Dysregulated Expression of Tim-3 and NKp30 Receptors on NK Cells of Patients with Chronic Lymphocytic Leukemia.

BACKGROUND: In this study, the expression pattern of NKp30 and T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3), as candidates for activating and inhibitory receptors of NK cells, were evaluated in patients with chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS: 24 CLL patients and 19 healthy controls were enrolled. Fresh peripheral blood was collected from all subjects and stained with fluorochrome-conjugated antibodies. The frequency of CD56+/CD3-/NKp30+ and CD56+/CD3-/Tim-3+ cells was determined by multicolor flow cytometry. RESULTS: Our results revealed that Tim-3 is significantly upregulated on natural killer (NK) cells of CLL patients in comparison to healthy controls. NK cells of CLL patients showed lower expression of NKp30-activating receptor compared to controls. Tim-3 expression pattern on NK cells of CLL patients was correlated with poor prognostic factors including low hemoglobin level, high absolute lymphocyte count, and high serum C-reactive protein level. CONCLUSION: Dysregulated expression of Tim-3 and NKp30 receptors confirms the exhaustion state of NK cells in CLL. Our data introduce Tim-3 as a promising biomarker and potential target for immunotherapy of CLL.

Spontaneous Immunity Against the Receptor Tyrosine Kinase ROR1 in Patients with Chronic Lymphocytic Leukemia.

BACKGROUND: ROR1 is a receptor tyrosine kinase expressed in chronic lymphocytic leukemia (CLL) and several other malignancies but absent in most adult normal tissues. ROR1 is considered an onco-fetal antigen. In the present study we analysed spontaneous humoral and cellular immunity against ROR1 in CLL patients. MATERIALS AND METHODS: Antibodies against ROR1 were analysed in 23 patients and 20 healthy donors by ELISA and Western blot. Purified serum IgG from patients was tested for cytotoxicity against CLL cells using the MTT viability assay. A cellular immune response against ROR1 derived HLA-A2 restricted 9 aa and 16 aa long peptides were analysed using peptide loaded dendritic cells co-cultured with autologous T cells from CLL patients (n = 9) and healthy donors (n = 6). IFN-γ, IL-5 and IL-17A-secreting T cells were assessed by ELISPOT and a proliferative response using a H3-thymidine incorporation assay. RESULTS: The majority of CLL patients had antibodies against ROR1. Significantly higher titers of anti-ROR1 antibodies were noted in patients with non-progressive as compared to progressive disease. The extracellular membrane-close ROR1 KNG domain seemed to be an immunodominant epitope. Ten patients with high titers of anti-ROR1 binding antibodies were tested for cytotoxicity. Five of those had cytotoxic anti-ROR1 antibodies against CLL cells. ROR1-specific IFN-γ and IL-17A producing T cells could be detected in CLL patients, preferentially in non-progressive as compared to patients with progressive disease (p<0.05). CONCLUSION: ROR1 seemed to spontaneously induce a humoral as well as a T cell response in CLL patients. The data support the notion that ROR1 might be a specific neo-antigen and may serve as a target for immunotherapy.

Alteration in frequency and function of CD4⁺CD25⁺FOXP3⁺ regulatory T cells in patients with immune thrombocytopenic purpura.

Immune thrombocytopenic purpura (ITP) is an autoimmune bleeding disorder characterized by production of auto-antibodies against platelet antigens. It is obvious that regulatory T cells (Tregs) have a major role in controlling immune homeostasis and preventing autoimmunity.To investigate the frequency and functions of Tregs, twenty ITP patients and twenty age- and sex-matched healthy controls were recruited. The peripheral blood mononuclear cells were isolated and the proportion of Tregs was defined by flow cytometry method. The expression of immune-regulatory markers, cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and glucocorticoid induced tumor necrosis factor receptor (GITR) were also assessed by quantitative Real-time PCR TaqMan method. For evaluation of Treg function, Tregs were enriched and their ability to inhibit proliferation of T cells was measured and levels of immune-regulatory cytokines IL-10 and TGF-ß were also measured.Results showed that the frequency of Tregs and the mean fluorescence intensity of FOXP3 protein significantly decreased in ITP patients compared to those in healthy controls. In addition, there was a significant reduction in relative expression of both CTLA-4 and GITR mRNA in ITP patients (P=0.02 and P=0.006, respectively). The suppressive function of Tregs also diminished in ITP patients compared to that in controls. Both IL-10 and TGF-ß cytokines were produced in lower amounts in ITP patients than controls.It could be concluded that alteration in Treg frequency and functional characteristics might be responsible for loss of self-tolerance and subsequently destructive immune responses observed in ITP patients.

Pro-inflammatory cytokine profile of critically ill septic patients following therapeutic plasma exchange.

UNLABELLED: Severe sepsis involves a generalized inflammatory response, mediated by a number of various cytokines and factors. Plasma exchange (PE) has been proposed as a therapeutic approach to improve survival of patients with severe sepsis and septic shock. The theory is that removing harmful excessive endogenous inflammatory mediators is beneficial. Upon establishment of a diagnosis of severe sepsis, twelve patients received PE plus conventional sepsis treatment. Interleukin (IL)-6, IL-1ß and tumor necrosis factor (TNF)-α were assayed before and after each session of PE. RESULTS: There were no significant changes in cytokine plasma levels after each PE session compared to pre-procedure levels. Among measured pro-inflammatory cytokines, only the plasma levels of IL-6 before the 2nd and 3rd PE sessions were lower than baseline levels (p=0.011 and p=0.012, respectively). All patients tolerated PE therapy well without any adverse effects or homodynamic instability. The results of this study showed that PE does not have a direct and rapid effect on plasma level of TNF-α, IL-1ß and IL-6.

Reduced frequency of NKT-like cells in patients with progressive chronic lymphocytic leukemia.

Natural killer T (NKT) cells are a subset of innate immune cells displaying a limited repertoire of antigen specificities and CD1d restriction. Little is known about contribution of NKT cells in cancer initiation and progression. In this study, the frequencies of NKT-like cells, B cells expressing CD1d molecule and CD4(+) regulatory (Treg) cells were analyzed in 40 patients with chronic lymphocytic leukemia (CLL) and 15 healthy subjects by flow cytometry. Our results showed that the frequency of CD3(+)CD56(+) NKT-like cells is significantly decreased in progressive (4.9 ± 0.8 % of total CD3(+) T cells) compared with indolent (8.1 ± 1.2 %, p = 0.036) patients and healthy subjects (10.6 ± 1.7 %, p = 0.003). However, no association was found between NKT-like cell frequency and immunoglobulin heavy chain variable region gene (IGHV) mutation or CD38 and ZAP70 expression. On the other hand, expression of CD1d molecule was significantly higher in leukemic B cells of patients with CLL (75 ± 1.5 % of total CD19(+) B cells) compared to B cells from healthy subjects (59.6 ± 2.2 %, p < 0.001), with no significant difference between progressive and indolent patients. Interestingly, the frequency of Treg cells was inversely correlated with that of NKT-like cells in patients with CLL (r = -0.4, p = 0.002). Our results suggest a protective role for NKT-like cells in patients with CLL, which seems to be downregulated presumably by Treg cells.

Expression profile of galectin-1 and galectin-3 molecules in different subtypes of chronic lymphocytic leukemia.

Galectin-1 (Gal-1) and galectin-3 (Gal-3) molecules are involved in many vital, biological, and pathological processes. In this study the expression pattern of these two molecules was investigated in leukemic cells from 85 Iranian chronic lymphocytic leukemia (CLL) patients, classified into indolent, progressive, mutated, and unmutated subtypes by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry methods. Our results showed significant downregulation of Gal-3, but not Gal-1, in CLL patients compared with normal subjects (p < .001). Higher representation of Gal-3 mRNA was observed in indolent patients compared with the progressive group (p < .05). Our findings imply a regulatory role for Gal-3 gene in initiation and/or progression of CLL.

Immunoglobulin heavy chain variable region gene usage and mutational status of the leukemic B cells in Iranian patients with chronic lymphocytic leukemia.

The mutational status of the immunoglobulin variable region heavy chain genes (IGHV) is an important prognostic marker in chronic lymphocytic leukemia (CLL). The data accumulated in the literature has largely been derived from studies conducted on Caucasian Western populations. Little is known about Asian CLL patients. In this study the IGHV genes usage and somatic hypermutation status have been investigated in 87 Iranian CLL patients. Based on a cut-off of 98% nucleotide sequence homology, 64.4% and 35.6% of the patients expressed mutated and unmutated IGHV genes, respectively, with most non-progressive patients being in the mutated group (35/44 vs 19/40; P = 0.009). Progression-free survival (PFS) and time to first treatment (TTFT) were significantly higher in our mutated and non-progressive patients compared to unmutated and progressive subtypes, respectively. The most frequently used IGHV gene was IGHV3-7 (12.6%) followed by IGHV3-30 (11.4%), IGHV3-48 (9.2%), IGHV4-39 (6.9%), and IGHV1-8 (6.9%) genes, which taken together comprised nearly half of the IGHV genes expressed in the Iranian CLL patients. Of the IGHV genes, IGHV3-7 was significantly over-represented in non-progressive compared to progressive CLL patients (P = 0.036), whereas IGHV1-69 and IGHV1-2 were expressed at a higher frequency in unmutated compared to mutated CLL patients (P < 0.03). Comparison of IGHV gene usage in our patients with that of Western CLL patients revealed significant differences in expression of IGHV1-69, IGHV3-7, IGHV3-21, and IGHV4-34 genes. Analysis of the IGHV third complementary determining region (HCDR3) sequences revealed a high frequency use of certain HCDR3 motifs, such as YYYGMDV, in our samples. These findings imply contribution of antigen selection and regional (ethnic/geographic) parameters in the leukomogenesis of CLL.

Variation in WNT genes expression in different subtypes of chronic lymphocytic leukemia.

The Wnt molecules are a family of secretory glycoproteins implicated in proliferation and differentiation of both normal and malignant cells. Despite extensive investigation of the WNT genes expression profile in various tumors, little is known about their expression in chronic lymphocytic leukemia (CLL). In this study, the expression profile of 14 WNT genes was investigated in a large number of Iranian patients with CLL. Semi-quantitative RT-PCR was performed on peripheral blood leukemic cells obtained from 62 patients with CLL. Peripheral blood mononuclear cells isolated from 11 age matched normal subjects served as control to determine baseline expression level of these genes. Our results have demonstrated significant up-regulation of WNT-3, WNT-4, WNT-5B, WNT-7B, WNT-9A, WNT-10A, and WNT-16B in patients with CLL compared to normal subjects (p < 0.05 to p < 0.0001). WNT gene expression analysis in different CLL subtypes showed a similar pattern of expression in progressive and indolent clinical subtypes. Over-expression of WNT-5A and WNT-9A genes was observed in patients with no mutation in their immunoglobulin (Ig) variable region heavy chain (Ig VH) genes compared to those with mutated Ig VH genes. Comparison between patients expressing VH1 (n = 9), VH3 (n = 40) and VH4 (n = 12) gene families, revealed down-regulation of WNT-3 and WNT-9A in VH3 positive patients. Our results indicate up-regulation of many members of the WNT gene family in CLL suggesting involvement of the Wnt canonical and/or noncanonical signaling pathways in CLL tumorigenesis.

Human leukocyte antigen class II allele association to disease progression in Iranian patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most frequent type of leukemia in Western countries, but its incidence is low in Asian populations. In the present study we determined the frequency of DRB1 and DQB1 alleles in 87 Iranian CLL patients and 100 healthy controls using a polymerase chain reaction (PCR) technique. An increased frequency of DRB1*07 (p = 0.04), DQB1*06 (p = 0.01) alleles, and DRB1*13/DQB1*03 haplotype (p = 0.01) and decreased frequency of the DQB1*03 (p = 0.01) allele were observed in our patients compared with healthy controls. Comparison between patients with indolent (n = 42) and progressive (n = 38) disease revealed a significant increase in DRB1*04 and DRB5 alleles in progressive patients. Similarly, a higher frequency of DRB5 (p = 0.01) allele was observed in CD38(+) compared with CD38(-) patients. Classification of the patients into immunoglobulin variable region heavy-chain genes mutated and unmutated subtypes did not reveal significant differences for the expression of any of the HLA alleles or haplotypes between these two subtypes. Our findings observed in an Iranian population indicate that CLL could be associated with distinct HLA class II alleles and haplotypes of which the DQB1*06 allele and DRB1*13/DQB1*03 haplotype have not already been reported in CLL patients from other ethnic backgrounds. Some HLA class II alleles may contribute to disease progression in CLL.

Fc receptor-like 1-5 molecules are similarly expressed in progressive and indolent clinical subtypes of B-cell chronic lymphocytic leukemia.

Fc receptor-like (FCRL) 1-5 molecules are exclusively expressed in B-cells and have recently been considered as potential targets for immunotherapy of B-cell malignancies. In this study, the expression pattern of FCRL1-5 molecules was investigated in Iranian patients with B-cell chronic lymphocytic leukemia (B-CLL). Our RT-PCR results have demonstrated that all FCRL molecules, except FCRL4, were expressed in the vast majority of the patients with B-CLL. However, comparison of the relative mRNA expression levels of FCRL between B-CLL (n = 86) and elderly normal subjects (n = 10) revealed significantly lower expression levels of FCRL1 (p < 0.0001), FCRL3 (p = 0.01) and FCRL4 (p = 0.002), but not FCRL2 or FCRL5, in cases with B-CLL. No significant differences were observed between the indolent and progressive subtypes of patients with B-CLL. Comparison between the mutated and unmutated subtypes revealed a significantly higher expression level of FCRL3 (p = 0.017) in patients with mutated CLL. Surface and intracytoplasmic expression of FCRL1, 2, 4 and 5 in leukemic cells of 12 patients by flow cytometry revealed similar results to those obtained by RT-PCR with a few exceptions. Thus, while FCRL4 was expressed in only 2 samples at intracytoplasmic level, FCRL1 and 2 were expressed in the majority of samples, both at surface and intracytoplasm. FCRL5 protein was also detected in 10 samples, but surface expression was confirmed in only 2. Analysis of B-cells from 5 normal subjects by flow cytometry revealed higher expression levels of FCRL molecules compared to CLL. Our results indicate differential expression of FCRL molecules in B-CLL and suggest the potential implication of FCRL1 and 2 for immunotherapeutic interventions.

Immunophenotypic characterization of the leukemic B-cells from Iranian patients with chronic lymphocytic leukemia: association between CD38 expression and disease progression.

BACKGROUND: Patients with B-cell chronic lymphocytic leukemia (B-CLL) have heterogeneous clinical courses, thus several biological parameters need to be added to the current clinical staging systems to predict disease outcome. Recent immunophenotypic studies performed mainly in Western populations have demonstrated the prognostic value of CD38 and ZAP-70 expression in B-CLL. OBJECTIVES: To investigate the expression pattern of a variety of membrane antigens on leukemic cells from Iranian patients with CLL and to find out if there are any differences in the expression of these markers between indolent and progressive groups. METHODS: In the present study, peripheral blood samples from 87 Iranian patients with B-CLL were analysed by flow cytometry. RESULTS: In all cases, the neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/sIg+). The vast majority of the cases expressed CD23, but failed to stain for CD3 or CD14. The leukemic cells of most patients expressed CD27 (84/87, 95.4%) and CD45RO (74/87, 83.9%) molecules, suggesting a memory B-cell phenotype. Comparison between the indolent (n=42) and progressive (n=37) patients revealed significantly higher frequency and intensity of CD38 expression in progressive group (40.5%) compared to indolent (11.9%) patients (p<0.05). None of the other membrane antigens were differentially expressed in these two groups of patients. CONCLUSION: Our results obtained in an Asian ethnic population confirm and extend previous findings obtained from Western populations regarding the association of CD38 expression and disease progression in B-CLL.

Assessment of in vitro cytokine response in hemophilia A patients with or without factor VIII inhibitory antibody.

Factor VIII (FVIII) inhibitor antibodies are produced in a proportion of hemophilia A patients. Development of anti-FVIII inhibitor antibodies is a T cell-dependent response, mediated by FVIII specific CD4(+) T cells. This study was performed to investigate the contribution of T helper (Th) cell-mediated cytokine response in inhibitor production. Peripheral blood mononuclear cells (PBMCs) were obtained from hemophilia A patients with (n = 14) or without inhibitor (n = 14) and from normal individuals (n = 14). Following stimulation of PBMCs with rFVIII and phytohemagglutinin (PHA) mitogen, the secreted cytokines, interferon-gamma (IFN-gamma), interleukin-10 (IL-10), and transforming growth factor-beta1 (TGF-beta1), in culture supernatant and the proliferative response were assessed using sandwich ELISA and (3)H-thymidine incorporation, respectively. No significant proliferative response to FVIII was observed, whereas PHA induced a strong response in all groups. No cytokine secretion was observed in response to FVIII stimulation. Although PHA induced IL-10, TGF-beta1 and IFN-gamma secretion in all groups, the level of IFN-gamma was significantly lower in hemophilia A patients than in normal individuals (p < 0.0001). The levels of TGF-beta1 and IL-10 were similarly higher in patients compared with normal subjects, but the difference was not statistically significant. Lack of FVIII-induced proliferative response and cytokine production together with reduced secretion of PHA-induced IFN-gamma in both groups of patients suggest involvement of nonspecific immunosuppression possibly due to hepatitis C virus (HCV) infection observed in the majority of patients.

Expression profile of Wnt molecules in leukemic cells from Iranian patients with acute myeloblastic leukemia.

BACKGROUND: Wnt molecules play a key role in growth, proliferation and development of some embryonic and adult organs as well as hematopoietic stem cells. Wnt signaling pathways are aberrantly activated in many tumor types, including solid tumors and hematologic malignancies. OBJECTIVE: To investigate the expression profile of a large number of Wnt genes in leukemic cells from Iranian patients with acute myeloblastic leukemia. METHODS: RT-PCR method was used to determine the Wnt genes expression in bone marrow (BM) and/or peripheral blood (PB) samples from 16 patients with AML and PB samples of 36 normal subjects. RESULTS: Among 14 Wnt molecules included in this study, Wnt-7A and Wnt-10A were significantly down-regulated (p = 0.002 and p < 0.0001, respectively) and Wnt-3 was significantly over-expressed (p < 0.02) in AML patients compared to normal subjects. No significant association was found between Wnt expression and FAB classification of the patients. CONCLUSION: Our results demonstrated for the first time aberrant expression of Wnt-7A, Wnt-10A and Wnt-3 genes in Iranian AML patients. This may be of relevance to the tumorigenesis process in this malignancy.

Comparative measurement of anti-factor VIII antibody by Bethesda assay and ELISA reveals restricted isotype profile and epitope specificity.

Factor VIII (FVIII) inhibitor antibodies are produced against functional epitopes of FVIII in about 30% of severe hemophilia A patients leading to inhibition of its procoagulant activity. The Bethesda assay, the most commonly used method to measure FVIII inhibitors, based on inhibition of coagulant activity of FVIII, is neither able to detect noninhibitory antibodies nor their isotype. In this study we employed an indirect enzyme-linked immunosorbent assay (ELISA) to measure dif ferent isotypes and IgG subclasses of anti-FVIII anti body in the plasma of hemophiliacs (with and without inhibitor) and normal individuals using recombinant FVIII-coated microtiter plates. The results showed a predominance of IgG and IgG4, though IgA was slightly elevated in a few inhibitor-positive patients and IgM was hardly detectable. A highly significant correlation was found between the Bethesda titer and the optical density values of total Ig, IgG and IgG4 anti-FVIII antibodies obtained by ELISA (p<0.0001). These findings suggest a restricted specificity of anti-FVIII response in hemophiliacs towards functional epitopes of the molecule. Furthermore, high specificity and reasonable sensitivity of the ELISA, together with other technical advantages, suggest this method as a suitable supplementary technique for rapid large-scale screening of inhibitor-positive samples, though ELISA-negative samples need to be rechecked by the Bethesda assay to identify patients with a low inhibitor titer.

Fibromodulin, an extracellular matrix protein: characterization of its unique gene and protein expression in B-cell chronic lymphocytic leukemia and mantle cell lymphoma.

Fibromodulin is an extracellular matrix protein normally produced by collagen-rich tissues; the fibromodulin gene has been found to be the most overexpressed gene in B-cell chronic lymphocytic leukemia. In this study, fibromodulin was expressed at the gene level (reverse transcription-polymerase chain reaction [RT-PCR]) in all patients with B-CLL (n = 75) and in most (5 of 7) patients with mantle cell lymphoma (MCL). No mutations in the fibromodulin gene were detected. Fibromodulin was also detected at the protein level in the cytoplasm of the B-CLL cells and in the supernatant after in vitro cultivation, but not at the cell surface. Fibromodulin was not found in patients with T-cell chronic lymphocytic leukemia (T-CLL), B-cell prolymphocytic leukemia (B-PLL), T-cell prolymphocytic leukemia (T-PLL), hairy cell leukemia, follicular lymphoma, lymphoplasmacytic lymphoma, multiple myeloma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), or chronic myelogenous leukemia (CML) or in 36 hematologic cell lines. Normal blood mononuclear cells (T and B lymphocytes, monocytes), tonsil B cells, and granulocytes did not express fibromodulin. Activation (phorbol 12-myristate 13-acetate [PMA]/ionomycin) of normal T and B lymphocytes induced weak fibromodulin gene expression, but not to the extent seen in freshly isolated B-CLL cells. The reason for the exclusive ectopic expression of fibromodulin in B-CLL and MCL is unknown. However, its unique protein expression makes it likely that fibromodulin is involved in the pathobiology of B-CLL and MCL.