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.

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.

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.

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.

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.

Patients with the most advanced rheumatoid arthritis remain with Th1 systemic defects after TNF inhibitors treatment despite clinical improvement.

Systemic immune defects might reflect severely dysregulated control of chronic inflammation related to disease progression. Th17/Treg cell imbalance has been demonstrated to be involved in rheumatoid arthritis (RA) pathogenesis. Despite controversial results, a growing anti-inflammatory role in this process has been recently attributed to Th1 responses. The aim of the study was to estimate the extent of Th1/Th17/Treg imbalance in peripheral blood (PB) of patients with short- and long-term RA in relation to cytokine milieu and its reversal after therapy with methotrexate and/or TNF inhibitors, respectively. Patients with different duration of RA (median 6 vs. 120 months) in the active phase of RA were enrolled in this study. We performed flow cytometric analysis of PB Th1, Th17, and Treg populations together with estimation of serum cytokine concentrations using cytometric bead array. Disease activity was calculated on the basis of clinical and biochemical indices of inflammation (DAS28, ESR, CRP). All parameters were measured and correlated with each other before and after 6 months therapy. Elevated levels of circulating Th17 cells and IL-6 were found in all active patients, of which Th17 cells were down-regulated by the treatment. Significantly reduced Th1 and functional CTLA-4+ Treg cell frequencies as well as Th1 cytokines observed only in progressive RA seemed to be irreversible. Although therapy induced clinical improvement in almost all patients, those with advanced RA remained with signs of inflammation. Our report demonstrates that both the extent of systemic immune abnormalities and their restoration are dependent on duration of the active RA.

rs2072135, a low-penetrance variant for chronic lymphocytic leukaemia?

Recent multi-stage genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) that are robustly associated with chronic lymphocytic leukaemia (CLL) risk. Given that most of these SNPs map to non-coding regions of the genome, it suggests that the functional basis of many GWAS signals will be through differential gene expression. By referencing publically accessible expression quantitative trait loci (eQTL) data on lymphoblastoid cells lines (LCLs) we have globally demonstrated an association between GWAS P-values and eQTLs, consistent with much of the variation in CLL risk being defined by variants impacting on gene expression. To explore using eQTL data to select GWAS SNPs for replication, we genotyped rs2072135 (GWAS P-value = 0·0024, eQTL P-value = 1·510(-19)) in five independent case-control series totalling 1968 cases and 3538 controls. While not attaining statistical significance (combined P-value = 1 × 10(-4)), rs2072135 defines a promising risk locus for CLL. Incorporating eQTL information offers an attractive strategy for selecting SNPs from GWAS for validation.

PPARg2 Ala¹² variant protects against Graves' orbitopathy and modulates the course of the disease.

Orbital fibroblast differentiation to adipocytes is a peroxisome proliferator-activated receptor g (PPARg)-dependent process essential for pathogenic tissue remodeling in Graves' orbitopathy (GO). PPARg2 Pro¹²Ala polymorphism modulates expression and/or function of the molecule encoded by this gene and is a promising locus of GO. Here, we analyzed associations of PPARg2 Pro¹²Ala with clinical manifestation of GO in 742 Polish Caucasians including 276 Graves' disease (GD) patients. In our study, the Ala¹² allele and Ala¹² variant (Ala¹²Ala and/or Pro¹²Ala genotype) decreased the risk of GO (p = 0.000012 and p = 0.00013). Moreover, Ala¹²Ala genotype was observed only in patients without GO (p = 0.002). GD patients with Ala¹² variant had less active and less severe eye symptoms. Female carriers of the Ala¹² allele rarely developed GO, but the marker was not related to symptoms of GO. The opposite finding was recorded in males, in whom the studied polymorphism was related to activity, but not to the development, of GO. In Ala¹² variant carriers without familial history of thyroid disease, risk of GO was lower than in persons with a familial background. The Ala¹² allele seemed to protect smokers from GO, but in nonsmokers, such a relation was not obvious. A multivariate analysis indicated the Pro¹²Ala marker as an independent risk factor of eye symptoms (p = 0.0001) and lack of Ala increases the risk of GO 3.24-fold. In conclusion, the gain-of-function Ala¹² variant protects against GO and modulates the course of the disease.

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.

Common genetic variation at 15q25.2 impacts on chronic lymphocytic leukaemia risk.

A genome-wide association study of chronic lymphocytic leukaemia (CLL) suggested that common variants at 15q25.2 (rs783540) and 18q21.1 (rs1036935) influence CLL. To validate these associations and explore their relationship with CLL risk we genotyped case-control datasets from Poland, UK and Italy totalling 1428 cases and 1920 controls. Combined data from these and previously genotyped series (2503 cases and 5789 controls) provided evidence for an association between 15q25.2 and 18q21.1 loci and CLL risk (P(combined) = 1·10 × 10(-7) and 1·30 × 10(-5) respectively). These data provide further evidence for the involvement of common genetic variants in CLL risk and insight into the biological basis of disease development.

Zeta chain expression in T and NK cells in peripheral blood of children with nephrotic syndrome.

The idiopathic nephrotic syndrome (INS) has been related to cellular immune disturbances. The zeta (zeta) chain, a component of the T-cell receptor/CD3 (TCR) complex and CD16 heterodimer in NK cells, plays a crucial role in T and NK cell activation and proliferation. The aim of our study was to examine zeta chain expression in CD4+, CD8+ T lymphocytes and NK cells in the peripheral blood of children with INS and to evaluate the effect of anti-CD3+rIL-2 stimulation on the level of zeta chain expression in the INS pediatric population. The study group consisted of 15 children with INS in relapse, 16 patients with INS in clinical remission, and 17 controls. The percentage of zeta-positive cells and the values of mean fluorescence intensity (MFI) were determined by flow cytometry. Compared with that in the controls, the percentage of zeta+ freshly isolated NK cells in children with INS in relapse was significantly lower, whereas, in CD3+/CD4+ and CD3+/CD8+ populations, no alteration was observed. There were no differences in the MFI values between the populations of freshly isolated cells either. Stimulation with anti-CD3+rIL-2 decreased the percentage of zeta+/CD4+ T cells and NKzeta+ cells in a significant way in all the groups analysed, whereas the percentage of zeta+/CD8+ T cells decreased significantly only in patients with INS in relapse. The altered pattern of zeta expression in fresh NK cells from children with INS in relapse, and the disturbed response of zeta+/CD8+ T cells to anti-CD3+rIL-2 stimulation in relapse, suggests the possible role of this chain in immune dysregulation in INS, particularly with regard to cytotoxic cells.

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.

The CTLA-4 gene polymorphisms are associated with CTLA-4 protein expression levels in multiple sclerosis patients and with susceptibility to disease.

Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is an important molecule in the down-regulation of T-cell activation. A study was undertaken to evaluate the association of the CTLA-4 gene polymorphisms -319C/T, +49A/G, (AT)(n), CT60A/G and Jo31G/T with the levels of membrane CTLA-4 (mCTLA-4) and cytoplasmic CTLA-4 (cCTLA-4) in CD4(+) T lymphocytes from patients with multiple sclerosis (MS) and with susceptibility to MS, and the course of the disease. It was found that the Jo31GG and CT60GG genotypes were associated with decreased mean fluorescence intensity (MFI) of total CTLA-4 (mCTLA-4 + cCTLA-4) molecules in CD4(+) T cells from both relapsing-remitting (RR) and secondary progressive (SP) MS patients compared with others. Consequently, possessing the Jo31G allele and/or the CT60G allele were associated with susceptibility to MS. The percentages of cells expressing mCTLA-4 and cCTLA-4 in RR patients were higher in carriers of the alleles non-predisposing to MS (namely CT60A and Jo31T), but the percentages of corresponding cells were unexpectedly significantly lower in SP patients than in RR patients. Increased risk of paresthesia and pyramidal signs as a first manifestation of disease, and earlier transition to the SP form in those patients, was also noted. It is hypothesized that the decreasing frequencies of cells expressing immunosuppressive mCTLA-4 and cCTLA-4 in carriers of alleles non-predisposing to MS (i.e. CT60A and Jo31T) may lead to inadequate down-regulation of ongoing T-cell responses in these patients and, as a consequence, earlier progression of disease from the RR form to the SP form.

High intracellular content of cyclin-dependent kinase inhibitor p27(Kip1) in early- and intermediate stage B-cell chronic lymphocytic leukemia lymphocytes predicts rapid progression of the disease.

Previous studies showed that peripheral blood lymphocytes of B-cell chronic lymphocytic leukemia (B-CLL) displayed a high intracellular level of cell cycle inhibitory protein p27(Kip1). It has been suggested that its' high expression may confer them survival advantage and lead to unfavorable prognosis, but the prognostic significance of p27(Kip1) expression for previously untreated, non-advanced stage B-CLL was not established. We studied a relationship between the intracellular level of p27(Kip1) of lymphocytes of early- and intermediate stage B-CLL patients and their spontaneous apoptosis in vitro, as well as prognostic significance of p27(Kip1) in B-CLL lymphocytes for the risk of disease progression. Intracellular p27(Kip1) content of peripheral blood lymphocytes obtained from 48 previously untreated 0-II Rai stage B-CLL patients was determined by flow cytometry. The viability and apoptosis of those lymphocytes after 72-h culture were also assessed. During the follow-up period (6-71 months, median 59.5), we recorded the time elapsed to the doubling of lymphocyte count, progression to a higher Rai stage and the appearance of indications for cytostatic treatment. The p27(Kip1) expression was neither correlated with initial lymphocyte count, CD38 expression, cell viability nor spontaneous apoptosis ratio after 72-h culture. Higher p27(Kip1) level was related to the probability of earlier occurrence of each of three above-mentioned events. We did not find a prognostic significance of in vitro cell viability nor apoptosis as to the risk of disease progression. Our results indicate that elevated intracellular p27(Kip1) level in leukemic lymphocytes of early- and intermediate stage B-CLL patients contributes to rapid progression of the disease.

Is cyclin D2 a marker of B-cLL cell activation?

The most recent studies emphasize a link between B-cell proliferation in vivo and clinical outcome of B-cell chronic lymphocytic leukemia (B-CLL). The expression of cyclin D2 in B-CLL cells isolated from the peripheral blood of 27 untreated patients in relation to the apoptosis ratio both before and 72 h after culture in the absence of growth factors was analyzed by immunocytochemistry. The significant associations between cell death in culture and both cyclin D2 expression in freshly isolated cells and the rate of its decrement in culture found in this study confirm the special role of cyclin D2 in enhancing the longevity of these cells in vivo. As cyclin D2 is inducible in the early G1 phase, its increased expression might also reflect the activation of cells attempting to replicate in vivo. Furthermore, the finding that B-CLL progression positively correlates with the gradual increase in the proportion of apoptotic B lymphocytes in culture seems to support the notion of cells striving to undergo division in the absence of growth factors. All together, these results indicate the possibility that cyclin D2+ cells represent a pool of leukemic cells with the potential to enter the dividing compartment.

Impaired zeta chain expression and IFN-gamma production in peripheral blood T and NK cells of patients with advanced lung cancer.

Recent studies show that low expression of zeta chain in T and NK cell leads to impaired anti-tumour immunity in patients with cancer, poor prognosis, and shorter overall survival. Therefore, monitoring zeta chain expression may be useful in assessing immune competence in lung cancer patients and in following changes during anticancer therapies. Such studies concerning small-cell and non-small cell lung cancer (SCLC and NSCLC, respectively) have not been published so far. The expression of zeta chain and IFN-gamma in peripheral blood (PB) T and NK cells from SCLC and NSCLC patients at advanced (III, IV) stages were analysed before and after chemotherapy with etoposide and cisplatin using flow cytometry. Serum concentrations of TGF-beta1 and IL-10 were also estimated at each time point tested. Before therapy, impaired zeta chain expression was observed in all the patients corresponding with increased levels of immuno-suppressive cytokines in sera compared with controls. Decreased IFN-gamma production in T cells from all patients was also demonstrated. In NK cells, IFN-gamma was secreted at lower levels in NSCLC patients, while in the SCLC group it was normal. After chemotherapy, restoration of zeta expression in NK cells and its insignificant increase in T cells in SCLC patents corresponding with normalization of TGF-beta secretion were noted. In contrast, NSCLC patients retained impaired zeta expression in T and NK cells. SCLC and NSCLC patients showed a profound defect in IFN-gamma secretion in T and NK cells upon treatment. There were no differences in studied parameters between NSCLC and SCLC groups before and after chemotherapy. This is the first report of impaired zeta expression in PB T and NK cells in patients with SCLC and NSCLC in advanced stages, which may result from higher levels of immunosuppressive cytokines in sera. After cytostatic treatment, all the studied patients, including those with initial good response to chemotherapy, remained with profound abnormalities in T and NK cells, which could have dramatic consequences regarding severely impaired anti-tumour immunity.

The significance of Treg cells in defective tumor immunity.

Regulatory T cells (Treg) enriched in FoxP3+, glucocorticoid-induced TNF receptor+, and cytotoxic T-lymphocyte-associated antigen-4+ exert a potential to suppress effector T cells in the periphery. These cells exist in markedly higher proportions within tumor-infiltrating lymphocytes, peripheral blood lymphocytes, and/or regional lymph node lymphocytes of patients with cancer and their frequencies are suggested to be strongly related to tumor progression and inversely correlated with the efficacy of treatment. Tumor-specific Treg cells require ligand-specific activation and cell-to-cell contact to exert their suppressive activity on tumor-specific effector cells (CD8+ cytotoxic T lymphocytes and CD4+ Th cells), which includes decreased cytotoxity, proliferation, and Th1 cytokine secrection. Depletion or blockade of Treg cells can enhance immune protection from tumor-associated antigens that are expressed as self antigens. Recent studies revealed that lymphoma T cells might adopt a Treg profile as well. Studies assessing the influence of chemotherapy on Treg cells have also been included in this review.

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.

Cytotoxic T-lymphocyte associated antigen 4 gene polymorphisms and autoimmune thyroid disease: a meta-analysis.

CONTEXT: Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) polymorphisms have been widely examined for their associations with autoimmune thyroid diseases [Graves' disease (GD) and Hashimoto thyroiditis (HT)], but their relative population effect remains unclear. OBJECTIVE: The aim was to generate large-scale evidence on whether the CTLA-4 polymorphisms (A49G and CT60) and haplotypes thereof increase the susceptibility to GD and/or HT. DESIGN, SETTING, AND PARTICIPANTS: Meta-analyses of group-level data were reviewed from 32 (11,019 subjects) and 12 (4,479) published and unpublished studies for the association of the A49G polymorphism with GD and HT, respectively (PubMed and HuGeNet search until July 2006). There were 15 (n = 7246) and six (n = 3086) studies available for the CT60 polymorphism, respectively. Meta-analyses of individual-level data from 10 (4906 subjects) and five (2386) collaborating teams for GD and HT, respectively, were also reviewed. MAIN OUTCOME MEASURES: Association of gene variants and haplotypes with GD and HT was measured. RESULTS: Group-level data suggested significant associations with GD and HT for both A49G [odds ratios 1.49 (P = 6 x 10(-14)) and 1.29 (P = 0.001) per G allele, respectively] and CT60 [1.45 (P = 2 x 10(-9)) and 1.64 (P = 0.003) per G allele, respectively]. Results were consistent between Asian and Caucasian descent subjects. Individual-level data showed that compared with the AA haplotype, the risk conferred by the GG haplotype was 1.49 (95% confidence interval 1.31,1.70) and 1.36 (95% confidence interval 1.16,1.59) for GD and HT, respectively. Data were consistent with a dose-response effect for the G allele of CT60. CONCLUSION: The CT60 polymorphism of CTLA-4 maps an important genetic determinant for the risk of both GD and HT across diverse populations.