Leishmania-reactive CD4+ and CD8+ T cells associated with cure of human cutaneous leishmaniasis.

Fourteen patients suffering from American cutaneous leishmaniasis were studied. Assays of the lymphocyte proliferative response induced in vitro by Leishmania braziliensis antigens were performed. After 5 days in culture, L. braziliensis-stimulated blast T cells were harvested for CD4+ and CD8+ phenotype analysis. When results before and at the end of therapy were compared, leishmaniasis patients showed an increase in the percentage of CD8+ blast T cells and a decline in the proportion of CD4+ blast T cells in cultures. The levels of gamma interferon in T-cell culture supernatants showed a tendency to increase when the patients were cured. These results show a pattern of higher proportions of Leishmania-reactive CD8+ T cells and lower proportions of Leishmania-reactive CD4+ T cells after cure.     

Th1 cytokine pattern in sarcoidosis is expressed by bronchoalveolar CD4+ and CD8+ T cells

The pathogenesis of pulmonary sarcoidosis has been related to an increased production of Th1-like cytokines. However, cytokine expression in sarcoidosis has not been systematically studied at a single-cell level. We therefore investigated the expression of IL-2, IL-4, IL-13, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) intracellularly in bronchoalveolar lavage (BAL) and peripheral blood CD3+ T lymphocytes from patients with pulmonary sarcoidosis (radiologic stage II-III, n = 8) and normal controls (n = 9) by flow cytometry. In contrast to IL-4 and IL-13, the percentage of T lymphocytes expressing intracellular IL-2 (49.3 +/- 21.3% versus 14.5 +/- 15.6%), IFN-gamma (75.5 +/- 14.9% versus 32.6 +/- 18.7%) and TNF-alpha (68.3 +/- 18.7% versus 36.8 +/- 20.8%) was significantly higher in patients with sarcoidosis than in normal controls (each P < 0.005). In contrast to BAL lymphocytes, expression of these cytokines in peripheral blood lymphocytes did not differ between patients with sarcoidosis and normal controls. Close correlations were observed between the percentages of BAL lymphocytes expressing intracellular IL-2, IFN-gamma and TNF-alpha, but not for IL-4 or IL-13. Analysis of the expression of these cytokines in T lymphocyte subsets revealed IL-2, IFN-gamma, and TNF-alpha in CD4+ as well as CD8+ T lymphocytes, suggesting a contribution of TC1 cells to the production of proinflammatory cytokines in sarcoidosis. We conclude that a Th1-like cytokine pattern can be observed in CD4+ as well as in CD8+ BAL T lymphocytes in patients with pulmonary sarcoidosis.     

CD45 isoform expression is associated with different susceptibilities of human naive and effector CD4+ T cells to respond to IL-4

Interleukin-4 (IL-4) is the major factor promoting the development of T helper type 2 (Th2) cells from naive precursor T cells. Minute amounts of IL-4 produced by naive T cells seem to be sufficient; however, the molecular mechanisms explaining this efficient utilization of IL-4 are not yet known. Here, we show that human CD4+ CD45RA+ naive T cells, in contrast to CD4+ CD45R0+ effector T cells, show responsiveness to endogenous as well as exogenous IL-4 to proliferate and differentiate towards Th2 cells in vitro. Despite production levels of IL-4 below conventional detection limits, CD45RA+ T cell-derived IL-4 could clearly activate STAT6. Although the expression levels of IL-4R and STAT6 were not different between naive and effector T cells, only naive T cells responded to IL-4 in a STAT6-dependent reporter gene assay. Transfecting a trans-dominant negative form of STAT6 abrogated IL-4-induced proliferation in CD45RA+ cells. A significantly higher protein tyrosine phosphatase (PTPase) activity was detected in CD45R0+ T cells as compared to CD45RA+ T cells. Cross-linking CD45 potently reduced PTPase activity in CD45R0+ T cells and restored their ability to proliferate in response to IL-4. Thus, CD45 PTPase activity contributes to the susceptibility of naive and memory T cells to respond to IL-4.     

CD46 engagement on human CD4+ T cells produces T regulatory type 1-like regulation of antimycobacterial T cell responses

Understanding the regulation of human immune responses is critical for vaccine development and treating infectious diseases. We have previously shown that simultaneous engagement of the T cell receptor (TCR) and complement regulator CD46 on human CD4(+) T cells in the presence of interleukin-2 (IL-2) induces potent secretion of the immunomodulatory cytokine IL-10. These T cells mediate IL-10-dependent suppression of bystander CD4(+) T cells activated in vitro with anti-CD3 and anti-CD28 costimulation, reflecting a T regulatory type 1 (Tr1)-like phenotype. However, CD46-mediated negative regulation of pathogen-specific T cells has not been described. Therefore, we studied the ability of CD46-activated human CD4(+) T cells to suppress T cell responses to Mycobacterium bovis BCG, the live vaccine that provides infants protection against the major human pathogen Mycobacterium tuberculosis. Our results demonstrate that soluble factors secreted by CD46-activated human CD4(+) T cells suppress mycobacterium-specific CD4(+), CD8(+), and γ(9)δ(2) TCR(+) T cells. Dendritic cell functions were not downregulated in our experiments, indicating that CD46-triggered factors directly suppress pathogen-specific T cells. Interestingly, IL-10 appeared to play a less pronounced role in our system, especially in the suppression of γ(9)δ(2) TCR(+) T cells, suggesting the presence of additional undiscovered soluble immunoregulatory factors. Blocking endogenous CD46 signaling 3 days after mycobacterial infection enhanced BCG-specific T cell responses in a subset of volunteers. Taken together, these results indicate that CD46-dependent negative regulatory mechanisms can impair T cell responses vital for immune defense against mycobacteria. Therefore, modulating CD46-induced immune regulation could be integral to the development of improved tuberculosis therapeutics or vaccines.     

Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells

Aside from an intermediate stage in thymic T-cell development, the expression of CD4 and CD8 is generally thought to be mutually exclusive, associated with helper or cytotoxic T-cell functions, respectively. Stimulation of CD8+ T cells, however, induces the de novo expression of CD4. We demonstrate that while superantigen (staphylococcal enterotoxin B, SEB) and anti-CD3/CD28 costimulation of purified CD8+ T cells induced the expression of CD4 on CD8+ T cells by 30 and 17%, respectively, phytohaemagglutinin (PHA) stimulation did not induce CD4 expression on purified CD8+ T cells but significantly induced the expression of both CD4 on CD8 (CD4dimCD8bright) and CD8 on CD4 (CD4brightCD8dim) T cells in unfractionated peripheral blood mononuclear cells (PBMC). The level of the PHA-mediated induction of CD4dimCD8bright and CD4brightCD8dim was at 27 and 17%, respectively. Depletion of CD4+ T cells from PBMC abrogated this PHA-mediated effect. Autologous CD4+ and CD8+ T-cell co-cultures in the presence of PHA induced this CD4dimCD8bright T-cell expression by 33%, demonstrating a role for CD4 cells in the PHA-mediated induction of the double positive cells. The induction of CD4dimCD8bright was independent of a soluble factor(s). Phenotypic analysis of CD4dimCD8bright T cells indicated significantly higher levels of CD95, CD25, CD38, CD69, CD28, and CD45RO expression than their CD8+CD4- counterparts. CD4dimCD8bright T cells were also negative for CD1a expression and were predominantly T-cell receptor (TCR) alphabeta cells. Our data demonstrate that CD4dimCD8bright T cells are an activated phenotype of CD8+ T cells and suggest that CD4 upregulation on CD8+ T cells may function as an additional marker to identify activated CD8+ T cells.     

CD8+ T cells are associated with severe gastritis in Helicobacter pylori-infected mice in the absence of CD4+ T cells

Helicobacter pylori infection results in the development of chronic gastritis, and CD4⁺ T cells are a major component of the gastric cellular infiltrate. To examine whether CD4⁺ T cells are important in initiating and maintaining H. pylori-induced gastritis, mice deficient in CD4⁺ T cells (B6.BM1.GK 1.5 mice [GK 1.5 mice]) were infected with H. pylori. We found that as in normal mice, H. pylori-specific antibodies, mostly of the immunoglobulin M isotype, developed in GK 1.5 mice but were unable to cure H. pylori infection. Further, while the stomachs of H. pylori-infected GK 1.5 mice were more heavily infiltrated with CD8⁺ T cells and B cells, mice deficient in both CD4⁺ and CD8⁺ T cells developed mild inflammation comparable to the level observed for C57BL/6 mice. These observations suggest that CD4⁺ T cells may play an important role in regulating or suppressing gastric CD8⁺ T cells which, in the absence of CD4⁺ T cells, may mediate more-severe disease. These studies have revealed a potentially important role for CD8⁺ T cells in the gastric disease resulting from H. pylori infection.     

Absence of CD4+CD25+ regulatory T cells is associated with a loss of regulation leading to increased pathology in Helicobacter pylori-infected mice

Helicobacter pylori induces symptomatic chronic gastritis in a subpopulation of infected individuals. The mechanism(s) determining the development and severity of pathology leading to symptoms are not fully understood. In a mouse model of H. pylori infection we analysed the influence of immunoregulatory CD4+CD25+ T cells on H. pylori colonization and gastritis. Athymic C57BL/6 nu/nu mice were reconstituted with (a) lymph node (LN) cells (b) LN cells depleted of CD25+ T cells (CD25(-) LN) or (c) not reconstituted at all. Mice were then infected orally with 3 x 10(8)H. pylori SS1 bacteria. At 2 and 6 weeks after the inoculation there was a significant (P < 0.001) reduction in H. pylori colonization in athymic mice transferred with CD25(-) LN cells compared to mice transferred with LN cells. Colonization was still reduced at 12 weeks after inoculation. Mice transferred with CD25(-) LN cells showed an earlier onset and increased severity of gastritis as compared to mice receiving LN cells. Splenic cells isolated from mice receiving CD25(-) LN cells produced the highest level of IFN-gamma on stimulation with H. pylori antigens in vitro, had a higher H. pylori-specific DTH response and increased infiltration of CD4+ T cells and macrophages in the gastric mucosa. Athymic mice not transferred with T cells had persistent high H. pylori colonization and displayed a normal gastric epithelium without inflammatory cells. In conclusion, CD4+CD25+ cells reduce immunopathology in H. pylori infection, possibly by reducing the activation of IFN-gamma producing CD4+ T cells, even at the expense of a higher H. pylori load in the gastric mucosa.     

Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

CD4(+) CD25(+) regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4(+) CD25(+) regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4(+) CD25(+) regulatory T cells anergize CD4(+) T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4(+) CD25(+) T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4(+) and CD8(+) T cells. This suppression only occurs when CD4(+) CD25(+) T cells are preactivated. Furthermore, we could demonstrate that CD4(+) T-cell clones stop secreting interferon-gamma (IFN-gamma), start to produce interleukin-10 and transforming growth factor-beta after coculture with preactivated CD4(+) CD25(+) T cells and become suppressive themselves. Surprisingly preactivated CD4(+) CD25(+) T cells affect CD8(+) T cells differently, leading to reduced proliferation and reduced production of IFN-gamma. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8(+) T cells, unlike CD4(+) T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4(+) CD25(+) T cells.     

Gene expression patterns associated with tumor-infiltrating CD4+ and CD8+ T cells in invasive breast carcinomas

BackgroundBreast carcinoma is one of the most common tumors in women. The immune microenvironment, especially T cell infiltration, is related to the occurrence and prognosis of breast carcinoma.ObjectiveThis study investigated the gene expression patterns associated with tumor-infiltrating CD4+ and CD8+ T cells in invasive breast carcinomas.MethodsThe gene expression data and corresponding clinical phenotype data from the Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) were downloaded. The stromal and immune score were calculated using ESTIMATE. The differentially expressed genes (DEGs) with a high vs. low stromal score and a high vs. low immune score were screened and then functionally enriched. The tumor-infiltrating immune cells were investigated using the Cibersort algorithm, and the CD4+ and CD8+ T cell-related genes were identified using a Spearman correlation test of infiltrating abundance with the DEGs. Moreover, the miRNA-mRNA pairs and lncRNA-miRNA pairs were predicted to construct the competing endogenous RNAs (ceRNA) network. Kaplan-Meier (K-M) survival curves were also plotted.ResultsIn total, 478 DEGs with a high vs. low stromal score and 796 DEGs with a high vs. low immune score were identified. In addition, 39 CD4+ T cell-related genes and 78 CD8+ T cell-related genes were identified; of these, 14 genes were significantly associated with the prognosis of BRCA patients. Moreover, for CD4+ T cell-related genes, the chr22-38_28785274-29006793.1–miR-34a/c-5p–CAPN6 axis was identified from the ceRNA network, whereas the chr22-38_28785274-29006793.1–miR-494-3p–SLC9A7 axis was identified for CD8+ T cell-related genes.ConclusionsThe chr22-38_28785274-29006793.1–miR-34a/c-5p–CAPN6 axis and the chr22-38_28785274-29006793.1–miR-494-3p–SLC9A7 axis might regulate cellular activities associated with CD4+ and CD8+ T cell infiltration, respectively, in BRCA.     

Differential kinetics of intracellular caspase-1-like and caspase-3-like enzyme activity in human alloreactive CD4(+) and CD8(+) T cells undergoing apoptosis

The ICE-like families of serine proteases (caspases) have integral roles in apoptosis. These studies were performed to further understand the role of two critical caspases in relation to apoptotic regulation of the alloimmune response. A novel three-color cytofluorographic technique was utilized for measuring intracellular (in situ) caspase-1-like and caspase-3-like enzyme activity in responding CD4(+) and CD8(+) T cells over several time points of human mixed lymphocyte reactions (MLR). We found that activity levels of caspase 3 in both CD4(+) and CD8(+) responder cells began rising at day 10 of the MLR and peaked at day 14. By comparison, caspase 1 demonstrated the highest activity at day 7 in both cell subpopulations. These results coincided with the appearance of apoptotic cells among the alloreactive cells in the MLR. These findings demonstrate that intracellular caspase-1- and -3-like enzyme activity increases in both CD4(+) and CD8(+) alloreactive T cells as the primary response to allostimulatory cells progresses. While the kinetic profiles for these enzymes differed, both had a temporal association with the appearance of apoptosis in the MLR-generated cells. In all cases, the highest enzyme activity and presence of apoptosis was seen subsequent to the peak proliferative period. These results support the concept that changes in the rate and amount of apoptosis in alloreactive T cells is one mechanism by which the response to alloantigens is attenuated (i.e., tolerance) or sustained.     

Recovery from experimental allergic encephalomyelitis is TGF-beta dependent and associated with increases in CD4+LAP+ and CD4+CD25+ T cells

SJL mice are highly susceptible to proteolipid protein (PLP) 139-151-induced experimental allergic encephalomyelitis (EAE). The disease is characterized by a relapsing-remitting type of paralysis. However, the mechanism by which animals recover from EAE is poorly understood. Here, we investigated the role of regulatory T cells in the recovery from disease. We found that Forkhead box P3-expressing CD4+CD25+ T cells were increased in the blood, draining lymph node and spleen of EAE-recovered SJL mice. These cells were anergic and inhibited proliferation of CD4+CD25- T cells to PLP 139-151 or anti-CD3 antibody stimulation. Depletion of CD4+CD25+ T cells during the recovery phase exacerbated disease, resulted in the expansion of IA(s)/PLP 139-151-tetramer-positive cells and enhanced IFN-gamma production. In addition, transforming growth factor-beta (TGF-beta) was shown to be involved in the recovery from EAE as the percentage of CD4+ cells expressing TGF-beta latency-associated peptide (LAP) on the cell surface increased significantly in blood and spleen of EAE-recovered mice as compared with the naive mice and in vivo neutralization of TGF-beta abolished recovery from disease. Taken together, our results demonstrate that both CD4+CD25+ and CD4+LAP+ regulatory T cells mediate recovery from PLP 139-151-induced EAE in SJL mice in which TGF-beta plays an important role.     

Generation of peptide-specific CD8+ T cells by phytohemagglutinin-stimulated antigen-mRNA-transduced CD4+ T cells

Functional analysis of antigen-specific CD8(+) T cells is important for understanding the immune response in various immunological disorders. To analyze CD8(+) T cell responses to a variety of antigens with no readily defined peptides available, we developed a system using CD4(+) phytohemagglutinin (PHA) blasts transduced with mRNA for antigen molecules. CD4(+) PHA blasts express MHC class I and II, and also CD80 and CD86 and are thus expected to serve as potent antigen presenting cells. EGFP mRNA could be transduced into and the protein expressed by more than 90% of either LCL or CD4(+) PHA blasts. Its expression stably persisted for more than 2 weeks after transduction. In experiments with HLA-A*2402 restricted CD8(+) CTL clones for either EBNA3A or a cancer-testis antigen, SAGE, mRNA-transduced lymphoid cells were appropriate target cells in ELISPOT assays or (51)Cr releasing assays. Finally, using CD4(+) PHA blasts transduced with mRNA of a cancer-testis antigen MAGE-A4, we successfully generated specific CTL clones that recognized a novel HLA-B*4002 restricted epitope, MAGE-A4(223-231). Messenger RNA-transduced CD4(+) PHA blasts are thus useful antigen presenting cells for analysis of CD8(+) T cell responses and induction of specific T cells for potential immunotherapy.     

Antigen-specific CD8+ T cells inhibit IgE responses and interleukin-4 production by CD4+ T cells

There is a growing body of evidence which suggests that CD8⁺ T cells play an important part in regulating the IgE response to non-replicating antigens. In this study we have systematically investigated their role in the regulation of IgE and of CD4⁺ T cell responses to ovalbumin (OVA) by CD8⁺ T cell depletion in vivo. Following intraperitoneal immunization with alum-precipitated OVA, OVA-specific T cell responses were detected in the spleen and depletion of CD8⁺ T cells in vitro significantly enhanced the proliferative response to OVA. Depletion of CD8⁺ T cells in vivo 7 days after immunization failed to enhance IgE production, while depletion of CD8⁺ T cells on days 12–18 greatly enhanced the IgE response, which rose to 26 μ/ml following a second injection of anti-CD8 on day 35 and remained in excess of 1 μ/ml over 300 days afterwards. Reconstitution on day 21 of rats CD8-depleted on day 12 with purified CD8⁺ T cells from animals immunized on day 12 completely inhib ited the IgE response. This effect was antigen specific; CD8⁺ T cells from OVA-primed animals had little effect on the IgE response of bovine serum albumin immunized rats. In vivo, CD8⁺ T cell depletion decreased interferon (IFN)-γ production but enhanced interleukin (IL)-4 production by OVA-stimulated splenic CD4⁺ T cells. Furthermore, CD8⁺ T cell depletion and addition of anti-IFN-γ antibody enhanced IgE production in vitro in an IL-4-supplemented mixed lymphocyte reaction. These data clearly show that antigen-specific CD8⁺ T cells inhibit IgE in the immune response to non-replicating antigens. The data indicate two possible mechanisms: first, CD8⁺ T cells have direct inhibitory effects on switching to IgE in B cells and second, they inhibit OVA-specific IL-4 production but enhance IFN-γ production by CD4⁺ T cells.     

Lymphatic tissue fibrosis is associated with reduced numbers of naive CD4+ T cells in human immunodeficiency virus type 1 infection

The organized structure of lymphatic tissues (LTs) constitutes a microenvironment referred to as a niche that plays a critical role in immune system homeostasis by promoting cellular interactions and providing access to cytokines and growth factors on which cells are dependent for survival, proliferation, and differentiation. In chronic human immunodeficiency virus type 1 (HIV-1) infection, immune activation and inflammation result in collagen deposition and disruption of this LT niche. We have previously shown that these fibrotic changes correlate with a reduction in the size of the total population of CD4+ T cells. We now show that this reduction is most substantial within the na?ve CD4+ T-cell population and is in proportion to the extent of LT collagen deposition in HIV-1 infection. Thus, the previously documented depletion of na?ve CD4+ T cells in LTs in HIV-1 infection may be a consequence not only of a decreased supply of thymic emigrants or chronic immune activation but also of the decreased ability of those cells to survive in a scarred LT niche. We speculate that LT collagen deposition might therefore limit repopulation of na?ve CD4+ T cells with highly active antiretroviral therapy, and thus, additional treatments directed to limiting or reversing inflammatory damage to the LT niche could potentially improve immune reconstitution.     

Neoplastic thymic epithelial cells of human thymoma support T cell development from CD4−CD8− cells to CD4+CD8+ cells in vitro

Human thymoma is a thymic epithelial cell tumour which often contains a large number of immature T cells and is frequently associated with autoimmune diseases. Since thymic epithelial cells play key roles in the development and selection of T cells in the normal thymus, we hypothesized that the neoplastic thymic epithelial cells of thymoma may support T cell differentiation in the tumour. We characterized CD4^?CD8^? cells in thymoma and applied an in vitro reconstitution culture system using the CD4^?CD8^? cells and the neoplastic epithelial cells isolated from thymoma. CD34, a stem cell marker, was expressed on 29.9 ± 12.2% of CD4^?CD8^? cells in thymoma. TCRγδ was expressed on 27.4 ± 15.1% of CD4^?CD8^? cells and CD19, a B cell marker, was expressed on 14.1 ± 23.1% of CD4^?CD8^? cells. CD4^?CD8^? cells expressed both IL-7R α-chain and common γ-chain. Purified CD4^?CD8^? cells from thymomas were cultured with the neoplastic epithelial cells, and their differentiation into CD4⁺CD8⁺ cells via CD4 single-positive intermediates was observed within 9 days' co-culture in the presence of recombinant IL-7. Furthermore, we examined the reconstitution culture using CD34⁺CD4^?CD8^? cells purified from normal infant thymus. The CD34⁺CD4^?CD8^? cells in normal thymus also differentiated to CD4⁺CD8⁺ cells in the allogeneic co-culture with the neoplastic epithelial cells of thymoma. These results indicate that the tumour cells of thymoma retain the function of thymic epithelial cells and can induce differentiation of T cells in thymoma.     

Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells

We investigated the function of CD56+ CD8+ T cells (CD56+ T cells) and CD56- CD57+ CD8+ T cells (CD57+ T cells; natural killer (NK)-type T cells) and compared them with those of normal CD56- CD57- CD8+ T cells (CD8+ T cells) and CD56+ NK cells from healthy volunteers. After the stimulation with immobilized anti-CD3 antibodies, both NK-type T cells produced much larger amounts of interferon-gamma (IFN-gamma) than CD8+ T cells. Both NK-type T cells also acquired a more potent cytotoxicity against NK-sensitive K562 cells than CD8+ T cells while only CD56+ T cells showed a potent cytotoxicity against NK-resistant Raji cells. After the stimulation with a combination of interleukin (IL)-2, IL-12 and IL-15, the IFN-gamma amounts produced were NK cells > or = CD56+ T cells > or = CD57+ T cells > CD8+ T cells. The cytotoxicities against K562 cells were NK cells > CD56+ T cells > or = CD57+ T cells > CD8+ T cells while cytotoxicities against Raji cells were CD56+ T cells > CD57+ T cells > or = CD8+ T cells > or = NK cells. However, the CD3-stimulated proliferation of both NK-type T cells was smaller than that of CD8+ T cells partly because NK-type T cells were susceptible to apoptosis. In addition to NK cells, NK-type T cells but not CD8+ T cells stimulated with cytokines, expressed cytoplasmic perforin and granzyme B. Furthermore, CD3-stimulated IFN-gamma production from peripheral blood mononuclear cells (PBMC) correlated with the proportions of CD57+ T cells in PBMC from donors. Our findings suggest that NK-type T cells play an important role in the T helper 1 responses and the immunological changes associated with ageing.