Anti-HIV state but not apoptosis depends on IFN signature in CD4+ T cells

To gain insights into the molecular mechanisms underlying early host responses to HIV in the CD4(+) T cell target population, we examined gene expression in CD4(+) T cells isolated 24 h after ex vivo HIV infection of lymphocyte aggregate cultures derived from human tonsils. Gene profiling showed a distinct up-regulation of genes related to immune response and response to virus, notably of IFN-stimulated genes (ISGs), irrespective of the coreceptor tropism of the virus. This mostly IFN-alpha-dependent gene signature suggested the involvement of plasmacytoid dendritic cells, a principal component of the antiviral immune response. Indeed, depletion of plasmacytoid dendritic cells before HIV inoculation abrogated transcriptional up-regulation of several ISGs and resulted in increased levels of HIV replication. Treatment with a blocking anti-IFN-alphaR Ab yielded increased HIV replication; conversely, HIV replication was decreased in pDC-depleted cultures treated with IFN-alpha. Among up-regulated ISGs was also TRAIL, indicating a potential role of the IFN signature in apoptosis. However, a blocking anti-TRAIL Ab did not abrogate apoptosis of CD4(+) T cells in CXCR4-tropic HIV-infected cultures, suggesting the involvement of pathways other than TRAIL mediated. We conclude that acute HIV infection of lymphoid tissue results in up-regulation of ISGs in CD4(+) T cells, which induces an anti-HIV state but not apoptosis.

CD4 T cells are required for both development and maintenance of disease in a new mouse model of reversible colitis

Current therapies to treat inflammatory bowel diseases have limited efficacy, significant side effects, and often wane over time. Little is known about the cellular and molecular mechanisms operative in the process of mucosal healing from colitis. To study such events, we developed a new model of reversible colitis in which adoptive transfer of CD4(+)CD45RB(hi) T cells into Helicobacter typhlonius-colonized lymphopenic mice resulted in a rapid onset of colonic inflammation that was reversible through depletion of colitogenic T cells. Remission was associated with an improved clinical and histopathological score, reduced immune cell infiltration to the intestinal mucosa, altered intestinal gene expression profiles, regeneration of the colonic mucus layer, and the restoration of epithelial barrier integrity. Notably, colitogenic T cells were not only critical for induction of colitis but also for maintenance of disease. Depletion of colitogenic T cells resulted in a rapid drop in tumor necrosis factor α (TNFα) levels associated with reduced infiltration of inflammatory immune cells to sites of inflammation. Although neutralization of TNFα prevented the onset of colitis, anti-TNFα treatment of mice with established disease failed to resolve colonic inflammation. Collectively, this new model of reversible colitis provides an important research tool to study the dynamics of mucosal healing in chronic intestinal remitting-relapsing disorders.Mucosal Immunology advance online publication 16 September 2015; doi:10.1038/mi.2015.93.

Destruction of lymphoid organ architecture and hepatitis caused by CD4+ T cells

Immune responses have the important function of host defense and protection against pathogens. However, the immune response also causes inflammation and host tissue injury, termed immunopathology. For example, hepatitis B and C virus infection in humans cause immunopathological sequel with destruction of liver cells by the host's own immune response. Similarly, after infection with lymphocytic choriomeningitis virus (LCMV) in mice, the adaptive immune response causes liver cell damage, choriomeningitis and destruction of lymphoid organ architecture. The immunopathological sequel during LCMV infection has been attributed to cytotoxic CD8(+) T cells. However, we now show that during LCMV infection CD4(+) T cells selectively induced the destruction of splenic marginal zone and caused liver cell damage with elevated serum alanin-transferase (ALT) levels. The destruction of the splenic marginal zone by CD4(+) T cells included the reduction of marginal zone B cells, marginal zone macrophages and marginal zone metallophilic macrophages. Functionally, this resulted in an impaired production of neutralizing antibodies against LCMV. Furthermore, CD4(+) T cells reduced B cells with an IgM(high)IgD(low) phenotype (transitional stage 1 and 2, marginal zone B cells), whereas other B cell subtypes such as follicular type 1 and 2 and germinal center/memory B cells were not affected. Adoptive transfer of CD4(+) T cells lacking different important effector cytokines and cytolytic pathways such as IFNγ, TNFα, perforin and Fas-FasL interaction did reveal that these cytolytic pathways are redundant in the induction of immunopathological sequel in spleen. In conclusion, our results define an important role of CD4(+) T cells in the induction of immunopathology in liver and spleen. This includes the CD4(+) T cell mediated destruction of the splenic marginal zone with consecutively impaired protective neutralizing antibody responses.

Tracking virus-specific CD4+ T cells during and after acute hepatitis C virus infection

BACKGROUND: CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C. CONCLUSIONS/SIGNIFICANCE: During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1+ patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists.

Lack of TNFR2 expression by CD4(+) T cells exacerbates experimental colitis

TNF plays fundamental roles in the induction and perpetuation of inflammation. The effects of TNF are mediated through TNF receptor (TNFR) 1 or 2. As these two receptors mediate different functions, selective targeting of one receptor may represent a more specific treatment for inflammatory disorders than the complete blocking of TNF. TNFR2 expression is up-regulated in inflammatory bowel disease. Hence, we directly assessed the role of TNFR2 signaling in the CD4(+) T-cell transfer model of colitis using TNFR2(-/-) or WT mice as donors of colitogenic CD4(+)CD45RB(hi) T cells for transfer into syngeneic RAG2(-/-) or RAG2(-/-)TNFR2(-/-) recipient mice. Although the absence of TNFR2 expression by non-lymphoid cells of the recipient mice does not influence the course of colitis, transfer of TNFR2(-/-) CD4(+) T cells leads to an accelerated onset of disease and to more severe signs of inflammation. The enhanced colitogenic potential of TNFR2(-/-) CD4(+) T cells is associated with reduced activation-induced cell death, resulting in an increased accumulation of TNFR2(-/-) CD4(+) T cells. Hence, TNFR2 signaling is crucial for the TNF-dependent contraction of the disease-inducing T cells. Therefore, a selective blocking of TNFR2 may lead to exacerbation rather than attenuation of T-cell-mediated inflammatory disorders.

CX3CR1+ cells facilitate the activation of CD4 T cells in the colonic lamina propria during antigen-driven colitis

Dendritic cells (DCs) and macrophages populate the intestinal lamina propria to initiate immune responses required for the maintenance of intestinal homeostasis. To investigate whether CX3CR1(+) phagocytes communicate with CD4 T cells during the development of transfer colitis, we established an antigen-driven colitis model induced by the adoptive transfer of DsRed OT-II cells in CX3CR1(GFP/+) × RAG(-/-) recipients challenged with Escherichia coli expressing ovalbumin (OVA) fused to a cyan fluorescent protein (CFP). After colonization of CX3CR1(GFP/+) × RAG(-/-) animals with red fluorescent E. coli pCherry-OVA, colonic CX3CR1(+) cells but not CD103(+) DCs phagocytosed E. coli pCherry-OVA. Degraded bacterial-derived antigens are transported by CD103(+) DCs to mesenteric lymph nodes (MLNs), where CD103(+) DCs prime naive T cells. In RAG(-/-) recipients reconstituted with OT II cells and gavaged with OVA-expressing E. coli, colonic CX3CR1(+) phagocytes are in close contact with CD4 T cells and presented bacterial-derived antigens to CD4 T cells to activate and expand effector T cells.

A novel regulatory macrophage induced by a helminth molecule instructs IL-10 in CD4+ T cells and protects against mucosal inflammation

Immunomodulation is a common feature of chronic helminth infections and mainly attributed to the secretion of bioactive molecules, which target and modify host immune cells. In this study, we show that the helminth immunomodulator AvCystatin, a cysteine protease inhibitor, induces a novel regulatory macrophage (Mreg; AvCystatin-Mreg), which is sufficient to mitigate major parameters of allergic airway inflammation and colitis in mice. A single adoptive transfer of AvCystatin-Mreg before allergen challenge suppressed allergen-specific IgE levels, the influx of eosinophils into the airways, local and systemic Th2 cytokine levels, and mucus production in lung bronchioles of mice, whereas increasing local and systemic IL-10 production by CD4(+) T cells. Moreover, a single administration of AvCystatin-Mreg during experimentally induced colitis strikingly reduced intestinal pathology. Phenotyping of AvCystatin-Mreg revealed increased expression of a distinct group of genes including LIGHT, sphingosine kinase 1, CCL1, arginase-1, and costimulatory molecules, CD16/32, ICAM-1, as well as PD-L1 and PD-L2. In cocultures with dendritic cells and CD4(+) T cells, AvCystatin-Mreg strongly induced the production of IL-10 in a cell-contact-independent manner. Collectively, our data identify a specific suppressive macrophage population induced by a single parasite immunomodulator, which protects against mucosal inflammation.

In vivo TCR Signaling in CD4(+) T Cells Imprints a Cell-Intrinsic, Transient Low-Motility Pattern Independent of Chemokine Receptor Expression Levels, or Microtubular Network, Integrin, and Protein Kinase C Activity

Intravital imaging has revealed that T cells change their migratory behavior during physiological activation inside lymphoid tissue. Yet, it remains less well investigated how the intrinsic migratory capacity of activated T cells is regulated by chemokine receptor levels or other regulatory elements. Here, we used an adjuvant-driven inflammation model to examine how motility patterns corresponded with CCR7, CXCR4, and CXCR5 expression levels on ovalbumin-specific DO11.10 CD4(+) T cells in draining lymph nodes. We found that while CCR7 and CXCR4 surface levels remained essentially unaltered during the first 48-72 h after activation of CD4(+) T cells, their in vitro chemokinetic and directed migratory capacity to the respective ligands, CCL19, CCL21, and CXCL12, was substantially reduced during this time window. Activated T cells recovered from this temporary decrease in motility on day 6 post immunization, coinciding with increased migration to the CXCR5 ligand CXCL13. The transiently impaired CD4(+) T cell motility pattern correlated with increased LFA-1 expression and augmented phosphorylation of the microtubule regulator Stathmin on day 3 post immunization, yet neither microtubule destabilization nor integrin blocking could reverse TCR-imprinted unresponsiveness. Furthermore, protein kinase C (PKC) inhibition did not restore chemotactic activity, ruling out PKC-mediated receptor desensitization as mechanism for reduced migration in activated T cells. Thus, we identify a cell-intrinsic, chemokine receptor level-uncoupled decrease in motility in CD4(+) T cells shortly after activation, coinciding with clonal expansion. The transiently reduced ability to react to chemokinetic and chemotactic stimuli may contribute to the sequestering of activated CD4(+) T cells in reactive peripheral lymph nodes, allowing for integration of costimulatory signals required for full activation.

Methodologies for the Analysis of HCV-Specific CD4(+) T Cells

Virus-specific CD4(+) T cells play a major role in viral infections, such as hepatitis C virus (HCV). Viral clearance is associated with vigorous and multi-specific CD4(+) T-cell responses, while chronic infection has been shown to be associated with weak or absent T-cell responses. Most of these studies have used functional assays to analyze virus-specific CD4(+) T-cell responses; however, these and other detection methods have various limitations. Therefore, the important question of whether virus-specific CD4(+) T cells are completely absent or primarily impaired in specific effector functions during chronic infection, has yet to be analyzed in detail. A novel assay, in which virus-specific CD4(+) T-cell frequencies can be determined by de novo CD154 (CD40 ligand) expression in response to viral antigens, can help to overcome some of the limitations of functional assays and restrictions of multimer-based methods. This and other current established methods for the detection of HCV-specific CD4(+) T cells will be discussed in this review.

Biomedical nanoparticles modulate specific CD4+ T cell stimulation by inhibition of antigen processing in dendritic cells

Understanding how nanoparticles may affect immune responses is an essential prerequisite to developing novel clinical applications. To investigate nanoparticle-dependent outcomes on immune responses, dendritic cells (DCs) were treated with model biomedical poly(vinylalcohol)-coated super-paramagnetic iron oxide nanoparticles (PVA-SPIONs). PVA-SPIONs uptake by human monocyte-derived DCs (MDDCs) was analyzed by flow cytometry (FACS) and advanced imaging techniques. Viability, activation, function, and stimulatory capacity of MDDCs were assessed by FACS and an in vitro CD4+ T cell assay. PVA-SPION uptake was dose-dependent, decreased by lipopolysaccharide (LPS)-induced MDDC maturation at higher particle concentrations, and was inhibited by cytochalasin D pre-treatment. PVA-SPIONs did not alter surface marker expression (CD80, CD83, CD86, myeloid/plasmacytoid DC markers) or antigen-uptake, but decreased the capacity of MDDCs to process antigen, stimulate CD4+ T cells, and induce cytokines. The decreased antigen processing and CD4+ T cell stimulation capability of MDDCs following PVA-SPION treatment suggests that MDDCs may revert to a more functionally immature state following particle exposure.

Equine CD4(+) CD25(high) T cells exhibit regulatory activity by close contact and cytokine-dependent mechanisms in vitro

Horses are particularly prone to allergic and autoimmune diseases, but little information about equine regulatory T cells (Treg) is currently available. The aim of this study therefore was to investigate the existence of CD4(+) Treg cells in horses, determine their suppressive function as well as their mechanism of action. Freshly isolated peripheral blood mononuclear cells (PBMC) from healthy horses were examined for CD4, CD25 and forkhead box P3 (FoxP3) expression. We show that equine FoxP3 is expressed constitutively by a population of CD4(+) CD25(+) T cells, mainly in the CD4(+) CD25(high) subpopulation. Proliferation of CD4(+) CD25(-) sorted cells stimulated with irradiated allogenic PBMC was significantly suppressed in co-culture with CD4(+) CD25(high) sorted cells in a dose-dependent manner. The mechanism of suppression by the CD4(+) CD25(high) cell population is mediated by close contact as well as interleukin (IL)-10 and transforming growth factor-beta1 (TGF-beta1) and probably other factors. In addition, we studied the in vitro induction of CD4(+) Treg and their characteristics compared to those of freshly isolated CD4(+) Treg cells. Upon stimulation with a combination of concanavalin A, TGF-beta1 and IL-2, CD4(+) CD25(+) T cells which express FoxP3 and have suppressive capability were induced from CD4(+) CD25(-) cells. The induced CD4(+) CD25(high) express higher levels of IL-10 and TGF-beta1 mRNA compared to the freshly isolated ones. Thus, in horses as in man, the circulating CD4(+) CD25(high) subpopulation contains natural Treg cells and functional Treg can be induced in vitro upon appropriate stimulation. Our study provides the first evidence of the regulatory function of CD4(+) CD25(+) cells in horses and offers insights into ex vivo manipulation of Treg cells.

CD4(+)CD25(+) T cells expressing FoxP3 in Icelandic horses affected with insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-beta1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Culicoides extract or a control antigen (Tetanus Toxoid). There were no differences between healthy and IBH horses either in the proportion of FoxP3(+)CD4(+)CD25(+) cells in freshly isolated PBMC or in the following stimulation with Tetanus Toxoid. However, upon stimulation of PBMC with the allergen, expression of FoxP3 by CD4(+)CD25(+high) and CD4(+)CD25(+dim) cells was significantly higher in healthy than in IBH horses. Addition of recombinant IL-4 to PBMC from healthy horses stimulated with the allergen significantly decreased the proportion of FoxP3 expressing cells within CD4(+)CD25(+high). These results suggest that IBH is associated with a decreased number of allergen-induced Tregs. This could be a consequence of the increased IL-4 production by PBMC of IBH-affected horses.

Interferon-gamma regulates idiopathic pneumonia syndrome, a Th17+CD4+ T-cell-mediated graft-versus-host disease

RATIONALE: Pulmonary complications of hematopoietic stem cell transplantation include infections and graft-versus-host diseases, such as idiopathic pneumonia syndrome (IPS). Conflicting data exist regarding the role of the interferon (IFN)-gamma-producing Th1 CD4(+) T-cell subset and IL-17A in IPS. OBJECTIVES: To determine the role of IFN-gamma and IL-17A in the establishment of pulmonary graft-versus-host disease. METHODS: A semiallogeneic murine model based on C57BL/6 x BALB/c as recipients with transplantation of BALB/c RAG2(-/-) bone marrow and transfer of different genetic knockout T cells (T-bet(-/-), IFN-gamma(-/-), IFN-gammaR(-/-)) on a BALB/c background. Lung tissue was examined for parenchymal changes and infiltrating cells by histology and fluorescence-activated cell sorter analysis. MEASUREMENTS AND MAIN RESULTS: After transfer of semiallogeneic bone marrow together with donor CD4(+) T cells lacking IFN-gamma or T-bet-a T-box transcription factor controlling Th1 commitment-we found severe inflammation in the lungs, but no enhancement in other organs. In contrast, wild-type donor CD4(+) T cells mediated minimal inflammation only, and donor CD8(+) T cells were not required for IPS development. Mechanistically, the absence of IFN-gamma or IFN-gamma signaling in pulmonary parenchymal cells promoted expansion of IL-17A-producing CD4(+) T cells and local IL-17A release. In vivo depletion of IL-17A reduced disease severity. CONCLUSIONS: One mechanism of IFN-gamma protection against IPS is negative regulation of the expansion of pathogenic IL-17A-producing CD4(+) T cells through interaction with the IFN-gamma receptor on the pulmonary parenchymal cell population.

Low current and nadir CD4+ T-cell counts are associated with higher hepatitis C virus RNA levels in the Swiss HIV cohort study

BACKGROUND: The aim of this study was to evaluate the effect of CD4+ T-cell counts and other characteristics of HIV-infected individuals on hepatitis C virus (HCV) RNA levels. METHODS: All HIV-HCV-coinfected Swiss HIV Cohort Study participants with available HCV RNA levels and concurrent CD4+ T-cell counts before starting HCV therapy were included. Potential predictors of HCV RNA levels were assessed by multivariate censored linear regression models that adjust for censored values. RESULTS: The study included 1,031 individuals. Low current and nadir CD4+ T-cell counts were significantly associated with higher HCV RNA levels (P = 0.004 and 0.001, respectively). In individuals with current CD4+ T-cell counts < 200/microl, median HCV RNA levels (6.22 log10 IU/ml) were +0.14 and +0.24 log10 IU/ml higher than those with CD4+ T-cell counts of 200-500/microl and > 500/microl. Based on nadir CD4+ T-cell counts, median HCV RNA levels (6.12 log10 IU/ml) in individuals with < 200/microl CD4+ T-cells were +0.06 and +0.44 log10 IU/ml higher than those with nadir T-cell counts of 200-500/microl and > 500/microl. Median HCV RNA levels were also significantly associated with HCV genotype: lower values were associated with genotype 4 and higher values with genotype 2, as compared with genotype 1. Additional significant predictors of lower HCV RNA levels were female gender and HIV transmission through male homosexual contacts. In multivariate analyses, only CD4+ T-cell counts and HCV genotype remained significant predictors of HCV RNA levels. Conclusions: Higher HCV RNA levels were associated with CD4+ T-cell depletion. This finding is in line with the crucial role of CD4+ T-cells in the control of HCV infection.