Dainius Characiejus

CD8+ CD28− and CD8+ CD57+ T cells and their role in health and disease

Chronic antigenic stimulation leads to gradual accumulation of late‐differentiated, antigen‐specific, oligoclonal T cells, particularly within the CD8+ T‐cell compartment. They are characterized by critically shortened telomeres, loss of CD28 and/or gain of CD57 expression and are defined as either CD8+CD28− or CD8+CD57+ T lymphocytes. There is growing evidence that the CD8+CD28− (CD8+CD57+) T‐cell population plays a significant role in various diseases or conditions, associated with chronic immune activation such as cancer, chronic intracellular infections, chronic alcoholism, some chronic pulmonary diseases, autoimmune diseases, allogeneic transplantation, as well as has a great influence on age‐related changes in the immune system status. CD8+CD28− (CD8+CD57+) T‐cell population is heterogeneous and composed of various functionally competing (cytotoxic and immunosuppressive) subsets thus the overall effect of CD8+CD28− (CD8+CD57+) T‐cell‐mediated immunity depends on the predominance of a particular subset. Many articles claim that CD8+CD28− (CD8+CD57+) T cells have lost their proliferative capacity during process of replicative senescence triggered by repeated antigenic stimulation. However recent data indicate that CD8+CD28− (CD8+CD57+) T cells can transiently up‐regulate telomerase activity and proliferate under certain stimulation conditions. Similarly, conflicting data is provided regarding CD8+CD28− (CD8+CD57+) T‐cell sensitivity to apoptosis, finally leading to the conclusion that this T‐cell population is also heterogeneous in terms of its apoptotic potential. This review provides a comprehensive approach to the CD8+CD28− (CD8+CD57+) T‐cell population: we describe in detail its origins, molecular and functional characteristics, subsets, role in various diseases or conditions, associated with persistent antigenic stimulation.

A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample

BackgroundPresently available flow cytometric methods of bromodeoxyuridine (BrdUrd) labelling do not provide information on the cell cycle time (TC) and the growth fraction (GF). In this paper, we describe a novel and simple method to estimate TC and GF from flow cytometric analysis of a single tumour sample after BrdUrd labelling.MethodsThe proposed method is based on two assumptions: (1) the number of labelled cells traversing the cell cycle per unit time is constant and (2) the total number of labelled cells is constant throughout the cycle, provided that cells produced after division are excluded. The total numbers of labelled divided G1 cells, labelled divided S cells, labelled undivided S cells, and labelled undivided G2 cells were obtained for DNA histograms of BrdUrd-positive cells in a collected sample. These cell numbers were used to write equations to determine the durations of cell cycle phases, TC and GF. To illustrate the application of the proposed formulae, cell cycle kinetic parameters were analysed in solid SL2 tumours growing in DBA/2 mice and in human T-leukaemia Jurkat cells in culture.ResultsThe suitability of the proposed method for estimating durations of the cell cycle phases, TC and GF was demonstrated. TC in SL2 tumours was found to be relatively constant at 4 and 10 days after tumour implantation (20.3 ± 1.1 h and 21.6 ± 0.9 h, respectively). GF in tumours at day 10 was lower than GF at day 4 (54.2 ± 7.7% vs. 79.2 ± 5.9%, p = 0.0003). Approximate values of TC and GF of cultured Jurkat cells were 23.9 h and 79.3%, respectively.ConclusionThe proposed method is relatively simple and permits estimation of the cell cycle parameters, including TC and GF, from a single tumour sample after labelling with BrdUrd. We have shown that this method may be useful in preclinical studies, allowing estimation of changes in GF during growth of murine tumours. Experiments with human Jurkat cells suggest that the proposed method might also prove suitable for measurement of cell kinetics in human tumours. Development of suitable software enabling more objective interpretation of the DNA profile in this method would be desirable.

Does the "new philosophy" in predictive, preventive and personalised medicine require new ethics?

This paper maps the ethical issues that arise in the context of personalised medicine. First, it highlights the ethical problems related to increased predictive power of modern diagnostic interventions. Such problems emerge because the ability to identify individuals or groups of individuals that can potentially benefit from a particular therapeutic intervention also raises a question of personal responsibility for health-related behaviour and lifestyle. The second major area of ethical concern is related to health prevention and distributive justice. The paper discusses the ethical challenges brought by the personalised medicine in the context of the Additional Protocol to the Convention on Human Rights and Biomedicine concerning Genetic Testing for Health Purposes. Finally, it notes that the issue of consent in the context of biobanks, the need to rethink the prevalent models of research designs and to communicate relevant findings to the donors of biological materials deserve further discussion.

Could the complement component C4 or its fragment C4d be a marker of the more severe conditions in patients with primary Sjögren’s syndrome?

Our aim is to evaluate the complement component C4 (C4) and its fragment C4d (C4d) levels, focusing on their associations with other markers of B cells’ activity in patients with primary Sjögren’s syndrome (pSS). Humoral factors C4, C4d, B cell-activating factor (BAFF), κ and λ free light chains (FLCs) and IgG (by immunoassay) were investigated in 58 patients with pSS and in 28 healthy controls. We observed significantly higher levels of BAFF, κ and λ FLC and IgG, and significantly lower level of C4 in pSS patients, while the level of C4d was similar in the both groups. Significantly higher levels of BAFF, κ and λ FLCs, IgG, and significantly lower C4 level were found in anti-SSA/SSB antibodies (Abs) seropositive pSS patients’ group comparing with healthy controls. Level of C4d was significantly lower in anti-SSA/SSB Abs seropositive pSS patients comparing with seronegative pSS patients and healthy controls. C4d correlated with C4, anti-SSB Abs level and κ/λ ratio. Significantly higher κ FLC and IgG levels were found in anti-SSA/SSB Abs seronegative pSS patients comparing with healthy controls. Anti-SSA/SSB seropositivity in pSS patients is associated with the decreased level of C4d. These results show that C4d can be an appropriate marker of antibody response and complement activation in pSS patients with Abs, and possibly may show the more severe condition—exhaustion of C4. Further studies are required to determine whether C4d assessment could be a relevant biomarker for the more severe condition and the worse prognosis of pSS.

New Ethical Paradigm in Preventive, Predictive and Personalised Medicine

This chapter deals with the ethical issues that arise in the context of personalised medicine. First, it highlights the ethical problems related to increased predictive power of modern diagnostic interventions. Further, it raises a question of personal responsibility for health-related behaviour and lifestyle, and it poses ethical questions related to health prevention and distributive justice. Finally, it emphasises the necessity to rethink current models of research designs and communication of relevant findings to the donors of biological materials.

Intratumoral Accumulation and Clonal Expansion May Not Be Decisive for Rejection of Allogeneic Tumors by CD8+T-Lymphocytes

Background: The aim of this study was to analyze the spatial distribution and proliferation of adoptively transferred CD8+ T-lymphocytes sensitized against allogeneic tumors. Materials and Methods: Transgenic β-actin-luc mice that express luciferase were sensitized against allogeneic SL2 lymphoma. CD8+ T-lymphocytes from these mice were transferred to lymphocyte-deficient, recombination activating gene-deficient (Rag−/−) mice bearing SL2 tumors and were tracked using bioluminescence imaging. Results: Two out of six Rag−/− mice rejected their tumors. There were no apparent differences in spatial distribution and proliferative intensity of adoptively-transferred CD8+ T-lymphocytes between the two Rag−/− mice that rejected allogeneic SL2 tumors and the four Rag−/− mice that did not. Conclusion: The pattern of distribution in the mouse body and proliferative intensity of CD8+ T-lymphocytes do not seem to be decisive factors influencing allogeneic tumor rejection.

Quantitative changes within the peripheral blood CD8highCD57+ T-cell subpopulation of patients with advanced renal cell carcinoma

Background. Antitumor immunotherapy strategies, such as cytokine- based or cell-based immunotherapies, are designed to activate immune response against cancer cells, but considering that malignancy may be associated with the expansion of immunosuppressive components of antitumor immunity, it is likely that in such cases activation of the immune system would further enhance activity of these components, leading to more severe suppression of antitumor immune response thus making more favourable conditions for tumor progression. Materials and methods. We studied the expression of biomarkers, representing immunosuppressive (FOXP3) and cytotoxic (perforin, IFN-γ) CD8 high CD57 + T-cell subpopulation functions in the peripheral blood of 34 patients with advanced clear cell renal cell carcinoma (RCC) and 26 controls by multicolour flow cytometry. Results. CD8 high CD57 + T cell subpopulation of all CD8 + T cells in RCC patients was significantly higher compared to age-matched healthy controls (p = 0.003). It was found that the mean percentage of suppressive CD8 high CD57 + FOXP3 + T-cell subset and cytotoxic CD8 high CD57 + Perforin + T-cell subset in the CD8 high CD57 + T-cell subpopulation was significantly increased in RCC patients compared to controls (p = 0.0004 and p = 0.008, respectively). There was no strong and biologically relevant negative correlation between the expression of FOXP3 and Perforin in the peripheral blood CD8 high CD57 + T-cell subpopulation of RCC patients. Conclusions. Subsets of immunosuppressive FOXP3+ T cell and tumor-attacking (cytotoxic) Perforin+ T cells in the CD8 high CD57 + T-cell subpopulation are significantly increased in RCC patients compared to controls. These quantitative rearrangements are independent and individual for each RCC patient.

Quantitative changes of functionally different CD8 h CD57 + T-cell subsets in the peripheral blood of advanced renal cell carcinoma or high-risk melanoma patients@@@Skirtingomis funkcijomis pasižyminčių CD8 h CD57 + T limfocitų subpopuliacijų kiekybiniai pokyčiai išplitusia inkstų ląstelių karcinoma ar didelės rizikos melanoma sergančių pacientų periferiniame kraujyje

Correspondence to: Marius Strioga, Laboratory of Immunology, Institute of Oncology, Vilnius University, P. Baublio 3B, LT-08660, Vilnius, Lithuania. E-mail: marius.strioga@vuoi.lt Introduction. CD8hCD57+ T-cell subpopulation and its functionally diff erent subsets play an important role in antitumour immunity. Th e relation of competitive subsets may infl uence the overall eff ect of CD8hCD57+ T-cell mediated antitumour immunity and determine an individual response to antitumour immunotherapy. Th e aim of this study was to evaluate the proportion of cytotoxic, immunomodulating and immunosuppressive subsets of the CD8hCD57+ T-cell subpopulation in the peripheral blood of cancer patients and agematched healthy controls. Materials and methods. We studied the expression of biomarkers representing the cytotoxic (perforin), immunosuppressive (FOXP3, NKG2A) and immunomodulating (IFNγ) properties of CD8+CD57+ T cells in the peripheral blood of 49 cancer patients: 30 with clear cell renal cell carcinoma (age median 58, range 43–81), 19 with high risk cutaneous melanoma (age median 68, range 45–86) and 26 controls (age median 55, range 41–81) by multicolour fl ow cytometry. Results. Th e percentage of immunosuppressive CD8hCD57+FOXP3+ T-cell subset in CD8hCD57+ T-cell population varied. It was absent in 65% of controls, while only 23% and 26% of such patients were observed in renal cell carcinoma (RCC) and melanoma groups, respectively. Even 40% of RCC and 37% of melanoma patients had a high percentage of CD8hCD57+FOXP3+ T-cell subset, while in the control group we found no such subjects. Th e cytotoxic CD8hCD57+Perforin+ T-cell subset was signifi cantly increased in RCC patients, but showed no relevant rise in melanoma patients, whereas the immunomodulating CD8hCD57+IFNγ+ subset was signifi cantly increased in melanoma patients but showed no relevant rise in RCC patients when compared to controls. Conclusions. Th e amount of various functionally diff erent subsets in CD8hCD57+ T-cell subpopulation varies greatly among cancer patients. Th ese diff erences may infl uence the overall CD8hCD57+ T-cell mediated antitumour immune response and determine an individual response to antitumour immunotherapy.

Relationship between growth fraction and clonogenic survival after ionizing irradiation in pancreatic MiaPaCa2 cells@@@Kasos MiaPaCa2 ląstelių augimo frakcijos sąsaja su klonogeniniu išgyvenimu po jonizuojančiosios spinduliuotės

Background. The prediction of cell radiosensitivity still remains a matter of great interest in the field of radiation biology and oncology. The aim of the present study was to examine the relationship between the value of growth fraction (GF) prior to irradiation and the clonogenic survival of the pancreatic cancer cell line MiaPaCa2. Materials and methods. Growth fraction prior to irradiation was determined by a novel method using bromodeoxyuridine-flow cytometry data from a single sample. The cells were irradiated with 2 and 8 Gy using a 60 Co source. Cell survival after ionizing radia tion was measured using a clonogenic assay. Pearson’ product–moment correlation coefficient was used to estimate the correlation of GF and clonogenic survival fraction values. Results. A positive correlation between GF values and cell survival fraction was obtained after irradiation with 8 Gy (r = 0.67, P = 0.0002). We observed a trend of a higher GF and better clonogenic survival of cells after irradiation with 2 Gy. Conclusions. A higher growth fraction prior to irradiation is related to a better clonogenic survival of the pancreatic cell line MiaPaCa2. Whether or not this finding is applica ble to other cell types remains to be established.