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.     

Immune modulation through 4-1BB enhances SIV vaccine protection in non-human primates against SIVmac251 challenge

Costimulatory molecules play a central role in the development of cellular immunity. Understanding how costimulatory pathways can be directed to positively influence the immune response may be critical for the generation of an effective HIV vaccine. Here, we evaluated the ability of intravenous administration of a blocking monoclonal antibody (mAb) directed against the negative costimulatory molecule CTLA-4, and an agonist mAb directed against the positive costimulatory molecule 4-1BB, either alone or in combination, to augment intramuscular SIV DNA immunizations. We then tested the ability these of these responses to impact a high-dose SIVmac251 challenge. Following immunization, the groups infused with the anti-4-1BB mAb exhibited enhanced IFN-γ responses compared to the DNA vaccine only group. Interestingly, although CTLA-4 blockade alone did not enhance IFN-γ responses it did increase the proliferative capacity of the CD4⁺ and CD8⁺ T cells. The combination of both mAbs enhanced the magnitude of the polyfunctional CD8⁺ T cell response. Following challenge, the group that received both mAbs exhibited a significant, ∼2.0 log, decrease in plasma viral load compared to the naïve group the included complete suppression of viral load in some animals. Furthermore, the use of the CTLA-4 blocking antibody resulted in significantly higher viral loads during chronic infection compared to animals that received the 4-1BB mAb, likely due to the higher CD4⁺ T cell proliferative responses which were driven by this adjuvant following immunization. These novel studies show that these adjuvants induce differential modulation of immune responses, which have dramatically different consequences for control of SIV replication, suggesting important implications for HIV vaccine development.     

Age-related differences in naturally acquired T cell memory to Plasmodium falciparum merozoite surface protein 1

Naturally acquired immunity to Plasmodium falciparum malaria in malaria holoendemic areas is characterized by the gradual, age-related development of protection against high-density parasitemia and clinical malaria. Animal studies, and less commonly, observations of humans with malaria, suggest that T-cell responses are important in the development and maintenance of this immunity, which is mediated primarily by antibodies that slow repeated cycles of merozoites through erythrocytes. To advance our rather limited knowledge on human T-cell immunity to blood stage malaria infection, we evaluated CD4 and CD8 T-cell effector memory subset responses to the 42 kDa C-terminal fragment of Merozoite Surface Protein 1 (MSP1₄₂), a malaria vaccine candidate, by 49 healthy 0.5 to ≥18 year old residents of a holoendemic area in western Kenya. The proportion of individuals with peripheral blood mononuclear cell MSP1₄₂ driven IFN-γ ELISPOT responses increased from 20% (2/20) among 0.5–1 year old children to 90% (9/10) of adults ≥18 years (P = 0.01); parallel increases in the magnitude of IFN-γ responses were observed across all age groups (0.5, 1, 2, 5 and ≥18 years, P = 0.001). Less than 1% of total CD4 and CD8 T-cells from both children and adults produced IFN-γ in response to MSP1₄₂. However, adults had higher proportions of MSP1₄₂ driven IFN-γ secreting CD4 and CD8 effector memory (CD45RA⁻ CD62L⁻) T-cells than children (CD4: 50.9% vs. 28.8%, P = 0.036; CD8: 52.1% vs. 18.3%, respectively P = 0.009). In contrast, MSP1₄₂ driven IFN-γ secreting naïve-like, transitional (CD45RA⁺ CD62L⁺) CD4 and CD8 cells were the predominant T-cell subset among children with significantly fewer of these cells in adults (CD4: 34.9% vs. 5.1%, P = 0.002; CD8: 47.0% vs. 20.5%, respectively, P = 0.030). These data support the concept that meaningful age-related differences exist in the quality of T-cell immunity to malaria antigens such as MSP1.     

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.     

Signaling signatures and functional properties of anti-human CD28 superagonistic antibodies

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3-complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4⁺ T cells but not in cynomolgus T lymphocytes. The sustained Ca⁺⁺-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-γ and TNF-α. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-γ and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells.     

Down-regulated NOD2 by immunosuppressants in peripheral blood cells in patients with SLE reduces the muramyl dipeptide-induced IL-10 production

Background

Pattern recognition receptors (PRRs) such as Toll-like receptors are aberrantly expressed of peripheral blood mononuclear cells (PBMCs) in systemic lupus erythematosus (SLE) patients, for playing immunopathological roles.

Methodology/Principal Findings

We investigated the expression and function of the PRR nucleotide-binding oligomerization domain (NOD2) in SLE. NOD2 expression in T, B lymphocytes, monocytes, myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) was assessed in SLE patients and healthy controls (HCs) using flow cytometric analysis. Ex vivo production of cytokines from PBMCs upon NOD2 agonist muramyl dipeptide (MDP) stimulation was assessed using Cytometric Bead Array. Over-expression of NOD2 in monocytes was observed in immunosuppressant naïve SLE patients, and was positively associated with longer disease duration. Immunosuppressive therapy was an independent explanatory variable for downregulating NOD2 expression in CD8+ T, monocytes, mDCs and pDCs. Ex vivo basal productions of cytokines (IL-6, IL-8 and IL-10) were significantly increased in immunosuppressant naïve patients and patients with active disease despite immunosuppressants compared with HCs. Upon MDP stimulaiton, relative induction (%) of cytokines (IL-1β) from PBMC was significantly increased in immunosuppressant naïve patients with inactive disease, and patients with active disease despite immunosuppressant treatment compared with HCs. Immunosuppressant usage was associated with a decreased basal production and MDP induced relative induction (%) of IL-10 in patients with inactive disease compared with immunosuppressant naïve patients and HCs.

Conclusions/Significance

Bacterial exposure may increase the NOD2 expression in monocytes in immunosuppressant naïve SLE patients which can subsequently lead to aberrant activation of PBMCs to produce proinflammatory cytokines, implicating the innate immune response for extracellular pathogens in the immunopathological mechanisms in SLE. Immunosuppressant therapy may downregulate NOD2 expression in CD8+ T lymphocytes, monocytes, and DCs in SLE patients which subsequently IL-10 reduction, contributing towards the regulation of immunopathological mechanisms of SLE, at the expense of increasing risk of bacterial infection.     

Detailed kinetics of the direct allo-response in human liver transplant recipients: new insights from an optimized assay

Conventional assays for quantification of allo-reactive T-cell precursor frequencies (PF) are relatively insensitive. We present a robust assay for quantification of PF of T-cells with direct donor-specificity, and establish the kinetics of circulating donor-specific T cells after liver transplantation (LTx). B cells from donor splenocytes were differentiated into professional antigen-presenting cells by CD40-engagement (CD40-B cells). CFSE-labelled PBMC from LTx-recipients obtained before and at several time points after LTx, were stimulated with donor-derived or 3^rd party CD40-B cells. PF of donor-specific T cells were calculated from CFSE-dilution patterns, and intracellular IFN-γ was determined after re-stimulation with CD40-B cells. Compared to splenocytes, stimulations with CD40-B cells resulted in 3 to 5-fold higher responding T-cell PF. Memory and naïve T-cell subsets responded equally to allogeneic CD40-B cell stimulation. Donor-specific CD4⁺ and CD8⁺ T-cell PF ranged from 0.5 to 19% (median: 5.2%). One week after LTx, PF of circulating donor-specific CD4⁺ and CD8⁺ T cells increased significantly, while only a minor increase in numbers of T cells reacting to 3^rd party allo-antigens was observed. One year after LTx numbers of CD4⁺ and CD8⁺ T cells reacting to donor antigens, as well as those reacting to 3^rd party allo-antigens, were slightly lower compared to pre-transplant values. Moreover, CD4⁺ and CD8⁺ T cells responding to donor-derived, as well as those reacting to 3^rd party CD40-B cells, produced less IFN-γ. In conclusion, our alternative approach enables detection of allo-reactive human T cells at high frequencies, and after application we conclude that donor-specific T-cell PF increase immediately after LTx. However, no evidence for a specific loss of circulating T-cells recognizing donor allo-antigens via the direct pathway up to 1 year after LTx was obtained, underscoring the relative insensitiveness of previous assays.     

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.     

Phenotypic characterization of HIV-specific CD8+ T cells during early and chronic infant HIV-1 infection

Although CD8⁺ T cells play an important role in the containment of adult HIV-1 replication, their role in infant HIV-1 infection is not as well understood. Impaired HIV-specific CD8⁺ T cell responses may underlie the persistently high viral loads observed in infants. We examined the frequency and phenotype of infant HIV-specific CD8⁺ T cells in 7 HIV-infected antiretroviral therapy-naïve infants during the first 2 years of life, using class I HLA tetramers and IFN-γ-ELISPOT. The frequency (0.088–3.9% of CD3⁺CD8⁺ cells) and phenotype (CD27⁺CD28⁻, CD45RA^+/−, CD57^+/−, HLA-DR⁺, CD95⁺) of infant HIV-specific CD8⁺ T cells were similar to reports in adults undergoing early infection. Unlike adults, at 23–24 months post-infection a high frequency of HIV-specific CD8⁺ T cells expressed HLA-DR (mean 80%, range 68–85%) and CD95 (mean 88%, range 79–96%), suggesting sustained activation and vulnerability to apoptosis. Despite comparable expansion of HIV-specific CD8⁺ T cells of a similar phenotype to adults during early infection, infant T cells failed to contain HIV-1 replication, and remained persistently activated and vulnerable to apoptosis during chronic infection.     

Helios expression is a marker of T cell activation and proliferation

Foxp3+ T-regulatory cells (Tregs) normally serve to attenuate immune responses and are key to maintenance of immune homeostasis. Over the past decade, Treg cells have become a major focus of research for many groups, and various functional subsets have been characterized. Recently, the Ikaros family member, Helios, was reported as a marker to discriminate naturally occurring, thymic-derived Tregs from those peripherally induced from naïve CD4+ T cells. We investigated Helios expression in murine and human T cells under resting or activating conditions, using well-characterized molecules of naïve/effector/memory phenotypes, as well as a set of Treg-associated markers. We found that Helios-negative T cells are enriched for naïve T cell phenotypes and vice versa. Moreover, Helios can be induced during T cell activation and proliferation, but regresses in the same cells under resting conditions. We demonstrated comparable findings using human and murine CD4+Foxp3+ Tregs, as well as in CD4+ and CD8+ T cells. Since Helios expression is associated with T cell activation and cellular division, regardless of the cell subset involved, it does not appear suitable as a marker to distinguish natural and induced Treg cells.     

Lack of functional selectin ligand interactions compromises long term tumor protection by CD8+ T cells

Central memory CD8⁺ T cells expressing the adhesion molecule CD62L (L-selectin) are potent mediators of anti-cancer immunity due to their ability to proliferate extensively upon antigen re-stimulation. The interaction of selectin with its ligands mediates leukocyte rolling along high endothelial venules. Mice deficient in α(1,3) Fucosyltransferase IV and VII (FtDKO) lack functional L, P and E selectin ligands. Thus, we addressed whether the lack of selectin ligand interactions alters tumor protection by CD8⁺ T cells in FtDKO mice. Listeria monocytogenes-OVA (LM-OVA) infection evoked potent OVA-specific CD8⁺ T cells that proliferated and contracted at similar kinetics and phenotype in FtDKO and wild-type mice. Additionally, OVA-specific CD8⁺ T cells in both mouse strains exhibited similar phenotypic differentiation, in vivo cytolytic activity and IFN-γ expression. However, FtDKO mice succumbed to B16-OVA tumors significantly earlier than wild-type mice. In contrast, FtDKO mice evoked strong recall memory CD8⁺ T cell responses and protection to systemic LM-OVA re-challenge. The diminished tumor protection in FtDKO mice was not related to defective antigen presentation by dendritic cells or reduced proliferation of antigen-specific CD8⁺ T cells. However, WT or FtDKO OVA-specific CD8⁺ T cells showed significantly reduced ability to traffic to lymph nodes upon adoptive transfer into naïve FtDKO recipients. Furthermore, FtDKO OVA-specific CD8⁺ T cells displayed poor ability to infiltrate tumors growing in WT mice. These results reveal that selectin ligand expression on host endothelium as well CD8⁺ T cells may be important for their efficient and continued extravasation into peripheral tumors.     

Tentative T cells: memory cells are quick to respond, but slow to divide

T cell memory is a cornerstone of protective immunity, and is the key element in successful vaccination. Upon encountering the relevant pathogen, memory T cells are thought to initiate cell division much more rapidly than their naïve counterparts, and this is thought to confer a significant biological advantage upon an immune host. Here, we use traceable TCR-transgenic T cells to evaluate this proposed characteristic in CD4⁺ and CD8⁺ memory T cells. We find that, even in the presence of abundant antigen that was sufficient to induce in vivo IFNγ production by memory T cells, both memory and naïve T cells show an extended, and indistinguishable, delay in the onset of proliferation. Although memory cells can detect, and respond to, virus infection within a few hours, their proliferation did not begin until ∼3 days after infection, and occurred simultaneously in all anatomical compartments. Thereafter, cell division was extraordinarily rapid for both naïve and memory cells, with the latter showing a somewhat accelerated accumulation. We propose that, by permitting memory T cells to rapidly exert their effector functions while delaying the onset of their proliferation, evolution has provided a safeguard that balances the risk of infection against the consequences of severe T cell–mediated immunopathology.     

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.     

A phase IIA randomized clinical trial of a multiclade HIV-1 DNA prime followed by a multiclade rAd5 HIV-1 vaccine boost in healthy adults (HVTN204)

Background

The safety and immunogenicity of a vaccine regimen consisting of a 6-plasmid HIV-1 DNA prime (envA, envB, envC, gagB, polB, nefB) boosted by a recombinant adenovirus serotype-5 (rAd5) HIV-1 with matching inserts was evaluated in HIV-seronegative participants from South Africa, United States, Latin America and the Caribbean.

Methods

480 participants were evenly randomized to receive either: DNA (4 mg IM by Biojector) at 0, 1 and 2 months, followed by rAd5 (10¹⁰ PU IM by needle/syringe) at 6 months; or placebo. Participants were monitored for reactogenicity and adverse events throughout the 12-month study. Peak and duration of HIV-specific humoral and cellular immune responses were evaluated after the prime and boost.

Results

The vaccine was well tolerated and safe. T-cell responses, detected by interferon-γ (IFN-γ) ELISpot to global potential T-cell epitopes (PTEs) were observed in 70.8% (136/192) of vaccine recipients overall, most frequently to Gag (54.7%) and to Env (54.2%). In U.S. vaccine recipients T-cell responses were less frequent in Ad5 sero-positive versus sero-negative vaccine recipients (62.5% versus 85.7% respectively, p = 0.035). The frequency of HIV-specific CD4+ and CD8+ T-cell responses detected by intracellular cytokine staining were similar (41.8% and 47.2% respectively) and most secreted ≥2 cytokines. The vaccine induced a high frequency (83.7%–94.6%) of binding antibody responses to consensus Group M, and Clades A, B and C gp140 Env oligomers. Antibody responses to Gag were elicited in 46% of vaccine recipients.

Conclusion

The vaccine regimen was well-tolerated and induced polyfunctional CD4+ and CD8+ T-cells and multi-clade anti-Env binding antibodies.

Trial Registration:

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00125970","term_id":"NCT00125970"}}NCT00125970     

Association of differentiation state of CD4+ T cells and disease progression in HIV-1 perinatally infected children

Background

In the USA, most HIV-1 infected children are on antiretroviral drug regimens, with many individuals surviving through adolescence and into adulthood. The course of HIV-1 infection in these children is variable, and understudied.

Methodology/Principal Findings

We determined whether qualitative differences in immune cell subsets could explain a slower disease course in long term survivors with no evidence of immune suppression (LTS-NS; CD4%≥25%) compared to those with severe immune suppression (LTS-SS; CD4%≤15%). Subjects in the LTS-NS group had significantly higher frequencies of naïve (CCR7+CD45RA+) and central memory (CCR7+CD45RA−) CD4+ T cells compared to LTS-SS subjects (p = 0.0005 and <0.0001, respectively). Subjects in the rapid progressing group had significantly higher levels of CD4+ T_EMRA (CCR7−CD45RA+) cells compared to slow progressing subjects (p<0.0001).

Conclusions/Significance

Rapid disease progression in vertical infection is associated with significantly higher levels of CD4+ T_EMRA (CCR7−CD45RA+) cells.     

Adaptive immunity against Leishmania nucleoside hydrolase maps its c-terminal domain as the target of the CD4+ T cell-driven protective response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199–314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5–88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.     

Pre-existing adenovirus immunity modifies a complex mixed Th1 and Th2 cytokine response to an Ad5/HIV-1 vaccine candidate in humans

The results of the recent Step Study highlight a need to clarify the effects of pre-existing natural immunity to a vaccine vector on vaccine-induced T-cell responses. To investigate this interaction, we examined the relationship between pre-existing Ad5 immunity and T-cell cytokine response profiles in healthy, HIV-uninfected recipients of MRKAd5 HIV-1 gag vaccine (HVTN 050, ClinicalTrials.gov #{"type":"clinical-trial","attrs":{"text":"NCT00849732","term_id":"NCT00849732"}}NCT00849732). Participants were grouped by baseline Ad5 neutralizing antibody titer as either Ad5-seronegative (titer ≤18; n = 36) or Ad5-seropositive (titer >200; n = 34). Samples from vaccine recipients were analyzed for immune responses to either HIV-1 Gag peptide pools or Ad5 empty vector using an ex vivo assay that measures thirty cytokines in the absence of long-term culture. The overall profiles of cytokine responses to Gag and Ad5 had similar combinations of induced Th1- and Th2-type cytokines, including IFN-γ, IL-2, TNF-α, IP-10, IL-13, and IL-10, although the Ad5-specific responses were uniformly higher than the Gag-specific responses (p<0.0001 for 9 out of 11 significantly expressed analytes). At the peak response time point, PBMC from Ad5-seronegative vaccinees secreted significantly more IP-10 in response to Gag (p = 0.008), and significantly more IP-10 (p = 0.0009), IL-2 (p = 0.006) and IL-10 (p = 0.05) in response to Ad5 empty vector than PBMC from Ad5-seropositive vaccinees. Additionally, similar responses to the Ad5 vector prior to vaccination were observed in almost all subjects, regardless of Ad5 neutralizing antibody status, and the levels of secreted IFN-γ, IL-10, IL-1Ra and GM-CSF were blunted following vaccination. The cytokine response profile of Gag-specific T cells mirrored the Ad5-specific response present in all subjects before vaccination, and included a number of Th1- and Th2-associated cytokines not routinely assessed in current vaccine trials, such as IP-10, IL-10, IL-13, and GM-CSF. Together, these results suggest that vector-specific humoral responses may reduce vaccine-induced T-cell responses by previously undetected mechanisms.     

Chronic myeloid leukemia patients in prolonged remission following interferon-α monotherapy have distinct cytokine and oligoclonal lymphocyte profile

Before the era of tyrosine kinase inhibitors (TKIs), interferon-alpha (IFN-α) was the treatment of choice in chronic myeloid leukemia (CML). Curiously, some IFN-α treated patients were able to discontinue therapy without disease progression. The aim of this project was to study the immunomodulatory effects of IFN-α in CML patients in prolonged remission and isolate biological markers predicting response. Due to rarity of patients on IFN-α monotherapy, a relatively small cohort of patients still on treatment (IFN-ON, n = 10, median therapy duration 11.8 years) or had discontinued IFN-α therapy but remained in remission for >2 years (IFN-OFF, n = 9) were studied. The lymphocyte immunophenotype was analyzed with a comprehensive flow cytometry panel and plasma cytokine levels were measured with multiplex bead-based assay. In addition, the clonality status of different lymphocyte subpopulations was analyzed by TCR γ/δ rearrangement assay. Median NK-cell absolute number and proportion from lymphocytes in blood was higher in IFN-OFF patients as compared to IFN-ON patients or controls (0.42, 0.19, 0.21×10⁹/L; 26%, 12%, 11%, respectively, p<0.001). The proportion of CD8+ T-cells was significantly increased in both patient groups and a larger proportion of T-cells expressed CD45RO. Most (95%) patients had significant numbers of oligoclonal lymphocytes characterized by T-cell receptor γ/δ rearrangements. Strikingly, in the majority of patients (79%) a distinct clonal Vγ9 gene rearrangement was observed residing in γδ⁺ T-cell population. Similar unique clonality pattern was not observed in TKI treated CML patients. Plasma eotaxin and MCP-1 cytokines were significantly increased in IFN-OFF patients. Despite the limited number of patients, our data indicates that IFN-α treated CML patients in remission have increased numbers of NK-cells and clonal γδ⁺ T-cells and a unique plasma cytokine profile. These factors may relate to anti-leukemic effects of IFN-α in this specific group of patients and account for prolonged therapy responses even after drug discontinuation.