Probiotic bacteria produce conjugated linoleic acid locally in the gut that targets macrophage PPAR γ to suppress colitis

Background

Inflammatory bowel disease (IBD) therapies are modestly successful and associated with significant side effects. Thus, the investigation of novel approaches to prevent colitis is important. Probiotic bacteria can produce immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anti-inflammatory effects. This study aimed to investigate the cellular and molecular mechanisms underlying the anti-inflammatory efficacy of probiotic bacteria using a mouse model of colitis.

Methodology/Principal Findings

The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were investigated in a mouse model of DSS colitis. Colonic specimens were collected for histopathology, gene expression and flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen, blood and colonic lamina propria cells were phenotypically and functionally characterized. Fecal samples and colonic contents were collected to determine the effect of VSL#3 and CLA on gut microbial diversity and CLA production. CLA and VSL#3 treatment ameliorated colitis and decreased colonic bacterial diversity, a finding that correlated with decreased gut pathology. Colonic CLA concentrations were increased in response to probiotic bacterial treatment, but without systemic distribution in blood. VSL#3 and CLA decreased macrophage accumulation in the MLN of mice with DSS colitis. The loss of PPAR γ in myeloid cells abrogated the protective effect of probiotic bacteria and CLA in mice with DSS colitis.

Conclusions/Significance

Probiotic bacteria modulate gut microbial diversity and favor local production of CLA in the colon that targets myeloid cell PPAR γ to suppress colitis.     

Lysate of probiotic Lactobacillus casei DN-114 001 ameliorates colitis by strengthening the gut barrier function and changing the gut microenvironment

Background

Probiotic bacteria can be used for the prevention and treatment of human inflammatory diseases including inflammatory bowel diseases (IBD). However, the nature of active components and exact mechanisms of this beneficial effects have not been fully elucidated. Our aim was to investigate if lysate of probiotic bacterium L. casei DN-114 001 (Lc) could decrease the severity of intestinal inflammation in a murine model of IBD.

Methodology/Principal Findings

The preventive effect of oral administration of Lc significantly reduces the severity of acute dextran sulfate sodium (DSS) colitis in BALB/c but not in SCID mice. In order to analyze how this beneficial effect interferes with well-known phases of intestinal inflammation pathogenesis in vivo and in vitro, we evaluated intestinal permeability using the FITC-labeled dextran method and analysed tight junction proteins expression by immunofluorescence and PCR. We also measured CD4⁺FoxP3⁺ regulatory T cells proportion by FACS analysis, microbiota composition by pyrosequencing, and local cytokine production by ELISA. Lc leads to a significant protection against increased intestinal permeability and barrier dysfunction shown by preserved ZO-1 expression. We found that the Lc treatment increases the numbers of CD4⁺FoxP3⁺ regulatory T cells in mesenteric lymph nodes (MLN), decreases production of pro-inflammatory cytokines TNF-α and IFN-γ, and anti-inflammatory IL-10 in Peyer''s patches and large intestine, and changes the gut microbiota composition. Moreover, Lc treatment prevents lipopolysaccharide-induced TNF-α expression in RAW 264.7 cell line by down-regulating the NF-κB signaling pathway.

Conclusion/Significance

Our study provided evidence that even non-living probiotic bacteria can prevent the development of severe forms of intestinal inflammation by strengthening the integrity of intestinal barrier and modulation of gut microenvironment.     

Probiotic Bacteria Regulate Intestinal Epithelial Permeability in Experimental Ileitis by a TNF-Dependent Mechanism

Background

We previously showed that the probiotic mixture, VSL#3, prevents the onset of ileitis in SAMP/YitFc (SAMP) mice, and this effect was associated with stimulation of epithelial-derived TNF. The aim of this study was to determine the mechanism(s) of VSL#3-mediated protection on epithelial barrier function and to further investigate the “paradoxical” effects of TNF in preventing SAMP ileitis.

Methods

Permeability was evaluated in SAMP mice prior to the onset of inflammation and during established disease by measuring transepithelial electrical resistance (TEER) on ex vivo-cultured ilea following exposure to VSL#3 conditioned media (CM), TNF or VSL#3-CM + anti-TNF. Tight junction (TJ) proteins were assessed by qRT-PCR, Western blot, and confocal microscopy, and TNFRI/TNFRII expression measured in freshly isolated intestinal epithelial cells (IEC) from SAMP and control AKR mice.

Results

Culture with either VSL#3-CM or TNF resulted in decreased ileal paracellular permeability in pre-inflamed SAMP, but not SAMP with established disease, while addition of anti-TNF abrogated these effects. Modulation of the TJ proteins, claudin-2 and occludin, occurred with a significant decrease in claudin-2 and increase in occludin following stimulation with VSL#3-CM or TNF. TNF protein levels increased in supernatants of SAMP ilea incubated with VSL#3-CM compared to vehicle, while IEC-derived TNFR mRNA expression decreased in young, and was elevated in inflamed, SAMP versus AKR mice.

Conclusions

Our data demonstrate that the previously established efficacy of VSL#3 in preventing SAMP ileitis is due to direct innate and homeostatic effects of TNF on the gut epithelium, modulation of the TJ proteins, claudin-2 and occludin, and overall improvement of intestinal permeability.     

Local induction of immunosuppressive CD8+ T cells in the gut-associated lymphoid tissues

Background

In contrast to intestinal CD4⁺ regulatory T cells (T_regs), the generation and function of immunomodulatory intestinal CD8⁺ T cells is less well defined. To dissect the immunologic mechanisms of CD8⁺ T cell function in the mucosa, reactivity against hemagglutinin (HA) expressed in intestinal epithelial cells of mice bearing a MHC class-I-restricted T-cell-receptor specific for HA was studied.

Methodology and Principal Findings

HA-specific CD8⁺ T cells were isolated from gut-associated tissues and phenotypically and functionally characterized for the expression of Foxp3⁺ and their suppressive capacity. We demonstrate that intestinal HA expression led to peripheral induction of HA-specific CD8⁺Foxp3⁺ T cells. Antigen-experienced CD8⁺ T cells in this transgenic mouse model suppressed the proliferation of CD8⁺ and CD4⁺ T cells in vitro. Gene expression analysis of suppressive HA-specific CD8⁺ T cells revealed a specific up-regulation of CD103, Nrp1, Tnfrsf9 and Pdcd1, molecules also expressed on CD4⁺ T_reg subsets. Finally, gut-associated dendritic cells were able to induce HA-specific CD8⁺Foxp3⁺ T cells.

Conclusion and Significance

We demonstrate that gut specific antigen presentation is sufficient to induce CD8⁺ T_regs in vivo which may maintain intestinal homeostasis by down-modulating effector functions of T cells.     

Increased bone marrow interleukin-7 (IL-7)/IL-7R levels but reduced IL-7 responsiveness in HIV-positive patients lacking CD4+ gain on antiviral therapy

Background

The bone marrow (BM) cytokine milieu might substantially affect T-lymphocyte homeostasis in HIV-positive individuals. Interleukin-7 (IL-7) is a bone marrow-derived cytokine regulating T-cell homeostasis through a CD4+-driven feedback loop. CD4+ T-lymphopenia is associated with increased free IL-7 levels and reduced IL-7R expression/function, which are only partially reverted by highly active antiretroviral therapy (HAART). We investigated the BM production, peripheral expression and signaling (pStat5+ and Bcl-2+ CD4+/CD8+ T cells) of IL-7/IL-7Rα in 30 HAART-treated HIV-positive patients who did not experience CD4+ recovery (CD4+ ≤200/µl) and who had different levels of HIV viremia; these patients included 18 immunological nonresponders (INRs; HIV-RNA≤50), 12 complete failures (CFs; HIV-RNA>1000), and 23 HIV-seronegative subjects.

Methods

We studied plasma IL-7 levels, IL-7Rα+CD4+/CD8+ T-cell proportions, IL-7Rα mRNA expression in PBMCs, spontaneous IL-7 production by BM mononuclear cells (BMMCs), and IL-7 mRNA/IL-7Rα mRNA in BMMC-derived stromal cells (SCs). We also studied T-cell responsiveness to IL-7 by measuring the proportions of pStat5+ and Bcl-2+ CD4+/CD8+ T cells.

Results

Compared to HIV-seronegative controls, CFs and INRs presented elevated plasma IL-7 levels and lower IL-7Rα CD4+/CD8+ cell-surface expression and peripheral blood production, confirming the most relevant IL-7/IL-7R disruption. Interestingly, BM investigation revealed a trend of higher spontaneous IL-7 production in INRs (p = .09 vs. CFs) with a nonsignificant trend toward higher IL-7-Rα mRNA levels in BMMC-derived stromal cells. However, upon IL-7 stimulation, the proportion of pStat5+CD4+ T cells did not increase in INRs despite higher constitutive levels (p = .06); INRs also displayed lower Bcl-2+CD8+ T-cell proportions than controls (p = .04).

Conclusions

Despite severe CD4+ T-lymphopenia and a disrupted IL-7/IL-7R profile in the periphery, INRs display elevated BM IL-7/IL-7Rα expression but impaired T-cell responsiveness to IL-7, suggesting the activity of a central compensatory pathway targeted to replenish the CD4+ compartment, which is nevertheless inappropriate to compensate the dysfunctional signaling through IL-7 receptor.     

Preterm labor and chorioamnionitis are associated with neonatal T cell activation

Background

Preterm parturition is characterized by innate immune activation and increased proinflammatory cytokine levels. This well established association leads us to hypothesize that preterm delivery is also associated with neonatal T lymphocyte activation and maturation.

Methodology/Principal Findings

Cord blood samples were obtained following term, preterm, and deliveries complicated by clinical chorioamnionitis. Activation marker expression was quantitated by flow cytometric analysis. Infants born following preterm delivery demonstrated enhanced CD4⁺ T lymphocyte activation, as determined by CD25 (Term 9.72% vs. Preterm 17.67%, p = 0.0001), HLA-DR (Term 0.91% vs. Preterm 1.92%, p = 0.0012), and CD69 expression (Term 0.38% vs. Preterm 1.20%, p = 0.0003). Neonates delivered following clinical chorioamnionitis also demonstrated increased T cell activation. Preterm neonates had an increased frequency of CD45RO⁺ T cells.

Conclusion/Significance

Preterm parturition is associated with neonatal CD4⁺ T cell activation, and an increased frequency of CD45RO⁺ T cells. These findings support the concept that activation of the fetal adaptive immune system in utero is closely associated with preterm labor.     

Interleukin-6 induces Gr-1+CD11b+ myeloid cells to suppress CD8+ T cell-mediated liver injury in mice

Background

Agonist antibodies against CD137 (4–1BB) on T lymphocytes are used to increase host anti-tumor immunity, but often leading to severe liver injury in treated mice or in patients during clinical trials. Interleukin-6 (IL-6) has been reported to protect hepatocyte death, but the role of IL-6 in protecting chronic T cell-induced liver diseases is not clearly defined due to lack of relevant animal models. We aimed to define the role of IL-6 in CD8+ T cell-mediated liver injury induced by a CD137 agonistic mAb (clone 2A) in mice.

Methods/Principal Findings

We expressed IL-6 in the liver by hydrodynamic gene delivery in mice treated with 2A or control mAb and studied how IL-6 treatment affected host immunity and T cell-mediated liver injury. We found that ectopic IL-6 expression in the liver elevated intrahepatic leukocyte infiltration but prevented CD8+ T cell-mediated liver injury. In IL-6 treated mice, CD8+ T cells proliferation and IFN-γ expression were inhibited in the liver. We discovered that IL-6 increased accumulation of Gr-1+CD11b+ myeloid derived suppressor cells (MDSCs) in the liver and spleen. These MDSCs had the ability to inhibit T cells proliferation and activation. Finally, we showed that the MDSCs were sufficient and essential for IL-6-mediated protection of anti-CD137 mAb-induced liver injury.

Conclusions/Significance

We concluded that IL-6 induced Gr-1+CD11b+ MDSCs in the liver to inhibit T cell-mediated liver injury. The findings have defined a novel mechanism of IL-6 in protecting liver from CD8+ T cell-mediated injury.     

Cellular and molecular mechanisms underlying the strong neonatal IL-12 response of lamb mesenteric lymph node cells to R-848

Background

Comparative studies on the response of neonates and adults to TLR stimulation have been almost exclusively limited to comparisons of human neonatal cord blood cells with peripheral blood from adults, and analyses of spleen cell responses in mice. We need to extend these studies and gain further information regarding such responses at mucosal sites.

Methodology/Principal Findings

We used sheep as a large animal model to study TLR agonist responses in the lymph nodes draining the intestine, an organ that must adapt to profound changes after birth. In response to the imidazoquinoline compound R-848, neonatal mesenteric lymph node (MLN) and spleen cells produced more IL-12 and, consequently, more IFNγ than their adult counterparts. This difference was age-related for both organs, but the preferential IL-12 response decreased more rapidly in the MLN, with young animals producing similar amounts of this cytokine to adults, from the age of 20 days onwards. Intracellular assays and depletion experiments identified CD14⁺CD11b⁺CD40⁺ cells as the main producer of IL-12. These cells accounted for a greater proportion of neonatal than of adult MLN cells, and also produced, in direct response to R-848, more IL-12 after isolation. This strong IL-12 response in neonates occurred despite the production of larger amounts of the regulatory cytokine IL-10 and the stronger upregulation of SOCS-1 and SOCS-3 mRNA levels than in adult cells, and was correlated with an increase in p38/MAPK phosphorylation.

Conclusions/Significance

This is the first attempt to decipher the mechanism by which neonatal MLN cells produce more IL-12 than adult cells in response to the TLR8 agonist R-848. CD14⁺CD11b⁺CD40⁺ IL-12-producing cells were more numerous in neonate than in adult MLN cells and displayed higher intracellular responsiveness upon R-848 stimulation. This work provides relevant information for future vaccination or immunostimulation strategies targeting neonates.     

TIM-3 expression characterizes regulatory T cells in tumor tissues and is associated with lung cancer progression

Background

T cell immunoglobulin-3 (TIM-3) has been established as a negative regulatory molecule and plays a critical role in immune tolerance. TIM-3 is upregulated in exhausted CD8⁺ T cells in both chronic infection and tumor. However, the nature of TIM-3⁺CD4⁺ T cells in the tumor microenvironment is unclear. This study is to characterize TIM-3 expressing lymphocytes within human lung cancer tissues and establish clinical significance of TIM-3 expression in lung cancer progression.

Methodology

A total of 51 human lung cancer tissue specimens were obtained from pathologically confirmed and newly diagnosed non-small cell lung cancer (NSCLC) patients. Leukocytes from tumor tissues, distal normal lung tissues, and peripheral blood mononuclear cells (PBMC) were analyzed for TIM-3 surface expression by flow cytometry. TIM-3 expression on tumor-infiltrating lymphocytes (TILs) was correlated with clinicopathological parameters.

Conclusions

TIM-3 is highly upregulated on both CD4⁺ and CD8⁺ TILs from human lung cancer tissues but negligibly expressed on T cells from patients'' peripheral blood. Frequencies of IFN-γ⁺ cells were reduced in TIM-3⁺CD8⁺ TILs compared to TIM-3⁻CD8⁺ TILs. However, the level of TIM-3 expression on CD8⁺ TILs failed to associate with any clinical pathological parameter. Interestingly, we found that approximately 70% of TIM-3⁺CD4⁺ TILs expressed FOXP3 and about 60% of FOXP3⁺ TILs were TIM-3⁺. Importantly, TIM-3 expression on CD4⁺ T cells correlated with poor clinicopathological parameters of NSCLC such as nodal metastasis and advanced cancer stages. Our study reveals a new role of TIM-3 as an important immune regulator in the tumor microenvironment via its predominant expression in regulatory T cells.     

Probiotics modulate intestinal expression of nuclear receptor and provide counter-regulatory signals to inflammation-driven adipose tissue activation

Background

Adipocytes from mesenteric white adipose tissue amplify the inflammatory response and participate in inflammation-driven immune dysfunction in Crohn''s disease by releasing proinflammatory mediators. Peroxisome proliferator-activated receptors (PPAR)-α and -γ, pregnane x receptor (PXR), farnesoid x receptor (FXR) and liver x-receptor (LXR) are ligand-activated nuclear receptor that provide counter-regulatory signals to dysregulated immunity and modulates adipose tissue.

Aims

To investigate the expression and function of nuclear receptors in intestinal and adipose tissues in a rodent model of colitis and mesenteric fat from Crohn''s patients and to investigate their modulation by probiotics.

Methods

Colitis was induced by TNBS administration. Mice were administered vehicle or VSL#3, daily for 10 days. Abdominal fat explants obtained at surgery from five Crohn''s disease patients and five patients with colon cancer were cultured with VSL#3 medium.

Results

Probiotic administration attenuated development of signs and symptoms of colitis, reduced colonic expression of TNFα, IL-6 and IFNγ and reserved colonic downregulation of PPARγ, PXR and FXR caused by TNBS. Mesenteric fat depots isolated from TNBS-treated animals had increased expression of inflammatory mediators along with PPARγ, FXR, leptin and adiponectin. These changes were prevented by VSL#3. Creeping fat and mesenteric adipose tissue from Crohn''s patients showed a differential expression of PPARγ and FXR with both tissue expressing high levels of leptin. Exposure of these tissues to VSL#3 medium abrogates leptin release.

Conclusions

Mesenteric adipose tissue from rodent colitis and Crohn''s disease is metabolically active and shows inflammation-driven regulation of PPARγ, FXR and leptin. Probiotics correct the inflammation-driven metabolic dysfunction.     

Monitoring of regulatory T cell frequencies and expression of CTLA-4 on T cells, before and after DC vaccination, can predict survival in GBM patients

Purpose

Dendritic cell (DC) vaccines have recently emerged as an innovative therapeutic option for glioblastoma patients. To identify novel surrogates of anti-tumor immune responsiveness, we studied the dynamic expression of activation and inhibitory markers on peripheral blood lymphocyte (PBL) subsets in glioblastoma patients treated with DC vaccination at UCLA.

Experimental Design

Pre-treatment and post-treatment PBL from 24 patients enrolled in two Phase I clinical trials of dendritic cell immunotherapy were stained and analyzed using flow cytometry. A univariate Cox proportional hazards model was utilized to investigate the association between continuous immune monitoring variables and survival. Finally, the immune monitoring variables were dichotomized and a recursive partitioning survival tree was built to obtain cut-off values predictive of survival.

Results

The change in regulatory T cell (CD3⁺CD4⁺CD25⁺CD127^low) frequency in PBL was significantly associated with survival (p = 0.0228; hazard ratio = 3.623) after DC vaccination. Furthermore, the dynamic expression of the negative co-stimulatory molecule, CTLA-4, was also significantly associated with survival on CD3⁺CD4⁺ T cells (p = 0.0191; hazard ratio = 2.840) and CD3⁺CD8⁺ T cells (p = 0.0273; hazard ratio = 2.690), while that of activation markers (CD25, CD69) was not. Finally, a recursive partitioning tree algorithm was utilized to dichotomize the post/pre fold change immune monitoring variables. The resultant cut-off values from these immune monitoring variables could effectively segregate these patients into groups with significantly different overall survival curves.

Conclusions

Our results suggest that monitoring the change in regulatory T cell frequencies and dynamic expression of the negative co-stimulatory molecules on peripheral blood T cells, before and after DC vaccination, may predict survival. The cut-off point generated from these data can be utilized in future prospective immunotherapy trials to further evaluate its predictive validity.     

Different capacity of monocyte subsets to phagocytose iron-oxide nanoparticles

Objective

To explore the capacity of human CD14⁺CD16⁺⁺ and CD14⁺⁺CD16^- monocytes to phagocyte iron-oxide nanoparticles in vitro.

Methods

Human monocytes were labeled with four different magnetic nanoparticle preparations (Ferumoxides, SHU 555C, CLIO-680, MION-48) exhibiting distinct properties and cellular uptake was quantitatively assessed by flow cytometry, fluorescence microscopy, atomic absorption spectrometry and Magnetic Resonance Imaging (MRI). Additionally we determined whether cellular uptake of the nanoparticles resulted in phenotypic changes of cell surface markers.

Results

Cellular uptake differed between the four nanoparticle preparations. However for each nanoparticle tested, CD14⁺⁺CD16^- monocytes displayed a significantly higher uptake compared to CD14⁺CD16⁺⁺ monocytes, this resulted in significantly lower T1 and T2 relaxation times of these cells. The uptake of iron-oxide nanoparticles further resulted in a remarkable shift of expression of cell surface proteins indicating that the labeling procedure affects the phenotype of CD14⁺CD16⁺⁺ and CD14⁺⁺CD16^- monocytes differently.

Conclusion

Human monocyte subsets internalize different magnetic nanoparticle preparations differently, resulting in variable loading capacities, imaging phenotypes and likely biological properties.     

Programmed Death-1 expression on Epstein Barr virus specific CD8+ T cells varies by stage of infection, epitope specificity, and T-cell receptor usage

Background

Programmed Death-1 (PD-1) is an inhibitory member of the CD28 family of molecules expressed on CD8+ T cells in response to antigenic stimulation. To better understand the role of PD-1 in antiviral immunity we examined the expression of PD-1 on Epstein-Barr virus (EBV) epitope-specific CD8+ T cells during acute infectious mononucleosis (AIM) and convalescence.

Methodology/Principal Findings

Using flow cytometry, we observed higher frequencies of EBV-specific CD8+ T cells and higher intensity of PD-1 expression on EBV-specific CD8+ T cells during AIM than during convalescence. PD-1 expression during AIM directly correlated with viral load and with the subsequent degree of CD8+ T cell contraction in convalescence. Consistent differences in PD-1 expression were observed between CD8+ T cells with specificity for two different EBV lytic antigen epitopes. Similar differences were observed in the degree to which PD-1 was upregulated on these epitope-specific CD8+ T cells following peptide stimulation in vitro. EBV epitope-specific CD8+ T cell proliferative responses to peptide stimulation were diminished during AIM regardless of PD-1 expression and were unaffected by blocking PD-1 interactions with PD-L1. Significant variability in PD-1 expression was observed on EBV epitope-specific CD8+ T cell subsets defined by V-beta usage.

Conclusions/Significance

These observations suggest that PD-1 expression is not only dependent on the degree of antigen presentation, but also on undefined characteristics of the responding cell that segregate with epitope specificity and V-beta usage.     

Minocycline inhibition of monocyte activation correlates with neuronal protection in SIV neuroAIDS

Background

Minocycline is a tetracycline antibiotic that has been proposed as a potential conjunctive therapy for HIV-1 associated cognitive disorders. Precise mechanism(s) of minocycline''s functions are not well defined.

Methods

Fourteen rhesus macaques were SIV infected and neuronal metabolites measured by proton magnetic resonance spectroscopy (¹H MRS). Seven received minocycline (4 mg/kg) daily starting at day 28 post-infection (pi). Monocyte expansion and activation were assessed by flow cytometry, cell traffic to lymph nodes, CD16 regulation, viral replication, and cytokine production were studied.

Results

Minocycline treatment decreased plasma virus and pro-inflammatory CD14+CD16+ and CD14^loCD16+ monocytes, and reduced their expression of CD11b, CD163, CD64, CCR2 and HLA-DR. There was reduced recruitment of monocyte/macrophages and productively infected cells in axillary lymph nodes. There was an inverse correlation between brain NAA/Cr (neuronal injury) and circulating CD14+CD16+ and CD14^loCD16+ monocytes. Minocycline treatment in vitro reduced SIV replication CD16 expression on activated CD14+CD16+ monocytes, and IL-6 production by monocytes following LPS stimulation.

Conclusion

Neuroprotective effects of minocycline are due in part to reduction of activated monocytes, monocyte traffic. Mechanisms for these effects include CD16 regulation, reduced viral replication, and inhibited immune activation.     

FcγRIIIa expression on monocytes in rheumatoid arthritis: role in immune-complex stimulated TNF production and non-response to methotrexate therapy

Objective

The expression of FcγRIIIa/CD16 may render monocytes targets for activation by IgG-containing immune complexes (IC). We investigated whether FcγRIIIa/CD16 was upregulated in rheumatoid arthritis (RA), associated with TNF production in response to IC-stimulation, and if this predicted response to methotrexate therapy.

Methods

FcγRIIIa/CD16 expression on CD14^low and CD14⁺⁺ monocytes was measured by flow cytometry in healthy controls and RA patients (early and long-standing disease). Intracellular TNF-staining was carried out after in vitro LPS or heat-aggregated immunoglobulin (HAG) activation. FcγRIIIa/CD16 expression pre- and post-steroid/methotrexate treatment was examined.

Results

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in long-standing RA patients compared to controls was demonstrated (p = 0.002) with intermediate levels in early-RA patients. HAG-induced TNF-production in RA patients was correlated with the percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 (p<0.001). The percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 at baseline in early DMARD-naïve RA patients was negatively correlated with DAS28-ESR improvement 14-weeks post-methotrexate therapy (p = 0.003) and was significantly increased in EULAR non-responders compared to moderate (p = 0.01) or good responders (p = 0.003). FcγRIIIa/CD16 expression was not correlated with age, presence of systemic inflammation or autoantibody titers.

Conclusion

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in RA may result in a cell that has increased responsiveness to IC-stimulation. This monocyte subset may contribute to non-response to methotrexate therapy.     

"Negative vaccination" by specific CD4 T cell tolerisation enhances virus-specific protective antibody responses

Background

Cooperation of CD4⁺ T helper cells with specific B cells is crucial for protective vaccination against pathogens by inducing long-lived neutralizing antibody responses. During infection with persistence-prone viruses, prolonged virus replication correlates with low neutralizing antibody responses. We recently described that a viral mutant of lymphocytic choriomeningitis virus (LCMV), which lacks a T helper epitope, counterintuitively induced an enhanced protective antibody response. Likewise, partial depletion of the CD4⁺ T cell compartment by using anti-CD4 antibodies enhanced protective antibodies.

Principal Findings

Here we have developed a protocol to selectively reduce the CD4⁺ T cell response against viral CD4⁺ T cell epitopes. We demonstrate that in vivo treatment with LCMV-derived MHC-II peptides induced non-responsiveness of specific CD4⁺ T cells without affecting CD4⁺ T cell reactivity towards other antigens. This was associated with accelerated virus-specific neutralizing IgG-antibody responses. In contrast to a complete absence of CD4⁺ T cell help, tolerisation did not impair CD8⁺ T cell responses.

Conclusions

This result reveals a novel “negative vaccination” strategy where specific CD4⁺ T cell unresponsiveness may be used to enhance the delayed protective antibody responses in chronic virus infections.     

Interferon-alpha administration enhances CD8+ T cell activation in HIV infection

Background

Type I interferons play important roles in innate immune defense. In HIV infection, type I interferons may delay disease progression by inhibiting viral replication while at the same time accelerating disease progression by contributing to chronic immune activation.

Methods

To investigate the effects of type I interferons in HIV-infection, we obtained cryopreserved peripheral blood mononuclear cell samples from 10 subjects who participated in AIDS Clinical Trials Group Study 5192, a trial investigating the activity of systemic administration of IFNα for twelve weeks to patients with untreated HIV infection. Using flow cytometry, we examined changes in cell cycle status and expression of activation antigens by circulating T cells and their maturation subsets before, during and after IFNα treatment.

Results

The proportion of CD38+HLA-DR+CD8+ T cells increased from a mean of 11.7% at baseline to 24.1% after twelve weeks of interferon treatment (p = 0.006). These frequencies dropped to an average of 20.1% six weeks after the end of treatment. In contrast to CD8+ T cells, the frequencies of activated CD4+ T cells did not change with administration of type I interferon (mean percentage of CD38+DR+ cells = 2.62% at baseline and 2.17% after 12 weeks of interferon therapy). As plasma HIV levels fell with interferon therapy, this was correlated with a “paradoxical” increase in CD8+ T cell activation (p<0.001).

Conclusion

Administration of type I interferon increased expression of the activation markers CD38 and HLA DR on CD8+ T cells but not on CD4+ T cells of HIV+ persons. These observations suggest that type I interferons may contribute to the high levels of CD8+ T cell activation that occur during HIV infection.     

Cytokine profiles, CTL response and T cell frequencies in the peripheral blood of acute patients and individuals recovered from hepatitis E infection

Background

Hepatitis E is a major public health problem in the developing countries. Pathogenesis of hepatitis E virus (HEV) infection is poorly understood.

Methods

This case-control study included 124 Hepatitis E patients (46 acute and 78 recovered), 9 with prior exposure to HEV and 71 anti-HEV negative healthy controls. HEV induced CTL response by Elispot, cytokines/chemokines quantitation by Milliplex assay and peripheral CD4+ & CD8+ T cell frequencies by flow cytometry were assessed.

Results

Among the patient categories, HEV specific IFN-γ responses as recorded by Elispot were comparable. Comparisons of cytokines/chemokines revealed significantly high levels of IL-1α and sIL-2Rα during acute phase. Circulating peripheral CD4/CD8+ T-cell subsets in acute and recovered individuals were comparable compared to controls, while among patient categories CD8+T cell subset was significantly higher in recovered individuals.

Conclusions

Our findings suggest that IL-1α and sIL-2Rα play a role in the pathogenesis of acute Hepatitis E infection. Lack of robust HEV ORF2-specific CTL response in the peripheral blood of HEV infected patients during the acute and recovered phases of the disease may be associated with involvement of innate immune cells/localization of the immune events at the site of infection.     

Progressive activation of CD127+132- recent thymic emigrants into terminally differentiated CD127-132+ T-cells in HIV-1 infection

Aim

HIV infection is associated with distortion of T-cell homeostasis and the IL-7/IL7R axis. Progressive infection results in loss of CD127+132− and gains in CD127−132+ CD4+ and CD8+ T-cells. We investigated the correlates of loss of CD127 from the T-cell surface to understand mechanisms underlying this homeostatic dysregulation.

Methods

Peripheral and cord blood mononuclear cells (PBMCs; CBMC) from healthy volunteers and PBMC from patients with HIV infection were studied. CD127+132−, CD127+132+ and CD127−132+ T-cells were phenotyped by activation, differentiation, proliferation and survival markers. Cellular HIV-DNA content and signal-joint T-cell receptor excision circles (sjTRECs) were measured.

Results

CD127+132− T-cells were enriched for naïve cells while CD127−132+ T-cells were enriched for activated/terminally differentiated T-cells in CD4+ and CD8+ subsets in health and HIV infection. HIV was associated with increased proportions of activated/terminally differentiated CD127−132+ T-cells. In contrast to CD127+132− T-cells, CD127−132+ T-cells were Ki-67+Bcl-2^low and contained increased levels of HIV-DNA. Naïve CD127+132− T-cells contained a higher proportion of sjTRECs.

Conclusion

The loss of CD127 from the T-cell surface in HIV infection is driven by activation of CD127+132− recent thymic emigrants into CD127−132+ activated/terminally differentiated cells. This process likely results in an irreversible loss of CD127 and permanent distortion of T-cell homeostasis.     

Prognostic impact of lymphocytes in soft tissue sarcomas

Purpose

The purpose of this study was to clarify the prognostic significance of lymphocyte infiltration in soft tissue sarcomas (STS). Prognostic markers in potentially curable STS should guide therapy after surgical resection. The immune status at the time of resection may be important, but the prognostic significance of tumor infiltrating lymphocytes is controversial as the immune system has conflicting roles during cancer development.

Experimental Design

Tissue microarrays from 249 patients with STS were constructed from duplicate cores of viable and representative neoplastic tumor areas. Immunohistochemistry was used to evaluate the CD3+, CD4+, CD8+, CD20+ and CD45+ lymphocytes in tumors.

Results

In univariate analyses, increased numbers of CD4+ (P = 0.008) and CD20+ (P = 0.006) lymphocytes in tumor correlated significantly with an improved disease-specific survival (DSS) in patients with wide resection margins (n = 108). In patients with non-wide resection margins (n = 141) increased numbers of CD3+ (P = 0.028) lymphocytes in tumor correlated significantly with shorter DSS. In multivariate analyses, a high number of CD20+ lymphocytes (HR = 5.5, CI 95%  = 1.6–18.6, P = 0.006) in the tumor was an independent positive prognostic factor for DSS in patients with wide resections margins.

Conclusions

High density of CD20+ lymphocytes in STS with wide resection margins is an independent positive prognostic indicator for these patients. Further research is needed to define if CD20+ cells can modify tumors in a way that reduces disease progression and metastatic potential.