Variation in the gene encoding the co-inhibitory molecule BTLA is associated with survival in patients treated for clear cell renal carcinoma - results of a prospective cohort study.

Introduction: The successful introduction of immune checkpoint blockade approaches to renal-cell carcinoma (RCC) treatment indicates the importance of molecules regulating the T cell response to RCC risk and progression. Material and methods: In this study, we evaluate the association of variations in the CTLA-4, BTLA and CD28 genes with overall survival (OS) of RCC patients and specifically clear cell RCC (ccRCC) patients. The following single nucleotide polymorphisms (SNPs) previously genotyped using the RFLP method or TaqMan SNP Genotyping Assays were analyzed: CTLA-4 gene: c.49A>G (rs231775), g.319C>T (rs5742909), g.*6230G>A (CT60; rs3087243), g.*10223G>T (Jo31; rs11571302); CD28 gene: c.17+3T>C (rs3116496), c.-1042G>A (rs3181098); BTLA gene: rs2705511, rs1982809, rs9288952, rs9288953, rs2705535 and rs1844089. Results: During long term observation (6.5 years) we discovered that possessing the A allele at BTLA rs1844089 SNP, together with advanced disease (stage ≥ 3, tumor grade > 3, tumor diameter ≥ 70 mm), is an independent risk factor of death which increases the hazard ratio (HR) of death by more than two-fold (HR = 2.21, 95% CI: 1.28-3.83). Furthermore, the OS of patients bearing this allele is 6 months shorter than for homozygous (GG) patients (42.5 vs. 48.2 months). Conclusions: Our results indicate for the first time that genetic variation within the gene encoding BTLA is significantly associated with overall survival in clear cell renal cell carcinoma patients.

Inappropriate Expression of PD-1 and CTLA-4 Checkpoints in Myeloma Patients Is More Pronounced at Diagnosis: Implications for Time to Progression and Response to Therapeutic Checkpoint Inhibitors.

Multiple myeloma (MM) is a hematologic malignancy characterized by severely profound immune dysfunction. Therefore, the efficacy of drugs targeting the immune environments, such as immune checkpoint inhibitors (ICIs), is of high clinical importance. However, several clinical trials evaluating ICIs in MM in different therapeutic combinations revealed underwhelming results showing a lack of clinical efficacy and excessive side effects. The underlying mechanisms of resistance to ICIs observed in the majority of MM patients are still under investigation. Recently, we demonstrated that inappropriate expression of PD-1 and CTLA-4 on CD4 T cells in active MM is associated with adverse clinical outcomes and treatment status. The aim of the current study was to determine the usefulness of immune checkpoint expression assessment as a predictive biomarker of the response to therapeutic inhibitors. For this purpose, along with checkpoint expression estimated by flow cytometry, we evaluated the time to progression (TTP) of MM patients at different clinical stages (disease diagnosis and relapse) depending on the checkpoint expression level; the cut-off point (dividing patients into low and high expressors) was selected based on the median value. Herein, we confirmed the defective levels of regulatory PD-1, CTLA-4 receptors, and the CD69 marker activation in newly diagnosed (ND) patients, whereas relapsed/refractory patients (RR) exhibited their recovered values and reactivity. Additionally, substantially higher populations of senescent CD4+CD28- T cells were found in MM, primarily in NDMM subjects. These observations suggest the existence of two dysfunctional states in MM CD4 T cells with the predominance of immunosenescence at disease diagnosis and exhaustion at relapse, thus implying different responsiveness to the external receptor blockade depending on the disease stage. Furthermore, we found that lower CTLA-4 levels in NDMM patients or higher PD-1 expression in RRMM patients may predict early relapse. In conclusion, our study clearly showed that the checkpoint level in CD4 T cells may significantly affect the time to MM progression concerning the treatment status. Therefore, when considering novel therapies and potent combinations, it should be taken into account that blocking PD-1 rather than CTLA-4 might be a beneficial form of immunotherapy for only a proportion of RRMM patients.

Deregulated Expression of Immune Checkpoints on Circulating CD4 T Cells May Complicate Clinical Outcome and Response to Treatment with Checkpoint Inhibitors in Multiple Myeloma Patients.

Unlike solid-tumor patients, a disappointingly small subset of multiple myeloma (MM) patients treated with checkpoint inhibitors derive clinical benefits, suggesting differential participation of inhibitory receptors involved in the development of T-cell-mediated immunosuppression. In fact, T cells in MM patients have recently been shown to display features of immunosenescence and exhaustion involved in immune response inhibition. Therefore, we aimed to identify the dominant inhibitory pathway in MM patients to achieve its effective control by therapeutic interventions. By flow cytometry, we examined peripheral blood (PB) CD4 T cell characteristics assigned to senescence or exhaustion, considering PD-1, CTLA-4, and BTLA checkpoint expression, as well as secretory effector function, i.e., capacity for IFN-γ and IL-17 secretion. Analyses were performed in a total of 40 active myeloma patients (newly diagnosed and treated) and 20 healthy controls. At the single-cell level, we found a loss of studied checkpoints' expression on MM CD4 T cells (both effector (Teff) and regulatory (Treg) cells) primarily at diagnosis; the checkpoint deficit in MM relapse was not significant. Nonetheless, PD-1 was the only checkpoint distributed on an increased proportion of T cells in all MM patients irrespective of disease phase, and its expression on CD4 Teff cells correlated with adverse clinical courses. Among patients, the relative defect in secretory effector function of CD4 T cells was more pronounced at myeloma relapse (as seen in declined Th1/Treg and Th17/Treg cell rates). Although the contribution of PD-1 to MM clinical outcomes is suggestive, our study clearly indicated that the inappropriate expression of immune checkpoints (associated with dysfunctionality of CD4 T cells and disease clinical phase) might be responsible for the sub-optimal clinical response to therapeutic checkpoint inhibitors in MM.

Imbalance in PB IL-17-Secreting and Regulatory Cells in Pars Planitis Is Associated with Dysregulation of IFN-γ-Secreting Cells, Especially in Patients with Clinical Complications.

RESULTS: In patients, an increase in the population of Th17-secreting cells negatively correlated with the abundance of both IFN-γ-producing and T regulatory as well as suppressor cells, regarding all the phenotypes studied. Although a strong dependence of the PB Th1 cell compartment on the duration of the disease was observed, it was limited to the subgroup of patients with macular edema only. The frequency of B regulatory cells was unchanged compared to controls. CONCLUSIONS: In pars planitis, the alterations in lymphocyte cell distribution affect primarily the T cell repertoire. The imbalance in PB Th1/Th17/Treg cells creates proinflammatory conditions, strengthening the suggestion that the immune background may play a role in pars planitis pathogenesis. Also, circulating Th1 level may be of potential clinical relevance in terms of prediction of a more severe course of the disease.

Dysregulation of the immune system as a driver of the critical course of novel coronavirus disease 2019.

Novel coronavirus disease 2019 (COVID­19) is a highly contagious, respiratory disease caused by the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2). Given that inflammatory immune cells may induce severe lung injury, the involvement of immunological factors in the pathogenesis of the disease cannot be overestimated. It has been demonstrated that coronaviruses have developed mechanisms of immune evasion, making themselves invisible to the immune system at an early stage of infection. The mechanism relies on inhibiting the antiviral response of type I interferons, which enhances uncontrolled viral replication in epithelial cells. There has been a growing body of evidence showing that fatal hyperinflammation (cytokine storm) responsible for the severe course of COVID­19 is a consequence of massive SARS­CoV­2 replication rather than inappropriate hyperresponsiveness of the immune system. Therefore, the suppressed antiviral innate immune response seems to be the primary cause of the delayed critical cascade of uncontrolled immune events leading to fulminant systemic inflammation. The occurrence of virus transmission even in asymptomatic individuals infected with SARS­CoV­2 clearly strengthens the evidence for the key role played by the sufficient immune control of viral replication in a subset of cases (eg, in children, a population with a highly effective innate immune response). Although administration of immunomodulatory drugs is recommended under certain conditions by the guidelines for COVID­19 management, controversies regarding treatment protocols in immunocompromised patients infected with SARS­CoV­2 still exist. Extending clinicians' knowledge on the dysregulated immune response, which is a driver of the COVID­19 outcome, may improve both therapeutic strategies and the prognosis of patients infected with SARS­CoV­2.

CD4+CD28null T cells are expanded in moderately active systemic lupus erythematosus and secrete pro-inflammatory interferon gamma, depending on the Disease Activity Index.

BACKGROUND: Pathogenic CD4+CD28null cells are characterized by inflammatory cytokine synthesis and tropism to the inflamed tissues. Recent studies showed the involvement of CD28null T cells in a severe clinical outcome of lupus. However, their role in moderately active disease is still unresolved. METHODS: We examined the levels of circulating CD4+CD28null cells and CD8+CD28null suppressor T cells. We also compared the CD4+CD28null and CD4+CD28+ T-cell functional properties, including the expression of interferon gamma (IFN-γ) and Ki67 among systemic lupus erythematosus (SLE) patients (n = 20) and healthy controls (n = 20). All the patients were under immunosuppressive treatment and exhibited moderate SLE activity (median SLE Disease Activity Index (SLEDAI) = 6). RESULTS: In patients, we found elevated CD4+CD28null and unchanged levels of suppressor CD8+CD28null T cells. There was no difference between patients and controls in IFN-γ and Ki67-expressing CD4+, CD4+CD28+, and CD4+CD28null T cells, except for higher IFN-γ levels in CD4+CD28+ T cells in SLE. In each studied group, we observed a higher preponderance of IFN-γ- and Ki67-expressing cells among CD4+CD28null T cells and lower levels of IFN-γ in CD4+CD28null T cells compared to the CD28+ subset. Similarly, Ki67 intensity was decreased in healthy CD4+CD28null cells, whereas in patients, comparably high expression was observed in both subsets. IFN-γ intensity in CD4+CD28null T cells correlated with SLEDAI. CONCLUSION: SLE with a moderately active clinical course is characterized by peripheral blood expansion of CD4+CD28null T cells and a normal abundance of suppressor CD8+CD28null T cells. The demonstration that these pathogenic CD4+ T cells, despite the lack of CD28, maintain the ability to produce pro-inflammatory IFN-γ positively correlated with disease activity as well as relatively high proliferative capacity may suggest their potentially predictive role in SLE flares.

The Influence of Genetic Variations in the CD86 Gene on the Outcome after Allogeneic Hematopoietic Stem Cell Transplantation.

CD86 molecule is the ligand for both costimulatory (CD28) and coinhibitory (CTLA-4) molecules, and it regulates immune response after allogeneic hematopoietic stem cell transplantation (alloHSCT). Therefore, we postulate that CD86 gene variations might influence the outcome after alloHSCT. Altogether, 295 adult patients (pts) undergoing related (105 pts) and unrelated (190 pts) donor-matched HSCT were genotyped for the following CD86 gene polymorphisms: rs1129055, rs9831894, and rs2715267. Moreover, the donors' rs1129055 polymorphism was determined. None of the investigated SNPs alone were associated with aGvHD and rate of relapse. However, we showed that rs2715267 SNP influenced overall survival (OS) after alloHSCT. The 24-month OS for the rs271526GG recipients was worse than that for the recipients possessing T allelle (TT or GT genotypes) (p = 0.009). Moreover, analysis of gene-gene interaction between CD86 and CTLA-4 showed that having both the A allele for CD86 rs1129055 and the CTLA-4 CT60GG genotype in recipients increased the risk of aGvHD about 3.5 times. Interestingly, the donors' rs1129055GG genotype and the recipients' CT60GG genotype also increased the risk of aGvHD about 2.7-fold. We postulate that recipients' CD86 gene polymorphisms influence the overall survival after alloHSCT and, together with CTLA-4 polymorphisms, might be considered a risk factor for aGvHD.

PD-1 gene polymorphic variation is linked with first symptom of disease and severity of relapsing-remitting form of MS.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), where inflammation, demyelination together with the axonopathy are the cardinal features on pathologic ground, with a combined genetic and environmental background. The associations of PD-1 single nucleotide polymorphisms (SNPs): PD-1.3 (in intron 4), PD-1.5 and PD-1.9 (both in exon 5) with clinical manifestation of MS in 479 south Polish individuals including 203 MS patients were analyzed. Presence of PD-1.5T allele was linked with the first manifestations of disease: diplopia and pyramidal signs - favored pyramidal signs but protected against of diplopia development. Farther, PD-1.3G/PD-1.5C/PD-1.9C haplotype significantly favored whereas GTC protected against diplopia. Besides, GTT haplotype strongly favored non-severe RRMS outcome and ATC haplotype was specific only for these MS patients. Our population-based case-control study, investigating selected three PD-1 SNPs: PD-1.3, PD-1.5 and PD-1.9, revealed that polymorphic variation may be rather disease-modifying than MS risk factor.

CD28/CTLA-4/ICOS haplotypes confers susceptibility to Graves' disease and modulates clinical phenotype of disease.

Graves' disease, an autoimmune disease with heterogeneous symptoms including Graves' orbitopathy, has a combined genetic/environmental background, where variations within CD28/CTLA-4/ICOS genes are considered as disease markers.Association of CD28c.17+3T>C(rs3116496), CTLA-4g.319C>T(rs5742909), CTLA-4c.49A>G(rs231775), CTLA-4g.*642AT(8_33), CT60(rs3087243), Jo31(rs11571302), ICOSc.1554+4GT(8_15) polymorphisms with susceptibility to Graves' disease and clinical outcome was investigated. The study group comprised of 561 Polish Caucasians, including 172 unrelated Graves' disease patients. CTLA-4c.49A>G, CTLA-4g.319C>T, and CT60 were genotyped by PCR-RFLP; Jo31 and CD28c.17+3C>T by minisequencing; CTLA-4g.*642AT(8_33) and ICOSc.1554+4GT(8_15)-PCR and fluorescence-based technique. CD28c.17+3T>C(rs3116496)T/CTLA-4g.319C>T(rs5742909)C/CTLA-4c.49A>G(rs231775)G/CTLA-4g.*642AT(8_33)(AT16-21)/CT60(rs3087243)G/Jo31(rs11571302)G/ICOSc.1554+4GT(8_15)(m) and TCA(AT<16)GT(m) haplotypes increased risk of Graves' disease, especially in males, as well as overall Graves' orbitopathy development with severe outcome. TCG(AT16-21)GG(l) haplotype increased risk of Graves' disease and reduced the chance of successful medical treatment. Although this haplotype was mainly observed in patients without signs of Graves' orbitopathy, if Graves' orbitopathy developed it favored a Graves' orbitopathy outcome. Haplotype TCA(AT>21)GT(m) increased Graves' disease risk in women and, in all patients, was linked to Graves' disease without Graves' orbitopathy. TCG(AT<16)GG(m) haplotype was predominantly observed in patients without Graves' orbitopathy, whereas TCA(AT16-21)GG(m) was absent in those patients. TCA(AT16-21)GG(m) occurred in patients with a mild Graves' orbitopathy outcome. The marker CTLA-4g.*642AT(8_33) was the only independent Graves' disease risk factor, whereas CT60 was an independent factor for disease progression. Sporadic Graves' disease was related to presence of CTLA-4c.49A>G[A] and the rare CTLA-4g.319C>T[T] allele variant. Familial background of the disease was exclusively associated with CTLA-4g.*642AT(8_33)[AT>21]/[AT>21] genotype. CD28/CTLA-4/ICOS loci may confer inherited susceptibility to Graves' disease or may be involved in susceptibility to Graves' disease and play a pathogenetic role.

Intragenic Variations in BTLA Gene Influence mRNA Expression of BTLA Gene in Chronic Lymphocytic Leukemia Patients and Confer Susceptibility to Chronic Lymphocytic Leukemia.

The aim of this study was to determine the association between polymorphisms in gene encoding B- and T-lymphocyte attenuator (BTLA) and susceptibility to chronic lymphocytic leukemia (CLL) and their influence on mRNA expression of BTLA gene in T and B cells from CLL patients (pts.). The following BTLA single-nucleotide polymorphisms (SNPs): rs2705511, rs1982809, rs9288952, rs76844316, rs16859633, rs9288953, rs2705535, rs1844089, rs2705565, rs2633580 were genotyped with use of TaqMan probes in 321 CLL pts. and in 470 controls. The mRNA levels of human BTLA were determined in subpopulations of T and B cells from 37 CLL patients with use of Applied Biosystems assays. Three SNPs: rs1982809, rs2705511 and rs9288953 were associated with susceptibility to CLL. The frequency of rs1982809[G] allele and rs2705511[C] allele carriers was higher in patients compared to the controls (0.51 vs. 0.41, OR 1.51, 95% CI 1.14-2.02, p = 0.004 and 0.56 vs. 0.44, OR 1.62, 95% CI 1.22-2.16, p = 0.0009, respectively). Furthermore, rs9288953[TT] genotype was overrepresented in CLL pts. compared to the controls (0.22 vs. 0.14, OR 1.74, 95% CI 1.20-2.53, p = 0.004). The evaluation of the influence of BTLA SNPs on BTLA mRNA expression in CLL pts. showed that the presence of rs1982809[G] allele was associated with lower median (±SD) BTLA mRNA expression in T cells (expressed as 2-delta Ct) in CLL pts. as compared to [AA] homozygotes (0.009 ± 0.013 vs. 0.026 ± 0.012, p = 0.03). Our results indicate that rs1982809 BTLA gene polymorphism is associated with mRNA expression level and that variations in the BTLA gene might be considered as potentially low-penetrating CLL risk factor.

Association of 3' nearby gene BTLA polymorphisms with the risk of renal cell carcinoma in the Polish population.

OBJECTIVE: T cells play an important role in antitumor immunity, and molecules regulating T-cell activity could influence cancer susceptibility. The distinct role of coinhibitory receptors in immunosurveillance has been considered. B- and T-lymphocyte attenuator (BTLA) is one of these receptors, which negatively regulate immune responses. The aim of this study was to investigate the association between BTLA gene polymorphisms and susceptibility to renal cell carcinoma (RCC) in the Polish population. METHODS: Altogether 282 patients with RCC and 480 healthy subjects were genotyped for the following polymorphisms: rs2705511, rs1982809, rs9288952, rs16859633, rs9288953, rs2705535, and rs1844089 using the TaqManSNP Genotyping Assays. RESULTS: Here, we found that the presence of rs1982809G allele (genotype GG+AG) is associated with increased risk of RCC (odds ratio = 1.38; 95% CI: 1.03-1.86; P = 0.03). In patients with clear-cell RCC (ccRCC) with high-grade (3 and 4) tumors, the frequency of rs1982809[GG] genotype was significantly higher as compared to those with low-grade (1 and 2) tumors and to the controls (0.14 vs. 0.06, P = 0.05 and 0.14 vs. 0.06, P = 0.04, respectively). Moreover, we have noticed the trend for overrepresentation of carriers of rs2705511C allele in patients with RCC as compared with the controls (0.51 vs. 0.44, P = 0.08) Haplotype rs2705511C/rs1982809G/rs9288952A/rs9288953T/rs2705535C/rs1844089G (CGATCG) increased the risk of RCC of 46% (odds ratio = 1.46; 95% CI: 1.08-1.96; Pcorrected = 0.05). CONCLUSION: Our results indicate that polymorphisms rs1982809 situated in 3' UTR nearby region of BTLA gene might be considered as low-penetrating risk factor for RCC, but results have to be confirmed in further studies.

Patients with chronic lymphocytic leukaemia (CLL) differ in the pattern of CTLA-4 expression on CLL cells: the possible implications for immunotherapy with CTLA-4 blocking antibody.

Recently, systemic administration of a human monoclonal antibody directed against cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expressed on circulating T cells in patients with chronic lymphocytic leukaemia (CLL) has been considered. Also, CLL cells have been shown to express CTLA-4, increased levels of which in the leukaemic compartment are a predictor of good clinical outcome. Since both CLL and Treg microenvironment cells can be targeted by the CTLA-4 blocking antibody in this immunotherapy approach, the investigation of the functional effect of CTLA-4 blockade on CLL cells might be of potential clinical relevance. The main aim of this study was to examine the effect of CTLA-4 blockade on proliferation activity and apoptosis of CLL cells in patients with low and high CTLA-4 expression. We found that in the high CTLA-4-expressing CLL group, CTLA-4 blockade on the CLL cell surface resulted in a significant increase in the median percentages of Ki67(+) cells and a tendency to decrease in the proportion of apoptotic cells. In contrast, in the low CTLA-4 expressors, CTLA-4 blockade did not affect the proliferation activity or the frequency of apoptosis. This study reports for the first time the different effect of CTLA-4 blockade on CLL cells in CLL patients depending on the levels of CTLA-4 expression. CTLA-4 blockade seems to induce pro-survival signals in leukaemic cells from CLL patients exhibiting high CTLA-4 expression, suggesting that an immunotherapy approach based on the systemic use of monoclonal anti-CTLA-4 antibodies could be an unfavourable strategy for some CLL patients.

CTLA-4 affects expression of key cell cycle regulators of G0/G1 phase in neoplastic lymphocytes from patients with chronic lymphocytic leukaemia.

Previously, we showed that cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is overexpressed in chronic lymphocytic leukaemia (CLL) and its expression is correlated with the expression of the major regulators of G1 phase progression: cyclins D2 and D3, and cyclin-dependent kinase inhibitory protein 1 (p27 (KIP1) ). In the present study, we blocked CTLA-4 on the surface of both CLL cells and normal B lymphocytes to investigate the impact of CTLA-4 on the expression of the mentioned G1 phase regulators. We found that in CLL patients and in healthy individuals, the median proportions of cyclin D2-positive cells as well as cyclin D3(+) cells significantly decreased following CTLA-4 blockade. Moreover, CTLA-4 blockade led to an increase in the median frequencies of p27 (KIP1) -positive cells, although this increase was marked only in CLL patients. Our study showed that CTLA-4 affects the expression of the key regulators of G1 phase progression in CLL cells as well as in normal B lymphocytes and may contribute to a better understanding of the role of CTLA-4 in the regulation of G1 phase progression.

A CT60G>A polymorphism in the CTLA-4 gene of the recipient may confer susceptibility to acute graft versus host disease after allogeneic hematopoietic stem cell transplantation.

T cell activation plays a crucial role in the development of acute graft versus host disease (aGvHD). Cytotoxic T cell antigen-4 (CTLA-4) is a co-inhibitory molecule that negatively regulates T cell activation, differentiation, and proliferation. Single-nucleotide polymorphisms (SNPs) in CTLA-4 gene may affect its function. Inconsistent observations have been reported regarding the associations of CTLA-4 SNPs with complications after hematopoietic stem cell transplantation (HSCT). Moreover, the majority of the observations were focused on the donors' SNPs. Recently, a few studies have shown that recipients' genetic variations in the CTLA-4 gene might influence HSCT results. The aim of our study was to determine the influence of the CTLA-4 gene polymorphisms of the donors and the recipients on the outcome of HSCT. Altogether, 312 donor-recipient pairs were genotyped for the CTLA-4c.49A>G (rs231775) and CT60G>A (rs3087243) SNPs using the TaqMan®SNP Genotyping Assays. In this study, it was shown that the recipients' CT60G>A[GG] genotype, the myeloablative conditioning regimen, and HSCT from an unrelated donor were independent aGvHD risk factors (odds ratio (OR) 2.63, 95% confidence intervals (95% CI) 1.45-4.59, p = 0.001; OR 2.68, 95% CI 1.65-4.07, p = 0.00003; and OR 1.87, 95 % CI 1.02-3.24, p = 0.04, respectively). Moreover, haplotype analysis revealed that possessing allele A in both of the SNPs decreased the risk of aGvHD approximately 1.5-fold (RR 0.69, p = 0.008). Our data suggest that the CT60G>A[GG] genotype in the recipient has an impact on aGvHD development, especially in patients receiving transplants from unrelated donors together with the myeloablative conditioning regimen.

Pretransplant donor and recipient CTLA-4 mRNA and protein levels as a prognostic marker for aGvHD in allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory T cells' receptor essential for maintaining T cell homeostasis and immunotolerance. The role of the co-stimulatory pathway in development of aGvHD has been studied mostly in animal models. To the best of our knowledge, there are no published data on the role of CTLA-4 in pathogenesis of aGvHD after hematopoietic stem cell transplantation (HSCT) in humans. Therefore the aim of our study was to determine the association of CTLA-4 mRNA and proteins level in HSCT donor-recipient pairs, prior to and after HSCT, with aGvHD risk. METHODS: Total CTLA-4 mRNA level in 51 donor-recipient pairs prior to and 56 days after HSCT was determined using real time PCR techniques, while membrane (m) and cytoplasmic (c) CTLA-4 expression in CD3+ cells were measured by flow cytometry in 40 donor-recipient pairs at the same time points. RESULTS: We found an association between the risk of aGvHD and high pre-transplant CTLA-4 mRNA expression level both in recipients and in donors, stronger in recipients (OR=2.02, CI95% 1.39-3.01), and less pronounced in donors (OR=1.57, CI95% 1.18-2.0). Moreover, we showed that proportion of CD3+ cells positive for mCTLA-4 in recipients prior to HSCT positively correlated with increased risk of aGvHD (OR=1.175, CI95% 1.024-1.311, p=0.018). CONCLUSION: Our results indicate that both donor and recipient CTLA-4 mRNA as well as recipient membrane protein expression levels measured before transplantation may be considered as prognostic factors for aGvHD development.

The function of a novel immunophenotype candidate molecule PD-1 in chronic lymphocytic leukemia.

Programmed death-1 (PD-1) is a negative receptor expressed on lymphocytes including malignant B cells in chronic lymphocytic leukemia (CLL). In this work, we found that patients with CLL had a higher expression of PD-1 transcript (PDCD1) than healthy volunteers (p < 0.0001). PDCD1 expression was comparable between CLL cells from accumulation (peripheral blood) and proliferation (bone marrow) disease compartments. In blood samples of patients with mutated IGHV genes PDCD1 expression was higher than with unmutated IGHV (p = 0.0299). We demonstrated that phosphorylation of SYK and LYN, key B-cell receptor signaling kinases, was independent of PD-1 expression in patients with CLL, while ZAP-70 phosphorylation in negative tyrosine residue 292 showed strong inverse correlation (r = - 0.8, p = 0.0019). No associations between five single nucleotide polymorphisms of PDCD1, their expressions and susceptibility to CLL were found. In conclusion, PD-1 might be an independent, universal marker of CLL cells and a part of their activated phenotype, and subsequently might modulate the function of ZAP-70.

Gene polymorphisms of novel immunotolerant molecule BTLA: distribution of alleles, genotypes and haplotypes in Polish Caucasian population.

B and T lymphocyte attenuator (BTLA) is one of the members of immunoglobulin superfamily which, like CTLA-4 and PD-1, is involved in down regulation of immune response. Despite the important role of BTLA in maintaining immune homeostasis, relatively little studies were devoted to the relationship of polymorphisms in the gene encoding BTLA with susceptibility to autoimmune disease and cancer. Moreover, all published works were done in Asian populations. BTLA gene is located on chromosome 3 in q13.2 and consists of five exons. The aim of this study was to investigate the alleles, genotypes and haplotypes frequency of selected BTLA gene polymorphisms in Caucasian population originating from Poland. For this study, the single-nucleotide polymorphisms (SNPs) were chosen on the basis of literature data. Additionally, the tag dSNP under linkage equilibrium r (2) > 0.8 and available at the National Center for Biotechnology Information (NCBI) for Caucasian population of rare alleles at a frequency greater than 5 % have been chosen using the NCBI database. The ten BTLA SNPs investigated were: rs1844089, rs2705535, rs9288952, rs9288953, rs1982809, rs2633580, rs2705511, rs2705565, rs76844316, rs16859633. For all SNPs selected on the basis of literature data the significantly different distributions of genotypes between Asian and Caucasian populations were observed.