Graves’ disease (GD) is an autoimmune inflammatory disease, and Graves’ ophthalmopathy (GO) occurs in 25–50% of patients with GD. Several susceptible genes were identified to be associated with GO in some genetic analysis studies, including the immune regulatory gene CTLA4. We aimed to find out the correlation of CTLA4 gene polymorphism and GO. A total of 42 participants were enrolled in this study, consisting of 22 patients with GO and 20 healthy controls. Chi-square or Fisher’s exact test were used to appraise the association between Graves’ ophthalmopathy and CTLA4 single nucleotide polymorphisms (SNPs). All regions of CTLA4 including promoter, exon and 3’UTR were investigated. There was no nucleotide substitution in exon 2 and exon 3 of CTLA4 region, and the allele frequencies of CTLA4 polymorphisms had no significant difference between patients with GO and controls. However, the genotype frequency of “TT” genotype in rs733618 significantly differed between patients with GO and healthy controls (OR = 0.421, 95%CI: 0.290–0.611, p = 0.043), and the “CC” and “CT” genotype in rs16840252 were nearly significantly differed in genotype frequency (p = 0.052). Haplotype analysis showed that CTLA4 Crs733618Crs16840252 might increase the risk of GO (OR = 2.375, 95%CI: 1.636–3.448, p = 0.043). In conclusion, CTLA4 Crs733618Crs16840252 was found to be a potential marker for GO, and these haplotypes would be ethnicity-specific. Clinical application of CTLA4 Crs733618Crs16840252 in predicting GO in GD patients may be beneficial.

In clinical practice, it is found that autoimmune thyroid disease often additionally occurs with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). In addition, several studies showed that eye-specific autoimmune diseases may have a strong relationship with systemic autoimmune diseases. We focused on Graves’ disease (GD) with ocular conditions, also known as Graves’ ophthalmopathy (GO), trying to find out the potential genetic background related to GO, RA, and SLE. There were 40 GO cases and 40 healthy controls enrolled in this study. The association between single-nucleotide polymorphisms (SNPs) of the co-stimulatory molecule genes and GO was analyzed using a chi-square test. It showed that rs11571315, rs733618, rs4553808, rs11571316, rs16840252, and rs11571319 of CTLA4, rs3181098 of CD28, rs36084323 and rs10204525 of PDCD1, and rs11889352 and rs4675379 of ICOS were significantly associated with GO based on genotype analysis and/or allele analysis (p < 0.05). After summarizing the GO data and the previously published SLE and RA data, it was found that rs11571315, rs733618, rs4553808, rs16840252, rs11571319, and rs36084323 were shared in these three diseases. Furthermore, the bio-function was confirmed by dual-luciferase reporter assay. It was shown that rs733618 T > C and rs4553808 A > G significantly decreased the transcriptional activity (both p < 0.001). This study is the first to confirm that these three diseases share genetically predisposing factors, and our results support the proposal that rs733618 T > C and rs4553808 A > G have bio-functional effects on the transcriptional activity of the CTLA4 gene.

Leukocytes and cytokines in blood units have been known to be involved in febrile non-hemolytic transfusion reaction (FNHTR), and these adverse reactions still occur while using pre-storage leukoreduced blood products. Blood transfusion is similar to transplantation because both implant allogeneic cells or organs into the recipient. CTLA4 gene polymorphism was found to be associated with graft-versus-host disease in hematopoietic stem cell transplantation. We performed a prospective cohort study at a major tertiary care center to investigate the correlation of CTLA4 gene polymorphism and transfusion reactions. Selected CTLA4 gene SNPs were genotyped and compared between patients with transfusion-associated adverse reactions (TAARs) and healthy controls. Nineteen patients and 20 healthy subjects were enrolled. There were 4 SNPs showing differences in allele frequency between patients and controls, and the frequency of “A” allele of rs4553808, “G” allele of rs62182595, “G” allele of rs16840252, and “C” allele of rs5742909 were significantly higher in patients than in controls. Moreover, these alleles also showed significantly higher risk of TAARs (OR = 2.357, 95%CI: 1.584–3.508, p = 0.02; OR = 2.357, 95%CI: 1.584–3.508, p = 0.02; OR = 2.462, 95%CI: 1.619–3.742, p = 0.008; OR = 2.357, 95%CI: 1.584–3.508, p = 0.02; OR = 2.357, 95%CI: 1.584–3.508, p = 0.02, respectively). The present study demonstrated the correlation of CTLA4 gene polymorphism and transfusion reaction, and alleles of 4 CTLA4 SNPs with an increased risk of TAARs were found. It is important to explore the potential immune regulatory mechanism affected by SNPs of costimulatory molecules, and it could predict transfusion reaction occurrence and guide preventive actions.

In a prior study, we discovered that hematopoietic stem cell transplantation (HSCT) and/or autoimmune diseases, such as systemic lupus erythematosus, were associated with the rs1234314 C/G and rs45454293 C/T polymorphisms of TNFSF4, the rs5839828 C > del and rs36084323 C > T polymorphisms of PDCD1, and the rs28541784C/T, rs200353921A/T, rs3181096C/T, and rs3181098 G/A polymorphisms of CD28. However, the association does not imply causation. These single nucleotide polymorphisms (SNPs) are all located in the promoter region of these genes, so we used the dual-luminescence reporter assay to explore the effect of single nucleotide polymorphisms (SNPs) on transcriptional activity. For each promoter–reporter with a single SNP mutation, more than 10 independent experiments were carried out, and the difference in transcription activity was compared using one-way ANOVA and Tukey’s honestly significant difference test. The results showed that the G-allele of rs1234314 had 0.32 ± 0.09 times the average amount of relative light units (RLU) compared to the C-allele (p = 0.003), the T-allele of rs45454293 had 4.63 ± 0.92 times the average amount of RLU compared to the C-allele (p < 0.001), the del-allele of rs5839828 had 1.37 ± 0.24 times the average amount of RLU compared to the G-allele (p < 0.001), and the T-allele of rs36084323 had 0.68 ± 0.07 times the average amount of RLU compared to the C-allele (p < 0.001). The CD28 SNPs studied here did not affect transcriptional activity. In conclusion, the findings of this study could only confirm that the SNP had a bio-functional effect on gene expression levels. According to the findings, several SNPs in the same gene have bio-functions that affect transcriptional activity. However, some increase transcriptional activity while others decrease it. Consequently, we inferred that the final protein level should be the integration result of the co-regulation of all the SNPs with the effect on transcriptional activity.

Background: Co-stimulatory and co-inhibitory molecules are critical to T cell responses and involved in the pathogenesis of systemic lupus erythematosus (SLE). This study aimed to comprehensively analyze the surface expression of these molecules in various phenotypic immune cells, comparing the differences between various levels of the severity in SLE and control groups. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque from blood samples of severe SLE patients (treatment with immunosuppressants), mild SLE patients (excluding those with persistent proteinuria or thrombocytopenia), and healthy controls (n = 10 each). PBMCs were stimulated for 48 h. The cells were stained with anti-CD3, CD4, CD28, PD-1, and CTLA-4 antibodies and analyzed by flow cytometry. Differences between groups were assessed using the Kruskal–Wallis test and Mann–Whitney U-test, with median values and statistical significance (p < 0.05) reported. Results: The results showed that CD28 expression was significantly higher in SLE patients compared to controls, with the highest levels in mild SLE. However, CD3⁺ CD28⁺ and CD4⁺ CD28⁺ cells were more prevalent in controls (p = 0.032 and 0.017, respectively). Mild SLE patients exhibited the highest CTLA-4 expression, with significant differences from severe SLE and controls (p = 0.030 and 0.037, respectively). PD-1 expression was lowest in severe SLE but highest in mild SLE within CD3⁺ CD4⁺ cells (p = 0.001). After 48 h of activation, CD4⁺ CTLA4⁺ and CD3⁺ CTLA4⁺ expression levels were significantly higher in controls compared to SLE groups. Conclusions: Our study highlighted that the expression of CD28, CTLA-4, and PD-1 in lymphocytes and specific T cell subsets was various according the severity of SLE in patients, underscoring their roles in disease pathogenesis.

B₃ is the most common subtype of blood group B in the Taiwanese population, and most of the B₃ individuals in the Taiwanese population have the IVS3 + 5 G > A (rs55852701) gene variation. Additionally, a typical mixed field agglutination is observed when the B₃ subtype is tested with anti-B antibody or anti-AB antibody. The molecular biology of the gene variation in the B₃ subtype has been identified, however, the mechanism of the mixed field agglutination caused by the type B₃ blood samples is still unclear. Therefore, the purpose of this study was to understand the reason for the mixed field agglutination caused by B₃. A micro-droplet platform was used to observe the agglutination of type B and type B₃ blood samples in different blood sample concentrations, antibody concentrations, and at reaction times. We found that the agglutination reaction in every droplet slowed down with an increase in the dilution ratio of blood sample and antibody, whether type B blood or type B₃ blood was used. However, as the reaction time increased, the complete agglutination in the droplet was seen in type B blood, while the mixed field agglutination still occurred in B₃ within 1 min. In addition, the degree of agglutination was similar in each droplet, which showed high reproducibility. As a result, we inferred that there are two types of cells in the B₃ subtype that simultaneously create a mixed field agglutination, rather than each red blood cell carrying a small amount of antigen, resulting in less agglutination.