Internalization of CD4 molecules in human T-cells demonstrated by immuno-electron microscopy.

Internalization of CD4 molecules on human CD4-enriched T-cells was demonstrated by immunocytochemical electron microscopy. CD4+ T-cell subclones were obtained from normal human peripheral blood, followed by one-way MLC screening and co-culturing with IL-2. Fixed and non-fixed T-cell samples were indirectly immunolabeled with mouse anti-human CD4 monoclonal antibody and goat anti-mouse IgG conjugated with peroxidase. Unfixed T-cells were immunolabeled at 4 degrees C and then re-incubated for 5-45 min at 37 degrees C. The selected CD4+ T-cell subclones showed strong CD4 binding on the cell surface after IL-2 incubation. However, fresh T-cells, monocytes, bone marrow cells and CD8+ T-cells all stained negative for CD4. The distribution of CD4 molecules on the fixed cell surface showed a homogeneous pattern. Capping and internalization of CD4-antibody-peroxidase complexes from the cell surfaces were observed follow a pathway of receptor-mediated endocytosis in unfixed T cells. Endocytotic vesicles, vacuoles of diverse sizes and shapes near the cell membrane or deep in the cell center were found to contain CD4 molecules. Negatively stained Golgi saccules were observed up to 45 min after re-incubation. These results suggest that increased CD4 molecules can be induced on the surface of normal human T-cells in vitro. Internalization and accumulation of CD4 molecules occurred in CD4-enriched T-cells with IL-2 pretreatment.     

Defects of mitogen-activated protein kinase in ICOS signaling pathway lead to CD4 and CD8 T-cell dysfunction in patients with active SLE

In this study, hypoproliferation and defects of effectors and cytokines in CD4⁺ and CD8⁺ T-cells via ICOS costimulation were found in active SLE patients, relative to normal individuals and RA patient controls. Exogenous IL-2 can partially reverse those defects. In addition, low level of ERK phosphorylation in ICOS-mediated signaling pathway was discovered in lupus CD4⁺ and CD8⁺ T-cells. When blocked with ERK-specific chemical inhibitor PD98059, cell proliferation and IL-2 production via ICOS costimulation from both CD4⁺ and CD8⁺ T-cells will be severely inhibited. These findings confirmed the dysfunction of both CD4⁺ and CD8⁺ T-cells after ICOS costimulation in lupus patients and most importantly pointed out that impairment of ERK activation might be one of the critical factors involved in ICOS-mediated IL-2 and T-cell hypoproliferation in active SLE.     

Toll-like receptor (TLR) expression on CD4 and CD8 T-cells in patients chronically infected with hepatitis C virus

Toll-like receptor (TLR) expression on T-cells and the signalling pathways that lead to the production of cytokines may limit antigen-specific T-cell responses. Here, expression of TLR and retinoic acid inducible gene I (RIG-I) on T-cells were evaluated in patients chronically infected with hepatitis C virus (HCV), before and during pegylated interferon-α and ribavirin therapy. Expression of TLR2,3,4,7,9 and retinoic acid inducible gene (RIG)-I on different CD4⁺ and CD8⁺ T-cell sub-populations (naïve: CD45RA⁺CD57⁻; central memory: T_CM CD45RA⁻CD57⁻; effector memory: T_EM CD45RA⁻CD57⁺ and terminally differentiated effector memory: T_EMRA CD45RA⁺CD57⁺) were measured by flow cytometry. TLR7, TLR9 and RIG-I expression on CD4⁺ T-cells and RIG-I expression on CD8⁺ T-cells was higher in patients than healthy controls. Therapy increased expression of TLR2, TLR4 and TLR9 and this was observed for all T-cell sub-populations. Evaluation of TLR expression at baseline did not identify patients able to achieve sustained virological response following therapy.     

Inhibitory Phenotype of HBV-Specific CD4+ T-Cells Is Characterized by High PD-1 Expression but Absent Coregulation of Multiple Inhibitory Molecules

Background

T-cell exhaustion seems to play a critical role in CD8⁺ T-cell dysfunction during chronic viral infections. However, up to now little is known about the mechanisms underlying CD4⁺ T-cell dysfunction during chronic hepatitis B virus (CHB) infection and the role of inhibitory molecules such as programmed death 1 (PD-1) for CD4⁺ T-cell failure.

Methods

The expression of multiple inhibitory molecules such as PD-1, CTLA-4, TIM-3, CD244, KLRG1 and markers defining the grade of T-cell differentiation as CCR7, CD45RA, CD57 and CD127 were analyzed on virus-specific CD4⁺ T-cells from peripheral blood using a newly established DRB1*01-restricted MHC class II Tetramer. Effects of in vitro PD-L1/2 blockade were defined by investigating changes in CD4⁺ T-cell proliferation and cytokine production.

Results

CD4⁺ T-cell responses during chronic HBV infection was characterized by reduced Tetramer⁺CD4⁺ T-cell frequencies, effector memory phenotype, sustained PD-1 but low levels of CTLA-4, TIM-3, KLRG1 and CD244 expression. PD-1 blockade revealed individualized patterns of in vitro responsiveness with partly increased IFN-γ, IL-2 and TNF-α secretion as well as enhanced CD4⁺ T-cell expansion almost in treated patients with viral control.

Conclusion

HBV-specific CD4⁺ T-cells are reliably detectable during different courses of HBV infection by MHC class II Tetramer technology. CD4⁺ T-cell dysfunction during chronic HBV is basically linked to strong PD-1 upregulation but absent coregulation of multiple inhibitory receptors. PD-L1/2 neutralization partly leads to enhanced CD4⁺ T-cell functionality with heterogeneous patterns of CD4⁺ T-cell rejunivation.     

Human Cytomegalovirus (HCMV)-Specific CD4+ and CD8+ T Cells Are Both Required for Prevention of HCMV Disease in Seropositive Solid-Organ Transplant Recipients

In solid-organ transplant recipients (SOTR) the protective role of human cytomegalovirus (HCMV)-specific CD4⁺, CD8⁺ and γδ T-cells vs. HCMV reactivation requires better definition. The aim of this study was to investigate the relevant role of HCMV-specific CD4⁺, CD8⁺ and γδ T-cells in different clinical presentations during the post-transplant period. Thirty-nine SOTR underwent virologic and immunologic follow-up for about 1 year after transplantation. Viral load was determined by real-time PCR, while immunologic monitoring was performed by measuring HCMV-specific CD4⁺ and CD8⁺ T cells (following stimulation with autologous HCMV-infected dendritic cells) and γδ T-cells by flow cytometry. Seven patients had no infection and 14 had a controlled infection, while both groups maintained CD4⁺ T-cell numbers above the established cut-off (0.4 cell/µL blood). Of the remaining patients, 9 controlled the infection temporarily in the presence of HCMV-specific CD8⁺ only, until CD4⁺ T-cell appearance; while 9 had to be treated preemptively due to a viral load greater than the established cut-off (3×10⁵ DNA copies/mL blood) in the absence of specific CD4⁺ T-cells. Polyfunctional CD8⁺ T-cells as well as Vδ2⁻ γδ T-cells were not associated with control of infection. In conclusion, in the absence of HCMV-specific CD4⁺ T-cells, no long-term protection is conferred to SOTR by either HCMV-specific CD8⁺ T-cells alone or Vδ2⁻ γδ T-cell expansion.     

Systemic expression of inflammatory mediators in patients with chronic rhinosinusitis and nasal polyps with and without Aspirin Exacerbated Respiratory Disease

BackgroundSystemic reactions are related to the pathogenesis of Aspirin Exacerbated Respiratory Disease (AERD). With this work we wanted to study the changes in the systemic levels of inflammatory mediators in both baseline and after oral aspirin challenge in patients with and without AERD.MethodsPatients with nasal polyposis and asthma with AERD (n = 20) and without (n = 18) were orally challenged with aspirin in a single-blind placebo controlled study. Serum samples and urine were collected before and 6 h after placebo and aspirin oral challenges. Serum levels of inflammatory mediators were assayed by using the Luminex technology and ELISA. The concentrations of 9-alpha, 11-beta prostaglandin F₂, and leukotriene E₄ (uLTE₄) were measured in urine samples by ELISA. The expression of T-cell surface markers was analyzed in peripheral blood mononuclear cells isolated before and after the challenges.ResultsAERD patients showed significantly higher baseline levels of s-IL-5R-alpha, uLTE₄ and percentage of CD4⁺CD25⁺CD127^pos and CD4⁺CD45RA⁻CD45RO⁺ but decreased levels of TGF-β₁ and number of CD4⁺CD25⁺CD127^neg cells. Aspirin challenge induced the release of uLTE₄, IL-6 and increased the number of CD4⁺CD45RA⁻CD45RO⁺ memory T-cells only in AERD patients but failed to reduce the levels of sCD40L as observed in non-AERD subjects. Further, IL-8 and sIL-5R-alpha levels directly correlated with the PD₂₀ASA and the effects of aspirin on IL-6 and number of memory T-cells was more pronounced in subjects showing more strong reaction (bronchial and nasal).ConclusionsAERD patients have a differential baseline inflammatory pattern that supports the role inflammation as underlying mechanism of the disease. Systemic response to oral aspirin challenge was related to an increase in serum IL-6 and the number of circulating memory T-cells in AERD patients.     

CD19 target-engineered T-cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models

Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19⁺ tumor lesions, and their ability to provide anti-tumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-γ in response to CD19, and lysed both Raji and Daudi CD19⁺ human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2^−/−γc^−/− immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma.     

Experimental Human Pneumococcal Carriage Augments IL-17A-dependent T-cell Defence of the Lung

Pneumococcal carriage is both immunising and a pre-requisite for mucosal and systemic disease. Murine models of pneumococcal colonisation show that IL-17A-secreting CD4⁺ T-cells (Th-17 cells) are essential for clearance of pneumococci from the nasopharynx. Pneumococcal-responding IL-17A-secreting CD4⁺ T-cells have not been described in the adult human lung and it is unknown whether they can be elicited by carriage and protect the lung from pneumococcal infection. We investigated the direct effect of experimental human pneumococcal nasal carriage (EHPC) on the frequency and phenotype of cognate CD4⁺ T-cells in broncho-alveolar lavage and blood using multi-parameter flow cytometry. We then examined whether they could augment ex vivo alveolar macrophage killing of pneumococci using an in vitro assay. We showed that human pneumococcal carriage leads to a 17.4-fold (p = 0.007) and 8-fold (p = 0.003) increase in the frequency of cognate IL-17A⁺ CD4⁺ T-cells in BAL and blood, respectively. The phenotype with the largest proportion were TNF⁺/IL-17A⁺ co-producing CD4⁺ memory T-cells (p<0.01); IFNγ⁺ CD4⁺ memory T-cells were not significantly increased following carriage. Pneumococci could stimulate large amounts of IL-17A protein from BAL cells in the absence of carriage but in the presence of cognate CD4⁺ memory T-cells, IL-17A protein levels were increased by a further 50%. Further to this we then show that alveolar macrophages, which express IL-17A receptors A and C, showed enhanced killing of opsonised pneumococci when stimulated with rhIL-17A (p = 0.013). Killing negatively correlated with RC (r = −0.9, p = 0.017) but not RA expression. We conclude that human pneumococcal carriage can increase the proportion of lung IL-17A-secreting CD4⁺ memory T-cells that may enhance innate cellular immunity against pathogenic challenge. These pathways may be utilised to enhance vaccine efficacy to protect the lung against pneumonia.     

Impact of cadmium in T lymphocyte subsets and cytokine expression: differential regulation by oxidative stress and apoptosis

Cadmium (Cd), a possible human carcinogen is a potent immunotoxicant. In rodents it causes thymic atrophy and splenomegaly, in addition to immuno-suppression and modulation of humoral and/or cellular immune response. Oxidative stress and apoptosis appear to be underlying mechanism of Cd induced thymic injury. To understand the involvement of reactive oxygen species (ROS), intracellular glutathione (GSH) and apoptosis in modulation of T-cell repertoire, we studied the effect of Cd (10, 25 and 50 μM) on primary T lymphocytes of BALB/c mice at different time intervals (6, 12 and 18 h). We observed a dose and time dependent decline in CD4⁺/CD8⁺ ratio (a bio-indicator of immunotoxicity) as a result of significant suppression of CD4⁺ subsets (helper T-cells) and enhancement in CD8⁺ cells (cytotoxic T-cells) At the same time, the CD4⁺CD8⁺ (DP) cell population was lowered while the CD4⁻CD8⁻ (DN) cells were increased. The oxidative stress and apoptotic data revealed almost similar ROS generation in both CD4⁺ and CD8⁺ cells, but relatively more marked GSH depletion and apoptosis in CD4⁺ than in CD8⁺ population. On further analysis of CD4⁺ T-subsets, cytokine release (IL-2 and IFNγ) by Th 1 cells and IL-4 by Th 2 cells were shown to be significantly suppressed in a dose responsive manner. The highest inhibition was observed in IFNγ, then IL-2 followed by IL-4. In conclusion, our data demonstrates that T-cell apoptosis by Cd, more in CD4⁺ than in CD8⁺ cells appear related to higher depletion of intracellular glutathione. Th 1 cells of CD4⁺ sub-population are more responsive to Cd than Th 2, leading to higher suppression of IL-2 and IFNγ than IL-4 and hence, the study unravels to some extend, the underlying events involved in Cd immunotoxicity.     

Adaptive immune responses during Shigella dysenteriae type 1 infection: an in vitro stimulation with 57 kDa major antigenic OMP in the presence of anti-CD3 antibody

An effort was made to understand the role of the 57 kDa major antigenic fraction of Shigella outer membrane protein (OMP) in the presence of T-cell antigen receptor in activation of adaptive immune responses of the cell mediated immune (CMI) restored patients. The expression of HLA-DR/CD4 out of CD3⁺ T-cells was significantly dominant over the HLA-DR/CD8 and comparable to unstimulated cells of infected or healthy controls. CD4⁺ T-cell activation together with HLA-DR is associated with the expression of CD25⁺ (IL2Rα) for IL-2 growth factors with decreased IL-4 levels, required for maintaining the homeostasis of CD4⁺ T cell. Furthermore, the positive expression of the CD45 antigen is possibly required for acquiring the memory for CD4⁺ cells signals and facilitates the interaction with CD54 antigen. As a result, antigen-specific secondary signal is generated for B-cell activation to produce IgG2a and IgG2b. This suggests that antibody mediated-adaptive immune responses are generated due to anti-CD3 induced helper T-cell activity. The above mentioned findings reflect that the antigen alone might not exacerbate the selective T-cell responses. But these antigens in the presence of anti-CD3 antibody might help to elicit adaptive immune response via T-cell receptor (TCR) activation.     

Loss of Function of Intestinal IL-17 and IL-22 Producing Cells Contributes to Inflammation and Viral Persistence in SIV-Infected Rhesus Macaques

In HIV/SIV-infected humans and rhesus macaques (RMs), a severe depletion of intestinal CD4⁺ T-cells producing interleukin IL-17 and IL-22 associates with loss of mucosal integrity and chronic immune activation. However, little is known about the function of IL-17 and IL-22 producing cells during lentiviral infections. Here, we longitudinally determined the levels and functions of IL-17, IL-22 and IL-17/IL-22 producing CD4⁺ T-cells in blood, lymph node and colorectum of SIV-infected RMs, as well as how they recover during effective ART and are affected by ART interruption. Intestinal IL-17 and IL-22 producing CD4⁺ T-cells are polyfunctional in SIV-uninfected RMs, with the large majority of cells producing four or five cytokines. SIV infection induced a severe dysfunction of colorectal IL-17, IL-22 and IL-17/IL-22 producing CD4⁺ T-cells, the extent of which associated with the levels of immune activation (HLA-DR⁺CD38⁺), proliferation (Ki-67⁺) and CD4⁺ T-cell counts before and during ART. Additionally, Th17 cell function during ART negatively correlated with residual plasma viremia and levels of sCD163, a soluble marker of inflammation and disease progression. Furthermore, IL-17 and IL-22 producing cell frequency and function at various pre, on, and off-ART experimental points associated with and predicted total SIV-DNA content in the colorectum and blood. While ART restored Th22 cell function to levels similar to pre-infection, it did not fully restore Th17 cell function, and all cell types were rapidly and severely affected—both quantitatively and qualitatively—after ART interruption. In conclusion, intestinal IL-17 producing cell function is severely impaired by SIV infection, not fully normalized despite effective ART, and strongly associates with inflammation as well as SIV persistence off and on ART. As such, strategies able to preserve and/or regenerate the functions of these CD4⁺ T-cells central for mucosal immunity are critically needed in future HIV cure research.     

Human CD8+ T-cells Recognizing Peptides from Mycobacterium tuberculosis (Mtb) Presented by HLA-E Have an Unorthodox Th2-like,Multifunctional, Mtb Inhibitory Phenotype and Represent a Novel Human T-cell Subset

Mycobacterial antigens are not exclusively presented to T-cells by classical HLA-class Ia and HLA-class II molecules, but also through alternative antigen presentation molecules such as CD1a/b/c, MR1 and HLA-E. We recently described mycobacterial peptides that are presented in HLA-E and recognized by CD8⁺ T-cells. Using T-cell cloning, phenotyping, microbiological, functional and RNA-expression analyses, we report here that these T-cells can exert cytolytic or suppressive functions, inhibit mycobacterial growth, yet express GATA3, produce Th2 cytokines (IL-4,-5,-10,-13) and activate B-cells via IL-4. In TB patients, Mtb specific cells were detectable by peptide-HLA-E tetramers, and IL-4 and IL-13 were produced following peptide stimulation. These results identify a novel human T-cell subset with an unorthodox, multifunctional Th2 like phenotype and cytolytic or regulatory capacities, which is involved in the human immune response to mycobacteria and demonstrable in active TB patients’ blood. The results challenge the current dogma that only Th1 cells are able to inhibit Mtb growth and clearly show that Th2 like cells can strongly inhibit outgrowth of Mtb from human macrophages. These insights significantly expand our understanding of the immune response in infectious disease.     

Decreases in Colonic and Systemic Inflammation in Chronic HIV Infection after IL-7 Administration

Despite antiretroviral therapy (ART), some HIV-infected persons maintain lower than normal CD4⁺ T-cell counts in peripheral blood and in the gut mucosa. This incomplete immune restoration is associated with higher levels of immune activation manifested by high systemic levels of biomarkers, including sCD14 and D-dimer, that are independent predictors of morbidity and mortality in HIV infection. In this 12-week, single-arm, open-label study, we tested the efficacy of IL-7 adjunctive therapy on T-cell reconstitution in peripheral blood and gut mucosa in 23 ART suppressed HIV-infected patients with incomplete CD4⁺ T-cell recovery, using one cycle (consisting of three subcutaneous injections) of recombinant human IL-7 (r-hIL-7) at 20 µg/kg. IL-7 administration led to increases of both CD4⁺ and CD8⁺ T-cells in peripheral blood, and importantly an expansion of T-cells expressing the gut homing integrin α4β7. Participants who underwent rectosigmoid biopsies at study baseline and after treatment had T-cell increases in the gut mucosa measured by both flow cytometry and immunohistochemistry. IL-7 therapy also resulted in apparent improvement in gut barrier integrity as measured by decreased neutrophil infiltration in the rectosigmoid lamina propria 12 weeks after IL-7 administration. This was also accompanied by decreased TNF and increased FOXP3 expression in the lamina propria. Plasma levels of sCD14 and D-dimer, indicative of systemic inflammation, decreased after r-hIL-7. Increases of colonic mucosal T-cells correlated strongly with the decreased systemic levels of sCD14, the LPS coreceptor - a marker of monocyte activation. Furthermore, the proportion of inflammatory monocytes expressing CCR2 was decreased, as was the basal IL-1β production of peripheral blood monocytes. These data suggest that administration of r-hIL-7 improves the gut mucosal abnormalities of chronic HIV infection and attenuates the systemic inflammatory and coagulation abnormalities that have been linked to it.     

TRP expression pattern and the functional importance of TRPC3 in primary human T-cells

TRP proteins form ion channels which are activated following receptor stimulation. In T-cell lines, expression data of TRP proteins have been published. However, almost no data about TRP expression is available in primary human T-cells. Using RT-PCR and quantitative RT-PCR, we compare the expression of TRP mRNA in 1) human peripheral blood lymphocytes, which are a mix of mostly mono-nuclear blood lymphocytes but contain other leucocytes, 2) a pure human CD4⁺ T-helper cell population in the resting (= naïve) and activated (= effector) state, and 3) two commonly used CD4⁺ Jurkat T-cell lines, E6-1 and parental. To mimic physiological cell stimulation, we analyzed TRP expression in primary human cells in a quantitative way over several days following formation of an immunological synapse through stimulation with antibody-coated beads. The TRP expression profile of primary human T-cells was significantly different from Jurkat T-cells. Among the TRP mRNAs of the TRPC, TRPM, and TRPV family, we found consistent expression of TRPC1, TRPC3, TRPV1, TRPM2, and TRPM7 in primary human CD4⁺ T-cells of all analyzed blood donors. Among these, TRPC3 and TRPM2 were strongly up-regulated following stimulation, but with different kinetics. We found that TRPC3 modulates Ca²⁺-dependent proliferation of primary CD4⁺ T-cells indicating that TRPC3 may be involved in Ca²⁺ homeostasis in T-cells besides the well-established STIM and ORAI proteins which are responsible for store-operated Ca²⁺ entry.     

Phosphorylation and down-regulation of CD4 and CD8 in human CTLs and mouse L cells

The CD4 and CD8 molecules are rapidly phosphorylated following exposure of CD4⁺ or CD8⁺ human cytotoxic T lymphocytes (CTL) clones to B-lymphoblastoid cell lines bearing the relevant target alloantigens. Treatment of CD4⁺ or CD8⁺ CTL clones with phorbol myristate acetate (PMA), phytohemagglutinin, or mitogenic combinations of CD2-specific antibodies also resulted in CD4 or CD8 phosphorylation. Down-regulation of the surface expression of these molecules could be demonstrated in both CD4⁺ and CD8⁺ clones following exposure to the relevant alloantigen or PMA. Parallel experiments were conducted using mouse L cells in which the human CD4 or CD8 antigens were stably expressed. Exposure of these transfectants to PMA induced rapid phosphorylation of the CD4 and CD8 molecules. As in CD4⁺ CTL clones, rapid modulation of the CD4 antigen could be demonstrated in L cells following PMA treatment. In contrast, there was no demonstrable down-regulation of the CD8 antigen in PMA-treated CD8⁺ L cell transfectants. These studies demonstrate a significant differential property of the CD4 and CD8 antigens and suggest that down-regulation of the CD8 antigen may require its expression in a T-cell environment and/or the association of CD8 with the T-cell receptor or other T cell-specific molecules.     

CCR5 Antagonism Impacts Vaccination Response and Immune Profile in HIV-1 Infection

Maraviroc (MVC) is the first licensed antiretroviral therapeutic agent to target a host cell surface molecule, and successful HIV-1 entry blockade by this C-C chemokine receptor type 5 (CCR5)-antagonist potentiates immunomodulation. We hypothesized that MVC intensification impacts immunization responses, T-cell phenotype, function and delayed type hypersensitivity (DTH) in HIV-1⁺ subjects. A 24-wk, double-blinded, placebo-controlled study of the addition of MVC to suppressive antiretroviral therapy in HIV-1⁺ persons was performed. Subjects received DTH tests, intramuscular tetanus, meningococcal and oral cholera immunizations. Antibody titers, T-cell function and phenotype were assessed. Of 157 patients referred, 47 were randomized 1:1; MVC:placebo. MVC enhanced meningococcal neo-immunization, blunted cholera response and expedited lymphoproliferation to tetanus boost, without affecting recall humoral response. Anti-HIV-1 group-specific antigen (Gag) and tetanus toxoid (TTox) function improved significantly, HIV-1-associated CD8 T-cell skewing normalized, and the percentage of late-stage and major histocompatibility complex (MHC) class II expressing CD4 T-cells increased. Activated CD4⁺ CD38⁺ human leukocyte antigen (HLA)-DR⁺ T-cells declined, and costimulation shifted to coinhibition. DTH was unchanged. Maraviroc intensification, through antagonism of the cell surface molecule CCR5, favorably influences immune profiles of HIV-1⁺ patients, supporting its immunomodulatory use in HIV-1 infection and potentially in other immunologically relevant settings.     

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4⁺T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4⁺ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4⁺ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4⁺ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4⁺ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4⁺ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4⁺ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4⁺ T-cell immunity.     

Interleukin-7 deficiency in rheumatoid arthritis: consequences for therapy-induced lymphopenia

We previously demonstrated prolonged, profound CD4⁺ T-lymphopenia in rheumatoid arthritis (RA) patients following lymphocyte-depleting therapy. Poor reconstitution could result either from reduced de novo T-cell production through the thymus or from poor peripheral expansion of residual T-cells. Interleukin-7 (IL-7) is known to stimulate the thymus to produce new T-cells and to allow circulating mature T-cells to expand, thereby playing a critical role in T-cell homeostasis. In the present study we demonstrated reduced levels of circulating IL-7 in a cross-section of RA patients. IL-7 production by bone marrow stromal cell cultures was also compromised in RA. To investigate whether such an IL-7 deficiency could account for the prolonged lymphopenia observed in RA following therapeutic lymphodepletion, we compared RA patients and patients with solid cancers treated with high-dose chemotherapy and autologous progenitor cell rescue. Chemotherapy rendered all patients similarly lymphopenic, but this was sustained in RA patients at 12 months, as compared with the reconstitution that occurred in cancer patients by 3–4 months. Both cohorts produced naïve T-cells containing T-cell receptor excision circles. The main distinguishing feature between the groups was a failure to expand peripheral T-cells in RA, particularly memory cells during the first 3 months after treatment. Most importantly, there was no increase in serum IL-7 levels in RA, as compared with a fourfold rise in non-RA control individuals at the time of lymphopenia. Our data therefore suggest that RA patients are relatively IL-7 deficient and that this deficiency is likely to be an important contributing factor to poor early T-cell reconstitution in RA following therapeutic lymphodepletion. Furthermore, in RA patients with stable, well controlled disease, IL-7 levels were positively correlated with the T-cell receptor excision circle content of CD4⁺ T-cells, demonstrating a direct effect of IL-7 on thymic activity in this cohort.     

The influence of immunoregulatory cytokines IL-2, IL-7, and IL-15 upon activation, proliferation, and apoptosis of immune memory T-cells in vitro

The influence of immunoregulatory cytokines IL-2, IL-7, and IL-15 on the activation, proliferation, and apoptosis of different subpopulations of an immune memory T-cell (CD45RO+) in healthy donors were investigated. It was demonstrated that rIL-2 equally affected both the activation and proliferation of CD4⁺ and CD8⁺ subpopulations of memory T-cells in vitro. High concentrations of rIL-2 increased the number of CD8⁺ memory cells expressing apoptotic marker CD95. Effect of rIL-7 and rIL-15 on the activation and proliferation of cytotoxic CD8⁺ memory cells in vitro was different. CD4⁺ memory lymphocytes exhibited relative resistance to activation and proliferation by rIL-7 and rIL-15 compared to rIL-2. This can provide them with relative resistance to apoptosis, as well as create the necessary conditions for accelerated implementation of their functional capacity in the development of a secondary immune response.     

T-cell autoreactivity to citrullinated autoantigenic peptides in rheumatoid arthritis patients carrying HLA-DRB1 shared epitope alleles

Introduction

Anti-citrullinated peptide antibodies are found in rheumatoid arthritis (RA) patients with HLA-DRβ chains encoding the shared epitope (SE) sequence. Citrullination increases self-antigen immunogenicity, through increased binding affinity to SE-containing HLA-DR molecules. To characterise T-cell autoreactivity towards citrullinated self-epitopes, we profiled responses of SE⁺ healthy controls and RA patients to citrullinated and unmodified epitopes of four autoantigens.

Methods

We compared T-cell proliferative and cytokine responses to citrullinated and native type II collagen 1,237 to 1,249, vimentin 66 to 78, aggrecan 84 to 103 and fibrinogen 79 to 91 in six SE⁺ healthy controls and in 21 RA patients with varying disease duration. Cytokine-producing cells were stained after incubation with peptide in the presence of Brefeldin-A.

Results

Although proliferative responses were low, IL-6, IL-17 and TNF were secreted by CD4⁺ T cells of SE⁺ RA patients and healthy controls, as well as IFNγ and IL-10 secreted by RA patients, in response to citrullinated peptides. Of the epitopes tested, citrullinated aggrecan was most immunogenic. Patients with early RA were more likely to produce IL-6 in response to no epitope or to citrullinated aggrecan, while patients with longstanding RA were more likely to produce IL-6 to more than one epitope. Cytokine-producing CD4⁺ T cells included the CD45RO⁺ and CD45RO^- and the CD28⁺ and CD28^- subsets in RA patients.

Conclusion

Proinflammatory cytokines were produced by CD4⁺ T cells in SE⁺ individuals in response to citrullinated self-epitopes, of which citrullinated aggrecan was most immunogenic. Our data suggest that the T-cell response to citrullinated self-epitopes matures and diversifies with development of RA.