of CD3-CD56+ NK cells to LPS may be driven by a MHC class II+ B7+ CD14- peripheral population, most likely blood dendritic cells.

Dendritic cells (DC) are the major APC in human peripheral blood (PB) and rheumatoid synovium. We previously identified in PB a population of CD33dim-CD14dim DC precursors, as well as a smaller population of CD33bright CD14dim mature DC. Neither PB DC population expressed the CD28/CTLA4 ligands, suggesting that additional signals are required for full functional DC differentiation. Because rheumatoid synovium is characterized by an ongoing immune response, the expression and function of CD80, CD86, and other markers of DC differentiation by rheumatoid arthritis synovial APC were examined. The phenotype of a large subset of freshly isolated rheumatoid arthritis synovial fluid (SF) DC resembled that of the mature PB DC. These DC expressed CD45R0, CD11c, CMRF-44, and high levels of CD33. Whereas CD80 expression by rheumatoid SF DC and monocytes was minimal, CD86 was expressed by a subset of SF monocytes and by CMRF-44+SF DC. Furthermore, sorted CD86- SF DC spontaneously up-regulated CD86 in vitro. CD80 was expressed diffusely and at low levels by rheumatoid synovial tissue cells, whereas CD86 was expressed by perivascular HLA-DR+HLA-DQ+CD80+CMRF-44+ DC, and by some CD14+ monocytes. Anti-CD86 mAb and CTLA4 Ig, but not anti-CD80 mAb, inhibited the MLR stimulated by SF DC. Both CMRF-44+ and CMRF-44- SF DC were efficient stimulators of the allogeneic MLR, which was in each case blocked by CTLA4 Ig. The data indicate that rheumatoid synovial DC can undergo full functional differentiation, associated with CD86 expression, in vitro and in situ. Synovial DC expressing high levels of MHC molecules and CD86 are strategically located to present arthritogenic Ag to T cells after transendothelial migration.

Recent genetic studies have led to identification of numerous loci that are associated with susceptibility to autoimmune diseases. The strategy of using information from these studies has facilitated the identification of novel juvenile idiopathic arthritis (JIA) susceptibility loci, specifically, PTPN22 and IL2RA. Several novel autoimmune susceptibility loci have recently been identified, and we hypothesise that single-nucleotide polymorphisms (SNPs) within these genes may also be JIA susceptibility loci. Five SNPs within the genes AFF3, IL2/IL21, IL7R, CTLA4 and CD226, previously associated with multiple autoimmune diseases were genotyped, in a large data set of Caucasian JIA patients and controls, and tested for association with JIA. We identified two susceptibility loci for JIA, AFF3 and the IL2/IL21 region and additional weak evidence supporting an association with the CTLA4 and IL7R genes, which warrant further investigation. All results require validation in independent JIA data sets. Further characterisation of the specific causal variants will be required before functional studies can be performed.

OBJECTIVE

To investigate the possible association of the CT60A/G marker with systemic lupus erythematosus (SLE) in Spanish patients, and to identify the possible CTLA4 haplotype responsible for the association, taking into account other polymorphisms described at positions -1722T/C, -319C/T, +49A/G, and the microsatellite (AT)(n) in the 3'-untranslated region (3'-UTR) of the CTLA4 gene.

METHODS

Genotyping of CT60 was performed in 395 patients with SLE and 293 healthy controls using polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis. Genotyping of the rest of the dimorphisms has been previously reported. Genotyping of microsatellite polymorphism (AT)(n) in the 3'-UTR was performed using PCR with a fluorescence-labeled primer.

RESULTS

With regard to CT60A/G, the frequency of the AA genotype was significantly decreased among the SLE patients (18.7% versus 28.3% in the control group; P = 0.003, corrected P [P(corr)] = 0.009, odds ratio [OR] = 0.58, 95% confidence interval [95% CI] 0.40-0.85). In other words, the frequency of individuals bearing the G phenotype was increased in the patient group compared with the control group (81.2% versus 71.7%; P = 0.003, P(corr) = 0.006, OR = 1.71, 95% CI 1.18-2.49). The distribution of allele frequency was also significantly different between patients and controls (P = 0.01, P(corr) = 0.02, OR [for allele G] = 1.32, 95% CI 1.06-1.65). After combining the data on the different polymorphisms, 2 neutral haplotypes were found: +49A;(AT)(7);CT60A and +49G;(AT)(8-19);CT60G. In addition, a susceptibility haplotype was found: +49A;(AT)(>19);CT60G.

CONCLUSIONS

The 3'-UTR of the CTLA4 gene is involved in susceptibility to SLE.

Genome-wide association studies have identified gene regions associated with type 1 diabetes. The aim of this study was to determine how the combined allele frequency of multiple susceptibility genes can stratify islet autoimmunity and/or type 1 diabetes risk. Children of parents with type 1 diabetes and prospectively followed from birth for the development of islet autoantibodies and diabetes were genotyped for single-nucleotide polymorphisms at 12 type 1 diabetes susceptibility genes (ERBB3, PTPN2, IFIH1, PTPN22, KIAA0350, CD25, CTLA4, SH2B3, IL2, IL18RAP, IL10 and COBL). Non-human leukocyte antigen (HLA) risk score was defined by the total number of risk alleles at these genes. Receiver operator curve analysis showed that the non-HLA gene combinations were highly effective in discriminating diabetes and most effective in children with a high-risk HLA genotype. The greatest diabetes discrimination was obtained by the sum of risk alleles for eight genes (IFIH1, CTLA4, PTPN22, IL18RAP, SH2B3, KIAA0350, COBL and ERBB3) in the HLA-risk children. Non-HLA-risk allele scores stratified risk for developing islet autoantibodies and diabetes, and progression from islet autoimmunity to diabetes. Genotyping at multiple susceptibility loci in children from affected families can identify neonates with sufficient genetic risk of type 1 diabetes to be considered for early intervention.

The transplantation of fetal porcine neurons is a potential therapeutic strategy for the treatment of human neurodegenerative disorders. A major obstacle to xenotransplantation, however, is the immune-mediated rejection that is resistant to conventional immunosuppression. To determine whether genetically modified donor pig neurons could be used to deliver immunosuppressive proteins locally in the brain, transgenic pigs were developed that express the human T cell inhibitory molecule hCTLA4-Ig under the control of the neuron-specific enolase promoter. Expression was found in various areas of the brain of transgenic pigs, including the mesencephalon, hippocampus and cortex. Neurons from 28-day old embryos secreted hCTLA4-Ig in vitro and this resulted in a 50% reduction of the proliferative response of human T lymphocytes in xenogenic proliferation assays. Transgenic embryonic neurons also secreted hCTLA4-Ig and had developed normally in vivo several weeks after transplantation into the striatum of immunosuppressed rats that were used here to study the engraftment in the absence of immunity. In conclusion, these data show that neurons from our transgenic pigs express hCTLA4-Ig in situ and support the use of this material in future pre-clinical trials in neuron xenotransplantation.

CTLA-4 is considered one of the most potent negative regulators of T-cell activation. To circumvent experimental limitations due to fatal lymphoproliferative disease associated with genetic ablation of CTLA-4, we have used radiation chimeras reconstituted with a mixture of CTLA-4+/+ and CTLA-4-/- bone marrow that retain a normal phenotype and allow the evaluation of long-term T-cell immunity under conditions of intrinsic CTLA-4 deficiency. Following virus infection, we profiled primary, memory, and secondary CD8+ and CD4+ T-cell responses directed against eight different viral epitopes. Our data demonstrate unaltered antigen-driven proliferation, acquisition of effector functions, distribution of epitope hierarchies, T-cell receptor repertoire selection, functional avidities, and long-term memory maintenance in the absence of CTLA-4. Moreover, regulation of memory T-cell survival and homeostatic proliferation, as well as secondary responses, was equivalent in virus-specific CTLA4+/+ and CTL-A-4-/- T-cell populations. Thus, lack of CTLA-4 expression by antigen-specific T cells can be compensated for by extrinsic factors in the presence of CTLA-4 expression by other cells. These findings have implications for the physiologic, pathological, and therapeutic regulation of costimulation.

BACKGROUND

CD154 blockade-based immunosuppression successfully prevents both humoral and cellular adaptive immune responses in baboons receiving α1,3-galactosyltransferase gene-knockout (GTKO) pig organs. Using a GTKO pig artery transplantation model in baboons, we evaluated the efficacy of CD28/B7 costimulatory pathway blockade in comparison with CD154 blockade.

METHODS

Baboons received artery patch grafts from GTKO pigs, with no (Group1), anti-CD154mAb-based (Group2), or CTLA4-Ig-based (Group3) immunosuppressive therapy. Anti-pig IgM and IgG antibody and cellular responses were monitored. Xenografts were immunohistologically evaluated for antibody and complement deposition, and cellular infiltration.

RESULTS

Group1 baboons developed increased IgM and IgG antibody and cellular responses against GTKO antigens. In Group2, anti-CD154mAb alone prevented the development of both IgM and IgG antibody and cellular responses,but not cellular infiltration of the graft. In the single baboon that received anti-thymocyte globulin (ATG) + mycophenolate mofetil (MMF) + anti-CD154mAb, cellular infiltration of the graft was not seen. In Group3, CTLA4-Ig with ATG + MMF inhibited the cellular proliferative response to pig antigens but did not prevent the IgG response or cellular infiltration.

CONCLUSIONS

(i) Artery patch transplantation is a simple model to monitor the adaptive immune response to xenografts; (ii) anti-CD154mAb prevents sensitization but not cellular infiltration (but, without anticoagulation, may result in early thrombosis of a pig xenograft); (iii) although in only one baboon, the addition of ATG and MMF prevents cellular infiltration and (iv) replacement of anti-CD154mAb by CTLA4-Ig (at the doses used), even in combination with ATG and MMF, prevents the cellular proliferative response to GTKO pig antigens but is insufficient to prevent the development of anti-pig antibodies.

Previous studies have shown complex roles for the B7 receptors in providing both positive and negative regulation of experimental autoimmune encephalomyelitis (EAE). B7 blockade can ameliorate clinical EAE by indirectly interfering with CD28 signaling. However, B7 blockade can also result in disease exacerbation, presumably by interfering with regulatory B7:CTLA-4 interactions. Therefore, we have directly targeted T cell CD28 with specific mAbs both during initial Ag priming and after the onset of clinical signs of EAE. We found that CD28 blockade ameliorated EAE during the efferent and afferent limbs of the immune response. Disease amelioration at disease onset was associated with suppression of TNF-alpha production. Finally, Ab blockade of T cell CD28 during the first disease episode resulted in significant attenuation of the subsequent disease course, with no significant relapses. In contrast to previous studies targeting APC B7 with CTLA4-Ig, reagents targeting CD28 can block ongoing disease. Therefore, the present results suggest a clinically relevant therapeutic scenario for human diseases, such as multiple sclerosis.

The protein kinase encoded by the Tpl2 protooncogene plays an obligatory role in the transduction of Toll-like receptor and death receptor signals in macrophages, B cells, mouse embryo fibroblasts, and epithelial cells in culture and promotes inflammatory responses in animals. To address its role in T cell activation, we crossed the T cell receptor (TCR) transgene 2C, which recognizes class I MHC presented peptides, into the Tpl2(-/-) genetic background. Surprisingly, the TCR2C(tg/tg)/Tpl2(-/-) mice developed T cell lymphomas with a latency of 4-6 months. The tumor cells were consistently TCR2C(+)CD8(+)CD4(-), suggesting that they were derived either from chronically stimulated mature T cells or from immature single positive (ISP) cells. Further studies showed that the population of CD8(+) ISP cells was not expanded in the thymus of TCR2C(tg/tg)/Tpl2(-/-) mice, making the latter hypothesis unlikely. Mature peripheral T cells of Tpl2(-/-) mice were defective in ERK activation and exhibited enhanced proliferation after TCR stimulation. The same cells were defective in the induction of CTLA4, a negative regulator of the T cell response, which is induced by TCR signals via ERK. These findings suggest that Tpl2 functions normally in a feedback loop that switches off the T cell response to TCR stimulation. As a result, Tpl2, a potent oncogene, functions as a tumor suppressor gene in chronically stimulated T cells.

A key factor driving the underlying pathyphysiology of "chronic rejection" in organ transplantation is a persistent T cell-mediated alloimmune response. Members of both the B7 family (including CD28 and CTLA4) and the tumor necrosis factor (TNF) family, in which the CD40-CD154 pathway is preeminent, play key roles in the T cell response following alloantigen presentation. "Positive" costimulatory molecules promote full T cell activation, whereas a subgroup of costimulatory molecules delivers "negative" costimulatory signals that function to downregulate alloimmune responses. Emerging experimental data point to key differences between the various positive and negative costimulatory molecules in terms of their temporal and spatial expression profiles, their effects of T and B cell subsets, and on their relative importance within the hierarchy of costimulatory signals delivered to the T cell. In this review, we address the role of costimulatory pathways in allograft rejection and tolerance. We will address in particular the potential of the novel costimulatory pathways as targets for tolerance induction in CD28-independent alloresponses, and we will review emerging data that suggests a key role for parenchymal expression of negative costimulatory molecules in the termination of pathogenic immune responses.

Improving the immunological potency, particularly the Ab response, is a serious hurdle for the protective efficacy and hence broad application of DNA vaccines. We examined the immunogenicity and protective efficacy of a hemagglutinin-based influenza DNA vaccine that was targeted to antigen-presenting cells (APCs) by fusion to CTLA4. The targeted vaccine was shown to induce an accelerated and increased Ab response (as compared with those receiving the nontargeted control) that was predominated by IgG1 and recognized conformationally dependent viral epitopes. Moreover, mice receiving the APC-targeted DNA vaccine had significantly reduced viral titers (100-fold) after a nonlethal virus challenge. The increased protective efficacy was most likely because of increased Ab responses, as cytotoxic T lymphocyte responses were not enhanced. Targeting was demonstrated by direct binding studies of CTLA4 fusion proteins to the cognate ligand (B7; expressed on APCs in vivo). In addition, a targeted protein was detected at 4-fold higher levels in draining lymph nodes within 2-24 h of administration. Therefore, this study demonstrates that targeting DNA-encoded antigen to APCs results in enhanced immunity and strongly suggests that this approach may be useful in improving the protective efficacy of DNA vaccines.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Multiple genetic and environmental factors contribute to the pathogenesis of this disease. Recent genome-wide association studies have added substantially to the number of genes associated with SLE. To replicate some of these susceptibility loci, single-nucleotide polymorphisms reported to be associated to SLE were evaluated in a cohort of 245 well-phenotyped Canadian SLE trios. Our results replicate previously reported associations to alleles of interferon regulatory factor 5 (IRF5), major histocompatibility complex (MHC), tumor necrosis factor (ligand) superfamily member 4 (TNFSF4), Kell blood group complex subunit-related family member 6 (XKR6), B-cell scaffold protein with ankyrin repeats 1 (BANK1), protein tyrosine phosphatase non-receptor type 22 (PTPN22), ubiquitin-conjugating enzyme E2L 3 (UBE2L3) and islet cell autoantigen 1 (ICA1). We also identify putative associations to cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a gene associated with several autoimmune disorders, and ERBB3, a locus on 12q13 that was previously reported to be associated with type 1 diabetes. This study confirms the existence of multiple genetic risk factors for SLE, and supports the notion that some risk factors for SLE are shared with other inflammatory disorders.

Cytotoxic T-lymphocyte antigen 4 (CTLA4) polymorphisms located in the promotor region at positions -318 (C/T) and in exon 1 (49 A/ G) were investigated in 138 Spanish patients (37 men and 101 women) with rheumatoid arthritis and in 305 ethnically-matched healthy controls. When the allelic and genotypic frequencies corresponding to the CTLA4 -318 position were compared, no significant differences between patients and controls were found. However, when the CTLA4 49 A/G polymorphism was analysed, a significant increase of A/G heterozygous individuals among female patients (48.5% vs. 33.8% in controls; P=0.008; OR=2.0) was observed. This increase was absent among males (37.8%, P=NS). Analysis of the CTLA4 49 polymorphism with respect to HLA-DRB1 typing demonstrated a significant increase of A/G heterozygosity in the HLA-DR3-positive patient group compared with HLA-DR3-negative patient group (14/19, 74% vs. 49/119, 41%; P=0.009, OR=4.0). The increase of A/G genotype among HLA-DR3-positive patients was found in both males (4/6, 67%) and females' (10/13, 77%), although statistical differences were only reached in the female group. These results provide new insight into this complex association, confirm previous data from other studies, and suggest that the CTLA4 gene could be involved in the pathogenesis of rheumatoid arthritis.