Epithelial cells along human gastrointestinal mucosal surface express pathogen-recognizing receptors and actively participate in the regulation of inflammatory reactions in response to microbial infection. The NAD-dependent deacetylase sirtuin-1 (SIRT1), one member of the sirtuin family of proteins and an NAD-dependent deacetylase has been implicated in the regulation of multiple cellular processes, including inflammation, longevity, and metabolism. In this study, we demonstrated that infection of cultured human biliary epithelial cells (H69 cholangiocytes) with a parasitic protozoan, Cryptosporidium parvum, induced SIRT1 expression at the protein level without a change in SIRT1 mRNA content. Using real-time PCR and Northern blot analyses, we found that C. parvum infection decreased the expression of let-7i in infected H69 cells. Down-regulation of let-7i caused relief of miRNA-mediated translational suppression of SIRT1 and consequently, resulted in an increased SIRT1 protein level in infected H69 cell cultures. Moreover, gain- and loss-of-function studies revealed that let-7i could modulate NF-κB activation through modification of SIRT1 protein expression. Thus, our data suggest that let-7i regulates SIRT1 expression in human biliary epithelial cells in response to microbial challenge, suggesting a new role of let-7i in the regulation of NF-κB-mediated epithelial innate immune response.
let-7; SIRT1; C. parvum; Epithelium; NF-κB
Our work aimed to examine the potential influence of variants in interleukin/interleukin receptors genes on high-risk (HR-HPV) HPV clearance. Clearance of genital HR-HPV infection was evaluated for 134 HIV-1 seropositive African-American female adolescents from the Reaching for Excellence in Adolescent Care and Health (REACH) cohort. Genotyping targeted 225 single nucleotide polymorphisms (SNPs) within the exons, 5′ untranslated region (UTR) and 3′ UTR sequences of 27 immune-related candidate genes encoding interleukin family of cytokines. Cox proportional hazard models were used to determine the association of type- specific HPV clearance adjusting for time-varying CD4+ T-cell count and low-risk (LR-HPV) HPV co-infections. HR-HPV clearance rates were significantly (p< 0.001) associated with five SNPs (rs228942, rs419598, rs315950, rs7737000, rs9292618) mapped to coding and regulatory regions in three genes (IL2RB, IL1RN, and IL7R). These data suggest that the analyzed genetic variants in interleukin family of cytokines modulate HR-HPV clearance in HIV-1 seropositive African-Americans that warrants replication.
HPV clearance; genetic association; interleukins; HIV-1 seropositive; African American adolescents
Th1 immune responses are crucial for eliminating Leishmania parasites. However, despite strong Th1 responses, cutaneous leishmaniasis (CL) patients infected with L. braziliensis develop the disease, while milder Th1 responses are found in sub-clinical (SC) infections. Therefore, CL patients may experience impaired regulatory T cell (Treg) function, causing excessive Th1 responses and tissue damage. To address this hypothesis, we characterized the function of circulating Tregs in L. braziliensis infected CL patients and compared them to Tregs from uninfected controls (UC) and SC subjects. The frequency of circulating Tregs was similar in CL patients, UC and SC subjects. Moreover, CL patients Tregs suppressed lymphocyte proliferation and PBMC pro-inflammatory cytokine production more efficiently than UC Tregs, and also produced higher levels of IL-10 than UC and SC Tregs. Furthermore, PBMC and mononuclear cells from lesions of CL patients responded normally to Treg-induced suppression. Therefore, the lesion development in CL patients infected with L. braziliensis is not associated with impairment in Treg function or failure of cells to respond to immunomodulation. Rather, the increased Treg activation in CL patients may impair parasite elimination, resulting in establishment of chronic infection. Thus, immunological strategies that interfere with this response may improve leishmaniasis treatment.
Regulatory T cells; Leishmania braziliensis; cutaneous leishmaniasis
Shellfish allergy is an immune-mediated adverse reaction to allergenic shellfish and is responsible for significant morbidity and mortality. CD4 T cell responses play an important role in the pathophysiological mechanisms of sensitization and in production of IgE.
We sought to identify and validate CD4 T cell shrimp tropomyosin-derived epitopes and characterize CD4 T cell responses in subjects with a clinical history of shellfish allergy.
Using an in vitro MHC-peptide binding assay, we screened 91 overlapping peptides and identified 28 epitopes with moderate and strong binding capacities; 3 additional peptides were included based on MHC binding prediction score. These peptides were then examined in proliferation and cytokine release assays with T cells from allergic subjects.
17 epitopes restricted to DRB*01:01, DRB1*03:01, DRB1*04:01, DRB1*09:01, DQB1*02:01, DQB1*03:02 and DQB1*05:01 alleles were identified and validated by both the MHC binding and the functional assays. Two peptides showed specificities to more than one MHC class II allele. We demonstrated that these peptides exert functional responses in an epitope specific manner, eliciting predominantly IL-6 and IL-13.
The identified epitopes are specific to common MHC class II alleles in the general population. Our study provides important data for the design of peptide-based immunotherapy of shrimp-allergic patients.
Estimates of T regulatory cell populations in the periphery of patients with Crohn’s disease are confounded by disease activity and concomitant immunotherapeutic agents known to affect T cell proliferation and survival. We performed deuterium pulse/chase experiments in patients with quiescent Crohn’s disease on no immunotherapy and healthy control subjects to estimate T regulatory cell kinetics. Quantification of deuterated DNA isolated from T cell subsets over 10 days was determined by mass spectrophotometry. We demonstrate enhanced proliferation within the T regulatory cell population from patients with Crohn’s disease when compared to non-T regulatory cells and T regulatory cells from healthy control subjects. We speculate that T regulatory cells isolated from the periphery of patients with Crohn’s disease experience persistent antigen stimulation resulting in excess proliferative rates.
Recent studies strongly suggest an increasing role for immune responses against self-antigens (Ags) which are not encoded by the major histocompatibility complex in the immunopathogenesis of allograft rejection. Although, improved surgical techniques coupled with improved methods to detect and avoid sensitization against donor human leukocyte antigen (HLA) have improved the immediate and short term function of transplanted organs. However, acute and chronic rejection still remains a vexing problem for the long term function of the transplanted organ. Immediately following organ transplantation, several factors both immune and non immune mechanisms lead to the development of local inflammatory milieu which sets the stage for allograft rejection. Traditionally, development of antibodies (Abs) against mismatched donor HLA have been implicated in the development of Ab mediated rejection. However, recent studies from our laboratory and others have demonstrated that development of humoral and cellular immune responses against non-HLA self-Ags may contribute in the pathogenesis of allograft rejection. There are reports demonstrating that immune responses to self-Ags especially Abs to the self-Ags as well as cellular immune responses especially through IL17 has significant pro-fibrotic properties leading to chronic allograft failure. This review summarizes recent studies demonstrating the role for immune responses to self-Ags in allograft immunity leading to rejection as well as present recent evidence suggesting there is interplay between allo- and autoimmunity leading to allograft dysfunction.
Alloimmunity; Autoimmunity; Transplant rejection; Antigen presentation; Interleukin-17
We assessed the effects of sex, race and ethnicity on smallpox vaccine-induced immune responses in 1,071 armed forces members after primary Dryvax® smallpox vaccination, including 790 males and 281 females; 580 Caucasians, 217 African-Americans, and 217 Hispanics. Analysis of vaccinia-specific cytokine responses revealed that Caucasians had higher total IFNγ ELISPOT responses (median 57 spot-forming units/SFUs per 200,000 cells, p=0.01) and CD8+IFNγ ELISPOT responses (12 SFUs, p<0.001) than African-Americans (51 and 4 SFUs, respectively) and Hispanics (47 and 8 SFUs, respectively). Similarly, Caucasians secreted higher levels of vaccinia-specific IL-2 (p=0.003) and IFNα (p<0.001) compared to other racial/ethnic groups. Males had higher total IFNγ ELISPOT responses (median 55 SFUs) compared to females (41 SFUs, p<0.001). We observed statistically significant sex-related differences in the secretion of IL-2 (p<0.001), IL-1β (p<0.001) and IL-10 (p=0.017). These data suggest that vaccinia-specific cytokine responses following primary smallpox vaccination are significantly influenced by race and sex of vaccinees.
Smallpox Vaccine; Cytokine; Cellular Immunity; Race; Sex; Smallpox Vaccine; Cytokines; Immunity, Cellular; Sex; Hispanics; African Americans; Whites
Four genetic polymorphisms located at the promoter (C-257T) and coding regions of CFH gene (exon 2 G257A, exon 14 A2089G and exon 19 G2881T) were investigated in 121 dengue patients (DENV-3) in order to assess the relationship between allele/haplotypes variants and clinical outcomes. A statistical value was found between the CFH-257T allele (TT/TC genotypes) and reduced susceptibility to severe dengue (SD). Statistical associations indicate that individuals bearing a T allele presented significantly higher protein levels in plasma. The –257T variant is located within a NF-κB binding site, suggesting that this variant might have effect on the ability of the CFH gene to respond to signals via the NF-κB pathway. The G257A allelic variant showed significant protection against severe dengue. When CFH haplotypes effect was considered, the ancestral CG/CG promoter-exon 2 SNP genotype showed significant risk to SD either in a general comparison (ancestral × all variant genotypes), as well as in individual genotypes comparison (ancestral × each variant genotype), where the most prevalent effect was observed in the CG/CG × CA/TG comparison. These findings support the involvement of –257T, 257A allele variants and haplotypes on severe dengue phenotype protection, related with high basal CFH expression.
The immunoregulatory role of human donor bone marrow cells (DBMC) has been studied extensively in our laboratory using in vitro and ex vivo assays. However, new experimental systems that can overcome the limitations of tissue culture assays but with more clinical relevance than purely animal experimentation, needed to be generated. Therefore we have developed a new human peripheral blood lymphocyte (PBL) severe combined immunodeficient (SCID) mouse islet transplantation model without the occurrence of graft-versus-host disease (GvHD) and have used it to evaluate the tolerogenic effects of DBMC. Nonobese diabetogenic (NOD)–SCID mice were transplanted with human deceased donor islets and were reconstituted with human PBL (allogeneic to islets; denoted as recipient) with or without DBMC from the islet donor.
It was observed that the most cellularly economical dose was 3000 islets per animal and that injection into the portal vein was better than implantation under the kidney capsule. Even though maximal lymphoid reconstitution was observed with 40-million fresh and anti-CD3 activated recipient PBL (conventional method), the mice developed severe graft GvHD. However, with the new method of reconstitution where animals were injected with 20-million anti-CD3-activated plus 40-million anti– donor-activated recipient PBL, no discernible GvHD was observed. More importantly, this latter method was associated with islet transplant rejection, which in turn could be abrogated by co-injection of the animals with DBMC. These in vivo results confirmed our previous in vitro observations that human DBMC have regulatory activity.
SCID; Human islet model; Bone marrow; Transplantation; Tolerance
Inflammasomes are increasingly implicated in regulating immunity, but how their activation relates to function of human dendritic cells (DCs) is unknown. Here we show that DC maturation stimuli lead to rapid activation of caspase-1 in human monocyte-derived DCs. RNAi mediated inhibition of the inflammasome component ASC leads to marked inhibition of the capacity of lipopolysachharide (LPS)-matured DCs to stimulate antigen-specific T cells. RNAi mediated inhibition of Cathepsin B (CatB) also similarly inhibits the capacity of human DCs to stimulate immunity. The defective ability of ASC or CatB deficient DCs to stimulate T cells is independent of inflammasome-mediated processing of inflammatory cytokines and also includes DCs loaded with pre-processed peptide. Gene expression profiles of ASC or CatB deficient human DCs show marked overlap with downregulation of genes implicated in DC function. These data demonstrate an important role for ASC and CatB in regulating function of human DCs with overlapping effects on gene expression.
human; dendritic cells; maturation; immunity; inflammasome
The CD8 memory T cell repertoire to the influenza A derived M158–66 epitope shows a restricted V genes and CDR3 sequences usage. The repertoire is highly polyclonal and the clonotype distribution has been described as consisting of two components, one showing a power law-like distribution and the other composed of a few clonotypes with a very high relative frequency. The question is whether the complex repertoire defined by its ability to flourish in a short term recall culture corresponded to functional cells. Here we show that there is a relation between expression of the degranulation marker CD107 and cytotoxicity or IFN-γ production in CD8 T cell lines and clones. We then examine recently degranulated CD8 cells from recall cultures from four middle aged HLA-A2 subjects and show that these functional cells are polyclonal. The clonotype distributions of the CD8 + CD107+ repertoires are complex in the same manner as previously reported. The clonotype composition of CD8 + CD107+ repertoires is also very similar to CD8 only repertoires, and to CD8 + HLA-A2-M158–66 pentamer positive repertoires. We postulate that multiple exposures during childhood to this conserved influenza A epitope has generated a complex functional repertoire in HLA-A2 individuals.
The binding motif of human CTLA-4 is well known to be MYPPPY and for porcine CTLA-4 the binding motif is LYPPPY. Is this single amino acid difference of methionine (M) versus leucine (L) critical for the CTLA-4 binding? Recently, we have reported that the recombinant soluble porcine CTLA-4 was incapable of binding to human CD80. In this study we mutated L to M in the binding motif of the soluble porcine CTLA-4 and mutated M to L in the binding motif of the soluble human CTLA-4. We then analyzed how these mutations affected the binding affinity of the mutants to both porcine and human CD80+ cells. The soluble porcine CTLA-4-L97M mutant partially lost its binding affinity to porcine CD80 compared to the wild-type and conferred very weak binding ability to human CD80. These results indicate that the L in the binding motif of porcine CTLA-4 is important for determining its binding ability to porcine CD80. Wild-type soluble human CTLA-4 binds to both human and porcine CD80 with comparable affinity, however, the soluble human CTLA-4-M97L mutant almost lost its binding ability to human CD80 and increased its binding ability to porcine CD80. These results indicate that M in the human CTLA-4 binding motif is extremely critical for its binding to human CD80. Those data suggest that the human CTLA-4 based recombinant protein drugs such as human CTLA-4-Ig can be used and/or tested in a porcine model. Conversely, the use of porcine CTLA-4 based recombinant protein drugs such as porcine CTLA-4-Ig is restricted to swine models. The difference in binding specificity of CTLA-4 observed in this study may be useful for studies such as pig to nonhuman primate xeno-transplantation. Porcine CTLA-4- and human CTLA-4-M97L mutant-based recombinant protein drugs can be used to specifically block the direct presentation by donor antigen presenting cells in pig to nonhuman primate xeno-transplantation. Human CTLA-4-M97L mutant-based recombinant protein drugs will be more ideal as it is without immunogenicity to human being.
We compared peripheral blood immunophenotyping in 31 adult liver transplant recipients on differing long-term immunosuppressive (IS) monotherapy with and without peri-transplantation alemtuzumab (AL) induction. All patients had been stable on monotherapy with either sirolimus (SRL) (n = 10) or without SRL (tacrolimus (TAC) (n = 10), mycophenolate mofetil (MMF) (n = 11)) for more than 6 months. Five-color flow cytometry for putative “regulatory” T and dendritic cells as well as serum assays for soluble HLA-G (sHLA-G) were performed. The SRL monotherapy group had significantly higher percentages of CD4+CD25high+ Foxp3+ T cells (1.3 ± 1.0) compared with the non-SRL group (0.7 ± 0.6) (p = 0.04). The SRL effect was even higher in a subset with prior AL induction and no prior hepatitis C or rejection (1.7 ± 0.2) compared with all other subgroups (0.7 ± 0.6) (p = 0.02). TAC patients showed significantly higher “regulatory” DC2:DC1 ratios (10 ± 7.6) compared with non-TAC patients (4.1 ± 2.3) (p = 0.04). Although sHLA-G levels appeared higher in TAC patients, the differences were not statistically significant. In conclusion, IS monotherapy provides an opportunity to investigate regulatory roles of individual agents. SRL maintenance and prior AL induction in subsets of patients appeared to show a regulatory T cell immunophenotype. However, TAC patients may have other regulatory characteristics, supporting the need for larger, prospective studies to clarify differences.
Regulatory T cells; Dendritic cells; Immunophenotyping; Liver transplantation; Immunosuppression
We studied the effects of alemtuzumab on T-regulatory cells (Tregs) during alloactivation, first by differences in depletion of various naive versus alloactivated cell subsets in peripheral blood of healthy volunteers, then by adding serial concentrations to human leukocyte antigen (HLA)–DR–matched and –mismatched responding and stimulating cells in mixed lymphocyte reaction (MLR). Lymphoproliferation inhibition and the development of proliferating carboxyfluorescein succinimidyl ester (CFSE)–diluted CD4+CD25highCD127−FOXP3+ (phenotypic) Tregs by flow cytometry were measured. Also, the ability of alemtuzumab-treated versus nontreated MLR generated CD4+CD127− cells to allospecifically inhibit MLRs and recruit additional responding Tregs was tested. We found a more pronounced refractoriness of alloactivated versus naive CD4+CD25high cells to alemtuzumab induced lymphodepletion. Alemtuzumab dose dependently inhibited lymphoproliferation while amplifying percentages of MLR-generated Tregs. This was somewhat augmented by human complement addition. CD127−CD4+ cells immunoselected after 7 days from alemtuzumab-treated MLRs allospecifically inhibited lymphoproliferation and recruited additional Tregs in fresh MLR-responding cells, similar to modulators derived from MLRs without drug addition (media). Addition of tacrolimus and sirolimus to alemtuzumab further inhibited MLR proliferation. However, Treg percentages were markedly higher with sirolimus. These results support the notion that alemtuzumab induces immunoregulation in naïve T cells undergoing alloactivation absent presensitization, especially used in conjunction with maintenance SRL.
Mixed lymphocyte reaction; Regulatory T cells; FOXP3; Alemtuzumab; mTOR inhibition
Human leukocyte antigen A (HLA-A) genotypes were determined for samples from 283 multiplex, Caucasian, type 1 diabetes families from the Human Biological Data Interchange (HBDI) using an immobilized probe assay. Distribution of HLA-A alleles transmitted to patients was significantly different from that in affected family-based controls (AFBAC) (p = 0.004). Transmission disequilibrium test (TDT) analysis revealed differential transmission of several HLA-A alleles from parents to affected offspring. HLA class II DRB1 and DQB1 loci were also typed, allowing assignment of HLA-A alleles to haplotypes and calculation of linkage disequilibrium values. Some of the apparent effects of HLA-A alleles on type 1 diabetes susceptibility were attributable to linkage disequilibrium with DR and DQ alleles, although others were not. The differences in frequencies between patients and controls of alleles A*0101, A*2402, and A*3002 could not be explained by linkage disequilibrium alone. Our results suggest an important role for class I antigens in modulating susceptibility to type 1 diabetes.
HLA-A genotypes; type 1 diabetes
Alleles of human leukocyte antigen (HLA) class II genes are well known to affect susceptibility to type 1 diabetes (T1D), but less is known about the contribution of HLA class I alleles to T1D susceptibility. In this study, molecular genotyping was performed at the HLA-B and HLA-C loci for 283 multiplex Caucasian families, previously typed for HLA-A and the class II loci. Allele frequencies were compared between affected siblings and affected family-based controls. Linkage disequilibrium coefficients were calculated for HLA-B–HLA-C haplotypes and for class I–class II haplotypes. After adjustment for linkage disequilibrium, the following alleles remain associated with T1D: B*1801, B*3906, B*4403, C*0303, C*0802, and C*1601. B and C allele associations were tested for certain T1D-associated DRB1-DQB1 haplotypes, with the following results: B*3801 is protective on DRB1*0401-DQB1*0302 haplotypes, both C*0701 and C*0702 are predisposing on DRB1*0404-DQB1*0302 haplotypes, and B*3906 is predisposing on DRB1*0801-DQB1*0402 haplotypes. As with previous results for HLA-A, HLA-B and HLA-C are associated with age at T1D onset (mean 11.6 ± 0.3 years). The protective allele B*4403 was associated with older age at onset (15.1 years; p < 0.04), and the predisposing alleles C*0702 and B*3906 were associated with younger age at onset (9.5 years, p < 0.001; and 7.8 years, p < 0.002, respectively). These data support a role for HLA class I alleles in susceptibility to and age at onset of T1D.
type 1 diabetes; HLA class I; HLA-B; HLA-C; age at onset
The role of polymorphisms within the antiviral tripartite motif (TRIM) genes in measles vaccine adaptive immune responses was examined. A limited association was found between TRIM5 (rs7122620) and TRIM25 (rs205499) gene polymorphisms and measles-specific antibody levels. However, many associations were found between TRIM gene SNPs and variations in cellular responses (IFN-γ Elispot and secreted cytokines IL-2, IL-6, IL-10, IFN-γ, and TNF-α). TRIM22 rs2291841 was significantly associated with an increased IFN-γ Elispot response (35 vs. 102 SFC per 2×105 PBMC, p=0.009, q=0.71) in Caucasians. A non-synonymous TRIM25 rs205498 (in LD with other SNPs, r2≥0.56), as well as the TRIM25 AAAGGAAAGGAGT haplotype, was associated with a decreased IFN-γ Elispot response (t-statistic −2.32, p=0.02) in African-Americans. We also identified polymorphisms in the TRIM5, TRIM22, and TRIM25 genes that were associated with significant differences in cytokine responses.
Additional studies are necessary to replicate our findings and to examine the functional consequences of these associations.
Single-nucleotide polymorphisms; measles virus; measles vaccine immunity; TRIM genes; antiviral; innate; antibody; cytokines; Elispot; Caucasians; African-Americans
The C1858T single nucleotide polymorphism in PTPN22, which is the gene encoding lymphoid tyrosine phosphatase (LYP), confers increased risk for various autoimmune disorders in Caucasians. Although the disease-associated LYP allele (LYP*W620) is a gain-of-function variant that has higher catalytic activity than the major allele (LYP*R620), it is still unclear how LYP*W620 predisposes for autoimmunity. Here, we compared both T cell signaling and T cell function in healthy human donors homozygous for either LYP*R620 or LYP*W620. Generally, the presence of LYP*W620 caused reduced proximal T cell antigen receptor-mediated signaling (e.g. ζ chain phosphorylation) but augmented CD28-associated signaling (e.g. AKT activation). Altered ligand binding properties of the two LYP variants could explain these findings since LYP*R620 interacted more strongly with the p85 subunit of PI3K. Variation in signaling between cells expressing either LYP*R620 or LYP*W620 also affected the differentiation of conventional CD4+ T cells. For example, LYP*W620 homozygous donors displayed exaggerated Th1 responses (e.g. IFNγ production) and reduced Th17 responses (e.g. IL-17 production). Importantly, while regulatory T cells normally suppressed Th1-mediated IFNγ production in LYP*R620 homozygous individuals, such suppression was lost in LYP*W620 homozygous individuals. Altogether, these findings provide a molecular and cellular explanation for the autoimmune phenotype associated with LYP*W620.
LYP; PTPN22; TCR; autoimmunity
A novel MICA allele, MICA*070, was defined by sequencing. The new allele differs from the MICA*008:04 sequence in exon 2, encoding a C instead of G corresponding to cDNA nucleotide position 183. This nucleotide substitution is predicted to encode serine instead of arginine at residue 38 of the α1 domain of the MICA molecule.
Major histocompatibility complex class I chain-related A genotype; Sequence-based typing; Phasing
Although HLA alleles are associated with type 1 diabetes, association with microvascular complications remains controversial. We tested HLA association with complications in multiplex type 1 diabetes families.
Probands from 425 type 1 diabetes families from the Human Biological Data Interchange (HBDI) collection were analyzed. The frequencies of specific HLA alleles in patients with complications were compared with the frequencies in complications-free patients. The complications we examined were: retinopathy, neuropathy, and nephropathy. We used logistic regression models with covariates to estimate odds ratios.
We found that the DRB1*03:01 allele is a protective factor for complications (OR=0.58; p = 0.03), as is the DQA1*05:01-DQB1*02:01 haplotype found in linkage disequilibrium with DRB1*03:01 (OR= 0.59; p = 0.031). The DRB1*04:01 allele showed no evidence of association (OR=1.13; p = 0.624), although DRB1*04:01 showed suggestive evidence when the carriers of the protective DRB1*03:01 were removed from the analysis. The class II DQA1*03:01-DQB1*03:02 haplotype was not associated with complications, but the class I allele B*39:06 (OR=3.27; P = 0.008) suggested a strong positive association with complications.
Our results show that in type 1 diabetes patients, specific HLA alleles may be involved in susceptibility to, or protection from, microvascular complications.
Retinopathy; nephropathy; neuropathy; microvascular complications; genetics; type 1 diabetes; logistic regression; association; HLA
The Vitamin D Receptor (VDR) gene encodes a transcription factor which, on activation by vitamin D, modulates diverse biological processes including calcium homeostasis and immune function. Genetic variation involving VDR shows striking differences in allele frequency between populations and has been associated with disease susceptibility including tuberculosis and autoimmunity, although results have often been conflicting. We hypothesized that methylation of VDR may be population specific and that the combination of differential methylation and genetic variation may characterise TB predisposition. We use bisulphite conversion and/or pyrosequencing to analyse the methylation status of 17 CpGs of VDR and to genotype 7 SNPs in the 3′ CpG Island (CGI 1060), including the commonly studied SNPs ApaI (rs7975232) and TaqI (rs731236). We show that for lymphoblastoid cell lines from two ethnically diverse populations (Yoruba from HapMap, n=30 and Caucasians, n=30) together with TB cases (n=32) and controls (n=29) from the Venda population of South Africa there are methylation variable positions (MVPs) in the 3′ end that significantly distinguish ethnicity (9/17 CpGs) and TB status (3/17 CpGs). Moreover methylation status shows complex association with TaqI genotype highlighting the need to consider both genetic and epigenetic variants in genetic studies of VDR association with disease.
VDR (vitamin D (1,25- dihydroxyvitamin D3) receptor); gene polymorphism; CpG methylation; TB (tuberculosis); ethnic differences
The suppressive effects of CD4+CD25+ regulatory T cells (Tregs) on T cells have been well documented. Here we investigated whether human CD4+CD25+ Tregs can inhibit the pro-inflammatory properties of monocytes/macrophages. Monocytes and T cells were isolated from peripheral blood of healthy volunteers by magnetic cell separation, and co-cultured for 40 hours. Monocytes were analyzed directly for cytokine production and phenotypic changes, or re-purified and used in T cell stimulation and LPS challenge assays. Co-culture with CD4+CD25+ Tregs induced minimal cytokine production in monocytes, whereas co-culture with CD4+CD25− T cells resulted in large amounts of pro-inflammatory (TNF-α, IFN-γ IL-6) and regulatory (IL-10) cytokines. Importantly, when these CD4+CD25+ Treg-treated monocytes were re-purified after co-culture and challenged with LPS, they were severely inhibited in their capacity to produce TNF-α and IL-6 compared to control-treated monocytes. In addition, monocytes that were pre-cultured with CD4+CD25+ Tregs displayed limited up-regulation of HLA class II, CD40 and CD80, and down-regulation of CD86 compared to control-treated monocytes. This altered phenotype had functional consequences, as shown by the reduction in T cell-stimulatory capacity of Treg-treated monocytes. Together these data demonstrate that CD4+CD25+ Tregs can exert direct suppressive effects on monocytes/macrophages, thereby affecting subsequent innate and adaptive immune responses.
Suppression; antigen-presenting cell; rheumatoid arthritis
The production of anti-donor antibodies to HLA class I and class II antigens following transplantation is associated with development of transplant vasculopathy and graft loss. Antibodies against HLA class I (HLA-I) molecules are thought to contribute to transplant vasculopathy by triggering signals that elicit the activation and proliferation of endothelial cells. The proximal molecular events that regulate HLA-I dependent signal transduction are not well understood. We demonstrated a mutual dependency between HLA-I and integrin β4 to stimulate signal transduction and cell proliferation. Similarly, we found that integrin β4-mediated cell migration was dependent upon its interactions with HLA-I molecules. Since integrin β4 has been implicated in angiogenesis and tumor formation, associations between integrin β4 and HLA-I may play an important role in cancer. Further characterization of interactions between HLA-I and integrin β4 may lead to the development of therapeutic strategies for the treatment and prevention of chronic allograft rejection and cancer.
Antibody Mediated Rejection; Endothelial Cells; HLA class I; Integrin β4; Signal Transduction
Significant progress has been made in preventing acute allograft rejection following solid organ transplantation resulting in improved allograft survival. However, long term function still remains disappointing primarily due to chronic allograft rejection. Alloimmune responses primarily defined by the development of antibodies (Abs) to donor mismatched major histocompatibility antigens during the post-transplantation period have been strongly correlated to the development of chronic rejection. In addition, recent studies have demonstrated an important role for autoimmunity including the development of Abs to organ specific self-antigens in the pathogenesis of chronic allograft rejection. Based on this, a new paradigm has evolved indicating a possible cross-talk between the alloimmune responses and autoimmunity leading to chronic rejection. In this review, we will discuss the emerging concept for the role of cellular and humoral immune responses to self-antigens in the immunopathogenesis of chronic allograft rejection which has the potential to develop new strategies for the prevention and/or treatment of chronic rejection.
Alloimmunity; Autoimmunity; Chronic Rejection; Th17; regulatory T-cells and Organ transplantation