Gender effect on in vitro lymphocyte subset levels of healthy individuals

Differences in gender immune response have resulted in differences in immune protection and susceptibility to inflammatory diseases. Cultured peripheral blood mononuclear cells (PBMC) are widely used in immunomodulation studies, yet the influence of gender is usually not considered. We examined the effect of in vitro culture and phytohaemagglutinin (PHA) stimulation on PBMC lymphocyte subsets using flowcytometry. Full blood counts of whole blood showed higher levels of lymphocyte in male subjects. Lymphocyte subsets enumeration revealed higher NK cell counts in males and higher B cells in females. Cultured PBMC resulted in significant increases in B and total T cell percentages among females and NK cells among males. PHA stimulated significantly increased percentages of NK and total T cells in males and total activated T cells (CD69+) in females. Our results showed significant gender differences in lymphocyte subsets in cultured conditions. This may affect experimental outcome.     

Sex- and age-specific clinical and immunological features of coronavirus disease 2019

To simultaneously determine clinical and immunological responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old females and males, 681 coronavirus disease 2019 (COVID-19) patients and 369 normal controls (NCs) were analyzed based on age and sex classifications using multiple linear regression analysis. Compared to the age-matched NCs, both young and old male and female non-comorbid COVID-19 patients had lower lymphocyte counts and alanine aminotransferase (ALT) concentration, and only young male and female patients had lower neutrophil counts. Compared to young patients, both old males and females had significantly higher plasma ALT and AST concentrations. Compared to young and old females, age-matched males had higher plasma ALT and AST concentrations, but only young males had higher C-reactive protein (CRP) concentration. Compared to females, old males, but not young males, showed higher incidence of critical illness. Compared to young patients, old females had more leukocyte and neutrophil counts above the normal upper limit and B cell count below the normal lower limit (NLL), while old males had more lymphocyte and natural killer (NK) cell counts below the NLL. No sex or age associations with B cell and NK cell counts were observed. However, there were age-dependent decreases in CD8⁺ T-cell counts in both male and female COVID-19 patients. Age was negatively associated with CD8⁺ T cell counts but positively associated with neutrophil count, CRP, ALT, and AST concentrations, and sex (females) was negatively associated with neutrophil count, CRP, ALT, and AST concentrations. The present study suggests that SARS-CoV-2 infection mainly induced 1) beneficial sex (female)-related differences regarding reduced COVID-19 disease severity and negative associations with inflammatory responses and liver damage, and 2) harmful age-related differences relating to negative associations with CD8⁺ T cell count and positive associations with inflammatory responses and liver damage. Thus, sex and age are biological variables that should be considered in the prevention and treatment of COVID-19.     

Effects of labour and medication on major lymphocyte subsets in cord

It is envisaged that flow cytometric analysis of lymphocyte subsets in cord blood may be used as a biomarker for effects on the immune system of exposure to environmental factors. In order to investigate the possible application of this parameter, we first studied the effects of other factors that may influence the outcome of subset analysis in cord blood. FACS analysis was performed in 112 pairs of umbilical cord blood and of peripheral maternal blood sampled at labour. Whereas in maternal blood no statistically significant effects of medication during labour on T lymphocyte numbers and NK cells were found, in oxytocin and in oxytocin and prostaglandin treated mothers B cell numbers showed a statistically significant increase. In cord blood, the course of labour and or medication during labour were identified as the most important factors determining distribution of major lymphocyte subsets. In cord blood after deliveries without medication or after neuroplegic analgesia NPA, the mean percentage of cord blood T lymphocytes CD3 was highest 59 and that of NK lymphocytes CD3- CD16 56 lowest 20 . The mean percentage of T lymphocytes was significantly lower 52 and that of NK lymphocytes higher 28 in cord blood where deliveries were done under NPA in combination with infusion of oxytocin. The combination of NPA with oxytocin and induction of labour by prostaglandin E2 led to a further reduction of T lymphocytes and an increase of NK cells 39 and 38 respectively. The changes in ratio of T and NK lymphocytes were due both to decreasing absolute counts of T lymphocytes and increasing counts of NK lymphocytes. Thus, the effects of labour and or medication during labour must be taken into account when this parameter is applied as a potential biomarker of effects of environmental factors on the immune system.     

Effects of labour and medication on major lymphocyte subsets in cord

It is envisaged that flow cytometric analysis of lymphocyte subsets in cord blood may be used as a biomarker for effects on the immune system of exposure to environmental factors. In order to investigate the possible application of this parameter, we first studied the effects of other factors that may influence the outcome of subset analysis in cord blood. FACS analysis was performed in 112 pairs of umbilical cord blood and of peripheral maternal blood sampled at labour. Whereas in maternal blood no statistically significant effects of medication during labour on T lymphocyte numbers and NK cells were found, in oxytocin and in oxytocin and prostaglandin treated mothers B cell numbers showed a statistically significant increase. In cord blood, the course of labour and or medication during labour were identified as the most important factors determining distribution of major lymphocyte subsets. In cord blood after deliveries without medication or after neuroplegic analgesia NPA , the mean percentage of cord blood T lymphocytes CD3 was highest 59 and that of NK lymphocytes CD3- CD16 56 lowest 20 . The mean percentage of T lymphocytes was significantly lower 52 and that of NK lymphocytes higher 28 in cord blood where deliveries were done under NPA in combination with infusion of oxytocin. The combination of NPA with oxytocin and induction of labour by prostaglandin E2 led to a further reduction of T lymphocytes and an increase of NK cells 39 and 38 respectively . The changes in ratio of T and NK lymphocytes were due both to decreasing absolute counts of T lymphocytes and increasing counts of NK lymphocytes. Thus, the effects of labour and or medication during labour must be taken into account when this parameter is applied as a potential biomarker of effects of environmental factors on the immune system.     

山西省健康成年人淋巴细胞亚群正常参考值范围

目的:建立山西省健康成人外周血淋巴细胞亚群的正常参考值范围,为机体免疫状态的分析和肿瘤患者的免疫评估提供理论依据。方法:选取山西省1 238例健康成人体检人群,采用流式细胞术测定外周血淋巴细胞亚群的绝对计数和相对计数。结果:确定了健康成人外周血淋巴细胞表达水平,并发现CD3~+T细胞相对计数和绝对计数、CD4~+T细胞相对计数、CD8~+T细胞相对计数和绝对计数、NK细胞相对计数和绝对计数、CD19细胞相对计数和绝对计数、CD4/CD8比值在不同年龄组间存在显著差异性(P0.05);不同性别之间CD8~+T细胞相对计数、CD4~+T细胞绝对计数和CD19细胞绝对计数无统计学意义,CD3~+T细胞、CD8~+T细胞、NK细胞相对计数和绝对计数、CD4~+T细胞、CD19细胞相对计数均存在显著差异(P0.05)。结论:初步建立了山西省健康成年人外周血淋巴细胞亚群参考值范围,为机体免疫功能的评价和肿瘤免疫治疗、诊断提供了参考依据。     

多发性骨髓瘤患者外周血淋巴细胞亚群分析及与预后因子的相关性

目的通过对多发性骨髓瘤（MM）患者外周血淋巴细胞亚群的检测以评价MM患者机体的免疫功能状态。方法采用流式细胞术检测36例MM患者和25例健康志愿者外周血T、B淋巴细胞、NK细胞及CD4＋CD25＋T细胞的表达。结果与正常对照组相比,MM患者外周血的CD4、CD19细胞的表达显著下调,CD8细胞的表达显著上调,CD4/CD8比值则显著降低（P〈0.05或〈0.01）;MM患者外周血的CD4＋CD25＋T细胞占CD3＋T细胞的比例明显增高（P〈0.01）,且与血清中的β2-MG浓度成正相关（γ=0.56,P〈0.05）。结论 MM患者体内存在淋巴细胞亚群的异常表达、CD4＋CD25＋Treg细胞的异常扩增,可能是MM患者体内广泛存在免疫缺陷的一个主要原因。     

基于流式细胞术快速检测人鼻黏膜组织淋巴细胞亚群的实验研究

目的：基于流式细胞术检测正常人的鼻黏膜组织中的淋巴细胞亚群(CD3+T、CD4+T、CD8+T、NK、CD19+B)的比例,初步探讨 鼻局部黏膜免疫功能的意义,为鼻局部黏膜免疫性疾病的研究提供更多的参考。方法：用鼻黏膜刮匙获取21 例正常人的鼻黏膜 组织,并抽取其外周血2 mL。采用流式细胞术分别检测其鼻黏膜和外周血中的淋巴细胞亚群(包括CD3+T、CD4+T、CD8+T、NK、 CD19+B)分别所占的比例。结果：鼻黏膜和外周血的淋巴细胞亚群存在很大差异,与外周血相比,CD3+T 比例增加（t=15.34,P<0. 0001),CD4+T 比例降低(t=5.952,P<0.0001)、CD8+T 比例增加(t=12.44,P<0.0001)、NK 比例降低(t=4.865,P<0.0001)、CD19+B 比例降 低(t=15.56,P<0.0001),CD4+T/CD8+T 降低。结论：流式细胞术可以用来检测鼻黏膜的淋巴细胞亚群,鼻黏膜的淋巴细胞亚群和外 周血的淋巴细胞亚群存在很大差异,这种差异体现鼻黏膜组织独特的局部黏膜免疫功能,本方法为变应性鼻炎的研究提供了新 的研究途径。     

An unusual CD56(bright) CD16(low) NK cell subset dominates the early posttransplant period following HLA-matched hematopoietic stem cell transplantation

The expansion of the cytokine-producing CD56(bright) NK cell subset is a main feature of lymphocyte reconstitution after allogeneic hematopoietic stem cell transplantation (HSCT). We investigated phenotypes and functions of CD56(bright) and CD56(dim) NK subsets from 43 HLA-matched non-T cell-depleted HSCT donor-recipient pairs. The early expansion of CD56(bright) NK cells gradually declined in the posttransplant period but still persisted for at least 1 year and was characterized by the emergence of an unusual CD56(bright)CD16(low) subset with an intermediate maturation profile. The activating receptors NKG2D and NKp46, but also the inhibitory receptor NKG2A, were overexpressed compared with donor CD56(bright) populations. Recipient CD56(bright) NK cells produced higher amounts of IFN-gamma than did their respective donors and were competent for degranulation. Intracellular perforin content was increased in CD56(bright) NK cells as well as in T cells compared with donors. IL-15, the levels of which were increased in the posttransplant period, is a major candidate to mediate these changes. IL-15 serum levels and intracellular T cell perforin were significantly higher in recipients with acute graft-vs-host disease. Altogether, CD56(bright) NK cells postallogeneic HSCT exhibit peculiar phenotypic and functional properties. Functional interactions between this subset and T cells may be important in shaping the immune response after HSCT.     

Sustained impairment of IFN-gamma secretion in suppressed HIV-infected patients despite mature NK cell recovery: evidence for a defective reconstitution of innate immunity

The impairment of NK cell functions in the course of HIV infection contributes to a decreased resistance against HIV and other pathogens. We analyzed the proportion of mature and immature NK cell subsets, and measured subsets of IFN-gamma and TNF-alpha-producing NK and T cells in viremic or therapy-suppressed HIV-infected subjects, and noninfected control donors. Viremic HIV(+) individuals had significantly lower proportions of mature CD3(-)/CD161(+)/CD56(+) NK cells and of IFN-gamma-producing NK cells compared with noninfected donors, independent of CD4(+) T cell counts. HIV-infected subjects with undetectable viral load recovered mature CD3(-)/CD161(+)/CD56(+) NK cells and cytotoxicity against tumor (K562) and HSV-infected target cells to percentages comparable with those of uninfected individuals, but their NK cells remained impaired in their ability to produce IFN-gamma. In parallel to these ex vivo findings, in vitro NK cell differentiation of CD34-positive cord blood precursors in the presence of R5 or X4 HIV-1 resulted in the production of NK cells with a normal mature phenotype, but lacking the ability to produce IFN-gamma, whereas coculture of uninfected PBMC with HIV failed to affect mature NK cell properties or IFN-gamma secretion. Altogether, our findings support the hypothesis that mature NK cell phenotype may be uncoupled from some mature functions following highly active antiretroviral therapy-mediated suppression of HIV-1, and indicate that relevant innate immune functions of NK cell subsets may remain altered despite effective viral suppression following antiretroviral treatment.     

Active but not inactive granulomatosis with polyangiitis is associated with decreased and phenotypically and functionally altered CD56dim natural killer cells

BackgroundThe role of natural killer (NK) cells in granulomatosis with polyangiitis (GPA) is poorly understood. We recently reported that peripheral blood NK cell percentages correlate with the suppression of GPA activity (cohort I). The purpose of the current study was to further characterize NK cell subsets, phenotype and function in a second GPA cohort (cohort II).MethodsPeripheral blood lymphocyte subsets were analyzed at a clinical diagnostic laboratory. Clinical data were extracted from medical records and patients were grouped according to their activity state (remission vs. active/non-remission). Separate analysis (cohort II, n = 22) and combined analysis (cohorts I and II, n = 34/57) of NK cell counts/percentages was performed. NK cell subsets and phenotypes were analyzed by multicolor flow cytometry. Cytotoxicity assays were performed using ⁵¹Cr-labeled K562 target cells.ResultsIn cohort II, NK cell counts were lower than the lower limit of normal in active GPA, despite normal percentages due to lymphopenia. NK cell counts, but not other lymphocyte counts, were significantly higher in remission. Combined analysis of cohorts I and II confirmed decreased NK cell counts in active GPA and increased percentages in long-term remission. Follow-up measurements of six patients revealed increasing NK cell percentages during successful induction therapy. Multicolor analysis from cohort II revealed that in active GPA, the CD56^dim subset was responsible for decreased NK cell counts, expressed more frequently CD69, downregulated the Fc-receptor CD16 and upregulated the adhesion molecule CD54, the chemokine receptor CCR5 and the activating receptor NKG2C. In remission, these markers were unaltered or marginally altered. All other receptors investigated (NKp30, NKp44, NKp46, NKG2D, DNAM1, 2B4, CRACC, 41BB) remained unchanged. Natural cytotoxicity was not detectable in most patients with active GPA, but was restored in remission.ConclusionsNK cell numbers correlate inversely with GPA activity. Reduced CD56^dim NK cells in active GPA have an activated phenotype, which intriguingly is associated with profound deficiency in cytotoxicity. These data suggest a function for NK cells in the pathogenesis and/or modulation of inflammation in GPA. NK cell numbers, phenotype (CD16, CD69, NKG2C) or overall natural cytotoxicity are promising candidates to serve as clinical biomarkers to determine GPA activity.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-016-1098-7) contains supplementary material, which is available to authorized users.     

Distinctive lack of CD48 expression in subsets of human dendritic cells tunes NK cell activation

CD48 is a glycosyl phosphatidylinositol anchor protein known to be virtually expressed by all human leukocytes. Its ligand, 2B4, is a signaling lymphocyte activation molecule-related receptor involved in NK cell activation. Because dendritic cells (DCs) are strong inducers of NK cell functions, we analyzed the expression of CD48 in different human DC subsets. We observed that monocytes differentiating in DCs promptly down-regulate CD48. Similarly, DCs isolated from inflamed lymph nodes generally do not express CD48. Plasmocytoid DCs do not express CD48 either, whereas myeloid DCs harbored in blood, bone marrow, and thymus express it. In addition, we showed that CD48 expression in DCs affects NK cell functions during NK/DC cross-talk, because NK cells obtained from normal donors and from X-linked lymphoproliferative disease patients are, respectively, triggered or inhibited by DCs expressing surface CD48. Remarkably, IFN-gamma production by lymph node NK cells, in contrast to blood NK cells, can be negatively modulated by 2B4/CD48 interactions, indicating a 2B4 inhibitory pathway in lymph node NK cells. Therefore, the CD48 deficiency of DCs harbored in inflamed lymph nodes that we report in this study might be relevant to successfully activate lymph node NK cells in the early phase of the immune response. Our results show that distinct subsets of human DCs, differently from all other mononuclear hemopoietic cells, specifically do not express CD48. Moreover, the expression of CD48 depends on the anatomic location of DCs and might be related to the tissue-specific 2B4 function (activating or inhibitory) of the NK cells with which they interact.     

In vitro modulation of cytokine production by lymphocytes in human coccidioidomycosis

The modulation of the cytokine response to coccidioidal antigen by lymphocytes from donors with coccidioidomycosis was examined. In initial experiments, samples from 13 healthy immune donors and seven donors with active coccidioidomycosis anergic to the coccidioidal antigen T27K were assessed for CD3 lymphocyte expression of intracellular IFN-gamma using whole blood analysis. Addition of 10 ng/ml of recombinant IL-12 significantly increased response to T27K among immune and anergic subjects (p<0.05), but the percent of cells expressing IFN-gamma was still significantly greater for immune subjects. Among immune donors, the percentage of CD3 lymphocytes expressing IFN-gamma was significantly reduced with the addition of 10 ng/ml of recombinant IL-4, IL-10, TGF-beta, or their combination (for all, p<0.05). Among anergic donors, addition of 10 ng/ml of anti-IL-10 significantly increased IFN-gamma production (p<0.05), but addition of anti-IL-4 or anti-TGF-beta did not. Among immune donors, the percent of both CD3 lymphocytes and NK cells expressing IFN-gamma after 24h of T27K was increased above control (p<0.05), while the percent of NK cells producing TNF-alpha in response to T27K was not greater than control. Depletion of NK cells from peripheral blood mononuclear cells resulted in significant increases in TNF-alpha and IL-10 (for both, p<0.05) but resulted in no significant decrease in IFN-gamma or IL-2. These data demonstrate a differential response to stimulation with the coccidioidal antigen T27K among donors with coccidioidomycosis that can be manipulated by cell type and cytokine environment.     

Effect of medical castration on CD4+ CD25+ T cells, CD8+ T cell IFN-gamma expression, and NK cells: a physiological role for testosterone and/or its metabolites

The higher prevalence of autoimmune disease among women compared with men suggests that steroids impact immune regulation. To investigate how sex steroids modulate cellular immune function, we conducted a randomized trial in 12 healthy men aged 35-55 yr treated for 28 days with placebo, a GnRH antagonist, acyline to induce medical castration, or acyline plus daily testosterone (T) gel to replace serum T, followed by a 28-day recovery period. Serum hormones were measured weekly and peripheral blood lymphocytes (PBLs) were collected biweekly for analyses of thymus-derived lymphocyte (T cell) subtypes and natural killer (NK) cells. Compared with the other groups and to baseline throughout the drug exposure period, men receiving acyline alone had significant reductions in serum T (near or below castrate levels), dihydrotestosterone, and estradiol (P < 0.05). Medical castration significantly reduced the percentage of CD4+ CD25+ T cells (P < 0.05), decreased mitogen-induced CD8+ T cell IFN-gamma expression, and increased the percentage of NK cells without affecting the ratio of CD4+ to CD8+ T cells and the expression of NK cell-activating receptor NKG2D or homing receptor CXCR1. No changes in immune composition were observed in subjects receiving placebo or acyline with replacement T. These data suggest that T and/or its metabolites may help maintain the physiological balance of autoimmunity and protective immunity by preserving the number of regulatory T cells and the activation of CD8+ T cells. In addition, sex steroids suppress NK cell proliferation. This study supports a complex physiological role for T and/or its metabolites in immune regulation.     

Differential responses of cellular immunity in patients undergoing neoadjuvant therapy followed by surgery for carcinoma of the oesophagus

Background  To compare immune responses following neoadjuvant chemoradiation therapy in combination with hyperthermia plus surgery to those induced by surgery alone in patients with oesophageal cancer. Methods  Thirty-two patients with histopathologically proven oesophageal cancer, scheduled for potentially curative transhiatal or transthoracic oesophagectomy with (neo, n = 20) or without (control, n = 12) neoadjuvant thermochemoradiation therapy (ThCR) were included. Peripheral blood samples were obtained before ThCR, after 2 weeks of ThCR, 1 day before surgery, on postoperative days 1, 3, 7, and 6 weeks after surgery, for white blood cell counts, lymphocyte subsets and T helper type 1 (Th1) and type 2 (Th2) lymphocyte responses. Results  Neo patients showed a significant decrease in granulocytes and lymphocyte subsets, and T cell cytokines after 2 weeks of ThCR. Only CD8+ (cytotoxic) T cells recovered after ThCR to reach normal levels prior to surgery. In contrast, CD4+ T (helper) cells, and NK- and B cells in neo patients did not recover prior to surgery (all P < 0.05). Oesophagectomy induced a significant increase in granulocytes and a decrease in lymphocytes (and subsets). Only those subsets that had not recovered after ThCR (CD4+ T cells, NK and B cells but not CD8+ T cells), were significantly lower (all P < 0.05) during the entire postoperative study period. Postoperatively, the stimulated cytokine production capacity of Th1 and Th2 cells, corrected for number of T cells, was not significantly different between the groups. Conclusion  Neoadjuvant thermochemoradiation for oesophageal cancer caused significant disturbances of host cellular immunity with reduced T, NK and B cell counts, and differential recovery of cytotoxic and helper T cells leading to prolonged T cell imbalance that extends beyond the time of surgery. The functional and anti-tumour consequences of this immunodisturbance need further investigation, as recovery of T helper cytokine production towards surgery was less impaired than T helper cell counts.     

Adhesion molecules on peripheral blood lymphocyte subpopulations in the elderly

Adhesion molecules, such as CD49d, CD50 and CD62L, have important roles in many adhesive interactions involving cells of the immune system. Since it has been shown that many immunological alterations are present in aged subjects, we studied, by means of triple colour whole blood immunostaining and multiparametric flow cytometry, the expression and intensity level (MFI) of these molecules on peripheral blood lymphocyte subpopulations from 23 healthy elderly subjects and 13 young controls. In the elderly a decrease in total peripheral blood lymphocytes bearing CD62L antigen was observed (39 +/- 13% vs 63 +/- 6% and 745 +/- 312/mm3 vs 1,393 +/- 407/mm3; p<0.001), whereas the numbers of lymphocytes expressing CD49d and CD50 antigens were comparable in aged and young subjects. In addition, CD50 and CD62L MFI values on total peripheral blood lymphocytes were higher in elderly than in young subjects (5.23 +/- 1.03 vs 4.18 +/- 0.44, p = 0.001 and 2.60 +/- 0.35 vs 2.21 +/- 0.40, p = 0.005 respectively) while the intensity expression of CD49d was unchanged. The percentages and absolute numbers of T and B lymphocytes expressing CD62L were decreased in elderly compared to young subjects (CD62L+CD3+: 43 +/- 15% vs 66 +/- 9% and 581 +/- 257/mm3 vs 1,028 +/- 418/mm3, p<0.001; CD62L+CD19+: 78 +/- 12% vs 90 +/- 4%, p < 0.005 and 103 +/- 64/mm3 vs 207 +/- 98, p < 0.001). A decrease in the proportion of CD62L bearing NK cells was also observed in the elderly (25 +/- 14% vs 46 +/- 24%, p<0.005), although their absolute number was unchanged. No significant differences were detected in the proportion of T, B and NK lymphocytes expressing CD49d and CD50 antigens and only the absolute numbers of B cells expressing these adhesion molecules were lower in elderly (CD49d+CD19+: 121 +/- 71/mm3 and CD50+CD19+: 107 +/- 73/mm3) compared to young donors (CD49d+CD19+: 248 +/- 112/mm3 and CD50+CD19+: 235 +/- 120/mm3, p < 0.001). Moreover, the intensity of adhesion molecule expression was differentially modulated in the elderly depending on the specific lymphocyte cell population considered. The densities of CD49d, CD50 and CD62L antigens on B and NK lymphocytes from the two age groups were not different; on the contrary, T lymphocytes from elderly donors exhibited increased CD49d (1.69 +/- 0.09 vs 1.62 +/- 0.07, p < 0.05), CD50 (4.98 +/- 1.16 vs 3.77 +/- 0.46, p < 0.001) and CD62L (2.26 +/- 0.38 vs 1.99 +/- 0.37, p < 0.05) MFI values compared to young donors.     

Effects of human soluble BAFF synthesized in Escherichia coli on CD4+ and CD8+ T lymphocytes as well as NK cells in mice

B cell-activating factor belonging to the TNF family (BAFF, also called BLyS, TALL-1, zTNF-4, or THANK) is an important survival factor for B lymphocytes. In this study, we injected mouse abdominal cavity with human soluble BAFF (hsBAFF, 0.01, 0.1, 0.5, 2 mg/kg body mass) synthesized in Escherichia coli. On the 8th day after injection, we investigated the effects of hsBAFF on immune functional activities of splenic B lymphocytes, CD4(+) and CD8(+) T lymphocytes and natural killer (NK) cells in mice. The results showed that B lymphocyte proliferation significantly increased in hsBAFF-treated groups with dosages of 0.1 mg/kg (p<0.05), 0.5 and 2 mg/kg (p<0.01). We observed a dose-dependent increase of CD4(+) T lymphocyte percentage and significantly higher values in 0.5 and 2 mg/kg hsBAFF-treated groups (p<0.05 and p<0.001, respectively) compared to control group, but CD8(+) T lymphocyte percentage remained unchanged. The ratio of CD4(+) to CD8(+) T lymphocytes rose with increasing hsBAFF dosage (p<0.05 for 2 mg/kg hsBAFF vs. control). Significantly stronger NK cell activities were found in 0.5 and 2 mg/kg hsBAFF-treated groups (p<0.05). The main finding of this study is that the hsBAFF can enhance immune responses in the body by increasing B lymphocyte and CD4(+) T lymphocyte function as well as elevating NK cell activity.     

Humoral and cellular CMV responses in healthy donors; identification of a frequent population of CMV-specific, CD4+ T cells in seronegative donors

CMV status is an important risk factor in immune compromised patients. In hematopoeitic cell transplantations (HCT), both donor and recipient are tested routinely for CMV status by serological assays; however, one might argue that it might also be of relevance to examine CMV status by cellular (i.e., T lymphocyte) assays. Here, we have analyzed the CMV status of 100 healthy blood bank donors using both serology and cellular assays. About half (56%) were found to be CMV seropositive, and they all mounted strong CD8+ and/or moderate CD4+ T cell responses ex vivo against the immunodominant CMV protein, pp65. Of the 44 seronegative donors, only five (11%) mounted ex vivo T cell responses; surprisingly, 33 (75%) mounted strong CD4+ T cell responses after a brief in vitro peptide stimulation culture. This may have significant implications for the analysis and selection of HCT donors.     

Primary immune responses by cord blood CD4(+) T cells and NK cells inhibit Epstein-Barr virus B-cell transformation in vitro

Epstein-Barr virus (EBV) transformation of B cells from fetal cord blood in vitro varies depending on the individual sample. When a single preparation of EBV was simultaneously used to transform fetal cord blood samples from six different individuals, the virus transformation titer varied from less than zero to 10(5.9). We show that this variation in EBV transformation is associated with a marked primary immune response in cord blood samples predominately involving CD4(+) T cells and CD16(+) CD56(+) NK cells. After virus challenge both CD4(+) T cells and NK cells in fetal cord blood cultures expressed the lymphocyte activation marker CD69. The cytotoxic response against autologous EBV-infected lymphoblastoid cell line (LCL) targets correlated with the number of CD16(+) CD69(+) cells and was inversely correlated with the virus transformation titer. Although NK activity was detected in fresh cord blood and increased following activation by the virus, killing of autologous LCLs was detected only following activation by exposure to the virus. Both activated CD4(+) T cells and CD16(+) NK cells were independently able to kill autologous LCLs. Both interleukin-2 and gamma interferon were produced by CD4(+) T cells after virus challenge. The titer of EBV was lower when purified B cells were used than when whole cord blood was used. Addition of monocytes restored the virus titer, while addition of resting T cells or EBV-activated CD4(+) T-cell blasts reduced the virus titer. We conclude that there are primary NK-cell and Th1-type CD4(+) T-cell responses to EBV in fetal cord blood that limit the expansion of EBV-infected cells and in some cases eliminate virus infection in vitro.     

Apoptosis and oxidative status in peripheral blood mononuclear cells of diabetic patients

We have compared the concentrations of intracellular glutathione (GSH), glutathione-dependent antioxidative enzymes, the cell death rate and immunophenotype profile of peripheral blood mononuclear cells (PBMC) from healthy donors and from patients with insulin-dependent type I (IDDM) or non insulin-dependent type II (NIDDM) diabetes mellitus. The IDDM and NIDDM patients had above-normal absolute lymphocyte counts, whereas the percentages of CD3, CD4 and CD8 T lymphocytes were significantly reduced. In contrast, the absolute number and percentage of B lymphocytes was higher in diabetic patients than in healthy donors. The low intracellular reduced glutathione (GSH) and the unbalanced profile of key enzymes involved in GSH metabolism, gamma glutamyltransferase (-GT) and glutathione-S-transferase (GST), account for the increased oxidative status of PBMC from diabetic patients. The plasma membranes of PBMC from diabetic patients were less permeable to propidium iodide than those of PBMC from healthy donors, indicating that the apoptotic cell death rate was lower in the cells from diabetic patients. These differences are potentially useful markers of pathogenic metabolic changes which occur during clinical diabetes and if they are confirmed could be used to identify the onset of diabetes.     

Increased B Cell and Cytotoxic NK Cell Proportions and Increased T Cell Responsiveness in Blood of Natalizumab-Treated Multiple Sclerosis Patients

Background

Changes in the blood lymphocyte composition probably both mediate and reflect the effects of natalizumab treatment in multiple sclerosis, with implications for treatment benefits and risks.

Methods

A broad panel of markers for lymphocyte populations, including states of activation and co-stimulation, as well as functional T cell responses to recall antigens and mitogens, were assessed by flow cytometry in 40 patients with relapsing multiple sclerosis before and after one-year natalizumab treatment.

Results

Absolute numbers of all major lymphocyte populations increased after treatment, most markedly for NK and B cells. The fraction of both memory and presumed regulatory B cell subsets increased, as did CD3^-CD56^dim cytotoxic NK cells, whereas CD3^-CD56^bright regulatory NK cells decreased. The increase in cell numbers was further associated with a restored T cell responsiveness to recall antigens and mitogens in functional assays.

Conclusions

Our data confirms that natalizumab treatment increases the number of lymphocytes in blood, likely mirroring the expression of VLA-4 being highest on NK and B cells. This finding supports reduction of lymphocyte extravasation as a main mode of action, although the differential effects on subpopulation composition suggests that cell-signalling may also be affected. The systemic increase in T cell responsiveness reflects the increase in numbers, and while augmenting anti-infectious responses systemically, localized responses may become correspondingly decreased.