Type 1 vs. type 2 cytokine secretion in vitro and its regulation by hydrocortisone in humans subjected to 120-day anti-orthostatic bed-rest regime

Head-Down Bed-Rest (HDBR) mimics some of the physiological stress effects of microgravity. Six healthy volunteers were subjected to bed-rest for 120 days. Blood samples were collected one month before (PRE), on day 110 of HDBR (DAY 110), and on the 7th day after bed-rest regime ends (POST). Distribution of T-cell subsets, NK-, B-cells and monocytes was assessed in the whole blood. Distribution of cytokine secreting T-cells was assessed in PMA/ionomycin cell culture. Peripheral Blood Mononuclear Cells (PBMC) and whole blood cells (WB) were activated with a combination of PHA and LPS to assess cytokine secretion. In addition, PHA/LPS activated cell cultures were treated with 10(-6) M of hydrocortisone (HCS) in order to study stress-induced alterations in the cortisol-sensitivity of immunocytes. Results from HCS culture were compared to non-treated control cultures. Stress factors of HDBR affect immune responsiveness and immune-endocrine homeostatic interrelations in vitro as follow: 1) alter expression of surface receptor to IL-2 (CD25) by CD4+ and CD8+ T-cell subsets in PHA/LPS activated PBMC culture; 2) alter distribution of IL-2 and/or IFN-gamma producing CD4+ and CD8+ T-cells in PMA/ionomycin activated culture; 3) significantly affect secretion of IL-2, IFN-gamma, and IL-4, but not IL-10 and soluble IL-2 receptor alpha in PHA/LPS activated PBMC culture; 4) shift Type 1 vs. Type 2 cytokine balance in PHA/LPS activated culture toward to Type 1 response; 5) in vitro treatment with hydrocortisone unequally modulate expression of CD25 on CD4+, and CD8+ T-cells, as well as secretion of Type 1 and Type 2 cytokines in PHA/LPS activated PBMC culture during bed-rest regime; 6) assessment of immune profile depends from the cellular and humoral milieu of cell culture.     

The cell death regulator GRIM-19 is involved in HIV-1 induced T-cell apoptosis

One of the hallmarks of Human Immunodeficiency Virus-1 (HIV-1) infection is progressive depletion of the infected and bystander CD4+ T-cells by apoptosis. Different mitochondrial proteins have been implicated in this apoptotic process; however, the role of different subunits of mitochondrial oxidative phosphorylation (OXPHOS) complexes in apoptosis is not clearly understood. Some of the OXPHOS complex subunits seem to perform other functions in addition to their primary role in energy generating process. GRIM-19 (gene associated with retinoid-interferon-induced-mortality-19), a subunit of mitochondrial complex-I was previously implicated in Interferon-β and retionoic acid induced apoptosis in many tumor cells. In this study we report, using differential gene expression analysis, that GRIM-19 is up-regulated in HIV-1 infected apoptotic T-cells. A temporal up regulation of this subunit was observed in different HIV-1 infected T-cell lines and human PBMC and the extent of increase correlated to increasing apoptosis and virus production. Moreover, silencing GRIM-19 in HIV-1 infected cells reduced apoptosis, indicating its involvement in HIV-1 induced T-cell death.     

Activation of CD4+ T-lymphocytes in asymptomatic HIV infected patients induce the program action of lymphocyte death by apoptosis]

Activation-induced programmed cell death, or apoptosis, is a physiological cell suicide process involved in the negative thymic selection of the T-cell repertoire. We have proposed that the inappropriate re-emergence in the mature CD4+ T-cell population of such a death program could explain both the early dysfunction and the late depletion of CD4+ T-cells from human immunodeficiency virus (HIV)-infected individuals. We present evidence showing that the selective failure of T-cells from 10 HIV-infected asymptomatic individuals (with normal CD4+ T-cell counts) to proliferate in vitro to pokeweed mitogen and to self-major histocompatibility complex class-II T-cell receptor mobilization by superantigens is due to the induction by these stimuli of CD4+ T-cell death. This death process has characteristic features of apoptosis, including DNA fragmentation into multiples of a 200 base pair unit, and the preventive effect of the protein synthesis inhibitor cycloheximide. These findings suggest that in vivo CD4+ T-cell suicide upon activation might account, independently of any HIV-mediated cytopathic effect, for the progressive depletion of CD4+ T-cells that leads to AIDS.     

Functional CD4(+) and CD8(+) T-cell responses induced by autologous mitomycin C treated Epstein-Barr virus transformed lymphoblastoid cell lines

Epstein-Barr virus (EBV) gene expression in tumor cells of posttransplant lymphoproliferative disorder (PTLD) patients resembles that of EBV transformed B-cell lines (LCL). EBV-specific cytotoxic T-lymphocytes can be generated by stimulating peripheral blood lymphocytes with autologous LCL. We describe a standardized method for the growth inactivation and cryopreservation of LCL for optimal T-cell stimulation and analyzed the function and phenotype of responding T-cells. LCL growth was completely blocked by mitomycin C treatment (McLCL) and McLCL could be cryopreserved while retaining excellent APC function. McLCL stimulated both CD4(+) and CD8(+) T-cells as measured by HLA-DR and CD25 expression using FACS analysis. EBV-specific CTL activity and T-cell proliferation were induced and immunocytochemical staining showed CD4(+) and (granzyme B positive) CD8(+) T-cells rosetting with McLCL. Granzymes A and B, IFN-gamma, and IL-6 were detected at significant levels in the supernatant. Thus, ex vivo T-cell activation with cryopreserved McLCL results in activation of both CD4(+) and CD8(+) T-cells producing a Th1-like cytokine profile, making this a suitable protocol for adoptive therapy of PTLD.     

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25?% of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50?% of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.     

Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed ‘classic’ features of apoptosis following exposure to pneumococci. Conversely, purified CD3⁺ T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3⁺ T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3⁺ T-cells in PBMC cultures required ‘classical’ CD14⁺ monocytes, which enhanced T-cell activation. CD3⁺ T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3⁺ T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.     

Overexpression of Interleukin-1α Suppresses Liver Metastasis of Lymphoma: Implications for Antitumor Effects of CD8+ T-cells

Interleukin (IL)-1 plays a key role in carcinogenesis, tumor progression, and metastasis. Although IL-1 may enhance the expansion of CD8+ T-cells, the pathological contribution of IL-1-activated CD8+ T-cells to tumor metastasis remains unclear. This study used a liver metastasis model of the EL4 T-cell lymphoma cells transplanted into human IL (hIL)-1α conditional transgenic (hIL-1α cTg) mice. Overproduction of hIL-1α suppressed both macroscopic and histological liver metastasis of EL4 T-cell lymphoma. The hIL-1α-induced inflammatory state increased the number of CD8+ T-cells both within and around metastatic tumors. Moreover, larger numbers of CD8+ T-cells showed greater infiltration of liver blood vessels in hIL-1α cTg mice than in control wild-type mice. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining of liver tissue from hIL-1α cTg mice indicated increased apoptosis of cells in the tumor. Localization of apoptosis cells resembled that of CD8+ T-cells. In addition, cytotoxicity assay showed that CD8+ T-cell counts from tumor-bearing hIL-1α cTg mice correlated with cytotoxicity against EL4. In summary, IL-1α suppresses lymphoma metastasis, and IL-1α-activated CD8+ T-cells may play important roles in inhibiting both tumor metastasis and metastatic tumor growth:     

Human immunodeficiency virus type 1 gp120 induces anergy in human peripheral blood lymphocytes by inducing interleukin-10 production.

The effects of recombinant gp120 on the proliferative responses and cytokine production by normal peripheral blood mononuclear cells (PBMC) were investigated. gp120 inhibited in a dose-dependent fashion the anti-CD3 monoclonal antibody (MAb)- and concanavalin A-induced proliferative responses. The production of interleukin-2 (IL-2) and IL-4 was diminished by gp120 in the anti-CD3- and concanavalin A-stimulated cultures. In unstimulated PBMC, gp120 induced the production of considerable amounts of IL-10, gamma interferon, and tumor necrosis factor alpha. The gp120-induced reduction in the proliferative responses of PBMC was at least partially reversed by the addition of IL-2, anti-CD28 MAb, or transfectants expressing CD80, CD86, or CD40 but not with exogenous IL-4. Also, a neutralizing anti-IL-10 MAb reversed the inhibitory effect of gp120 on the proliferative responses whereas exogenous IL-10 further enhanced this inhibitory effect. These findings indicate that IL-10 plays an important role in the inhibitory effect of gp120 on PBMC proliferation. The ratio of CD3+CD4+ to CD3+CD8+ T cells was the same in gp120-treated and untreated cell cultures. No apoptosis in these two T-cell populations was observed. However, the number of activated CD3+CD4+ T cells and CD3+CD8+ T cells, as judged by CD25, CD69, and HLA-DR expression, was consistently reduced. gp120 induced the expression of IL-10 in the monocyte/macrophage population, and therefore gp120 also reduced the proliferative responses of CD4+ T-cell-depleted PBMC. Taken together, our observations point to the importance of the cytokine pattern changes and, in particular, the role of IL-10 (produced by the monocytes) in the inhibitory effect of gp120. This mechanism of gp120-induced immunosuppression, if operative in vivo, could contribute to the depressed immune responses associated with human immunodeficiency virus infection and thus have important implications for immunotherapeutic strategies to slow down disease progression in AIDS.     

Expression of the IL-7 Receptor Alpha-Chain Is Down Regulated on the Surface of CD4 T-Cells by the HIV-1 Tat Protein

HIV infection elicits defects in CD4 T-cell homeostasis in both a quantitative and qualitative manner. Interleukin-7 (IL-7) is essential to T-cell homeostasis and several groups have shown reduced levels of the IL-7 receptor alpha-chain (CD127) on both CD4 and CD8 T-cells in viremic HIV+ patients. We have shown previously that soluble HIV Tat protein specifically down regulates cell surface expression of CD127 on human CD8 T-cells in a paracrine fashion. The effects of Tat on CD127 expression in CD4 T-cells has yet to be described. To explore this effect, CD4 T-cells were isolated from healthy individuals and expression levels of CD127 were examined on cells incubated in media alone or treated with Tat protein. We show here that, similar to CD8 T-cells, the HIV-1 Tat protein specifically down regulates CD127 on primary human CD4 T-cells and directs the receptor to the proteasome for degradation. Down regulation of CD127 in response to Tat was seen on both memory and naive CD4 T-cell subsets and was blocked using either heparin or anti-Tat antibodies. Tat did not induce apoptosis in cultured primary CD4 T-cells over 72 hours as determined by Annexin V and PI staining. Pre-incubation of CD4 T-cells with HIV-1 Tat protein did however reduce the ability of IL-7 to up regulate Bcl-2 expression. Similar to exogenous Tat, endogenously expressed HIV Tat protein also suppressed CD127 expression on primary CD4 T-cells. In view of the important role IL-7 plays in lymphocyte proliferation, homeostasis and survival, down regulation of CD127 by Tat likely plays a central role in immune dysregulation and CD4 T-cell decline. Understanding this effect could lead to new approaches to mitigate the CD4 T-cell loss evident in HIV infection.     

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.     

Apoptosis of human T-cells: induction by glucocorticoids or surface receptor ligation in vitro and ex vivo.

Activated T-cells are susceptible to induction of apoptosis or programmed cell death in response to ligation of several cell surface structures, including CD2, CD3, and CD95/Fas. These mechanisms may be important in the regulation of immune responses and in prevention of autoimmunity. We used flow cytometric quantitation of DNA strand breaks to detect T-cells committed to programmed cell death. Activated human peripheral blood T-lymphocytes, and freshly isolated human thymocytes underwent apoptosis when exposed to dexamethasone or to monoclonal antibodies directed at CD2 or CD3. Interleukin-2 reduced spontaneous or dexamethasone-induced apoptosis, but augmented apoptosis due to ligation of CD2. A neutralizing anti-Fas antibody reduced the amount of DNA strand breakage, not only in T-cells exposed to antibodies to CD2 or CD3, but also in dexamethasone-treated cultures. In vivo activated T-cells, from inflammatory synovial fluids, were sensitive to immediate induction of DNA strand breaks without prior in vitro activation by lectin and IL-2. Taken together, the results indicated that: 1. Human lymphocytes, like murine thymocytes, are sensitive to glucocorticoid-induced apoptosis, as well as to programmed cell death triggered through surface receptors; 2. The effects of IL-2 on T-cell apoptosis depend on the apoptotic stimulus; 3. Fas/Fas ligand interactions may be relevant for both membrane receptor and glucocorticoid-induced cell death; and 4. Induction of T-cell apoptosis may be important in therapeutic effects of glucocorticoids in human disease.     

Functional and phenotypic modifications induced by IL-4, as single agent or in combination with IL-2, on PBMC preactivated in vivo by alpha-interferon + interleukin-2 therapy

The effect of human IL-4, used as a single agent or in combination with low or high dose IL-2, upon LAK-cell proliferation and activation has been tested on PBMC from patients treated with alpha 2-IFN and IL-2. Four days in vitro culture with IL-4 did not induce any LAK-cell activation; IL-4 induced the proliferation of CD3+ CD4+ T-cells, but decreased the percentage of NK cells in culture samples. When combined with high dose IL-2, IL-4 improved the recovery of MN cell without modification of T-cell subsets; however, IL-4 had no major effect on IL-2-induced NK or LAK cell activity. The combination of IL-4 and low dose IL-2 still significantly improved the total MN cell recovery but did not modify the distribution of T and NK lymphocytes; IL-4 inhibited low dose IL-2-induced NK and LAK cell activity, and increased the BL-esterase activity induced by high or low dose IL-2. The combination of IL-4 and IL-2 did not induce any large variation in the percentage of IL-2R (p55) expressing cells. In all tested conditions, IL-2R (p55) was mainly expressed on CD4+ T cells; less than 2% of the cells coexpressed the NK cell marker CD56 and IL-2R (p55). The effect of IL-4 upon IL-2-induced LAK cell expansion is thus very different on PBMC pre-activated in vivo by alpha IFN + IL-2 therapy than on PBMC pre-treated in vitro with IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibility to programmed cell death in T-lymphocytes from septic patients: a mechanism for lymphopenia and Th2 predominance

Sepsis causes lymphopenia which is inversely correlated with patient survival. The role of apoptosis-specific immune-activation and activation-induced cell-death in sepsis is incompletely understood. Fifteen septic patients and 20 healthy controls were included. T-cell proliferation was measured by [3H]thymidine uptake. Apoptosis and cell phenotype were determined by FACS. sTNFR1, sCD95, interleukin-1beta converting enzyme (sICE), and interleukin (IL)-10 were measured by ELISA. PHA and CD3-driven T-cell proliferation were significantly decreased in septic patients. The percentages of CD3(+) and CD4(+) T cells and CD19(+) B cells were significantly reduced. Percent memory T-cells (CD45RO(+)) and cells undergoing apoptosis (CD95(+)/annexin-V(+)) were significantly increased in sepsis. Moreover, sCD95, sTNFRI, and ICE were significantly increased. Anti-CD3 antibody triggering induced a 56% increase of CD4 T-cell death in septic patients vs. 7.5% in controls relative to IgG. Serum level of IL-10, a Th2 cytokine, was enhanced. These findings strongly suggest that in septic patients Th1 T-cells are selectively susceptible to undergo apoptosis. This observation provides an additional pathophysiological concept in the genesis of Th2 dominance.     

Expression of interleukin-4 in apoptotic cells: stimulation of the type-2 cytokine by different toxins in human peripheral blood mononuclear and tumor cells

BACKGROUND: Immunological reactivity is regulated by T-cell populations (type-1 and type-2 cells) via cytokine secretion, but their influence on apoptosis remains unclear. METHODS: Intracellular expression of type-1 (interferon [IFN]-gamma) and type-2 (interleukin [IL]-4) cytokines and apoptosis-related molecules (Apo2. 7, Bcl-2 protein) was studied by flow cytometry in human peripheral blood mononuclear cells (PBMC), myeloma (U-266), monocytic (THP-1), and T-leukemia cells (MOLT-4) in response to toxins, which act on different intracellular targets (actinomycin D, cycloheximide, the mistletoe lectins [ML]-1 and ML-3, brefeldin A, staurosporine). RESULTS: The apoptosis-inducing toxins stimulated intracellular IL-4 expression mainly in PBMC with high expression of the mitochondrial apoptosis marker, Apo2.7, but with decreased level of the anti-apoptotic Bcl-2 protein. Up-regulation of IL-4 coincided with a significant down-regulation of IFN-gamma in CD4(+) and CD8(+) cells. The inhibitor of oxidative phosphorylation, oligomycin, and the caspase inhibitor, z-VAD-fmk, abolished IL-4 expression and DNA fragmentation in the PBMC. Also in the myeloma, monocytic, and T-leukemia cells, IL-4 was mainly observed in the Apo2.7(+) apoptotic cells in response to the toxins. CONCLUSIONS: We suggest that the different apoptotic toxins activate a common pathway in which IL-4 production plays a yet unknown intracellular role further downstream during apoptosis.     

Progression of intracranial glioma disrupts thymic homeostasis and induces T-cell apoptosis in vivo

The thymus is the site where all T-cell precursors develop, mature, and subsequently leave as mature T-cells. Since the mechanisms that mediate and regulate thymic apoptosis are not fully understood, we utilized a syngenic GL261 murine glioma model to further elucidate the fate of T-cells in tumor bearing C57BL/6 mice. First, we found a dramatic reduction in the size of the thymus accompanied by a decrease in thymic cellularity in response to glioma growth in the brains of affected mice. There was a marked reduction of double positive subset and an increase in the frequency of CD4⁺ and CD8⁺ single positive T-cell subsets. Analysis of double negative thymocytes showed an increase in the accumulation of CD44⁺ cells. In contrast, there was a marked loss of CD44 and CD122 expression in CD4⁺ and CD8⁺ subsets. The growth of intracranial tumors was also associated with decreased levels of HO-1, a mediator of anti-apoptotic function, and increased levels of Notch-1 and its ligand, Jagged-1. To determine whether thymic atrophy could be due to the effect of Notch and its ligand expression by glioma in vivo, we performed a bone marrow transplant experiment. Our results suggest that Notch-1 and its ligand Jagged-1 can induce apoptosis of thymocytes, thereby influencing thymic development, immune system homeostasis, and function of the immune cells in a model of experimental glioma.     

Lipoic acid downmodulates CD4 from human T lymphocytes by dissociation of p56(Lck)

Lipoic acid is an antioxidant that suppresses and treats a model of multiple sclerosis, experimental autoimmune encephalomyelitis. We now demonstrate that treatment of human PBMC and T cell lines with LA downmodulated CD4 expression in a concentration-dependent manner. LA treatment of Con A stimulated PBMC specifically removed CD4 from the T-cell surface, but not CD3. Epitope masking by LA was excluded by using monoclonal antibodies targeting different domains of CD4. Incubation on ice inhibited CD4 removal following LA treatment, suggesting that endocytosis was involved in its downmodulation. LA is in a unique category of compounds that induce CD4 downmodulation by various mechanisms (e.g., gangliosides). We hypothesized that LA might induce dissociation of p56(Lck) from CD4, thus leading to its downmodulation. Immunoblot analyses demonstrated reduced co-precipitation of p56(Lck) from Jurkat T-cells following LA treatment and precipitation of CD4. This unique immunomodulatory effect of LA warrants further investigation.     

Cerebrospinal Fluid (CSF) CD8+ T-Cells That Express Interferon-Gamma Contribute to HIV Associated Neurocognitive Disorders (HAND)

BackgroundHIV associated neurocognitive disorders (HAND) continue to affect cognition and everyday functioning despite anti-retroviral treatment (ART). Previous studies focused on mechanisms related to monocyte/macrophage mediated inflammation. However, in the ART era, there is increasing evidence for the involvement of CD8+ T-cells in CNS pathogenesis.MethodsTo investigate the relationship between T-cell responses and neurocognitive impairment (NCI), cerebrospinal fluid (CSF) and peripheral blood CD4+ and CD8+ T-cell intracellular cytokine (IFNγ, IL-2, TNFα) and lytic marker (CD107a) expression were assessed in HIV infected subjects who underwent comprehensive neurocognitive (NC) evaluation and either initiated or changed ART.ResultsData were collected from 31 participants at 70 visits. The frequency of cytokine expressing T-cells in CSF was significantly higher than in peripheral blood for CD4+T-cells: TNFα, IL-2, IFNγ and CD8+T-cells: IL-2 and IFNγ. Analysis of T-cell activity and NCI as a function of CSF HIV RNA levels suggested a general association between NCI, high CSF CD8+ (but not CD4+T-cell) cytokine expression and CSF HIV RNA <10³ copies/ml (p<0.0001). Specifically, CSF CD8+ T-cell IFNγ expression correlated with severity of NCI (r = 0.57, p = 0.004). Multivariable analyses indicated that CSF CD8+T-cell IFNγ and myeloid activation (CD163) contributed equally and independently to cognitive status and a composite variable produced the strongest correlation with NCI (r = 0.83, p = 0.0001). In contrast, CD8+ cytolytic activity (CD107a expression) was negatively correlated with NCI (p = 0.05) but was dependent on CD4 levels >400/μl and low CSF HIV RNA levels (<10³ copies/ml). In our longitudinal analysis of 16 subjects, higher CSF CD8+IFNγ expression at baseline predicted NC decline at follow-up (p = 0.02). Severity of NCI at follow-up correlated with level of residual HIV RNA in CSF.ConclusionsPresence of IFNγ expressing CD8+ T-cells, absence of cytolytic CD8+ T-cells, high myeloid activation, and failure of ART to suppress HIV replication in CSF contribute to increased risk of HAND.     

Decreased memory T-cell response and function in human immunodeficiency virus-infected patients with tegumentary leishmaniasis

The effects of human immunodeficiency virus (HIV) on the immune response in patients with cutaneous leishmaniasis have not yet been fully delineated. This study quantified and evaluated the function of memory T-cell subsets in response to soluble Leishmania antigens (SLA) from patients coinfected with HIV and Leishmania with tegumentary leishmaniasis (TL). Eight TL/HIV coinfected subjects and 10 HIV seronegative subjects with TL were evaluated. The proliferative response of CD4+and CD8+T-cells and naïve, central memory (CM) and effector memory (EM) CD4+T-cells in response to SLA were quantified using flow cytometry. The median cell division indices for CD4+and CD8+T-cells of coinfected patients in response to SLA were significantly lower than those in patients with Leishmania monoinfection (p < 0.05). The proportions of CM and EM CD4+T-cells in response to SLA were similar between the coinfected patients and patients with Leishmania monoinfection. However, the median CM and EM CD4+T-cell counts from coinfected patients were significantly lower (p < 0.05). The reduction in the lymphoproliferative response to Leishmania antigens coincides with the decrease in the absolute numbers of both EM and CM CD4+T-cells in response to Leishmania antigens in patients coinfected with HIV/Leishmania.     

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 naive 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.     

Negative regulation of IL-17 production by OX40/OX40L interaction

The T-cell cytokine IL-17 is implicated in multiple inflammatory diseases through its induction of several pro-inflammatory cytokines and chemokines in a broad range of cell targets. Production of IL-17 defines the Th17 subset of helper T-cells associated with protection against microorganisms, a profile best characterized in the murine system. Multiple regulators of Th17 cell differentiation and IL-17 production are reported, but the impact of OX40L is not described. OX40 ligand (OX40L) is an early-stage activator of T-cells through its interaction with CD134 (OX40) that is up-regulated on antigen challenged T-cells. Here, we show that OX40L suppresses IL-17 production by PHA-stimulated human PBMC and purified CD4 and CD8 cells. In agreement with prior reports, OX40L signaling through CD134 increased IFNgamma and IL-4, both of which are reported to inhibit the production of IL-17. OX40L suppression of IL-17 was completely reversed by a neutralizing IFNgamma antibody while there was no effect with a neutralizing IL-4 antibody. Moreover, OX40L also suppressed IL-17 in the presence of IL-23, an established inducer of IL-17 and differentiation factor for Th17 cells. Presuming mediation by IFNgamma, we evaluated expression of this cytokine in the presence of OX40L and IL-23. Surprisingly, IL-23 also induced IFNgamma by PHA-stimulated T-cells and this effect was enhanced in the presence of OX40L. Addition of the IFNgamma antibody not only reversed the OX40L suppression of IL-17 in the presence of IL-23, it markedly enhanced the level of IL-17. These results further establish IFNgamma as a primary modulator of IL-17 production in the human cells, much as in the murine system.