Abnormal Expression of BTLA and CTLA-4 Immune Checkpoint Molecules in Chronic Lymphocytic Leukemia Patients.

Chronic lymphocytic leukemia (CLL) is characterized by the peripheral accumulation of neoplastic B cells and is frequently complicated by the systemic immunosuppression associated with an impairment in B and T lymphocyte activation. We hypothesized that the expression of immune checkpoint suppressors B and T lymphocyte attenuator (BTLA) and cytotoxic T lymphocyte antigen (CTLA-4) is disturbed in both lymphocyte subpopulations in CLL. The expression of CTLA-4 and BTLA mRNA was determined by real-time PCR, while CTLA-4 protein expression (surface or intracellular) was estimated in BTLA+ lymphocytes by flow cytometry. In CLL patients, we observed a higher gene transcript level of BTLA and CTLA-4 than in healthy individuals in both freshly isolated and PMA stimulated B and T cells. Remarkably, lower amounts of both inhibitory proteins were found in peripheral blood (PB) CLL B cells, whereas normal BTLA and elevated CTLA-4 were found in T cells. Consistently, there was a prevalence of CTLA-4+ cells within circulating BTLA+ T cells cells of patients confronting PB healthy cells. After in vitro stimulation, the only change found in CLL patients was a decrease in BTLA expression in B and T lymphocytes. In contrast, healthy lymphocytes responded more vigorously as regards the BTLA and CTLA expression with substantially higher frequency of CD69+ cells under the stimulating condition compared to corresponding cells from the CLL group. Our results indicate that CLL development is associated with the affected expression of BTLA and CTLA-4 checkpoint receptors in PB and its impaired expression might be associated with lowering of the threshold for B cell activation and proliferation, while upregulated CTLA-4 expression in CLL peripheral BTLA+ T cells may contribute to suppressed T cell effector functions. This hypothesis needs to be validated in future studies, which would allow us to explain how the increased or decreased expression of these molecules affects the cell function.

Stimulated peripheral production of interferon-gamma is related to fatigue and depression in multiple sclerosis.

OBJECTIVES: The aim of the study was to evaluate the stimulated production of interferon-gamma (IFNγ) by peripheral CD3+CD4+ T lymphocytes in patients with multiple sclerosis (MS) with regard to the degree of fatigue, and to investigate relationships between immunological parameters, level of depression and clinical variables. METHODS: Forty MS patients (30 women, 10 men, aged 22-60 years): 20 fatigued and 20 non-fatigued were involved in the study. Fatigue was evaluated using the Fatigue Severity Scale (FSS) and Modified Fatigue Impact Scale (MFIS), depression level - using Beck Depression Inventory (BDI). Production of IFNγ by stimulated peripheral blood CD3+CD4+ T lymphocytes, assessed using flow cytometry, was compared between MS patients with different levels of fatigue and controls. Correlations were searched out between immunological findings and BDI, age, duration and course of MS, relapse rate, disability (assessed in Expanded Disability Status Scale - EDSS) and its progression. RESULTS: Stimulated production of IFNγ by CD3+CD4+ T lymphocytes was higher in severely fatigued patients in comparison with non-fatigued ones and controls, tended to correlate with FSS and MFIS, and correlated with BDI. No relationships were found between immunological findings and disease-related variables. CONCLUSION: Stimulated production of IFNγ by peripheral CD3+CD4+ T lymphocytes is related to fatigue and depression in MS patients.

Different patterns of activation markers expression and CD4+ T-cell responses to ex vivo stimulation in patients with clinically quiescent multiple sclerosis (MS).

Patients with relapsing-remitting (RR) and secondary progressive (SP) forms of multiple sclerosis (MS), although in long-term clinical remission, showed different patterns of increased expressions of the activation markers: CD69, CD40L, and both membrane/surface and cytoplasmic CTLA-4 (mCTLA-4 and cCTLA-4, respectively) in freshly isolated peripheral blood (PB) CD4+ T cells compared with controls. Also observed were dysregulated responses to ex vivo stimulation in both groups of MS patients accompanied by increased IFN-gamma synthesis. Our findings may suggest that the mechanisms leading to each clinical form of the disease may be heterogeneous.

Possible association of cytotoxic T-lymphocyte antigen 4 gene promoter single nucleotide polymorphism with acute rejection of allogeneic kidney transplant.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) molecule is an important inhibitor of T-lymphocyte response. Polymorphisms in the CTLA-4 gene have been described to be associated with numerous autoimmune diseases. However, similar studies in solid organ transplantation have been scarce. Therefore, we examined the distribution of three single nucleotide dimorphisms, namely, -1147T/C, -318C/T, and +49A/G, in two groups of allogeneic kidney graft recipients: (1) those with at least one acute rejection episode ("rejectors"; n = 38) and (2) those with no signs of acute rejection ("nonrejectors"; n = 53). Allele frequencies in both groups of patients were similar in two positions, -1147T/C and +49A/G. However, rejectors showed slight differences from nonrejectors for allele and genotype frequencies in position -318. The -318T allele was two times less frequent among rejectors than nonrejectors, a difference that was close to statistical significance (P = .039; P corrected = .0583), and may reach it when greater numbers of patients are tested.

Alterations of the expression of T-cell-related costimulatory CD28 and downregulatory CD152 (CTLA-4) molecules in patients with B-cell chronic lymphocytic leukaemia.

In the present study, we have examined the kinetics and magnitude of expression of the CD28 and CD152 molecules on unstimulated and anti-CD3+rIL-2-stimulated peripheral blood CD4+ and CD8+ T cells in patients with chronic lymphocytic leukaemia (B-CLL) and controls. The mean percentages of both CD3+/CD4+/CD28+ and CD3+/CD8+/CD28+ cells were significantly lower in B-CLL than in controls before culture, decreased rapidly, reaching their lowest levels between 24 and 48 h, and returned to basal levels after 72 h of culture. In controls, the lowest proportions of CD3+/CD4+/CD28+ and CD3+/CD8+/CD28+ cells were found after 24 h and returned to prestimulation levels after 48 h of stimulation. We observed significantly higher proportions of unstimulated CD3+/CD4+/CD152+ and CD3+/CD8+/CD152+ cells in B-CLL patients than in controls. The highest percentages of CD3+/CD4+/CD152+ and CD3+/CD8+/CD152+ cells were observed in controls after 72 h, and in B-CLL patients after 24 h, and remained statistically higher after 48, 72 and 96 h of stimulation. CD152 molecule expression returned to prestimulation levels after 96 h of culture in controls, and after 120 h in B-CLL patients. The abnormal kinetics and levels of CD28 and CD152 expression on T cells in B-CLL may lead to a state of hyporesponsiveness or anergy and could be one of the mechanisms of immune deficiency in this disease.

Lactoferrin-induced up-regulation of zeta (zeta) chain expression in peripheral blood T lymphocytes from cervical cancer patients.

Alteration of T-cell-associated signal transduction molecules has recently been implicated in immune suppression in tumour-bearing hosts. Here we report the immunoregulatory effects of human lactoferrin (LF) on zeta-chain expression in peripheral blood T lymphocytes from cervical cancer patients and healthy donors. By quantitative flow cytometry analysis, we demonstrated that the mean zeta-chain expression was significantly higher in freshly-isolated T lymphocytes from healthy donors (69%), compared with the patients (38%). Following 3-day culture under standard conditions, zeta-chain expression in T lymphocytes from the patients increased significantly, whereas it dropped in the cells from healthy donors. Anti-CD3 MoAb as well as LF, significantly increased expression of zeta-chain in T cells both from patients and control subjects. The addition of LF to the anti-CD3 MoAb cell cultures resulted in an even higher stimulation of the zeta-chain expression. The results suggest that, in patients with cervical cancer, zeta-chain defects could be corrected by the therapeutic application of LF.

Cell cycle regulatory proteins and apoptosis in B-cell chronic lymphocytic leukemia.

BACKGROUND AND OBJECTIVES: The pathogeny of B-cell chronic lymphocytic leukemia (B-CLL) involves both deregulated proliferation and inhibition of cell death. A particular role in the regulation of these phenomena is played by proteins involved in early G1 phase regulation: pRb kinases: cyclin-dependent kinases (cdk): cdk4 and cdk6 activated by cyclins D, and universal cdk inhibitor p27(Kip1). DESIGN AND METHODS: We determined by flow cytometry the expression of p27(Kip1) and cyclins D (D2 and D3) in populations of peripheral blood lymphocytes obtained from 59 (for p27(Kip1)) and 31 (for cyclins D) previously untreated patients with B-CLL, and compared them with cell cycle parameters, cell viability and apoptosis in 72-hour cultures in medium only. As a control we determined the expression of p27(Kip1), cyclin D2 and D3 in peripheral blood CD5+/CD19+ lymphocytes from 15 healthy donors. RESULTS: p27(Kip1) was present in nearly 100% of lymphocytes in all B-CLL populations tested. Its cellular content estimated semiquantitatively by specific mean fluorescence intensity was higher than in normal CD5+/CD19+ lymphocytes, p27(Kip1) was inversely correlated with patients' age and not correlated with other clinical variables, cell cycle or apoptosis rate. Cyclin D2 was detectable in 25 out of 31, and cyclin D3 in all B-CLL lymphocytes populations studied. In contrast to p27Kip1 present in all CD5+/CD19+ lymphocytes, both cyclins were detected only in a subset of neoplastic cells: 27.5 to 87% (mean 51.2) for cyclin D2 and 20.3 to 98% (mean 76.5) for cyclin D3. In cyclin D2- and D3-positive normal CD5+/CD19+ lymphocytes and B-CLL cell populations, cyclin D3 was expressed in a higher percentage of cells than cyclin D2. Both cyclin D2-and cyclin D3-positive fractions of B-CLL cells were, on average, larger than corresponding fractions of normal CD5+/CD19+ peripheral blood lymphocytes. INTERPRETATION AND CONCLUSIONS: Our results indicate that cyclin D3 plays an important role in the regulation of normal and neoplastic CD5+/CD19+ cells, and point to the possibility of the exit of a number of CLL lymphocytes from quiescence.

The effect of peripheral blood lymphocyte stimulation on zeta chain expression and IL-2 production in Hodgkin's disease.

It has been reported that peripheral blood T cells and NK cells express reduced levels of the T-cell receptor signal-transducing zeta chain in Hodgkin's disease (HD). The zeta chain has emerged as a key subunit of the T-cell antigen receptor, which plays a central role in the signal-transducing events leading to T and NK-cell activation. We were interested in determining whether the low zeta chain expression in HD could be corrected by anti-CD3, anti-CD3-rIL-2 ex vivo stimulation. Zeta chain expression was analysed by dual immunofluorescence on permeabilized cells before and after 72 hours of culture. The IL-2 concentration in the culture supernatants was measured by ELISA. Zeta chain was significantly reduced on unstimulated CD4+, CD8+ and CD56+ cells from patients in active disease compared with normal subjects. In patients in complete remission, the values were normal except for CD8+ cells, on which zeta expression remained significantly reduced. Stimulation with anti-CD3 did not change zeta expression. Co-stimulation with rIL-2 increased but did not normalize the proportions of CD4(+)/zeta(+), CD8(+)/zeta(+)and CD56(+)/zeta(+)cells and IL-2 production in active disease. Stimulation of cells from patients in clinical remission with anti-CD3(+)rIL-2 increased the proportion of CD8(+)zeta(+)cells and normalized IL-2 production levels. Considering the pivotal role of CD3-zeta in immune response, our data suggest that successful immunotherapy approaches in active HD should consider inclusion of other potent cytokines, as well as genetically engineered tumour vaccines.

Expression and functional significance of CTLA-4, a negative regulator of T cell activation.

The generation of an effective immune response involves antigen-specific T cell expansion and differentiation of effector function. T cell activation requires at least two distinct signals, including signaling via the antigen-specific T cell receptor (TCR) and a costimulatory pathway. Antigen stimulation of T cells can lead either to a productive immune response, characterized by proliferation, differentiation, clonal expansion and effector function, or, in the absence of an appropriate costimulation, to a state of long-lasting unresponsiveness, termed anergy. Anergic T cells fail to proliferate and secrete cytokines in response to secondary stimulation. The interaction between the costimulatory molecule CD28 on T cells and members of the B7 family on antigen-presenting cell results in upregulation of T cell proliferation and cytokine production and induces the expression of the anti-apoptotic protein Bcl-xl. Based of these findings, the two-signal requirement model for T cell activation is generally accepted today. The negative regulatory mechanisms during T cell activation are not well understood, but they are crucial for the maintainance of lymphocyte homeostasis. For several years the functional role of the enigmatic CD28 homologue cytotoxic T lymphocyte antigen-4 (CTLA-4) in T cell activation has been both obscure and controversial. CTLA-4 was initially supposed to provide a costimulatory signal in conjunction with TCR/CD3 signaling. Today we know that CD28 and CTLA-4 molecules may have diametrically opposed functions: signaling via CD28, in conjunctive with TCR, is required for T cell activation, while signaling via CTLA-4 is a negative signal that inhibits T cell proliferation. How the T cell integrates signals through the TCR/CD3 complex, CD28 and CTLA-4 to initiate, maintain and terminate antigen-specific immune response is in fact not fully clarified. In this review, we will focus on the emerging role of CTLA-4 as a negative regulator of T lymphocyte activation and its role in the dynamic interplay of activatory and inhibitory signals.

Immunotherapy with recombinant human interleukin 2 in patients with hematological malignancies after bone marrow or peripheral blood stem cell transplantation.

High-dose chemotherapy in conjunction with bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT) is increasingly being used for treatment of patients with hematological malignancies. Residual tumor cells, resistant to high-dose chemoradiotherapy, are responsible for reccurence of the disease. Interleukin 2 (IL-2), a pleiotropic cytokine which plays a central role in immune response, has been introduced in several clinical trials in patients with hematological malignancies after BMT or PBSCT to increase immunocompetence of these patients and eradicate residual malignant cells. At present there is no general agreement on the optimum dosage or route of administration and clinical trials also gave conflicting results. Establishment of optimum dosage schedules and methods of administration should enable a better assessment of the place of IL-2 in the treatment of these patients.

Carp liver actin: isolation, polymerization and interaction with deoxyribonuclease I (DNase I).

The aim of this study was to isolate and to characterize actin from the carp liver cytosol and to examine its ability to polymerize and interact with bovine pancreatic DNase I. Carp liver actin was isolated by ion-exchange chromatography, followed by gel filtration and a polymerization/depolymerization cycle or by affinity chromatography using DNase I immobilized to agarose. The purified carp liver actin was a cytoplasmic beta-actin isoform as verified by immunoblotting using isotype specific antibodies. Its isoelectric point (pI) was slightly higher than the pI of rabbit skeletal muscle alpha-actin. Polymerization of purified carp liver actin by 2 mM MgCl(2) or CaCl(2) was only obtained after addition of phalloidin or in the presence of 1 M potassium phosphate. Carp liver actin interacted with DNase I leading to the formation of a stable complex with concomitant inhibition of the DNA degrading activity of DNase I and its ability to polymerize. The estimated binding constant (K(b)) of carp liver actin to DNase I was calculated to be 1.85x10(8) M(-1) which is about 5-fold lower than the affinity of rabbit skeletal muscle alpha-actin to DNase I.

Alterations in signal transducing molecule CD3 zeta in patients with neoplastic diseases.

The impairment of immune cell functions in cancer patients may result from impairment of signal transduction. Recent studies have demonstrated altered expression and function of signal transduction molecule CD3 zeta in T and natural killer cells in patients with neoplastic diseases. Tumor infiltrating lymphocytes are more affected than peripheral blood lymphocytes. CD3 zeta expression is more prominent in advanced stage of the disease and correlates with reduced proliferative response, IFN-gamma, IL-2, TNF-alpha production.