Snapin promotes HIV‐1 transmission from dendritic cells by dampening TLR8 signaling

HIV‐1 traffics through dendritic cells (DCs) en route to establishing a productive infection in T lymphocytes but fails to induce an innate immune response. Within DC endosomes, HIV‐1 somehow evades detection by the pattern‐recognition receptor (PRR) Toll‐like receptor 8 (TLR8). Using a phosphoproteomic approach, we identified a robust and diverse signaling cascade triggered by HIV‐1 upon entry into human DCs. A secondary siRNA screen of the identified signaling factors revealed several new mediators of HIV‐1 trans‐infection of CD4⁺ T cells in DCs, including the dynein motor protein Snapin. Inhibition of Snapin enhanced localization of HIV‐1 with TLR8⁺ early endosomes, triggered a pro‐inflammatory response, and inhibited trans‐infection of CD4⁺ T cells. Snapin inhibited TLR8 signaling in the absence of HIV‐1 and is a general regulator of endosomal maturation. Thus, we identify a new mechanism of innate immune sensing by TLR8 in DCs, which is exploited by HIV‐1 to promote transmission.     

Knock‐down of HEXA and HEXB genes correlate with the absence of the immunostimulatory function of HSC‐derived dendritic cells

In an attempt to investigate whether the genetic defect in the HEXA and HEXB genes (which causes the absence of the lysosomal β‐N‐acetyl‐hexosaminidase), are related to the wide inflammation in GM2 gangliosidoses (Tay‐Sachs and Sandhoff disease), we have chosen the dendritic cells (DCs) as a study model. Using the RNA interference approach, we generated an in vitro model of HEXs knock‐down immunogenic DCs (i‐DCs) from CD34⁺‐haemopoietic stem cells (CD34⁺‐HSCs), thus mimicking the Tay‐Sachs (HEXA?/?) and Sandhoff (HEXB?/?) cells. We showed that the absence of β‐N‐acetyl‐hexosaminidase activity does not alter the differentiation of i‐DCs from HSCs, but it is critical for the activation of CD4⁺T cells because knock‐down of HEXA or HEXB gene causes a loss of function of i‐DCs. Notably, the silencing of the HEXA gene had a stronger immune inhibitory effect, thereby indicating a major involvement of β‐N‐acetyl‐hexosaminidase A isoenzyme within this mechanism. Copyright © 2011 John Wiley & Sons, Ltd.     

Human CD8+ T cells transduced with an additional receptor bispecific for both Mycobacterium tuberculosis and HIV‐1 recognize both epitopes

Tuberculosis (TB) and human immunodeficiency virus type 1 (HIV‐1) infection are closely intertwined, with one‐quarter of TB/HIV coinfected deaths among people died of TB. Effector CD8⁺ T cells play a crucial role in the control of Mycobacterium tuberculosis (MTB) and HIV‐1 infection in coinfected patients. Adoptive transfer of a multitude of effector CD8⁺ T cells is an appealing strategy to impose improved anti‐MTB/HIV‐1 activity onto coinfected individuals. Due to extensive existence of heterologous immunity, that is, T cells cross‐reactive with peptides encoded by related or even very dissimilar pathogens, it is reasonable to find a single T cell receptor (TCR) recognizing both MTB and HIV‐1 antigenic peptides. In this study, a single TCR specific for both MTB Ag85B_199‐207 peptide and HIV‐1 Env_120‐128 peptide was screened out from peripheral blood mononuclear cells of a HLA‐A*0201⁺ healthy individual using complementarity determining region 3 spectratype analysis and transferred to primary CD8⁺ T cells using a recombinant retroviral vector. The bispecificity of the TCR gene‐modified CD8⁺ T cells was demonstrated by elevated secretion of interferon‐γ, tumour necrosis factor‐α, granzyme B and specific cytolytic activity after antigen presentation of either Ag85B_199‐207 or Env_120‐128 by autologous dendritic cells. To the best of our knowledge, this study is the first report proposing to produce responses against two dissimilar antigenic peptides of MTB and HIV‐1 simultaneously by transfecting CD8⁺ T cells with a single TCR. Taken together, T cells transduced with the additional bispecific TCR might be a useful strategy in immunotherapy for MTB/HIV‐1 coinfected individuals.     

HIV Malaria Co-Infection Is Associated with Atypical Memory B Cell Expansion and a Reduced Antibody Response to a Broad Array of Plasmodium falciparum Antigens in Rwandan Adults

HIV infected individuals in malaria endemic areas experience more frequent and severe malaria episodes compared to non HIV infected. This clinical observation has been linked to a deficiency in antibody responses to Plasmodium falciparum antigens; however, prior studies have only focused on the antibody response to <0.5% of P. falciparum proteins. To obtain a broader and less-biased view of the effect of HIV on antibody responses to malaria we compared antibody profiles of HIV positive (HIV+) and negative (HIV-) Rwandan adults with symptomatic malaria using a microarray containing 824 P. falciparum proteins. We also investigated the cellular basis of the antibody response in the two groups by analyzing B and T cell subsets by flow cytometry. Although HIV malaria co-infected individuals generated antibodies to a large number of P. falciparum antigens, including potential vaccine candidates, the breadth and magnitude of their response was reduced compared to HIV- individuals. HIV malaria co-infection was also associated with a higher percentage of atypical memory B cells (MBC) (CD19+CD10-CD21-CD27-) compared to malaria infection alone. Among HIV+ individuals the CD4⁺ T cell count and HIV viral load only partially explained variability in the breadth of P. falciparum-specific antibody responses. Taken together, these data indicate that HIV malaria co-infection is associated with an expansion of atypical MBCs and a diminished antibody response to a diverse array of P. falciparum antigens, thus offering mechanistic insight into the higher risk of malaria in HIV+ individuals.     

IL‐35Ig–expressing dendritic cells induce tolerance via Arginase 1

The cytokine interleukin IL‐35 is known to exert strong immunosuppressive functions. Indoleamine 2,3‐dioxygenase 1 (IDO1) and Arginase 1 (Arg1) are metabolic enzymes that, expressed by dendritic cells (DCs), contribute to immunoregulation. Here, we explored any possible link between IL‐35 and the activity of those enzymes. We transfected a single chain IL‐35Ig gene construct in murine splenic DCs (DC₃₅) and assessed any IDO1 and Arg1 activities as resulting from ectopic IL‐35Ig expression, both in vitro and in vivo. Unlike Ido1, Arg1 expression was induced in vitro in DC₃₅, and it conferred an immunosuppressive phenotype on those cells, as revealed by a delayed‐type hypersensitivity assay. Moreover, the in vivo onset of a tolerogenic phenotype in DC₃₅ was associated with the detection of CD25⁺CD39⁺, rather than Foxp3⁺, regulatory T cells. Therefore, Arg1, but not Ido1, expression in DC₃₅ appears to be an early event in IL‐35Ig–mediated immunosuppression.     

Thymus‐derived regulatory T cells restrain pro‐inflammatory Th1 responses by downregulating CD70 on dendritic cells

The severity and intensity of autoimmune disease in immune dysregulation, polyendocrinopathy, enteropathy, X‐linked (IPEX) patients and in scurfy mice emphasize the critical role played by thymus‐derived regulatory T cells (tTregs) in maintaining peripheral immune tolerance. However, although tTregs are critical to prevent lethal autoimmunity and excessive inflammatory responses, their suppressive mechanism remains elusive. Here, we demonstrate that tTregs selectively inhibit CD27/CD70‐dependent Th1 priming, while leaving the IL‐12‐dependent pathway unaffected. Immunized mice depleted of tTregs showed an increased response of IFN‐γ‐secreting CD4⁺ T cells that was strictly reliant on a functional CD27/CD70 pathway. In vitro studies revealed that tTregs downregulate CD70 from the plasma membrane of dendritic cells (DCs) in a CD27‐dependent manner. CD70 downregulation required contact between Tregs and DCs and resulted in endocytosis of CD27 and CD70 into the DC. These findings reveal a novel mechanism by which tTregs can maintain tolerance or prevent excessive, proinflammatory Th1 responses.     

Membrane‐bound HSP70‐engineered myeloma cell‐derived exosomes stimulate more efficient CD8+ CTL‐ and NK‐mediated antitumour immunity than exosomes released from heat‐shocked tumour cells expressing cytoplasmic HSP70

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour‐derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T‐cell immune responses. To enhance EXO‐based antitumour immunity, we generated an engineered myeloma cell line J558_HSP expressing endogenous P1A tumour antigen and transgenic form of membrane‐bound HSP70 and heat‐shocked J558_HS expressing cytoplasmic HSP70, and purified EXO_HSP and EXO_HS from J558_HSP and J558_HS tumour cell culture supernatants by ultracentrifugation. We found that EXO_HSP were able to more efficiently stimulate maturation of DCs with up‐regulation of Ia^b, CD40, CD80 and inflammatory cytokines than EXO_HS after overnight incubation of immature bone‐marrow‐derived DCs (5 × 10⁶ cells) with EXO (100 μg), respectively. We also i.v. immunized BALB/c mice with EXO (30 μg/mouse) and assessed P1A‐specific T‐cell responses after immunization. We demonstrate that EXO_HSP are able to stimulate type 1 CD4⁺ helper T (Th1) cell responses, and more efficient P1A‐specific CD8⁺ cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXO_HS. In addition, we further elucidate that EXO_HSP‐stimulated antitumour immunity is mediated by both P1A‐specific CD8⁺ CTL and non‐P1A‐specific natural killer (NK) responses. Therefore, membrane‐bound HSP70‐expressing tumour cell‐released EXO may represent a more effective EXO‐based vaccine in induction of antitumour immunity.     

The Microvesicle Component of HIV-1 Inocula Modulates Dendritic Cell Infection and Maturation and Enhances Adhesion to and Activation of T Lymphocytes

HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45⁺ microvesicles (MV) which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, de novo HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1_BaL or matched non-depleted preparations, the presence of CD45⁺ MVs was shown to enhance DC maturation and ICAM-1 (CD54) expression, which is involved in DC∶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24⁺) MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DC∶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs) and nef were the likely DC maturation candidates. Recombinant HSP90α and β and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs in vitro and in vivo, particularly as MVs have been detected in the circulation of HIV-1 infected subjects.     

Aging is associated with increased regulatory T‐cell function

Regulatory T‐cell (Treg, CD4⁺CD25⁺) dysfunction is suspected to play a key role in immune senescence and contributes to increased susceptibility to diseases with age by suppressing T‐cell responses. FoxP3 is a master regulator of Treg function, and its expression is under control of several epigenetically labile promoters and enhancers. Demethylation of CpG sites within these regions is associated with increased FoxP3 expression and development of a suppressive phenotype. We examined differences in FoxP3 expression between young (3–4 months) and aged (18–20 months) C57BL/6 mice. DNA from CD4⁺ T cells is hypomethylated in aged mice, which also exhibit increased Treg numbers and FoxP3 expression. Additionally, Treg from aged mice also have greater ability to suppress effector T‐cell (Teff) proliferation in vitro than Tregs from young mice. Tregs from aged mice exhibit greater redox remodeling–mediated suppression of Teff proliferation during coculture with DCs by decreasing extracellular cysteine availability to a greater extent than Tregs from young mice, creating an adverse environment for Teff proliferation. Tregs from aged mice produce higher IL‐10 levels and suppress CD86 expression on DCs more strongly than Tregs from young mice, suggesting decreased T‐cell activity. Taken together, these results reveal a potential mechanism of higher Treg‐mediated activity that may contribute to increased immune suppression with age.     

Characterization of age‐associated exhausted CD8+ T cells defined by increased expression of Tim‐3 and PD‐1

Aging is accompanied by altered T‐cell responses that result in susceptibility to various diseases. Previous findings on the increased expression of inhibitory receptors, such as programmed cell death protein 1 (PD‐1), in the T cells of aged mice emphasize the importance of investigations into the relationship between T‐cell exhaustion and aging‐associated immune dysfunction. In this study, we demonstrate that T‐cell immunoglobulin mucin domain‐3 (Tim‐3), another exhaustion marker, is up‐regulated on aged T cells, especially CD8⁺ T cells. Tim‐3‐expressing cells also produced PD‐1, but Tim‐3⁺PD‐1⁺ CD8⁺ T cells had a distinct phenotype that included the expression of CD44 and CD62L, from Tim‐3^?PD‐1⁺ cells. Tim‐3⁺PD‐1⁺ CD8⁺ T cells showed more evident properties associated with exhaustion than Tim‐3^?PD‐1⁺ CD8⁺ T cells: an exhaustion‐related marker expression profile, proliferative defects following homeostatic or TCR stimulation, and altered production of cytokines. Interestingly, these cells produced a high level of IL‐10 and induced normal CD8⁺ T cells to produce IL‐10, which might contribute to immune dysregulation in aged mice. The generation of Tim‐3‐expressing CD8⁺ T cells in aged mice seems to be mediated by encounters with antigens but not by specific infection, based on their high expression of CD49d and their unbiased TCR Vβ usage. In conclusion, we found that a CD8⁺ T‐cell population with age‐associated exhaustion was distinguishable by its expression of Tim‐3. These results provide clues for understanding the alterations that occur in T‐cell populations with age and for improving dysfunctions related to the aging of the immune system.     

Identification of HLA‐A*11:01‐restricted Mycobacterium tuberculosis CD8+ T cell epitopes

New vaccines are needed to combat Mycobacterium tuberculosis (MTB) infections. The currently employed Bacillus Calmette‐Guérin vaccine is becoming ineffective, due in part to the emergence of multidrug‐resistant tuberculosis (MDR‐TB) strains and the reduced immune capacity in cases of HIV coinfection. CD8⁺ T cells play an important role in the protective immunity against MTB infections, and the identification of immunogenic CD8⁺ T cell epitopes specific for MTB is essential for the design of peptide‐based vaccines. To identify CD8⁺ T cell epitopes of MTB proteins, we screened a set of 94 MTB antigens for HLA class I A*11:01‐binding motifs. HLA‐A*11:01 is one of the most prevalent HLA molecules in Southeast Asians, and definition of T cell epitopes it can restrict would provide significant coverage for the Asian population. Peptides that bound with high affinity to purified HLA molecules were subsequently evaluated in functional assays to detect interferon‐γ release and CD8⁺ T cell proliferation in active pulmonary TB patients. We identified six novel epitopes, each derived from a unique MTB antigen, which were recognized by CD8⁺ T cells from active pulmonary TB patients. In addition, a significant level of epitope‐specific T cells could be detected ex vivo in peripheral blood mononuclear cells from active TB patients by an HLA‐A*11:01 dextramer carrying the peptide Rv3130c_194‐204 (from the MTB triacylglycerol synthase Tgs1), which was the most frequently recognized epitope in our peptide library. In conclusion, this study identified six dominant CD8⁺ T cell epitopes that may be considered potential targets for subunit vaccines or diagnostic strategies against TB.     

High‐dose wogonin exacerbates DSS‐induced colitis by up‐regulating effector T cell function and inhibiting Treg cell

Wogonin exerts anti‐tumour activities via multiple mechanisms. We have identified that high‐dose wogonin (50 or 100 mg/kg) could inhibit the growth of transplanted tumours by directly inducing tumour apoptosis and promoting DC, T and NK cell recruitment into tumour tissues to enhance immune surveillance. However, wogonin (20–50 μM) ex vivo prevents inflammation by inhibiting NF‐κB and Erk signalling of macrophages and epithelial cells. It is elusive whether high‐dose wogonin promotes or prevents inflammation. To investigate the effects of high‐dose wogonin on murine colitis induced by dextran sodium sulphate (DSS), mice were co‐treated with DSS and various doses of wogonin. Intraperitoneal administration of wogonin (100 mg/kg) exacerbated DSS‐induced murine colitis. More CD4⁺ CD44⁺ and CD8⁺ CD44⁺ cells were located in the inflamed colons in the wogonin (100 mg/kg) treatment group than in the other groups. Frequencies of CD4⁺ CD25⁺ CD127^? and CD4⁺ CD25⁺ Foxp3⁺ cells in the colons and spleen respectively, were reduced by wogonin treatment. Ex vivo stimulations with high‐dose wogonin (50–100 μg/ml equivalent to 176–352 μM) could synergize with IL‐2 to promote the functions of CD4⁺ and CD8⁺ cells. However, regulatory T cell induction was inhibited. Wogonin stimulated the activation of NF‐κB and Erk but down‐regulated STAT3 phosphorylation in the CD4⁺ T cells. Wogonin down‐regulated Erk and STAT3‐Y705 phosphorylation in the regulatory T cells but promoted NF‐κB and STAT3‐S727 activation. Our study demonstrated that high‐dose wogonin treatments would enhance immune activity by stimulating the effector T cells and by down‐regulating regulatory T cells.     

Aging impacts CD103+CD8+ T cell presence and induction by dendritic cells in the genital tract

As women age, susceptibility to systemic and genital infections increases. Tissue‐resident memory T cells (TRMs) are CD103⁺CD8⁺ long‐lived lymphocytes that provide critical mucosal immune protection. Mucosal dendritic cells (DCs) are known to induce CD103 expression on CD8⁺ T cells. While CD103⁺CD8⁺ T cells are found throughout the female reproductive tract (FRT), the extent to which aging impacts their presence and induction by DCs remains unknown. Using hysterectomy tissues, we found that endometrial CD103⁺CD8⁺ T cells were increased in postmenopausal compared to premenopausal women. Endometrial DCs from postmenopausal women were significantly more effective at inducing CD103 expression on allogeneic naïve CD8⁺ T cells than DCs from premenopausal women; CD103 upregulation was mediated through membrane‐bound TGFβ signaling. In contrast, cervical CD103⁺ T cells and DC numbers declined in postmenopausal women with age. Decreases in DCs correlated with decreased CD103⁺ T cells in endocervix, but not ectocervix. Our findings demonstrate a previously unrecognized compartmentalization of TRMs in the FRT of postmenopausal women, with loss of TRMs and DCs in the cervix with aging, and increased TRMs and DC induction capacity in the endometrium. These findings are relevant to understanding immune protection in the FRT and to the design of vaccines for women of all ages.     

Alteration of inhibitory and activating natural killer cell receptor expression on T cells in human immunodeficiency virus-infected Chinese

T cell expression of NKRs can trigger or inhibit cell‐mediated cytotoxicity. However, few studies on T lymphocyte NKR expression in HIV infection exist. Here, we examined the expression patterns of NKG2D, NKG2A, and KIR3DL1 on CD8⁺ and CD3⁺CD8^? cells by multicolor flow cytometry in groups of patients with HIV, AIDS or HAART‐treated AIDS, as well as HIV‐negative normal controls. Individual analysis of KIR3DL1 on CD3⁺CD8⁺ or CD3⁺CD8^? cells revealed no significant differences among any of the groups (P > 0.05). In contrast, the percentage of NKG2A⁺NKG2D^?CD8⁺ T cells was higher in the AIDS group than in the HIV‐negative normal control group (P < 0.01). Meanwhile, the prevalence of NKG2D⁺NKG2A^?CD8⁺T cells was lower in the AIDS group than in HIV‐negative normal controls (P < 0.001). Similar results were also observed for the percentage of NKG2A⁺NKG2D^? on CD3⁺CD8^?cells. However, in contrast to CD8⁺ T cells, the frequencies of NKG2D⁺NKG2A^? on CD3⁺CD8^? cells were higher in AIDS and HIV patients than in HIV‐negative normal controls (P < 0.01, P < 0.05, respectively). The percentage of NKG2A⁺NKG2D^?CD8⁺ T cells was negatively correlated with CD4⁺ T cell counts (r=?0.499, P < 0.01), while the percentage of NKG2D⁺NKG2A^?CD8⁺ T cells was positively correlated with CD4⁺ T cell counts (r= 0.494, P < 0.01). The percentage of NKG2D⁺NKG2A^?CD3⁺CD8^? T cells was also positively correlated with viral load (r= 0.527, P < 0.01) and negatively correlated with CD4⁺ T cell counts (r=?0.397, P < 0.05). Finally, HAART treatment reversed the changes in NKR expression caused by HIV infection. These results indicate that the expression of NKRs on T cells may be correlated with HIV disease progression.     

Role of T‐bet,the master regulator of Th1 cells,in the cytotoxicity of murine CD4+ T cells

Although CD4⁺ T cells are generally regarded as helper T cells, some activated CD4⁺ T cells have cytotoxic properties. Given that CD4⁺ cytotoxic T lymphocytes (CTLs) often secrete IFN‐γ, CTL activity among CD4⁺ T cells may be attributable to Th1 cells, where a T‐box family molecule, T‐bet serves as the “master regulator”. However, although the essential contribution of T‐bet to expression of IFN‐γ has been well‐documented, it remains unclear whether T‐bet is involved in CD4⁺ T cell‐mediated cytotoxicity. In this study, to investigate the ability of T‐bet to confer cytolytic activity on CD4⁺ T cells, the T‐bet gene (Tbx21) was introduced into non‐cytocidal CD4⁺ T cell lines and their cytolytic function analyzed. Up‐regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4⁺ T cells but not in untransfected parental cells. In one cell line, T‐bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas^? target cells. Although T‐bet was found to repress up‐regulation of CD40L (CD154), which controls FasL‐mediated cytolysis, the extent of CD40L up‐regulation on in vitro‐differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T‐bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells.

     

Development of CD8+ T cells expressing two distinct receptors specific for MTB and HIV‐1 peptides

The immune response in individuals co‐infected with Mycobacterium tuberculosis (MTB) and the human immunodeficiency virus (MTB/HIV) gradually deteriorates, particularly in the cellular compartment. Adoptive transfer of functional effector T cells can confer protective immunity to immunodeficient MTB/HIV co‐infected recipients. However, few such effector T cells exist in vivo, and their isolation and amplification to sufficient numbers is difficult. Therefore, enhancing immune responses against both pathogens is critical for treating MTB/HIV co‐infected patients. One approach is adoptive transfer of T cell receptor (TCR) gene‐modified T cells for the treatment of MTB/HIV co‐infections because lymphocyte numbers and their functional avidity is significantly increased by TCR gene transfer. To generate bispecific CD8⁺ T cells, MTB Ag85B_199–207 peptide‐specific TCRs (MTB/TCR) and HIV‐1 Env_120–128 peptide‐specific TCRs (HIV/TCR) were isolated and introduced into CD8⁺ T cells simultaneously using a retroviral vector. To avoid mispairing among exogenous and endogenous TCRs, and to improve the function and stability of the introduced TCRs, several strategies were employed, including introducing mutations in the MTB/TCR constant (C) regions, substituting part of the HIV/TCR C regions with CD3ζ, and linking gene segments with three different 2A peptides. Results presented in this report suggest that the engineered T cells possessed peptide‐specific specificity resulting in cytokine production and cytotoxic activity. This is the first report describing the generation of engineered T cells specific for two different pathogens and provides new insights into TCR gene therapy for the treatment of immunocompromised MTB/HIV co‐infected patients.     

The Polyunsaturated Fatty Acids Arachidonic Acid and Docosahexaenoic Acid Induce Mouse Dendritic Cells Maturation but Reduce T-Cell Responses In Vitro

Long-chain polyunsaturated fatty acids (PUFAs) might regulate T-cell activation and lineage commitment. Here, we measured the effects of omega-3 (n-3), n-6 and n-9 fatty acids on the interaction between dendritic cells (DCs) and naïve T cells. Spleen DCs from BALB/c mice were cultured in vitro with ovalbumin (OVA) with 50 μM fatty acids; α-linolenic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid or oleic acid and thereafter OVA-specific DO11.10 T cells were added to the cultures. Fatty acids were taken up by the DCs, as shown by gas chromatography analysis. After culture with arachidonic acid or DHA CD11c⁺ CD11b⁺ and CD11c⁺ CD11b^neg DCs expressed more CD40, CD80, CD83, CD86 and PDL-1, while IA^d remained unchanged. However, fewer T cells co-cultured with these DCs proliferated (CellTrace Violet^low) and expressed CD69 or CD25, while more were necrotic (7AAD⁺). We noted an increased proportion of T cells with a regulatory T cell (Treg) phenotype, i.e., when gating on CD4⁺ FoxP3⁺ CTLA-4⁺, CD4⁺ FoxP3⁺ Helios⁺ or CD4⁺ FoxP3⁺ PD-1⁺, in co-cultures with arachidonic acid- or DHA-primed DCs relative to control cultures. The proportion of putative Tregs was inversely correlated to T-cell proliferation, indicating a suppressive function of these cells. With arachidonic acid DCs produced higher levels of prostaglandin E₂ while T cells produced lower amounts of IL-10 and IFNγ. In conclusion arachidonic acid and DHA induced up-regulation of activation markers on DCs. However arachidonic acid- and DHA-primed DCs reduced T-cell proliferation and increased the proportion of T cells expressing FoxP3, indicating that these fatty acids can promote induction of regulatory T cells.     

Expression of PD‐1/LAG‐3 and cytokine production by CD4+ T cells during infection with Plasmodium parasites

CD4⁺ T cells play critical roles in protection against the blood stage of malarial infection; however, their uncontrolled activation can be harmful to the host. In this study, in which rodent models of Plasmodium parasites were used, the expression of inhibitory receptors on activated CD4⁺ T cells and their cytokine production was compared with their expression in a bacterial and another protozoan infection. CD4⁺ T cells from mice infected with P. yoelii 17XL, P yoelii 17XNL, P. chabaudi, P. vinckei and P. berghei expressed the inhibitory receptors, PD‐1 and LAG‐3, as early as 6 days after infection, whereas those from either Listeria monocytogenes‐ or Leishmania major‐infected mice did not. In response to T‐cell receptor stimulation, CD4⁺ T cells from mice infected with all the pathogens under study produced high concentrations of IFN‐γ. IL‐2 production was reduced in mice infected with Plasmodium species, but not in those infected with Listeria or Leishmania. In vitro blockade of the interaction between PD‐1 and its ligands resulted in increased IFN‐γ production in response to Plasmodium antigens, implying that PD‐1 expressed on activated CD4⁺ T cells actively inhibits T cell immune responses. Studies using Myd88^?/?, Trif^?/? and Irf3^?/? mice showed that induction of these CD4⁺ T cells and their ability to produce cytokines is largely independent of TLR signaling. These studies suggest that expression of the inhibitory receptors PD‐1 and LAG‐3 on CD4⁺ T cells and their reduced IL‐2 production are common characteristic features of Plasmodium infection.

     

Natural Killer Cell Receptor+ T‐Lymphocytes in Normal and Helicobacter pylori‐Infected Human Gastric Mucosa

Background: Helicobacter pylori infection is associated with development of chronic inflammation and infiltration of immune cells into the gastric mucosa. As unconventional T‐lymphocytes expressing natural killer cell receptors are considered to play central roles in the immune response against infection, a study investigating their frequencies in normal and H. pylori‐infected gastric mucosa was undertaken. Materials and Methods: Flow cytometry was used to quantify T‐cells expressing the natural killer cell markers CD161, CD56, and CD94 in freshly isolated lymphocytes from the epithelial and lamina propria layers of gastric mucosa. Thirteen H. pylori‐positive and 24 H. pylori‐negative individuals were studied. Results: CD94⁺ T‐cells were the most abundant (up to 40%) natural killer receptor‐positive T‐cell population in epithelial and lamina propria layers of H. pylori‐negative gastric mucosa. CD161⁺ T‐cells accounted for about one‐third of all T‐cells in both compartments, but the lowest proportion were of CD56⁺ T‐cells. Compared with H. pylori‐negative mucosa, in H. pylori‐infected mucosa the numbers of CD161⁺ T‐cells were significantly greater (p = .04) in the epithelium, whereas the numbers of CD56⁺ T‐cells were lower (p = .01) in the lamina propria. A minor population (< 2%) of T‐cells in both mucosal layers of H. pylori‐negative subjects were natural killer T‐cells, and whose proportions were not significantly different (p > .05) to those in H. pylori‐infected individuals. Conclusions: The predominance, heterogeneity, and distribution of natural killer cell receptor‐positive T‐cells at different locations within the gastric mucosa reflects a potential functional role during H. pylori infection and warrants further investigation.