Self-Reactive CD4+ T Cells and B Cells in the Blood in Health and Autoimmune Disease: Increased Frequency of Thyroglobulin-Reactive Cells in Graves’ Disease

The mechanisms underlying activation of potentially self-reactive circulating B cells and T cells remain unclear. We measured the uptake of a self-antigen, thyroglobulin, by antigen presenting cells, and the subsequent proliferation of CD4⁺ T cells and B cells from healthy controls and patients with autoimmune thyroiditis. In Hashimoto's thyroiditis, B cells bound increased amounts of thyroglobulin in a complement- and autoantibody-dependent manner, and the thyroglobulin-elicited proliferation of CD4⁺ T cells and B cells was complement dependent. Increased proportions of Tg-responsive CD4⁺ T cells and B cells were found in patients with Graves’ disease. Notably, both patient groups and healthy controls exhibited higher proliferative responses to thyroglobulin than to a foreign recall antigen, tetanus toxoid. Our results suggest that self-tolerance can be broken by exposure of circulating lymphocytes to high local concentrations of self-antigen, and that complement plays a role in the maintenance of autoimmune processes, at least in Hashimoto's thyroiditis.     

Cytokine Modulation with Immune γ-Globulin in Peripheral Blood of Normal Children and Its Implications in Kawasaki Disease Treatment

Intravenous immune -globulin (IVIG) is used successfully in the treatment of Kawasaki disease, with dose-dependent rapid resolution of symptoms such as fever and irritability and a decrease in ESR, WBCs, and platelets. The mode of action of IVIG in reducing this inflammatory response is not clearly understood. Recently anticytokine antibodies in IVIG have been demonstrated. Serum levels of proinflammatory cytokines have been shown to be elevated in patients with Kawasaki disease. The cytokine interleukin-6 (IL-6) is involved in the de novo production of acute-phase proteins by hepatocytes and cause thrombocytosis and fever in response to tissue injury. Patients receiving parenteral recombinant human IL-6 have dose-dependently experienced fever, malaise, chills, and acute-phase reaction. With high IL-6 concentrations, central nervous system toxicity has also been reported and IL-6 has been thought to mediate endothelial damage. We evaluated the response of stimulated blood cells of 12 normal children to IVIG in the release of the cytokines IL-6, IL-8, TNF-, and IL-6 receptor (sIL-6R). The levels of cytokines IL-6, IL-8, and TNF- (but not sIL-6R) in peripheral blood induced by stimulation with LPS were markedly reduced (P < 0.008) within 3 hr when incubated with IVIG compared to without IVIG. Thus we demonstrated that cells of normal children respond to IVIG in vitro by reducing cytokines such as IL-8, TNF-, and IL-6 without affecting the level of receptor sIL-6R during an acute inflammatory response. We also found significantly higher IL-6 levels in children with Kawasaki disease compared to children with blood culture-negative febrile illnesses. In five children with Kawasaki disease we measured serum IL-6 before and after IVIG and assessed the clinical response to IVIG therapy. Therapy with IVIG was followed by a rapid resolution of symptoms in Kawasaki disease, with a significant decrease in serum IL-6. The attenuation of proinflammatory cytokine responses, especially IL-6, following infusions of IVIG may play an integral role in the rapid resolution of symptoms and decrease in the acute-phase proteins in children with Kawasaki disease. Cells of normal children were found to respond to the IVIG in a manner similar to that of the Kawasaki children.     

Engineering Human Peripheral Blood Stem Cell Grafts that Are Depleted of Naïve T Cells and Retain Functional Pathogen-Specific Memory T Cells

Graft-versus-host disease (GVHD) is a frequent major complication of allogeneic hematopoietic cell transplantation (HCT). Approaches that selectively deplete T cells that cause GVHD from allogeneic stem cell grafts and preserve T cells specific for pathogens may improve HCT outcomes. It has been hypothesized that the majority of T cells that can cause GVHD reside within the naïve T cell (T_N) subset, and previous studies performed in mouse models and with human cells in vitro support this hypothesis. As a prelude to translating these findings to the clinic, we developed and evaluated a novel 2-step clinically compliant procedure for manipulating peripheral blood stem cells (PBSC) to remove T_N, preserve CD34⁺ hematopoietic stem cells, and provide for a fixed dose of memory T cells (T_M) that includes T cells with specificity for common opportunistic pathogens encountered after HCT. Our studies demonstrate effective and reproducible performance of the immunomagnetic cell selection procedure for depleting T_N. Moreover, after cell processing, the CD45RA-depleted PBSC products are enriched for CD4⁺ and CD8⁺ T_M with a central memory phenotype and contain T_M cells that are capable of proliferating and producing effector cytokines in response to opportunistic pathogens.     

Signals Analysis and Clinical Validation of Blood and Oxygen Data in Human Brain

INTRODUCTION Ithadlongbeenbelievedthatthescatteringandabsorptionofnearinfrared throughhumantissuescanonlyofferthesurfaceinformationoftissues〔1〕,butJob-sis〔2〕foundthatinnerinformationofbraincouldbeacquiredwhennearinfraredare usedtoirradiatebrainin1977.Thistechnologyhasstimulatedresearcherstostudy theactivesofhumanbrainwithscatteringofnearinfraredlightandhasdifferent namesindifferenttimes,likenearinfraredspectrum(NIRS),diffusionoftomogra-phy(DOT)andnearinfraredimaging(NIRI).Recentde…     

Distinct Pattern of Human Vδ1 γδ T Cells Recognizing MICA

γδT cells represent one unique recognition pattern,the limited recognition,which distinguishes from the specificrecognition for αβT cells and pattern recognition for macrophages.Vδ1 γδT cell is the major subset of human γδT cells,which predominates in mucosal tissue including the intestinal epithelia.Presently,a few antigens thathuman Vδ1TCR can recognize have been identified.Among them,MHC class I chain-related molecules A (MICA)have been studied most intensively.Besides Vδ1TCR,MICA is also the ligand of NKG2D,a C-type lectin-likeactivating immunoreceptor.In human,only Vδ1 cells can simultaneously express both types of receptors of MICAwhile NK cells,αβT cells and other subsets of γδT cells likewise express NKG2D.Although the precisemechanisms are still enigmatic,this distinct pattern of Vδ1 cells recognizing MICA predicts unique biologicalsignificance of Vδ1 cells in immune defense.Recent years,some progresses have been made in this issue.In thisreview we summarize the related reports and put forward some novel views based on our group's studies.Cellular& Molecular Immunology.2005;2(4):253-258.     

Analysis of the CDR3 Length Repertoire and the Diversity of TCRα Chain in Human Peripheral Blood T Lymphocytes

Analysis of complementarity determining region 3 （CDR3） length of T lymphocyte receptors （TCRs） by immunoscope spectratyping technique has been used successfully to investigate the diversity of TCR in autoimmune diseases and infection diseases. In this study, we investigated the patterns of CDR3 length distribution for all 32 TCR AV gene families in human peripheral blood lymphocytes of four normal volunteers by the immunoscope spectratyping technique. It was found that PCR products exhibited an obscure band on 1.5% agarose gel electrophoresis. Each TCR AV family exhibited more than 8 bands on 6% sequencing gel electrophoresis. The CDR3 spectratyping of all TCR AV families showed a standard Gaussian distribution with different CDR3 length, and the expression frequency of CDR3 was similar among the gene families. Most of CDR3 in TCR AV family recombine in frame. However, some of the CDR3 showed out-of frame gene rearrangement. Additionally, we found that in some of TCR AV families there were 18 amino acid discrepancies between the longest CDR3 and shortest CDR3. These results may be helpful to further study the recombination mechanism of human TCR genes, the TCR CDR3 gene repertoire, and the repertoire drift in health people and disease state. Cellular ＆ Molecular Immunology.     

Antigen-Driven Clonal Accumulation of Peritoneal γδ T Cells in vivo

How the clonality of γδ T cells changes in response to exogenous antigens is uncertain. Here we analyzed kinetics of Vγ1.1 and Vγ2 T cell clonality after intraperitoneal injection of purified protein derivatives (PPD) by the heterogeneity of the third complementarity determining region (CDR3) length in Vγ1.1-Jγ4-Cγ4 and Vγ2-Jγ1-Cγ1 junctions. The V-J junctions were analyzed in intrahepatic lymphocytes (IHL), spleen cells, and peritoneal exudate cells (PEC) by polyacrylamide gel electrophoresis. γδ T cells expressing Vγ1.1 and Vγ2 genes were heterogeneous in normal mice. Accumulation of specific Vγ1.1 T cell clones was transiently detected 7 days after the injection in PEC, but no accumulation was observed in IHL and spleen cells. The accumulated clones disappeared by 4 weeks. Transient accumulation of Vγ2 T cell clones was also observed in PEC at the early phase after the injection. These results suggest that γδ T cells with specific TCR respond to PPD and temporary accumulate in the peritoneal cavity, but not in liver and spleen.     

Distinctive Pattern of Cytokine Production and Adhesion Molecule Expression in Peripheral Blood Memory CD4+ T Cells from Patients with Active Crohn’s Disease

An expansion of both circulating and intestinal lamina propria CD4⁺CD45RO⁺ T cells has been described in patients with Crohn’s disease. We studied both the cytokine profile and the expression of adhesion molecules on this T-cell subset. Peripheral blood CD4⁺CD45RO⁺ T cells from patients with Crohn’s disease (n=45) were assessed by flow cytometry and RT-PCR methods. The cytokine profile was also measured in intestinal lamina propria from seven patients. They were classified according to the CDAI and the results were compared with those of patients with ulcerative colitis (n=21) and noninflammatory intestinal conditions (n=15), and healthy controls (n=39). The mean percentage of circulating CD4⁺CD45RO⁺ T cells producing intracellular TNF was higher in active than in inactive Crohn’s disease patients (p < 0.001), active (p = 0.49) and inactive ulcerative colitis (p = 0.019), and healthy controls (p =0. 017). TNF expression correlated with CDAI (p < 0.001). An increased expression of intracellular IL-2, IL-6, and IL-10 in active Crohn’s disease patients was also found. CD62L was downregulated in active Crohn’s disease patients while no differences were observed in CD49d and CD11a expression. Lamina propria CD4⁺CD45RO⁺ T cells from active Crohn’s disease lesions showed an increased intracellular staining of TNF, IFN-γ, and IL-10. Both peripheral and intestinal mucosa CD4⁺CD45RO⁺ T cells from active Crohn’s disease patients show an increased production of TNF. In addition, the circulating CD4⁺CD45RO⁺ T-cell subset expresses a pattern of adhesion molecules that promotes homing to extranodal lymphoid tissues. This T-cell subset may play a relevant role in the immunopathogenesis of Crohn’s disease.Dr. García de Tena and Dr. Manzano are joint first authors     

Regulation of Interleukin‐2 Induced Soluble Fas Ligand Release from Human Peripheral Blood Mononuclear Cells

Adjuvant interleukin (IL)‐2 immunotherapy has been used in the treatment of different malignant dieseases. However, clinical results have been rather disappointing. Therefore, further investigations on IL‐2‐induced mediators of cytotoxicity seem to be necessary in order to possibly create cytokine cocktails which could enhance the IL‐2‐induced cytotoxicity. We therefore investigated the regulation of IL‐2‐induced release of soluble Fas Ligand (sFasL), since this factor is known to possess anti‐tumor activities. In CD3‐stimulated peripheral blood mononuclear cells IL‐2 induced sFasL in a dose‐dependent fashion. Maximum sFasL concentrations were obtained after stimulation of MNC for 120 hrs. Inhibition of endogenous IL‐12 production significantly reduced IL‐2‐mediated sFasL release by about 25%. In contrast, addition of IL‐12 enhanced the IL‐2‐induced sFasL about 1,5‐fold. IL‐10 and IL‐4 reduced the IL‐2‐stimulated sFasL by about 30%. Interestingly, these suppressive effects could be antagonized by the addition of IL‐12. Not only exogenous IL‐10 but also endogenously produced IL‐10 decreased the sFasL release to that extent which had been stimulated by IL‐12. Since IL‐12 and IL‐10 only marginally influenced the IL‐2‐mediated cell proliferation as well as the IL‐2‐induced cell death, the IL‐12‐ and IL‐10‐controlled sFasL release seems to be based on an enhanced production per cell. However, the increase in cell numbers as well as the decrease of viability during cell culture might additionally contribute to the IL‐2‐induced increase of sFasL release. This secondary effect might explain why IL‐2‐mediated sFasL production is only partially controlled by regulatory cytokines such as IL‐4, IL‐10 or IL‐12. In conclusion, addition of IL‐12 might increase the efficacy of IL‐2 immunotherapy by inhibition of the IL‐10‐mediated negative feed‐back loop on IL‐2‐mediated sFasL release.     

CD4+CD25+CD127low/? T Cells: A More Specific Treg Population in Human Peripheral Blood

The quantitative identification and enrichment of viable regulatory T cells (Treg) requires reliable surface markers that are selectively expressed on Treg. Foxp3 is the accepted marker of nTreg, but it cannot be used to isolate cells for functional studies. In this study, we compared four staining profiles of Treg, including CD4⁺CD25^high T cells, CD4⁺CD39⁺ T cells, CD4⁺CD73⁺ T cells, and CD4⁺CD25⁺CD127^low/? T cells. We found that CD4⁺CD25⁺CD127^low/? T cells expressed the highest level of Foxp3 and had the strongest correlation with CD4⁺CD25⁺Foxp3⁺ T cells, the accepted identifying characteristics for ??real?? nTreg cells. Moreover, functional data showed that CD4⁺CD25⁺CD127^low/? T cells could effectively suppress the proliferation of CD4⁺CD25^? T cells, suggesting that compared with the other three populations, CD4⁺CD25⁺CD127^low/? T cells best fit the definition of naturally occurring regulatory T cells in human peripheral blood. Finally, we showed that CD4⁺CD25⁺CD127^low/? can be used to quantitate Treg cells in individuals with systemic lupus erythematosus supporting the use of CD4⁺CD25⁺CD127^low/? to identify human Treg cells.     

Kinetics of Effector Functions and Phenotype of Virus-Specific and γδ T Lymphocytes in Primary Human Cytomegalovirus Infection During Pregnancy

The T-cell response to human cytomegalovirus (HCMV) primary infection was analyzed in 27 pregnant women during the first year after primary HCMV infection. Pregnant women with remote HCMV infection were enrolled as controls. Interferon-γ-producing T cells were readily detected at levels comparable (CD4⁺) or higher (CD8⁺) than controls, whereas the CD4⁺ and CD8⁺ lymphoproliferative response as well as IL-2 production was significantly reduced with respect to controls for at least 9 months after infection. In addition, CD45RA re-expression as well as cytotoxic T lymphocyte activity and perforin expression were the major components of the adaptive CD4⁺ and CD8⁺ T-cell immune response, while Vδ2⁻ γδ T-cell expansion in response to HCMV infection followed kinetics similar to that of CD8⁺ T cells. Reduced CD45RA re-expression directly correlated with HCMV transmission to the fetus, thus providing an important prognostic parameter.     

Characteristics of the Vδ2 CDR3 Sequence of Peripheral γδ T Cells in Patients with Pulmonary Tuberculosis and Identification of a New Tuberculosis-Related Antigen Peptide

Antigen-specific γδ T cells may play an important role in the immune response to Mycobacterium tuberculosis. However, little is known about the characteristics of the length distribution of the δ2-chain complementarity determining region 3 (δ2 CDR3) of the γδ T-cell receptor (TCR) in patients with active pulmonary tuberculosis (TB) on a large scale. In addition, M. tuberculosis-activated γδ T cells potentially inhibit intracellular mycobacterial growth, but phosphoantigen-activated γδ T cells do not. Only a few M. tuberculosis-related antigen peptides or proteins that are recognized by γδ TCR have been identified. Twenty-four healthy donors (HDs) and 27 TB patients were included in the present study. The gene-scanning technique found that the δ2 CDR3 length distribution patterns of γδ TCR in TB patients were perturbed, and each pattern included different predominant CDR3 sequences. The predominant δ2 CDR3 sequences of γδ TCRs, which originated from TB patients and HD γδ T cells that were stimulated by M. tuberculosis heat resistance antigen (Mtb-HAg), were used as probes to screen peptides recognized by γδ TCR using a phage display library. We identified four peptides that bound to the predominant δ2 CDR3 fragments and showed homology to M. tuberculosis genes in a BLAST search. Notably, one peptide was related to M. tuberculosis H37Rv (QHIPKPP), and this fragment was confirmed as a ligand for the γδ TCR. Two fragments, Ag1 and Ag2, activated γδ T cells from HD or TB patients. In summary, the δ2 CDR3 lineage of TB patients apparently drifts, and the predominant δ2 CDR3 sequence that recognizes M. tuberculosis may exhibit specificity. The identified M. tuberculosis-related antigen peptides may be used as vaccines or adjuvants for protective immunity against M. tuberculosis.     

Human Umbilical Cord Mesenchymal Stromal Cells Support Viability of Umbilical Cord Blood Hematopoietic Stem Cells but not the “Stemness” of Their Progeny in Co-Culture

Bulletin of Experimental Biology and Medicine - Cell—cell interactions and the ability of mesenchymal stromal cells to support the expansion of hematopoietic progenitor cells were studied in...     

Trypanosoma cruzi-Induced Activation of Functionally Distinct αβ and γδ CD4? CD8? T Cells in Individuals with Polar Forms of Chagas' Disease

CD4⁻ CD8⁻ (double-negative [DN]) T cells have recently been shown to display important immunological functions in human diseases. They express γδ or αβ T-cell receptors that recognize lipid/glycolipid antigens presented via the nonclassical major histocompatibility complex molecules of the CD1 family. We recently demonstrated that while αβ DN T cells serve primarily to express inflammatory cytokines, γδ DN T cells express mainly interleukin-10 (IL-10) in patients with cutaneous leishmaniasis. We also demonstrated a correlation between DN T cells and the expression of gamma interferon in the acute phase of Trypanosoma cruzi experimental infection. In this work, we sought to investigate whether αβ or γδ DN T cells display distinct immunoregulatory potentials in patients with polar forms of human Chagas'' disease. Our data showed that in vitro infection with T. cruzi leads to expansion of DN T cells in patients with the indeterminate and severe cardiac clinical forms of the disease. However, while αβ DN T cells primarily produce inflammatory cytokines in both forms of the disease, γδ DN T cells display a marked, significant increase in antigen-specific IL-10 expression in indeterminate patients relative to cardiac patients. Finally, higher frequencies of the IL-10-producing γδ DN T cells were correlated with improved clinical measures of cardiac function in the patients, suggesting a protective role for these cells in Chagas'' disease. Taken together, these data show distinct functional characteristics for αβ and γδ DN T cells associated with distinct morbidity rates and clinical forms in human Chagas'' disease.T-cell activation is a key event in the establishment of immune responses directed toward intracellular pathogens. Depending on the functional capacity of the activated T cells, the fate of the infection may take different paths either toward a protective or a pathogenic outcome. While it is important that a strong, activated immune response is elicited early on in the infection in order to eliminate (or control) the pathogen, the further control of this activation is necessary to reestablish homeostasis, avoiding tissue damage (17, 25).One hallmark of most parasitic infections is that the great majority of individuals are able to trigger innate immunity and elicit an activated T-cell response during the acute infection, leading to the control of the parasite and establishment of a chronic infection. Interestingly, while many individuals develop severe forms of parasitic diseases once infection progresses to the chronic phase, most patients develop relatively mild forms, allowing for a host-parasite coexistence. One such example is observed upon human infection with the protozoan parasite Trypanosoma cruzi, which leads to Chagas'' disease. As a result of thousands of years of coevolution between human host and the parasite (6), most infected individuals develop an asymptomatic, or “indeterminate” (I), form of Chagas'' disease. This form is characterized by a lack of clinical signs and symptoms and has been associated predominantly with a modulatory cellular immune response based on cytokine profiles and downregulatory molecule expression (5, 20, 48, 49, 51). Chronic patients may also develop symptomatic clinical forms, mainly with digestive or cardiac alterations. Differential geographical prevalence of Chagas'' disease clinical forms has been reported. In Brazil, 15 to 30% of Chagas'' patients display the cardiac form, which is present in 20 states, while the digestive cases, observed in about 10% of infected individuals, have been reported in four states in the central region of the country (53). The digestive form is frequently found in Chile but is practically absent in Central America (42). These geographical differences might be related, in part, to host genetics and immune responses of local human populations, but it is believed that they are also related to the genetic diversity of T. cruzi strains (11). Different strains of parasite display tropism for different tissues, and, thus, an important factor determining the clinical course of disease might be the specific pool of infecting clones and their specific tropisms (29). However, a possible role for environmental, nutritional, and immunological aspects of the host cannot be discounted. While digestive and cardiac forms present significant morbidity, the cardiac form is the one associated with highest mortality. It is caused by neuronal and cardiomyocyte damage, ultimately resulting in ventricular dilation and subsequent functional heart failure, which can lead to death (44). Cardiac patients display a T-cell-mediated inflammatory response in situ (13, 24, 41), which is responsible for the pathology; this inflammatory profile is also observed in circulating activated T cells found at high frequencies in these patients (2, 16, 19, 32). Although it is clear that a plethora of parasite and host factors influences the clinical outcome of Chagas'' disease, recent studies have suggested that activation of functionally distinct T-cell populations in T. cruzi-infected individuals may be responsible for the establishment of different clinical forms (17, 20). Thus, identifying these populations and the factors responsible for their activation will be critical for driving immune-based interventions to prevent pathology.While the great majority of T cells express either the CD4 or the CD8 molecules, which are important for stabilizing the peptide-major histocompatibility complex (MHC) complex and which favor T-cell activation, a minority population of T cells that do not express CD4 or CD8 molecules has been identified in humans (8, 10, 27, 37). These double-negative (DN) T cells have been shown to be important sources of immunoregulatory cytokines in human leishmaniasis (4), to display modulatory functions (38), but also, under different circumstances, to display cytolytic activity (10, 36). A subpopulation of DN T cells is activated through the engagement of αβ or γδ T-cell receptors (TCRs) in the recognition of nonclassical MHC molecules of the CD1 family, presenting lipid or glycolipid antigens (36). This particular lipid/glycolipid antigenic recognition, as well as the immunoregulatory potential and susceptibility to chronic stimulation of these cells, highlights the important role these cells play in parasitic infections.In our work with Bottrel et al., we determined that DN lymphocytes were the second most prevalent cell type producing gamma interferon (IFN-γ) in human cutaneous leishmaniasis and that this IFN-γ production was seen after short-term cultures with medium alone, as well as after stimulation with soluble Leishmania antigen (SLA) (9). The novel work of Antonelli et al. went on to demonstrate that DN T cells composed of two different cell populations are present in the blood of individuals infected with Leishmania braziliensis and that DN T cells expressing the αβ TCR displayed a profile consistent with activation of leishmanicidal and inflammatory activities (higher IFN-γ and tumor necrosis factor alpha [TNF-α]) while the DN subpopulation expressing γδ TCR had a modulatory potential via higher production of interleukin-10 (IL-10) (4). Interestingly, IFN-γ production has been associated with pathogenic responses in human leishmaniasis in more than one clinical form (3, 7, 22). We recently demonstrated that rats infected with the CL-Brenner clone of T. cruzi displayed a marked increase in the frequency of circulating DN T cells during the acute phase of infection (33). Taken together, these data led to the question of the role that DN T-cell subpopulations play in the clinical dichotomy of chronic human Chagas'' disease.To answer these questions, we investigated the immunoregulatory potential of DN T cells in patients with the two polar forms of Chagas'' disease: indeterminate (I) and dilated cardiac (DC). Our data demonstrated that although no quantitative differences were seen with regard to the nonstimulated frequency of DN αβ and γδ T-cell subpopulations between patients and nonchagasic individuals, in vitro infection with trypomastigote forms of T. cruzi induced a marked increase in the frequency of these cells from chagasic patients. Moreover, the expanded αβ DN T cells displayed a greater inflammatory potential from cardiac patients than from indeterminate patients. This was accompanied by a greater down-modulatory ratio of IL-10 to inflammatory cytokine frequencies by γδ DN T cells from individuals with indeterminate disease, suggesting distinct roles for these cells in modulating the response in chronic Chagas'' disease. Finally, we observed a correlation between higher frequencies of IL-10-producing γδ DN T cells and improved clinical measures of cardiac function, suggesting a protective role for these cells in human Chagas'' disease. These data indicate that functionally distinct DN T cells are present in Chagas'' disease patients and that they are associated with the resulting morbidity of the disease.     

Production of TNF-α and IL-1β by Peripheral Blood Mononuclear Cells in Carriers of Different Allele Variants of the Gene

Associations of cytokine production by mononuclear cells and the TNF-α genetic polymorphism in positions -238, -308, -376, -857, -1031, and of IL-1β in positions -31 and +3954 were studied. The data on distribution of allele and genotype incidence and on the level of spontaneous and mitogen-induced production of these cytokines by donor mononuclear cells demonstrated a statistically significant association of TNF-α production by mononuclear cells with polymorphic variants of the gene promoter regions -238 > A (rs361525) and -857C > T (rs1799724). Carriers of -238GG/-308GG/-857CC/-1031NC genotype were characterized by low production of TNF-α, while carriers of -31TT/+3954CT genotype were characterized by low IL-1β stimulation index in response to mitogen in comparison with carriers of other genotype combinations.