Reduction in interleukin-2-producing cells but not Th1 to Th2 shift in moderate and advanced stages of human immunodeficiency virus type-1-infection: direct analysis of intracellular cytokine concentrations in CD4+ CD8- T cells

Previous studies have suggested that CD4+ T lymphocytes shift from the Th1 type to the Th2 type during disease progression in patients with the human immunodeficiency virus type-1 (HIV-1). In the present study, we used a modified method that allowed a direct measurement of intracellular cytokines in CD4+ CD8- T cells. A total of 48 HIV-1-infected (HIV+) and 16 HIV-1-uninfected (HIV-) individuals were studied. The percentages of CD4+ CD8- T cells producing interleukin-2 (IL-2), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), or interleukin-5 (IL-5) in HIV+ and HIV- subjects were 23.6% versus 34.9% (P < 0.01), 13.7% versus 13.2%, 1.3% versus 1.0%, and 1. 2% versus 0.9%, respectively. The population of IL-2-producing cells decreased proportionately with reductions in CD4 counts (< 200/mm3, 200-500/mm3, and > 500/mm3 to 18.0%, 23.5%, and 30.5%, P < 0.05, respectively). There was an inverse correlation between the percentage of IL-2-producing cells and plasma viral load (r = - 0. 446, P < 0.05). However, the percentages of CD4+ CD8- T cells producing other cytokines were not different between HIV+ and HIV-. Our cross-sectional study demonstrated a decrease in IL-2-producing cells but not the Th1 to the Th2 shift in the CD4+ CD8- T cell population in the moderate and advanced stages of HIV-1-infection.     

In vitro susceptibility of Thai seronegative donor CD8+ T lymphocytes to human immunodeficiency virus-1 (HIV-1) infection

To determine whether CD8+ T lymphocytes from Thai donor cells are susceptible to HIV-1 infection, undepleted peripheral blood mononuclear cells (PBMC) and CD8-enriched PBMC were infected with HIV-1 Thai subtype B and CRF01_AE (E) primary isolates. Virus kinetics in HIV-1 infection of CD4+ and CD8+ T lymphocytes peaked at day 7 or 10 post infection (pi); the TCID50 used for cell infection was proportional to the level of p24 production in the cultures. We also found that the level of p24 antigen in the supernatants of infected undepleted PBMC was significantly higher than that of infected CD8-enriched PBMC. Interestingly, both single positive T lymphocytes (CD4+ and CD8+ T lymphocytes) as well as double positive CD4+/CD8+ T lymphocytes were infected with HIV-1. The double positive T lymphocytes in PBMC were found only in the presence of both CD4+ and CD8+ T lymphocytes. The majority of p24+/CD4-/CD8- T lymphocytes were HIV-1 infected CD4 down-modulated PBMC. This report provides direct evidence that single positive CD8+ T lymphocytes and double positive CD4+/ CD8+ T lymphocytes from Thai donors can be infected with HIV-1 subtypes B and E in vitro.     

Increased sensitivity to IL-4 in cystic fibrosis patients with allergic bronchopulmonary aspergillosis

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is characterized by a heightened Th2 CD4+ T-cell response to Aspergillus fumigatus allergens and a hyper-immunoglobulin (Ig)E state compared with cystic fibrosis patients without ABPA. We hypothesize that one reason for this response is increased sensitivity to interleukin (IL)-4 in ABPA resulting in increased expression of CD23 and CD86 and leading to a positive amplification mechanism that increases Th2 CD4+ T cell responses. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from seven ABPA CF and 19 non-ABPA CF patients and 16 nonatopic controls and stimulated with rIL-4 (range 0.1-10 ng/ml) and rIL-13 (range 1-10 ng/ml) for 48 h. The number of CD23 molecules and percentages of CD23+ B cells were quantified by flow cytometry. Both phorbol 12-myristate 13-acetate (PMA)/ionomycin (IO) and antigen stimulated, toxoid and Asp f2/f3/f4, PBMC were examined for cytoplasmic cytokine synthesis enumerated by cytokine staining using flow cytometry to measure Th2 and Th1 CD3+ T cells. RESULTS: The numbers of CD23 molecules on B-cells were significantly elevated at time 0 in ABPA CF patients compared with both non-ABPA CF patients and nonatopic controls. Following IL-4 stimulation in vitro, the numbers and percentages of CD23 expression on B cells were significantly up-regulated in ABPA CF patients compared with non-ABPA CF patients and controls. The IL-13 stimulation up-regulated CD23 expression; however, there was no significant difference in ABPA CF patients compared with non-ABPA CF patients and controls. The percentages of interferon (IFN)-gamma+ CD3+T cells following PMA/IO stimulation were significantly decreased in both ABPA and non-ABPA CF patients compared with controls. There were no significant differences of IL-4+ and IL-13+ CD3+ T cells between ABPA and non-ABPA CF patients. When tetanus toxoid stimulated T cells were examined, both ABPA and non-ABPA CF patients had significantly decreased IFN-gamma+ CD3+ T cells compared with controls. In Asp f2/f3/f4 stimulated T cells, ABPA CF patients had significantly increased IL-4+ CD3+ T cells compared with non-ABPA CF patients and controls. CONCLUSIONS: ABPA CF patients have increased sensitivity to IL-4 but not to IL-13 up-regulation of CD23 molecules compared with non-ABPA CF patients. There were decreased percentages of IFN-gamma+ and IL-2+ Th1 T cells in CF patients compared with nonatopic controls but similar percentages of IL-4+ Th2 T cells in all three groups. However, ABPA CF patients had increased frequency of Aspergillus-stimulated Th2 T cells. This indicated that there is skewing of Th2 T cells in ABPA CF patients. Thus, in CF ABPA patients there is increased Th2 T cells and increased sensitivity to IL-4.     

Phenotype and Cytokine Profile ofSchistosoma mansoniSpecific T Cell Lines and Clones Derived from Schistosomiasis Patients with Distinct Clinical Forms

It is essential to distinguish the role of T lymphocytes on the physiopathology associated to more severe forms of schistosomiasis and on the immunomodulation that evolves in the majority of infected people. In this study, we generated Schistosoma mansoni-specific T cell lines and clones from patients with the acute and chronic (intestinal and hepatosplenic forms) phases of disease, from former ones, and from uninfected individuals sensitized to parasite soluble antigens. T cell lines derived from nontreated acute infected donors were capable of producing IL-4 and IL-5, while cells from treated patients secreted IFN-gamma. Lines from intestinal chronic and antigen-sensitized donors preferentially produced IFN-gamma, while those from hepatosplenic patients secreted all three cytokines. The cytokine analysis of CD4+ T cell clones revealed a Th2/Th0 pattern (clones producing IL-4 and IL-5 and clones producing all three cytokines) for those derived from infected patients, while cells from antigen-sensitized donors exhibited an opposite Th1/Th0 pattern (clones producing IFN-gamma and clones producing all three cytokines). The possible role of these T cell populations on human schistosomiasis mansoni is discussed.     

Selective induction of Th2 cells in murine Peyer's patches by oral immunization.

We have used three different methods to determine the T helper (Th) cell response, including Th1 and Th2 types, in murine Peyer's patches (PP) following oral immunization with sheep red blood cells (SRBC). These include: (i) use of cytokine-specific (IFN-gamma and IL-5), single cell assays to estimate the frequencies of Th1 and Th2 cells respectively, (ii) cytokine-specific mRNA--cDNA dot blot and Northern gel hybridizations to detect levels of specific mRNA, and (iii) T cell cloning techniques to determine the frequency of Th1 and Th2 clones. Mice were immunized with SRBC by either the oral or i.p. route. The PP and splenic (SP) CD3+ and CD3+ CD4+ T cell subsets were isolated and cultured with antigen, feeder cells, and IL-2, and were assessed at various intervals (days 0, 1, 3, and 6) for numbers of T cells producing either IFN-gamma or IL-5 by use of an enzyme-linked immunospot (ELISPOT) procedure. Cultures of T cells from PP or SP of mice given SRBC by the oral route had a high frequency of IL-5 spot forming cells (SFC), with lower numbers of IFN-gamma SFC. However, cultures of CD3+ T cells and CD3+ CD4+ Th cells from spleens of i.p. immunized mice exhibited predominantly IFN-gamma SFC, with smaller but significant numbers of IL-5 SFC. This distinct pattern of cytokine production was supported by mRNA analysis where high IL-5 specific mRNA levels were noted in PP T cell cultures of orally primed mice, while IFN-gamma mRNA was predominant in the SP CD3+ T cell and CD3+ CD4+ Th cell cultures from i.p. immunized mice. When the frequencies of IFN-gamma or IL-5 SFC were assessed among cloned Th cells from orally- or systemically-immunized mice, 74% of Th cell clones from PP of mice orally immunized with SRBC were IL-5 producers (Th2 type), while 67% of Th cell clones from SP of mice immunized by the i.p. route were IFN-gamma producers (Th1 type). Our studies show that higher frequencies of IFN-gamma producing Th1-type cells occur in SP of mice given antigen by the systemic route, while oral immunization results in predominantly IL-5 producing, Th2-type cells in PP.     

Assessment of selected co-stimulatory, adhesion and activatory molecules and cytokines of Th(1)/Th(2) balance in acute lymphoblastic leukemia in children

INTRODUCTION: Recent years have seen a rise in the importance of cytokine production and co-stimulatory/activatory molecule expression in the immune response in leukemia. The aim of our study was to assess the function of T lymphocytes in children with acute lymphoblastic leukemia (ALL) during remission induction based on selected cytokine and co-stimulatory/activatory molecule expression. MATERIAL/METHODS: The study group consisted of 50 children with ALL (B cell precursor). Peripheral blood samples were taken before treatment (day 0), after the prednisone prophase (day 8), and during (day 15) and after (day 33) remission induction. The percentages of T cells with interferon (IFN)-gamma (Th(1)), interleukin (IL)-4 (Th(2)) and IL-2 receptor (IL-2R), CD28, CTLA-4, CD38, ICAM-1, and HLA-DR expression were assessed by tricolor flow cytometry. RESULTS: At the time of diagnosis we noted higher percentages of T cells with adhesion molecule ICAM-1, activation molecule CD38 expression, and an increased population of Th(2 )cells (IL-4) compared with the control group. During and after remission induction we observed a decreased population of CD38(+) T cells, elevated percentages of helper T lymphocytes with IL-2R expression, and a rise in helper T lymphocytes producing IFN-gamma (Th(1)). During fever/infection, higher levels of activated T lymphocytes (CD4(+)HLA-DR(+), CD8(+)HLA-DR(+)), a rise in Th1, and no change in Th(2 )populations were observed. CONCLUSIONS: The results suggest T cell activation and Th(2 )predominance at the time of diagnosis and during remission induction in ALL in children. These results confirm the involvement of cellular immunity in the leukemic process and can be used in immune therapy in leukemia.     

Intracellular production of type I and type II cytokines during HIV-1 progression in Thai patients

A type I to type II cytokine switch on cells of the immune system has been suggested as a critical step in the etiology of HIV infection. In this study, type I and type II cytokine production of both CD4+ and CD8+ T cells activated by superantigen were investigated in 10 healthy donors and 39 HIV-1 infected patients. Patients were divided into 3 groups based on their CD4 count (< 200, 200-500, > 500 cells/microl). Whole blood from each subject was activated by staphylococcal enterotoxin B (SEB) and anti-CD28. Intracellular cytokine stainings for proinflamatory cytokine (TNF-alpha), type I cytokines (IFN-gamma and IL-2) and type II cytokines (IL-4 and IL-5) in CD4+ and CD8+ T lymphocytes were determined by flow cytometer. Type I cytokine (IFN-gamma) expression in CD4+ T cells co-expressing with CD69 were significantly increased in HIV infected patients, particularly in patients with CD4 counts < 200 and 200-500 cells/microl (means +/- S.D. of 20.7 +/- 18.7% and 10.5 +/- 5.9%, respectively) when compared with 4.8 +/- 1.8% in the normal group (p < 0.05). But IL-2 production in both groups of patients was significantly lower than the normal (3.8 +/- 2.6% and 3.2 +/- 1.4% in patients with < 200, 200-500 cells/microl, and 5.9 +/- 1.5% in the normal group) (p < 0.05). For type II cytokines, there was no difference in all groups of subjects when IL-4 was determined. However, IL-5 production was significantly higher in patients with a CD4 count < 200 cells/microl (0.6 +/- 0.5%) than that in the normal group (0.1 +/- 0.1%) (p < 0.005). CD8+ T cells also showed higher IFN-gamma production in patients with a CD4 count < 200 cells/microl (11.9 +/- 4.7%) and 200-500 cells/microl (12.0 +/- 4.3%) than the normal group (5.3 +/- 2.5%) (p < 0.005). In contrast, IL-2 production in CD8+ T cells was low in these HIV infected patients (0.3 +/- 0.2%, 0.3 +/- 0.2%, and 0.3 +/- 0.4% in patients with < 200, 200-500, and > 500 cells/microl, respectively), which was significantly different compared to the control group (1.2 +/- 0.8%) (p < 0.05). For type II cytokines, only IL-4 production in patients with a CD4 count < 200 cells/microl (0.1 +/- 0.1%) was significantly reduced when compared to the other groups (p < 0.05). This study shows that although HIV infection alters the production of both type I and type II cytokines, it does not induce a polarized type I or type II state in the course of HIV-1 progression in Thai patients.     

Expression of intracellular Th1 and Th2 cytokines in women with recurrent spontaneous abortion,implantation failures after IVF/ET or normal pregnancy

PROBLEM: We aimed to investigate absolute counts of intracellular T helper 1 (Th1) and Th2 cytokine expressing T-cell subpopulations in women with three or more recurrent spontaneous abortions (RSA), multiple implantation failures after in-vitro fertilization and embryo transfer (IVF/ET) (three or more) or during normal pregnancy. METHOD OF STUDY: Absolute cell counts and percentages of CD3+, CD3+/CD4+, and CD3+/CD8+ T-cell populations expressing intracellular cytokines [interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-4 and IL-10] was studied by four-color flow cytometry in 15 RSA and 13 implantation failure patients. Eighteen fertile non-pregnant and 47 normal pregnant women were also compared with regard to intracellular cytokine expression. RESULTS: Interleukin-10 producing CD3+/CD8+ T-cell counts were significantly lower in women with RSA (P < 0.05) and implantation failures (P < 0.05), and TNF-alpha producing CD3+/CD4+ T-cell counts were higher in women with RSA (P < 0.05) and implantation failures (P < 0.005) than those of non-pregnant fertile controls. During normal pregnancies, first trimester IL-4 expressing CD3+, CD3+/CD4+ T-cell counts (P < 0.05) and IFN-gamma expressing CD3+ T-cell counts (P < 0.05) were significantly higher than those of third trimester (P < 0.05). First trimester TNF-alpha expressing CD3+/CD8+ T-cell counts were significantly higher than those of second and third trimester women (P < 0.05). However, there are no differences in cytokine expression between non-pregnant and first trimester pregnant women. CONCLUSION: Absolute counts of IFN-gamma, IL-4, and TNF-alpha expressing T cells decrease with the progress of gestation (third trimester) during normal pregnancies. In women with implantation failures, absolute cell counts of TNF-alpha expressing CD3+/ 4- cells reflects the presence of dominant Th1 immune response. A significantly increased Th1 cytokine expression may be the underlying immune etiology for reproductive failures.     

Immunisation with recombinant modified vaccinia virus Ankara expressing HIV-1 gag in HIV-1-infected subjects stimulates broad functional CD4+ T cell responses

Virus-specific CD4+ T cells with IL-2-secreting and/or proliferative capacity are detected readily in HIV-1-infected long-term nonprogressors and rarely in persons with untreated progressive infection. The contribution of these cells to viraemia control is uncertain, but this question might be addressed in clinical therapeutic vaccination studies. However, the quality of T helper responses induced by currently available HIV-1 vaccine candidates has not been explored in depth. We determined the effect of vaccination with modified vaccinia virus Ankara (MVA) expressing HIV-1 gag p24/p17 (MVA.HIVA) on HIV-1-specific CD4+ T cell responses in 16 chronically infected, highly active antiretroviral therapy (HAART)-treated subjects using CD8-depleted IFN-gamma ELISPOT assays, intracellular cytokine staining assays for IL-2 and IFN-gamma, and a CFSE-based proliferation assay. Gag-specific CD4+ T cell responses were significantly increased in magnitude and breadth after vaccination and targeted both known and new epitopes, several of which were also recognised by healthy HIV-uninfected volunteers immunised with the same vaccines. The frequencies of CD4+ T cells expressing IL-2 or IFN-gamma, alone or simultaneously, were also augmented. These findings indicate that functional virus-specific T helper cells can be boosted by vaccination in chronic HIV-1 infection. Further evaluation of their role in viraemia control is warranted.     

Differential expression of the chemokine receptors by the Th1- and Th2-type effector populations within circulating CD4+ T cells

The in vitro studies have proposed that human Th1 cells favor expression of CXCR3 or CCR5, whereas Th2 cells favor CCR3 and CCR4. In this study, the in vivo relevance of expression of these chemokine receptors on Th cells was investigated in patients with atopic dermatitis (AD) as the Th2-dominated disorder and nonatopic normal individuals. Flow-cytometric analysis using monoclonal antibodies against CXCR3, CCR5, CCR3, and CCR4 disclosed that a substantial proportion of memory (CD45RO+) CD4+ T cells in the blood of AD and normal patients expressed CXCR3, CCR5, or CCR4, but expression of CCR3 on these cells was negligible. Stimulation studies combined with intracellular cytokine staining revealed that the cells capable of producing Th2 cytokines, such as interleukin-4 (IL-4), IL-5, and IL-13, were restricted to the CCR4-expressing population within memory CD4+ T cells. Concerning Th1 cytokine production, interferon-gamma (IFN-gamma)-producing cells resided exclusively in CXCR3-expressing memory CD4+ T cells, although IFN-gamma production was found in both memory CD4+ T cells with and without CCR5 expression. We observed that CCR4-expressing memory CD4+ T cells in the blood were more increased in AD patients as compared with normal patients, whereas CXCR3-expressing memory CD4+ T cells were present in a lower frequency in AD than seen in normal patients. These results suggest that CXCR3 and CCR4, but not CCR5 or CCR3, appear to serve as the useful markers for identification of circulating Th1 and Th2 effector populations.     

Rapid qualitative and quantitative analysis of T-cell responses in HIV-1-infected individuals receiving successful HAART and HIV-1 sero-negative controls: concomitant assessment of perforin, IFN-gamma and IL-4 secretion

The enzyme-linked immunospot (ELIspot) assay is a highly sensitive and valuable tool for determining the frequency of cytokine-secreting T cells. It is essential to determine both frequencies and functional capabilities of antigen-specific T cells, including cytokine secretion, degranulation, and cytotoxicity in order to obtain a fuller picture of the immune status of an individual. We describe here for the first time a perforin-release ELIspot assay which, when used in combination with IFN-gamma and IL-4 ELIspots, permits rapid assessment of these functional parameters for antigen-specific T cells. Whole antigen or peptides from HIV-1, recall and other viral antigens were used for in vitro stimulation. Anti-HIV-1 responses in treated chronically infected individuals were weak, both in terms of perforin and IFN-gamma production. Tetanus toxoid stimulation was associated with moderate perforin release and a predominantly type-2 IL-4 producing response, whilst herpes simplex virus antigen stimulation resulted in perforin release but only a weak type-1 IFN-gamma response. Anti-cytomegalovirus responses generated high levels of perforin in conjunction with IFN-gamma. Cytokines IL-2 and IL-12/IL-15 induced perforin release coupled with an IFN-gamma type-1 response. Perforin release strongly correlated with IFN-gamma production to individual influenza, Epstein-Barr virus or cytomegalovirus MHC class I restricted peptides, in an HIV-1 sero-negative cohort, indicating a cytolytic type-1 CD8+ T-cell response. Evaluation of immunogenicity and putative efficacy of candidate vaccines using IFN-gamma will not be as informative alone as when combined with perforin and IL-4 evaluations, which allow assessment of specific cytotoxic potential without extensive cell culture.     

Age-related differences in cell-specific cytokine production by acutely ill Malawian patients

Age-related changes in human cell-specific cytokine responses to acute illness have not been well examined. We therefore evaluated age-related differences in T, B and natural killer (NK) peripheral blood lymphocyte cytokine responses of 309 acutely ill hospitalized people in Malawi, Africa, < 1 month-61 years of age. We used four-colour flow cytometry and performed Wilcoxon rank sum and Kruskal-Wallis tests, Pearson (rp) and Spearman (rs) correlations, and linear and logistic regression analyses to control for human immunodeficiency virus infection (HIV) status, the percentages of lymphocytes expressing CD4, and the nature of the acute infection. The percentages of CD8- and CD8+ T cells producing induced IL-8 decreased with age (rs = -0.44 and -0.53). The percentages of T cells producing TNF-alpha were higher, and the percentages producing IL-10 were lower, in those > or =13 than those < 13 years old (medians: 17.7 versus 10.5 and 1.4 versus 3.0, respectively). The percentages of CD8- T cells producing IFN-gamma were higher and stable in those > or =1 year old compared to infants (medians: 23.5 versus 10.4); the percentages of NK producing IFN-gamma were higher post-infancy and then declined to relatively low levels with increasing age. The percentages of T cells producing IL-2 were highest in those 5- <31 years old (median 5.6) and lowest in those > or =31 years old (median 1.9). The ratios of the percentages of T cells producing IL-4 to those producing IL-8 and to those producing IL-10 both increased with age. These data suggest that innate immunity, represented by NK IFN-gamma production, dominates in early life. A number of shifts occur after infancy and before adolescence, including a proinflammatory shift from IL-8 to TNF-gamma and a type 2 shift from IL-10 to IL-4 dominance. These findings suggest distinct age-related differences in the human response to acute illness and may be useful in directing future efforts at immunomodulatory therapies.     

The absence of IFN-gamma leads to increased Th2 responses after influenza A virus infection characterized by an increase in CD4+ but not CD8+ T cells producing IL-4 or IL-5 in the lung

The cytokine IL-4 has been shown to be responsible for the switch of both CD4+ and CD8+ T cells to a Th2 or TC2 functional phenotype in vitro which both secrete IL-4 after stimulation. In contrast the presence of IFN-gamma interferes with the generation of Th2 and TC2 cells in vitro. Furthermore, it is well established that in the absence of IFN-gamma and the presence of IL-4 Th2 cells also develop in vivo. However, little is known about the conditions leading to the generation of TC2 cells in vivo. For this reason we investigated if Th2 and TC2 cells develop in the lung of IFN-gamma deficient mice which were infected with Influenza A virus. Surprisingly, we were only able to detect Th2 but not TC2 cells in the bronchoalveolar fluid and the mediastinal lymphnodes of IFN-gamma deficient mice infected with influenza A virus 1, 2 and 3 weeks by intracellular FACS staining for IL-4 or IL-5. In infected and uninfected wild type mice and uninfected IFN-gamma deficient mice we were not able to detect any Th2 or TC2 cells. These findings suggest that the prerequisites for Th2 and TC2 cell development are different in vivo than in vitro and may also explain why Th2 cells are more readily detected after immunisations or infections than TC2 cells in vivo.     

Efficient replication of human immunodeficiency virus type 1 in cytokine induced CD 4+ T helper 1 (Th 1)- and Th 2-type conditions]

Potent stimuli for CD 4+ T cell differentiation are cytokines. Among them, IL-12 or IL-4 induce naive CD 4+ T cells to Th 1 or Th 2 cells, respectively. In this study we found that macrophage-tropic (M-tropic) HIV-1 strains more efficiently replicated in interleukin 12 (IL-12) induced T helper 1 (Th 1)-type culture derived from normal CD 4+ T cells than T-cell-line-tropic (T-tropic) strains did. In contrast, T-tropic strains preferentially infected IL-4 induced Th 2-type culture derived from same donor CD 4+ T cells. Additional studies using chimeric viruses demonstrated that the V 3 region of gp 120 was the principle determinant for this efficient replication. It was also isolated T-tropic viruses from an acutely infected patient who had been evidenced as severe CD 4 depletion during short time course. These results indicate that HIV-1 isolates exhibit differences in the ability to infect CD 4+ T cell subset such as Th 1 or Th 2 cells, and that this difference may partly correlate with the viral pathogenesis. The findings suggest that immunological condition is one of the factors responsible for inducing selection of HIV-1 strains.     

Kinetics of adaptive immunity to Haemophilus influenzae type b meningitis in transgenic mice: evidence from diverse expression of double T1/T2 transgenes and Th1/Th2-related cytokines

To investigate the kinetic changes in adaptive immunity during experimental Haemophilus influenzae type b (Hib) meningitis, we established a murine meningitis model based on T1/T2 doubly transgenic mice. These mice carry two transgenes that express two distinct cell-surface markers: a human Thy1 transgene (hThy1) under the control of the murine IFN-gamma promoter, and a murine Thy1.1 transgene (mThy1.1) under the control of the murine IL-4 promoter, designated T1 and T2, respectively. Mice infected with Hib displayed severest symptoms and lowest total splenocyte counts on day 3 after infection. Simultaneously, we examined the significantly low percentage of CD19+ B cells, the relatively high level of CD4+ T cells and significantly high percentage of CD8+ T cells in Hib-infected mice. Furthermore, we observed the early induction of both Th1 and Th2 responses, in terms of the augmentation of Th1 cells (IFN-gamma-producing CD4+ T cells) and Th2 cells (IL-4-producing CD4+ T cells) in Hib-infected mice. On day 7 after infection, the Th1 response gradually declined and the Th2 response rather sustained. Two weeks after infection, both Th1 and Th2 cells were barely detectable. Moreover, we demonstrated using an antigen-specific re-stimulation test to analyze the effector function of lymphocyte subsets that CD8+ T cells contributed to more predominantly production of IFN-gamma than CD4+ T cells did; and CD4+ T cells partly contributed to the secretion of IL-4 from flowcytometry of intracellular cytokine staining. Our results support that these transgenic mice provide an available model to dissect the complex kinetic change of adaptive immunity in bacterial infectious diseases.     

T helper 1 (Th1)/Th2 cytokine expression shift of peripheral blood CD4+ and CD8+ T cells in patients at the post-acute phase of stroke

Local humoral and cellular immune responses modulate the inflammatory processes involved in the development of atherosclerotic lesions, as well as in the evolution of brain infarcts in stroke patients. The role of systemic adaptive immunity on the progression of such disease manifestations is less clear. In the current study, we evaluated the percentages of T helper 1 (Th1) [interleukin (IL)-2, interferon (IFN)-gamma] and Th2 (IL-4, IL-10) cytokine-producing peripheral blood CD4+ and CD8+ T cells in 23 patients with a history of ischaemic stroke (IS) at the chronic stable phase of the disease (median post-stroke time 34.5 months). Seven stroke-free individuals matched for age and vascular risk factors (matched controls, MC) were collected for comparison. To measure cytokine values at baseline and after stimulation, we used a flow cytometry method of intracellular cytokine staining. Intrinsic Th1 and Th2 cytokine production in unstimulated T cells was negligible in all study participants. Following mitogenic stimulation with phorbol 12-myristate13-acetate/ionomycin, both the IS and the MC groups exhibited a similarly strong Th1 response; IL-2 production predominated in the CD4+ T cells and IFN-gamma in the CD8+ T cells. However, when measuring the Th2 cytokine-production capacity post-stimulation, a significant increase in the percentage of IL-4-producing T cells was observed in the IS groups, compared with the MC group, resulting in a significantly lower ratio of IFN-gamma-/IL-4-producing T cells. No such Th2 enhancement could be confirmed for the case of IL-10. We propose that in IS patients there is a systemic shift of the immune system towards Th2 responses at the late post-acute phase of stroke.     

Infection by different HIV-1 subtypes (B and C) results in a similar immune activation profile despite distinct immune backgrounds.

We compared the immune activation profile of 46 HIV-negative and 75 HIV-positive Israelis infected with HIV-1 subtype B, with 85 HIV-negative and 102 HIV-positive Ethiopian immigrants to Israel, who were infected with HIV subtype C. The HIV-negative Ethiopians had exceedingly high blood levels of eosinophils, immunoglobulin E (IgE), and p75s tumor-necrosis factor receptors (p75sTNFR); secretion of interleukin-4 (IL-4) and IL-10 by peripheral blood mononuclear cells (PBMC); proportion of human leukocyte antigen (HLA)-DR+ cells within CD3+, CD4+, and CD8+ T-cell subsets; and proportion of CD45RO+ CD4+ cells; while having significantly lower secretion of interferon-gamma (IFN-gamma) by PBMC and percentage of CD45RA+ CD4+ and CD28+ CD8+ cells. HIV infection in both populations was associated with reduced IL-2, IL-4, IL-10, and IL-12 secretion, number of CD28+ and CD45RA+ CD8+ cells, and increased number of HLA-DR+-CD3+, CD4+, and CD8+ cells, and CD45RO+ CD8+ cells. Thus, infection with HIV-1 subtypes B and C of studied Israelis and Ethiopians, respectively, results in a similar immune activation profile at all stages of the infection when living in the same environment, despite the striking different immune profile observed in the HIV-negative Israeli and Ethiopian populations. Together with our previous observations, this indicates that HIV subtype is not a major determinant in the natural course of HIV infection.     

Leishmania-specific T cells expressing interferon-gamma (IFN-gamma) and IL-10 upon activation are expanded in individuals cured of visceral leishmaniasis.

Peripheral blood mononuclear cells (PBMC) from patients who have recovered from visceral leishmaniasis often respond to Leishmania antigens in vitro by production of both IL-4, IFN-gamma and IL-10. In order to establish the cellular sources of these cytokines, we activated cells from individuals with a history of visceral leishmaniasis with Leishmania antigen for 6 days in culture, and identified cytokine production at the single-cell level by flow cytometry. The cytokines were only found in CD3+ cells and among these mainly within the CD4+ subset. The percentage of cytokine-producing cells was compared in Leishmania-activated PBMC cultures from the previous patients and from individuals living in a village where leishmaniasis does not occur. The percentage of IL-10- and IFN-gamma-containing cells was significantly higher in the previous patients than in the controls, indicating that Leishmania-specific T cells producing IL-10 and/or IFN-gamma had been expanded as a result of the infection. The cytokine-producing cells in the previous patients could be divided into three types: (i) cells producing IFN-gamma only; (ii) cells producing IL-4 only; and (iii) cells producing IFN-gamma and IL-10 simultaneously. The first and second group of cells can be described as Th1- and Th2-type cells, respectively. The third group could be a regulatory subset of T cells important for maintaining a balance between Th1- and Th2-type cells in these individuals.