Alternaria-Induced Release of IL-18 from Damaged Airway Epithelial Cells: An NF-κB Dependent Mechanism of Th2 Differentiation?

Background

A series of epidemiologic studies have identified the fungus Alternaria as a major risk factor for asthma. The airway epithelium plays a critical role in the pathogenesis of allergic asthma. These reports suggest that activated airway epithelial cells can produce cytokines such as IL-25, TSLP and IL-33 that induce Th2 phenotype. However the epithelium-derived products that mediate the pro-asthma effects of Alternaria are not well characterized. We hypothesized that exposure of the airway epithelium to Alternaria releasing cytokines that can induce Th2 differentiation.

Methodology/Principal Finding

We used ELISA to measure human and mouse cytokines. Alternaria extract (ALT-E) induced rapid release of IL-18, but not IL-4, IL-9, IL-13, IL-25, IL-33, or TSLP from cultured normal human bronchial epithelial cells; and in the BAL fluids of naïve mice after challenge with ALT-E. Both microscopic and FACS indicated that this release was associated with necrosis of epithelial cells. ALT-E induced much greater IL-18 release compared to 19 major outdoor allergens. Culture of naïve CD4 cells with rmIL-18 induced Th2 differentiation in the absence of IL-4 and STAT6, and this effect was abrogated by disrupting NF- κB p50 or with a NEMO binding peptide inhibitor.

Conclusion/Significance

Rapid and specific release of IL-18 from Alternaria-exposed damaged airway epithelial cells can directly initiate Th2 differentiation of naïve CD4⁺ T-cells via a unique NF-κB dependent pathway.     

The anti-CD74 humanized monoclonal antibody, milatuzumab, which targets the invariant chain of MHC II complexes, alters B-cell proliferation, migration, and adhesion molecule expression

Introduction

Targeting CD74 as the invariant chain of major histocompatibility complexes (MHC) became possible by the availability of a specific humanized monoclonal antibody, milatuzumab, which is under investigation in patients with hematological neoplasms. CD74 has been reported to regulate chemo-attractant migration of macrophages and dendritic cells, while the role of CD74 on peripheral naïve and memory B cells also expressing CD74 remains unknown. Therefore, the current study addressed the influence of milatuzumab on B-cell proliferation, chemo-attractant migration, and adhesion molecule expression.

Methods

Surface expression of CD74 on CD27^- naïve and CD27⁺ memory B cells as well as other peripheral blood mononuclear cells (PBMCs) obtained from normals, including the co-expression of CD44, CXCR4, and the adhesion molecules CD62L, β7-integrin, β1-integrin and CD9 were studied after binding of milatuzumab using multicolor flow cytometry. The influence of the antibody on B-cell proliferation and migration was analyzed in vitro in detail.

Results

In addition to monocytes, milatuzumab also specifically bound to human peripheral B cells, with a higher intensity on CD27⁺ memory versus CD27^- naïve B cells. The antibody reduced B-cell proliferation significantly but moderately, induced enhanced spontaneous and CXCL12-dependent migration together with changes in the expression of adhesion molecules, CD44, β7-integrin and CD62L, mainly of CD27^- naïve B cells. This was independent of macrophage migration-inhibitory factor as a ligand of CD74/CD44 complexes.

Conclusions

Milatuzumab leads to modestly reduced proliferation, alterations in migration, and adhesion molecule expression preferentially of CD27^- naïve B cells. It thus may be a candidate antibody for the autoimmune disease therapy by modifying B cell functions.     

Restoration of regulatory and effector T cell balance and B cell homeostasis in systemic lupus erythematosus patients through vitamin D supplementation

Introduction

Systemic lupus erythematosus (SLE) is a T and B cell-dependent autoimmune disease characterized by the appearance of autoantibodies, a global regulatory T cells (Tregs) depletion and an increase in Th17 cells. Recent studies have shown the multifaceted immunomodulatory effects of vitamin D, notably the expansion of Tregs and the decrease of Th1 and Th17 cells. A significant correlation between higher disease activity and lower serum 25-hydroxyvitamin D levels [25(OH)D] was also shown.

Methods

In this prospective study, we evaluated the safety and the immunological effects of vitamin D supplementation (100 000 IU of cholecalciferol per week for 4 weeks, followed by 100 000 IU of cholecalciferol per month for 6 months.) in 20 SLE patients with hypovitaminosis D.

Results

Serum 25(OH)D levels dramatically increased under vitamin D supplementation from 18.7±6.7 at day 0 to 51.4±14.1 (p<0.001) at 2 months and 41.5±10.1 ng/mL (p<0.001) at 6 months. Vitamin D was well tolerated and induced a preferential increase of naïve CD4⁺ T cells, an increase of regulatory T cells and a decrease of effector Th1 and Th17 cells. Vitamin D also induced a decrease of memory B cells and anti-DNA antibodies. No modification of the prednisone dosage or initiation of new immunosuppressant agents was needed in all patients. We did not observe SLE flare during the 6 months follow-up period.

Conclusions

This preliminary study suggests the beneficial role of vitamin D in SLE patients and needs to be confirmed in randomized controlled trials.     

Apoptosis of purified CD4+ T cell subsets is dominated by cytokine deprivation and absence of other cells in new onset diabetic NOD mice

Background

Regulatory T cells (Treg) play a significant role in immune homeostasis and self-tolerance. Excessive sensitivity of isolated Treg to apoptosis has been demonstrated in NOD mice and humans suffering of type 1 diabetes, suggesting a possible role in the immune dysfunction that underlies autoimmune insulitis. In this study the sensitivity to apoptosis was measured in T cells from new onset diabetic NOD females, comparing purified subsets to mixed cultures.

Principal Findings

Apoptotic cells are short lived in vivo and death occurs primarily during isolation, manipulation and culture. Excessive susceptibility of CD25⁺ T cells to spontaneous apoptosis is characteristic of isolated subsets, however disappears when death is measured in mixed splenocyte cultures. In variance, CD25⁻ T cells display balanced sensitivity to apoptosis under both conditions. The isolation procedure removes soluble factors, IL-2 playing a significant role in sustaining Treg viability. In addition, pro- and anti-apoptotic signals are transduced by cell-to-cell interactions: CD3 and CD28 protect CD25⁺ T cells from apoptosis, and in parallel sensitize naïve effector cells to apoptosis. Treg viability is modulated both by other T cells and other subsets within mixed splenocyte cultures. Variations in sensitivity to apoptosis are often hindered by fast proliferation of viable cells, therefore cycling rates are mandatory to adequate interpretation of cell death assays.

Conclusions

The sensitivity of purified Treg to apoptosis is dominated by cytokine deprivation and absence of cell-to-cell interactions, and deviate significantly from measurements in mixed populations. Balanced sensitivity of naïve/effector and regulatory T cells to apoptosis in NOD mice argues against the concept that differential susceptibility affects disease evolution and progression.     

Interleukin-19: a constituent of the regulome that controls antigen presenting cells in the lungs and airway responses to microbial products

Background

Interleukin (IL)-19 has been reported to enhance chronic inflammatory diseases such as asthma but the in vivo mechanism is incompletely understood. Because IL-19 is produced by and regulates cells of the monocyte lineage, our studies focused on in vivo responses of CD11c positive (CD11c+) alveolar macrophages and lung dendritic cells.

Methodology/Principal Findings

IL-19-deficient (IL-19-/-) mice were studied at baseline (naïve) and following intranasal challenge with microbial products, or recombinant cytokines. Naïve IL-19-/- mixed background mice had a decreased percentage of CD11c+ cells in the bronchoalveolar-lavage (BAL) due to the deficiency in IL-19 and a trait inherited from the 129-mouse strain. BAL CD11c+ cells from fully backcrossed IL-19-/- BALB/c or C57BL/6 mice expressed significantly less Major Histocompatibility Complex class II (MHCII) in response to intranasal administration of lipopolysaccharide, Aspergillus antigen, or IL-13, a pro-allergic cytokine. Neurogenic-locus-notch-homolog-protein-2 (Notch2) expression by lung monocytes, the precursors of BAL CD11c+ cells, was dysregulated: extracellular Notch2 was significantly decreased, transmembrane/intracellular Notch2 was significantly increased in IL-19-/- mice relative to wild type. Instillation of recombinant IL-19 increased extracellular Notch2 expression and dendritic cells cultured from bone marrow cells in the presence of IL-19 showed upregulated extracellular Notch2. The CD205 positive subset among the CD11c+ cells was 3-5-fold decreased in the airways and lungs of naïve IL-19-/- mice relative to wild type. Airway inflammation and histological changes in the lungs were ameliorated in IL-19-/- mice challenged with Aspergillus antigen that induces T lymphocyte-dependent allergic inflammation but not in IL-19-/- mice challenged with lipopolysaccharide or IL-13.

Conclusions/Significance

Because MHCII is the molecular platform that displays peptides to T lymphocytes and Notch2 determines cell fate decisions, our studies suggest that endogenous IL-19 is a constituent of the regulome that controls both processes in vivo.     

Effector and naturally occurring regulatory T cells display no abnormalities in activation induced cell death in NOD mice

Background

Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff) and regulatory T cells (Treg) to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice.

Principal Findings

Both effector (CD25⁻, FoxP3⁻) and suppressor (CD25⁺, FoxP3⁺) CD4⁺ T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP tranegenes. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL) in both strains. The effector and suppressor CD4⁺ subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4⁺CD25⁻ T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice.

Conclusion

These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis.     

TSC1/2 Signaling Complex Is Essential for Peripheral Na?ve CD8+ T Cell Survival and Homeostasis in Mice

The PI3K-Akt-mTOR pathway plays crucial roles in regulating both innate and adaptive immunity. However, the role of TSC1, a critical negative regulator of mTOR, in peripheral T cell homeostasis remains elusive. With T cell-specific Tsc1 conditional knockout (Tsc1 KO) mice, we found that peripheral naïve CD8⁺ T cells but not CD4⁺ T cells were severely reduced. Tsc1 KO naïve CD8⁺ T cells showed profound survival defect in an adoptive transfer model and in culture with either stimulation of IL-7 or IL-15, despite comparable CD122 and CD127 expression between control and KO CD8⁺ T cells. IL-7 stimulated phosphorylation of Akt(S473) was diminished in Tsc1 KO naïve CD8⁺T cells due to hyperactive mTOR-mediated feedback suppression on PI3K-AKT signaling. Furthermore, impaired Foxo1/Foxo3a phosphorylation and increased pro-apoptotic Bim expression in Tsc1 KO naïve CD8⁺T cells were observed upon stimulation of IL-7. Collectively, our study suggests that TSC1 plays an essential role in regulating peripheral naïve CD8⁺ T cell homeostasis, possible via an mTOR-Akt-FoxO-Bim signaling pathway.     

In vivo expansion of naïve CD4+ CD25(high) FOXP3+ regulatory T cells in patients with colorectal carcinoma after IL-2 administration

Regulatory T cells (T_reg cells) are increased in context of malignancies and their expansion can be correlated with higher disease burden and decreased survival. Initially, interleukin 2 (IL-2) has been used as T-cell growth factor in clinical vaccination trials. In murine models, however, a role of IL-2 in development, differentiation, homeostasis, and function of T_reg cells was established. In IL-2 treated cancer patients a further T_reg-cell expansion was described, yet, the mechanism of expansion is still elusive. Here we report that functional T_reg cells of a naïve phenotype - as determined by CCR7 and CD45RA expression - are significantly expanded in colorectal cancer patients. Treatment of 15 UICC stage IV colorectal cancer patients with IL-2 in a phase I/II peptide vaccination trial further enlarges the already increased naïve T_reg-cell pool. Higher frequencies of T-cell receptor excision circles in naïve T_reg cells indicate IL-2 dependent thymic generation of naïve T_reg cells as a mechanism leading to increased frequencies of T_reg cells post IL-2 treatment in cancer patients. This finding could be confirmed in naïve murine T_reg cells after IL-2 administration. These results point to a more complex regulation of T_reg cells in context of IL-2 administration. Future strategies therefore might aim at combining IL-2 therapy with novel strategies to circumvent expansion and differentiation of naïve T_reg cells.     

Systemic immune activation in HIV infection is associated with decreased MDC responsiveness to TLR ligand and inability to activate naive CD4 T-cells

Background

HIV infection is characterized by ineffective anti-viral T-cell responses and impaired dendritic cell (DC) functions, including response to Toll-Like Receptor (TLR) ligands. Because TLR responsiveness may affect a host''s response to virus, we examined TLR ligand induced Myeloid and Plasmacytoid DC (MDC and PDC) activation of naïve T-cells in HIV+ subjects.

Methods

Freshly purified MDC and PDC obtained from HIV+ subjects and healthy controls were cultured in the presence and absence of TLR ligands (poly I∶C or R-848). We evaluated indices of maturation/activation (CD83, CD86, and HLA-DR expression), cytokine secretion (IFN-alpha and IL-6), and ability to activate allogeneic naïve CD4 T-cells to secrete IFN-gamma and IL-2.

Results

MDC from HIV+ subjects had increased spontaneous IL-6 production and increased CD83 and CD86 expression when compared to MDC of controls. MDC IL-6 expression was associated with plasma HIV level. At the same time, poly I∶C induced HLA-DR up-regulation on MDC was reduced in HIV+ persons when compared to controls. The latter finding was associated with impaired ability of MDC from HIV+ subjects to activate allogeneic naïve CD4 T-cells. PDC from HIV+ persons had increased spontaneous and TLR ligand induced IL-6 expression, and increased HLA-DR expression at baseline. The latter was associated with an intact ability of HIV PDC to activate allogeneic naïve CD4 T-cells.

Conclusion

These results have implications for the ability of the HIV+ host to form innate and adaptive responses to HIV and other pathogens.     

Signatures of T cells as correlates of immunity to Francisella tularensis

Tularemia or vaccination with the live vaccine strain (LVS) of Francisella tularensis confers long-lived cell-mediated immunity. We hypothesized that this immunity depends on polyfunctional memory T cells, i.e., CD4⁺ and/or CD8⁺ T cells with the capability to simultaneously express several functional markers. Multiparametric flow cytometry, measurement of secreted cytokines, and analysis of lymphocyte proliferation were used to characterize in vitro recall responses of peripheral blood mononuclear cells (PBMC) to killed F. tularensis antigens from the LVS or Schu S4 strains. PBMC responses were compared between individuals who had contracted tularemia, had been vaccinated, or had not been exposed to F. tularensis (naïve). Significant differences were detected between either of the immune donor groups and naïve individuals for secreted levels of IL-5, IL-6, IL-10, IL-12, IL-13, IFN-γ, MCP-1, and MIP-1β. Expression of IFN-γ, MIP-1β, and CD107a by CD4⁺CD45RO⁺ or CD8⁺CD45RO⁺ T cells correlated to antigen concentrations. In particular, IFN-γ and MIP-1β strongly discriminated between immune and naïve individuals. Only one cytokine, IL-6, discriminated between the two groups of immune individuals. Notably, IL-2- or TNF-α-secretion was low. Our results identify functional signatures of T cells that may serve as correlates of immunity and protection against F. tularensis.     

Autoregulatory function of interleukin-10-producing pre-na?ve B cells is defective in systemic lupus erythematosus

IntroductionPre-naïve B cells represent an intermediate stage in human B-cell development with some functions of mature cells, but their involvement in immune responses is unknown. The aim of this study was to determine the functional role of normal pre-naïve B cells during immune responses and possible abnormalities in systemic lupus erythematosus (SLE) that might contribute to disease pathogenesis.MethodsPre-naïve, naïve, and memory B cells from healthy individuals and SLE patients were stimulated through CD40 and were analyzed for interleukin-10 (IL-10) production and co-stimulatory molecule expression and their regulation of T-cell activation. Autoreactivity of antibodies produced by pre-naïve B cells was tested by measuring immunoglobulin M (IgM) autoantibodies in culture supernatants after differentiation.ResultsCD40-stimulated pre-naïve B cells produce larger amounts of IL-10 but did not suppress CD4⁺ T-cell cytokine production. Activated pre-naïve B cells demonstrated IL-10-mediated ineffective promotion of CD4⁺ T-cell proliferation and induction of CD4⁺FoxP3⁺ T cells and IL-10 independent impairment of co-stimulatory molecule expression and tumor necrosis factor-alpha (TNF-α) and IL-6 production. IgM antibodies produced by differentiated pre-naïve B cells were reactive to single-stranded deoxyribonucleic acid. SLE pre-naïve B cells were defective in producing IL-10, and co-stimulatory molecule expression was enhanced, resulting in promotion of robust CD4⁺ T-cell proliferation.ConclusionsThere is an inherent and IL-10-mediated mechanism that limits the capacity of normal pre-naïve B cells from participating in cellular immune response, but these cells can differentiate into autoantibody-secreting plasma cells. In SLE, defects in IL-10 secretion permit pre-naïve B cells to promote CD4⁺ T-cell activation and may thereby enhance the development of autoimmunity.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0687-1) contains supplementary material, which is available to authorized users.     

TGF-β Sensitivity Restrains CD8+ T Cell Homeostatic Proliferation by Enforcing Sensitivity to IL-7 and IL-15

The pleiotropic cytokine TGF-β has been implicated in the regulation of numerous aspects of the immune response, including naïve T cell homeostasis. Previous studies found that impairing TGF-β responsiveness (through expression of a dominant-negative TGF-β RII [DNRII] transgene) leads to accumulation of memory phenotype CD8 T cells, and it was proposed that this resulted from enhanced IL-15 sensitivity. Here we show naïve DNRII CD8 T cells exhibit enhanced lymphopenia-driven proliferation and generation of “homeostatic” memory cells. However, this enhanced response occurred in the absence of IL-15 and, unexpectedly, even in the combined absence of IL-7 and IL-15, which were thought essential for CD8 T cell homeostatic expansion. DNRII transgenic CD8 T cells still require access to self Class I MHC for homeostatic proliferation, arguing against generalized dysregulation of homeostatic cues. These findings suggest TGF-β responsiveness is critical for enforcing sensitivity to homeostatic cytokines that limit maintenance and composition of the CD8 T cell pool. (154 words).     

Lymphoid Tissue Damage in HIV-1 Infection Depletes Na?ve T Cells and Limits T Cell Reconstitution after Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can suppress HIV-1 replication and normalize the chronic immune activation associated with infection, but restoration of naïve CD4⁺ T cell populations is slow and usually incomplete for reasons that have yet to be determined. We tested the hypothesis that damage to the lymphoid tissue (LT) fibroblastic reticular cell (FRC) network contributes to naïve T cell loss in HIV-1 infection by restricting access to critical factors required for T cell survival. We show that collagen deposition and progressive loss of the FRC network in LTs prior to treatment restrict both access to and a major source of the survival factor interleukin-7 (IL-7). As a consequence, apoptosis within naïve T cell populations increases significantly, resulting in progressive depletion of both naïve CD4⁺ and CD8⁺ T cell populations. We further show that the extent of loss of the FRC network and collagen deposition predict the extent of restoration of the naïve T cell population after 6 month of HAART, and that restoration of FRC networks correlates with the stage of disease at which the therapy is initiated. Because restoration of the FRC network and reconstitution of naïve T cell populations are only optimal when therapy is initiated in the early/acute stage of infection, our findings strongly suggest that HAART should be initiated as soon as possible. Moreover, our findings also point to the potential use of adjunctive anti-fibrotic therapies to avert or moderate the pathological consequences of LT fibrosis, thereby improving immune reconstitution.     

Interleukin-7 enhances proliferation responses to T-cell receptor stimulation in naïve CD4+ T cells from human immunodeficiency virus-infected persons

Proliferation responses of naïve CD4⁺ T cells to T-cell receptor and interleukin-7 (IL-7) stimulation were evaluated by using cells from human immunodeficiency virus-positive (HIV⁺) donors. IL-7 enhanced responses to T-cell receptor stimulation, and the magnitude of this enhancement was similar in cells from healthy controls and from HIV⁺ subjects. The overall response to T-cell receptor stimulation alone or in combination with IL-7, however, was diminished among viremic HIV⁺ donors and occurred independent of antigen-presenting cells. Frequencies of CD127⁺ cells were related to the magnitudes of proliferation enhancement that were mediated by IL-7. Thus, IL-7 enhances but does not fully restore the function of naïve CD4⁺ T cells from HIV-infected persons.Interleukin-7 (IL-7) plays an important role in T-cell homeostasis by modulating thymic output (1, 16, 22) and by enhancing the peripheral expansion and survival of both naïve and memory T-cell subsets (12, 18, 20, 25, 26, 31, 32). Under normal circumstances, the homeostatic maintenance of naïve CD4⁺ T cells is regulated by at least two types of signals that include T-cell receptor (TCR) engagement and IL-7 (10, 26, 30). In addition, IL-7 may play an important role in the conversion of effector T cells into long-term memory cells (12, 14).Homeostasis of T cells is dysregulated in human immunodeficiency virus (HIV) infection such that there is a marked depletion of CD4⁺ cells and a progressive loss of naïve CD4 and CD8⁺ T cells (24). Although the mechanisms for these deficiencies are not fully understood, it is possible that impairments in T-cell proliferation and responsiveness to immunomodulatory cytokines could play a role. In HIV disease, IL-7 is increased in plasma (2, 5, 11, 15, 19, 21, 23) and the alpha chain of the IL-7 receptor, CD127, is less frequently expressed among T lymphocytes (2, 5, 11, 21, 23). The ability of patient T cells to respond to IL-7 stimulation may be diminished in HIV disease but may not be related to the density of CD127 expression as it is in T cells from healthy controls (4). Moreover, the responsiveness of T cells, including naïve CD4⁺ lymphocytes, to TCR stimulation is diminished in HIV disease (27-29). Thus, defects in responsiveness to cytokines or TCR stimulation could contribute to the perturbations in T-cell proliferation and survival in HIV disease.In these studies, we examined the responsiveness of naïve CD4⁺ T cells from viremic HIV-positive (HIV⁺) donors (median plasma HIV RNA level, 25,200 copies/ml [range, 1,015 to 1,000,000 copies/ml]; median CD4 cell count, 429 cells/μl [range, 41 to 950 cells/μl]; median age, 38 years [range, 22 to 64 years]; n = 25) and aviremic, highly active antiretroviral therapy (HAART)-treated HIV⁺ donors (plasma HIV RNA level, <400 copies/ml; median CD4 cell count, 309 cells/μl [range, 74 to 918 cells/μl]; median age, 48 years [range, 37 to 55 years]; n = 12) to the combined stimulus of recombinant IL-7 (Cytheris) plus agonistic anti-CD3 antibody. Peripheral blood mononuclear cells (PBMC) were depleted of CD45RO⁺ cells by magnetic bead depletion (>90% purity) and were incubated in medium alone or were stimulated with anti-CD3 antibody, IL-7, or anti-CD3 antibody plus IL-7. CD4⁺CD45RO⁻CD28⁺CD27⁺ cells were assessed for the expression of Ki67 2 days poststimulation by flow cytometric analyses. The addition of IL-7 to anti-CD3 antibody enhanced the induction of Ki67 expression in cells from both HIV⁺ and HIV-negative (HIV⁻) donors (Fig. ​(Fig.11 and Fig. ​Fig.2).2). A diminished response to anti-CD3 antibody was observed among naïve CD4⁺ T cells from viremic HIV⁺ donors. In contrast, cells from aviremic HIV⁺ donors (all receiving antiretroviral therapy) had normal responses to anti-CD3 antibody compared to cells from healthy donors (Fig. ​(Fig.2).2). Importantly, the addition of IL-7 to the cultures significantly improved the responses to above those observed with anti-CD3 alone in HIV⁻ and HIV⁺ donors, regardless of viremia (Wilcoxon signed ranks test; for each comparison, P was <0.04), and the magnitude of that enhancement, although slightly diminished in cells from HIV⁺ donors, was not significantly different between groups of subjects when measured as either the enhancement (n-fold; not shown) or as the change in percent Ki67⁺ cells above the background observed for cells stimulated with anti-CD3 alone (Fig. ​(Fig.3).3). Although IL-7 enhanced responses to TCR stimulation in HIV⁻ subjects, the overall magnitude of the responses among cells from HIV viremic subjects did not reach the levels seen with cells from healthy donors, even in the presence of IL-7 (Fig. ​(Fig.2).2). It should be noted, however, that these functional readouts were not related to clinical indices of plasma HIV RNA level, CD4 cell count, or age when considered as continuous variables, suggesting that the functional perturbations in naïve CD4⁺ T cells are probably undermined by complexities extending beyond HIV replication (not shown). Together, these results suggest that TCR responsiveness is diminished in naïve CD4⁺ T cells from viremic HIV⁺ subjects, whereas responsiveness to IL-7 stimulation is relatively preserved.Open in a separate windowFIG. 1.IL-7 enhances the induction of Ki67 expression in naïve CD4⁺ T cells from healthy controls and HIV⁺ donors. CD45RO-depleted PBMC were incubated with anti-CD3 antibody (100 ng/ml), IL-7 (50 ng/ml), anti-CD3 antibody plus IL-7, or medium alone (RPMI with 10% fetal bovine serum). Cells were gated on CD4⁺CD27⁺CD28⁺ lymphocytes and examined for Ki67 expression by intracellular flow cytometry.Open in a separate windowFIG. 2.IL-7 responsiveness in cells from viremic and aviremic HIV⁺ donors. Plotted values represent the percentages of CD4⁺CD27⁺CD28⁺CD45RO⁻ T cells that expressed Ki67 after a 2-day incubation with anti-CD3 or with anti-CD3 plus IL-7. Percentages of Ki67⁺ cells in cultures without stimulation or with IL-7 only were subtracted from the values shown. Responses of cells from healthy controls (n = 9), HIV⁺ subjects with plasma HIV RNA levels of >400 copies/ml (n = 25), and HIV⁺ subjects on HAART with suppressed viral replication (<400 copies/ml; n = 12) are shown. Statistically significant differences between cells from controls and HIV⁺ donors are indicated. Analyses included Kruskal-Wallis test (P = 0.002) for multigroup comparisons and Mann-Whitney U test for comparison of two groups (*, P < 0.05).Open in a separate windowFIG. 3.IL-7 enhances responses to anti-CD3 antibody stimulation to a similar degree in cells from HIV⁺ and HIV⁻ donors. Naïve CD4⁺ T cells were incubated with IL-7, anti-CD3, anti-CD3 plus IL-7, or medium alone for 2 days. Background division (percent Ki67⁺ cells) in medium alone or IL-7 alone was first subtracted from the responses observed with cells stimulated with anti-CD3 alone or with anti-CD3 plus IL-7, respectively. The magnitude of IL-7 enhancement was then calculated by subtracting the percentage of naïve CD4⁺ cells that expressed Ki67⁺ after anti-CD3 antibody stimulation from the percentage of naïve CD4⁺ cells that expressed Ki67 after stimulation with anti-CD3 plus IL-7. n = 9, 25, and 12 for healthy controls, viremic subjects, and aviremic subjects, respectively.Previous studies indicate that the frequency of CD127⁺ T cells, particularly memory T-cell subsets, is reduced in patients with HIV disease (5, 11, 21, 23). This could, in part, result from the modulation of receptor expression through increased exposure to IL-7 in vivo and also may reflect accumulation of CD127⁻ effector memory cells (21). We assessed the expression of CD127 in naïve CD4⁺CD45RA⁺CD28⁺CD27⁺ and memory CD4⁺CD45RO⁺ T cells in a subset of patients and asked if the frequencies of CD127⁺ cells were related to the induction of Ki67 expression by anti-CD3 or by anti-CD3 plus IL-7 among naïve CD4⁺ T cells. We reasoned that the ability of IL-7 to enhance responses to TCR stimulation might be limited if CD127 expression was diminished among naïve CD4⁺ T cells from HIV⁺ donors. Alternatively, a defect in functional responses also could be related to increased exposure to IL-7 in vivo, as may be reflected by the absence of CD127 receptor expression on memory T-cell subsets.In agreement with previous studies, our results suggest that CD127 expression is relatively preserved in naïve CD4⁺ T cells from HIV⁺ donors (representative histograms in Fig. ​Fig.4)4) (mean percentage of CD127⁺ cells, 87 and 83 for HIV⁻ donors [n = 5] and HIV⁺ donors [n = 17], respectively; P = 0.96) but is diminished in memory CD4⁺ T cells from HIV⁺ donors (mean percentage of CD127⁺ cells, 83 and 59 for HIV⁻ and HIV⁺ donors, respectively; P = 0.01). The frequencies of CD127⁺ naïve T cells were directly related to the frequencies of CD127⁺ memory T cells (Spearman''s correlations; r = 0.711, P = 0.001; n = 18) in HIV⁺ subjects. This result suggests that a similar mechanism modulates the expression of CD127 in these T-cell subsets, even though the loss of CD127 expression is clearly greater among the memory T cells in HIV disease. Neither CD127 expression among naïve CD4⁺ T cells nor CD127 expression among memory CD4⁺ T cells was related to the functional response of naïve CD4⁺ T cells to anti-CD3 (r = 0.238 and P = 0.36 for naïve CD127 expression; r = 0.293 and P = 0.25 for memory CD127 expression) or to anti-CD3 plus IL-7 (r = 0.32 and P = 0.21 for naïve CD127 expression; r = 0.31 and P = 0.22 for memory CD127 expression). There was a relationship between the percentage of CD127⁺ naïve T cells and the delta Ki67 expression that resulted from the addition of IL-7 to anti-CD3-treated cultures (percentage of Ki67⁺ cells in cultures treated with anti-CD3 plus IL-7 minus the percentage of Ki67⁺ cells in cultures treated with anti-CD3 alone) (Fig. ​(Fig.4).4). This relationship was statistically significant by Pearson''s correlation (r = 0.5, P = 0.041), the use of which was justified based on the normal distribution of the data. Spearman''s analysis, which is independent of data distribution, indicated a similar trend that was not statistically significant (r = 0.41, P = 0.1). The mean fluorescence intensity of CD127 expression on CD4⁺CD45RA⁺CD27⁺CD28⁺ T cells was not significantly related to the delta Ki67 expression induced by IL-7 but also suggested a trend consistent with a direct relationship between these indices (r = 0.45 and P = 0.07 by Pearson''s correlation; r = 0.34 and P = 0.18 by Spearman''s correlation). Despite the relative preservation of IL-7 receptor in naïve CD4⁺ T cells from HIV⁺ donors, the association between the frequencies of CD127⁺ cells and CD4⁺ T-cell proliferation responses to TCR plus IL-7 suggests that subtle IL-7 receptor perturbations might contribute to functional defects of naïve CD4⁺ T cells in HIV-infected persons.Open in a separate windowFIG. 4.CD127 receptor expression is related to enhancement of proliferation by IL-7. (A) Whole blood from a healthy control and an HIV-infected person was examined by flow cytometry for expression of CD127 on CD4⁺CD45RA⁺CD27⁺CD28⁺ (naïve) T cells. The gating strategy for identifying naïve cells involved an initial gate for lymphocyte forward and side scatter (SSC) characteristics (not shown) and then sequential gates for CD4 positive, CD45RA positive and, finally, CD28⁺CD27⁺ double-positive cells. (B) Plotted values indicating the relationship between the delta Ki67 expression in naïve CD4⁺ T cells and the percentage of CD127⁺ naïve T cells that was determined by using freshly isolated whole blood. The delta Ki67 expression was calculated by subtracting the percentage of naïve CD4⁺ cells that expressed Ki67⁺ after anti-CD3 antibody stimulation from the percentage of naïve CD4⁺ cells that expressed Ki67 after stimulation with anti-CD3 plus IL-7.To consider the possibility that antigen-presenting cells could contribute to the diminished response of T-cells to stimulation with TCR plus IL-7, we next asked if defects in TCR-plus-IL-7 stimulation could be detected in purified naïve CD4⁺ T-cell populations. CD4⁺CD45RO⁻ cells were negatively selected by magnetic bead depletion, achieving a purity of >90% as determined by flow cytometric analyses. Purified naïve CD4⁺ T cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) tracking dye and incubated with IL-7, anti-CD3 antibody that was immobilized on a plate, anti-CD3 plus IL-7, or medium alone. The induction of proliferation was measured 7 days later by the dilution of CFSE tracking dye among CD4⁺CD27⁺ cells by calculating the division index (average number of cell divisions of all CD4⁺CD27⁺ cells) and the proliferation index (average number of divisions of CD4⁺CD27⁺ cells that had diluted tracking dye; Flow-Jo analysis software). These purified CD4⁺ T cells proliferated poorly in response to anti-CD3 antibody stimulation alone, providing functional evidence that the samples were free of antigen-presenting cell contamination (Fig. ​(Fig.5A).5A). The combined treatment of anti-CD3 and IL-7 induced cellular expansion, whereas alone, neither stimulus induced cellular proliferation during the 7-day period (Fig. ​(Fig.5A).5A). Responses of cells from HIV⁺ donors were reduced compared to those of cells from healthy donors, confirming that the defects in naïve CD4⁺ T-cell expansion are independent of antigen-presenting cells and not fully corrected by IL-7 (Fig. ​(Fig.5B5B).Open in a separate windowFIG. 5.Diminished responses to TCR plus IL-7 in purified naïve CD4⁺ T cells from HIV⁺ donors. CD4⁺CD45RO⁻ cells were purified from PBMC by negative selection. Cells from HIV⁺ donors (n = 7) and healthy controls (n = 7) were labeled with CSFE and incubated with anti-CD3 immobilized on a plate (5 μg/ml, overnight at 4°C) plus IL-7 (10 ng/ml). CFSE dye dilution was measured among the CD4⁺CD27⁺ cells. (A) Representative histograms showing the dilution of CFSE and CD27 expression among cells incubated with anti-CD3 antibody alone, IL-7 alone, or the combination of anti-CD3 plus IL-7. Placements of quadrant gates were based on an isotype control antibody stain (for CD27 expression) and on cells that had been incubated in medium alone (for CFSE dye dilution). (B) Division indices (average number of cell divisions among CD4⁺CD27⁺ cells) and proliferation indices (average number of cell divisions among CD4⁺CD27⁺ cells that had diluted tracking dye) are shown.IL-7 is a promising candidate for therapeutic and vaccine adjuvant applications in HIV disease. This cytokine may be especially beneficial in circumstances of immune reconstitution, since it normally plays an essential role in T-cell proliferation and survival. Here, we demonstrate that IL-7 efficiently enhances TCR-triggered naïve CD4⁺ T-cell expansion in cells from healthy individuals and from HIV⁺ donors. The mechanism of IL-7 activity is not discerned in these experiments but may involve effects on survival, such as the induction of Bcl-2 (9), or may involve the enhancement of IL-2 or IL-2 receptor expression (6, 8). In any case, our studies provide evidence that IL-7 should provide an effective therapy for the regulation of naïve CD4⁺ T-cell homeostasis and may be useful for vaccine adjuvant applications in HIV disease. The potential of this approach has been illustrated by recent human trials of IL-7 that demonstrated the expansion of naïve T cells in vivo after IL-7 administration to HIV-infected persons (13) and by animal studies, wherein IL-7 administration enhanced T-cell responses to immunization in mice (17).Notably, the depletion studies and purification methods employed here did not necessarily eliminate terminally differentiated effector memory CD4⁺ T cells from our cultures; however, studies of CMV-specific terminally differentiated cells suggested that these cells are primarily CD27⁻ (3), and the use of three markers to identify naïve CD4⁺ T cells, including the ones used here (CD27, CD28, and CD45RO) is estimated to provide 98% assurance that the cells being examined are truly naïve (7). Thus, it is likely that terminally differentiated cells were largely removed from our analyses.Our observations provide confirmation of a significant defect in the responses of naïve CD4⁺ T cells to TCR triggering in HIV disease, and this defect is not fully corrected by IL-7, as shown here, or by IL-2, as we demonstrated previously (27). These deficiencies are reproduced even among naïve CD4⁺ T cells that are purified from professional antigen-presenting cells, indicating that the defects are intrinsic to the T cells and not a consequence of dysfunctional antigen-presenting cells. We propose that functional defects in naïve CD4⁺ T cells from HIV⁺ donors stem primarily from deficiencies in TCR signaling. Further studies that define the nature of naïve CD4⁺ T-cell defects in HIV disease will be required to address the underlying mechanisms.     

Extended interferon-alpha therapy accelerates telomere length loss in human peripheral blood T lymphocytes

Background

Type I interferons have pleiotropic effects on host cells, including inhibiting telomerase in lymphocytes and antiviral activity. We tested the hypothesis that long-term interferon treatment would result in significant reduction in average telomere length in peripheral blood T lymphocytes.

Methods/Principal Findings

Using a flow cytometry-based telomere length assay on peripheral blood mononuclear cell samples from the Hepatitis-C Antiviral Long-term Treatment against Cirrhosis (HALT-C) study, we measured T cell telomere lengths at screening and at months 21 and 45 in 29 Hepatitis-C virus infected subjects. These subjects had failed to achieve a sustained virologic response following 24 weeks of pegylated-interferon-alpha plus ribavirin treatment and were subsequently randomized to either a no additional therapy group or a maintenance dose pegylated-IFNα group for an additional 3.5 years. Significant telomere loss in naïve T cells occurred in the first 21 months in the interferon-alpha group. Telomere losses were similar in both groups during the final two years. Expansion of CD8⁺CD45RA⁺CD57⁺ memory T cells and an inverse correlation of alanine aminotransferase levels with naïve CD8⁺ T cell telomere loss were observed in the control group but not in the interferon-alpha group. Telomere length at screening inversely correlated with Hepatitis-C viral load and body mass index.

Conclusions/Significance

Sustained interferon-alpha treatment increased telomere loss in naïve T cells, and inhibited the accumulation of T cell memory expansions. The durability of this effect and consequences for immune senescence need to be defined.     

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.     

Genome Reshuffling for Advanced Intercross Permutation (GRAIP): simulation and permutation for advanced intercross population analysis

Background

Advanced intercross lines (AIL) are segregating populations created using a multi-generation breeding protocol for fine mapping complex trait loci (QTL) in mice and other organisms. Applying QTL mapping methods for intercross and backcross populations, often followed by naïve permutation of individuals and phenotypes, does not account for the effect of AIL family structure in which final generations have been expanded and leads to inappropriately low significance thresholds. The critical problem with naïve mapping approaches in AIL populations is that the individual is not an exchangeable unit.

Methodology/Principal Findings

The effect of family structure has immediate implications for the optimal AIL creation (many crosses, few animals per cross, and population expansion before the final generation) and we discuss these and the utility of AIL populations for QTL fine mapping. We also describe Genome Reshuffling for Advanced Intercross Permutation, (GRAIP) a method for analyzing AIL data that accounts for family structure. GRAIP permutes a more interchangeable unit in the final generation crosses – the parental genome – and simulating regeneration of a permuted AIL population based on exchanged parental identities. GRAIP determines appropriate genome-wide significance thresholds and locus-specific P-values for AILs and other populations with similar family structures. We contrast GRAIP with naïve permutation using a large densely genotyped mouse AIL population (1333 individuals from 32 crosses). A naïve permutation using coat color as a model phenotype demonstrates high false-positive locus identification and uncertain significance levels, which are corrected using GRAIP. GRAIP also detects an established hippocampus weight locus and a new locus, Hipp9a.

Conclusions and Significance

GRAIP determines appropriate genome-wide significance thresholds and locus-specific P-values for AILs and other populations with similar family structures. The effect of family structure has immediate implications for the optimal AIL creation and we discuss these and the utility of AIL populations.     

Differential susceptibility of na?ve and activated human gammadelta T cells to activation-induced cell death by T-cell receptor cross-linking.

BACKGROUND: T cells undergo activation-induced cell death (AICD) through repeated stimulation of their T cell receptors (TCRs). Activated human gammadelta T cells were found to die by apoptosis when their TCRs were cross-linked by antibodies, whereas naïve gammadelta T cells freshly isolated from blood did not. Therefore, we investigated the factors that could contribute to this differential susceptibility. MATERIALS AND METHODS: Gammadelta T cells were isolated from the peripheral blood of healthy human volunteers and their TCRs were cross-linked either directly (naïve) or after an in vitro incubation of 11 days (activated). Their cell cycle profiles, cytokine, Fas and FasL mRNA messages, and surface expression of Fas and FasL were determined. RESULTS: The naïve cells were cycling while the activated T cells exited from the G1 to subG1 phase upon TCR cross-linking. IL-2 and IL-4 mRNAs and surface expression of FasL were detected only in activated T cells in the time period examined. In addition, cFLIP mRNA expression was found only in naïve gammadelta T cells and activated T cells treated with cyclosporin A (CsA), which inhibited AICD in the activated T cells. CsA also downregulated the surface expression of FasL in activated T cells. CONCLUSIONS: The differential expression of cytokines, apoptotic inducers and inhibitors provide the basis for the differential susceptibility of naïve and activated gammadelta T cells to AICD upon TCR cross-linking. This contributes to our understanding of the regulation and maintenance of gammadelta T-cell homeostasis, which would be important in many infectious as well as autoimmune diseases, where gammadelta T cells have been implicated.