CD8+ lymphocyte phenotype and cytokine production in long-term non-progressor and in progressor patients with HIV-1 infection

In most HIV-1-infected patients, clinical and immunological progression develops within a few years. Few infected people, termed long-term non-progressors (LTNP), remain healthy and immunologically stable for a long time. The factors governing the maintenance of this condition are not well known, but it is conceivable that CD8⁺ lymphocytes, cells that play a central role in controlling in vitro HIV replication, may have a part in vivo in this process. The aim of this study was to characterize the phenotypic profile and the cytokine production of CD8⁺ cells in a group of LTNP patients who had stable CD4⁺ cell counts (>500/mm³) for at least 7 years. Their CD8⁺ absolute numbers were similar to a control group composed of HIV-1⁺ patients who have a progressive decline of their CD4⁺ cell counts. However, our multiparameter immunofluorescence studies show that a clinical and immunologically stable condition is associated with the presence of a CD28⁺, CD95 strongly positive CD8⁺ population, while disease progression is marked by the CD28⁻CD95⁺CD8⁺ subset. Purified CD8⁺ cells from LTNP retain their ability to produce IL-2, interferon-gamma (IFN-γ) and, to a lesser degree, to produce IL-10 and IL-4. In contrast, CD8⁺ cells from progressors are unable to secrete IL-2 and IL-10. Although CD8⁺ cytokine profile does not fit with the proposed T helper (Th)1/Th2 switch in progressive HIV infection, LTNP CD8⁺ T cells maintain their capacity to produce IL-2 and IL-10 (Th0-like), a pattern very similar to that observed in normal HIV healthy controls. We suggest that CD8⁺ cells expressing CD28, CD95 and having a Th0-like profile may be considered to be associated with long-term survival.     

Tetraspanin CD63 is a regulator of HIV-1 replication

Macrophages and CD4⁺ T-cells are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in HIV-1 replication events in primary human CD4⁺ T-cells, dendritic cells, and a CD4⁺ cell line. Most interestingly, we also show the evidences for the co-localization and internalization of CD63 and HIV-1 major receptor CD4 in primary human macrophages and CD4⁺ cell line by confocal microscopy and Co-Immunoprecipitation assay. Analysis revealed that CD63-depleted CD4⁺ T-cells, dendritic cells, and a cell line showed significant decrease in HIV-1 production. Further analysis showed that CD63 down regulation reduced production of the early HIV protein Tat, and affected HIV protein Gag by CD63-Gag interaction. In agreement, CD63 silencing also inhibited production of the late protein p24. Furthermore, we revealed that CD63 silencing has no effect on HIV-1 replication with extensive viral challenge (MOI > 0.2). These findings suggest that CD63 plays a dual-role both in early and late HIV-1 life cycle with a range of HIV-1 infection (MOI < 0.2).     

Dual immunoregulatory pathways of 4-1BB signaling

It is perhaps rare to encounter among the various immunologically competent receptor–ligand pairs that a single cell surface determinant unleashes both a hidden suppressive function and costimulation. 4-1BB, an activation-induced tumor necrosis factor receptor family member chiefly viewed as a powerful T-cell costimulatory molecule, is one such example. Accumulated evidence in recent years uncovered an unknown facet of in vivo 4-1BB signaling (i.e., “active suppression”). Although in vitro signaling via 4-1BB is shown to support both CD4⁺ and CD8⁺ T-cell responses, the same induces a predominant CD8⁺ T-cell response suppressing CD4⁺ T-cell function when applied in vivo. How, when, and why such dual immunoregulatory effect of anti-4-1BB monoclonal antibody (MAB) comes into play is currently the focus of intense research. Existing data, although not complete, uncover several important aspects of in vivo 4-1BB signaling in the amelioration or exacerbation of various immune disorders. Despite minor disagreements, a majority agree that upregulation of interferon (IFN)-γ is critical to anti-4-1BB MAB therapy in addition to immune modulators such as interleukin 2, transforming growth factor β, and indolamine 2,3-dioxygenase⁵, all of which contribute greatly to the success of anti-4-1BB MAB-based immunotherapy. Anti-4-1BB MAB-mediated expansion of novel CD11c⁺CD8⁺ T cells is additional weaponry that appears critical for its in vivo suppressive function. These CD11c⁺CD8⁺ T cells express high levels of IFN-γ, become effective killers, and mediate selective suppression of CD4⁺ T cells. In this review, we discuss the dual nature (costimulatory and suppressive) of 4-1BB-mediated immune regulation, its current status, future direction, and its impact on the immune system, with special reference to its immunotherapy.     

The HIV-1 Nef protein has a dual role in T cell receptor signaling in infected CD4+ T lymphocytes

The phenotypic changes that are induced by immune activation in CD4⁺ T lymphocytes provide an optimal environment for efficient HIV-1 replication in these cells. The pathogenic Nef protein of HIV-1 modulates the T cell receptor (TCR) signaling, but whether this has a positive or negative effect on cellular activation is a matter of debate. Here we have investigated the response to TCR stimulation of primary CD4⁺ T lymphocytes infected with wt or Nef-deficient HIV-1. Results show that, in freshly isolated quiescent T cells, Nef superinduces NFAT and IL-2 production bypassing early TCR effector molecules. Conversely, the early phosphorylation of PLC-γ1, the induction of NFAT, and the expression of IL-2 are impaired by Nef in sub-optimally activated/resting T cells. Our data indicate that Nef has a dual role in the modulation of TCR signaling aimed at favoring HIV-1 replication and spread in both quiescent and metabolically active CD4⁺ T lymphocytes.     

Overexpression of CD39 and high tumoral CD39+/CD8+ ratio are associated with adverse prognosis in resectable gastric cancer

CD39/ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1) is a cell surface-located, rate-limiting enzyme in the generation of adenosine, and plays a crucial role in tumor development. We examined co-expression of CD39 and CD8in gastric cancer (GC) and showed that the expression of CD39 and CD8 increased significantly in tumor tissues compared to paired peritumor tissues. The expression of tumoral CD39 (tCD39), but not tumoral CD8 (tCD8), was related to overall survival. Furthermore, the CD39⁺/CD8⁺ ratio was associated with poor prognosis in resected GC patients. Taken together, our data indicate that highCD39 expression and high tCD39⁺/CD8⁺ ratio in GC is a predictor of poor prognosis for GC patients after radical resection. Moreover, CD39 could serve as a potential target for cancer immunotherapy.     

The role of inducer cells in mediating in vitro suppression of feline immunodeficiency virus replication

CD8⁺ T-cell-mediated suppression of feline immunodeficiency virus (FIV) replication has been described by several groups, although the mechanisms of activation and conditions for viral suppression vary with the methodologies. We have previously reported that CD8⁺ T-cell-mediated suppression of FIV replication required inducer cell stimulation of the effector cells. The focus of the present study was to examine the essential role of inducer cells required for the induction of this soluble anti-FIV activity. Both FIV-PPR-infected T cells and feline skin fibroblasts (FSF) infected with an alphavirus vector expressing FIV capsid or the irrelevant antigen lacZ, stimulated autologous or heterologous effector cells to produce supernatants that suppressed FIV replication. Thus, induction of this suppression of FIV replication did not strictly require autologous inducer cells and did not require the presence of FIV antigen. Anti-viral activity correlated with the presence of CD8⁺ T cells. Suppression was maximal when the inducer cells and the effector cells were in contact with each other, because separation of the inducer and effector cells by a 0.45-μm membrane reduced FIV suppression by approximately 50%. These findings emphasize the importance for membrane antigen interactions and cytokines in the optimal induction of effector cell synthesis of the soluble anti-FIV activity.     

MicroRNAs are implicated in the suppression of CD4CD25 conventional T cell proliferation by CD4CD25 regulatory T cells

CD4⁺CD25⁺ regulatory T cells (Tregs) are critical for sustaining immunological homeostasis. CD4⁺CD25⁻ conventional T cells (Tcons) are the progenitors of populations including Th1, Th2, Th17, Tfh, and Treg cells. Suppression of Tcons proliferation by Tregs requires cell–cell contact and/or is mediated by immunosuppressive soluble factors. However, upon receiving suppressive signals from Tregs, the exact molecular responses in Tcons remain elusive. Here, by using microRNA (miRNA) microarray preliminary screening and quantitative RT-PCR (qRT-PCR) validation, we showed that paralleled with the suppression of the Tcons proliferation, miR-146a was induced but miR-106b and miR-21 were reduced in Tcons upon receiving suppressive signals from Tregs. Moreover, our results showed that either increase of miR-146a or decrease of miR-106b and miR-21 by using miRNA mimics or inhibitors in Tcons significantly enhanced the suppression triggered by Tregs. However, decrease of miR-146a or increase of miR-106b and miR-21 in Tcons impaired the suppression triggered by Tregs. Collectively, our findings demonstrate the roles of miR-146a, miR-106b and miR-21 in Tcons in regulating Treg-triggered immune-suppression.     

Functional discrepancies in HIV-specific CD8+ T-lymphocyte populations are related to plasma virus load

The potent ability of current antiretroviral drug regimens to control human immunodeficiency Virus-1 (HIV-1) replication, in conjunction with the clinical practice of structured therapeutic interruptions, provides a system in which virus levels are manipulated during a persistent infection in humans. Here, we exploit this system to examine the impact of variable plasma virus load (pVL) on the functionality of HIV-specific CD8⁺ T-lymphocyte populations. Using both ELISpot methodology and intracellular cytokine staining for interferon (IFN)- to assess functional status, together with fluorochrome-labeled peptide–major histocompatibility complex (pMHC) class I tetramer analysis to detect the physical presence of CD8⁺ T lymphocytes expressing cognate T-cell receptors (TCRs), we observed that the proportion of HIV-specific CD8⁺ T lymphocytes capable of mounting an effector response to antigen challenge directly ex vivo is related to the kinetics of virus exposure. Specifically, (a) after prolonged suppression of pVL with antiretroviral therapy (ART), physical and functional measures of HIV-specific CD8⁺ T-lymphocyte frequencies approximated; and (b) the percentage of functionally responsive cells in the HIV-specific CD8⁺ T lymphocyte populations declined substantially when therapy was discontinued and pVL recrudesced in the same patients. These results corroborate and extend observations in animal models that describe nonresponsive CD8⁺ T lymphocytes in the presence of high levels of antigen load and have implications for the interpretation of quantitative data generated by methods that rely on functional readouts.for the Swiss HIV Cohort Study     

Properties of HTLV-I transformed CD8 T-cells in response to HIV-1 infection

HIV-1 infection studies of primary CD8⁺ T-cells are hampered by difficulty in obtaining a significant number of targets for infection and low levels of productive infection. Further, there exists a paucity of CD8-expressing T-cell lines to address questions pertaining to the study of CD8⁺ T-cells in the context of HIV-1 infection. In this study, a set of CD8⁺ T-cell clones were originated through HTLV-I transformation in vitro, and the properties of these cells were examined. The clones were susceptible to T-cell tropic strains of the virus and exhibited HIV-1 production 20-fold greater than primary CD4⁺ T-cells. Productive infection resulted in a decrease in expression of CD8 and CXCR4 molecules on the surface of the CD8⁺ T-cell clones and antibodies to these molecules abrogated viral binding and replication. These transformed cells provide an important tool in the study of CD8⁺ T-cells and may provide important insights into the mechanism(s) behind HIV-1 induced CD8⁺ T-cell dysfunction.     

CD8+ T cell survival in lethal fungal sepsis was ameliorated by T-cell-specific mTOR deletion

Lethal fungal sepsis causes high morbidity and mortality in intensive care patients. Fungal infections have an immunological basis, and it has been shown in recent studies that decreased CD8⁺ T-cell count in fungal infections is related to prognosis, while the underlying mechanism is still unclear. Here, a lethal fungal sepsis model induced by candidemia was created and we found a decreased CD8⁺ T-cell count and exaggerated apoptosis. Simultaneously, expression of light chain (LC)3B in CD8⁺ T cells increased, along with increased autophagosomes and accumulation of p62 in infected mice. We regulated the activity of the mammalian target of rapamycin (mTOR) pathway using T-cell-specific mTOR/ TSC1 deletion mice. We observed increased number of autophagosomes and expression of LC3B in CD8⁺T cells after T-cell-specific mTOR knockout, while accumulation of p62 was not ameliorated, and there was no increase in the number of autolysosomes. Apoptosis rate and expression of BIM, a pro-apoptotic gene, decreased in CD8⁺ T cells in mTOR-deletion mice but increased in TSC1-deletion mice. Our results showed increased CD8⁺ T-cell death in spleen of lethal fungal sepsis mice, and decreased expression of mTOR ameliorated CD8⁺ T-cell survival. mTOR may be a possible target to reverse CD8⁺ T-cell immune dysfunction in lethal fungal sepsis.     

Too much of a good thing: Sustained type 1 interferon signaling limits humoral responses to secondary viral infection

The induction of a pan‐immunosuppressive state is a central feature of persistent viral infections. Over the past decade, multiple pathways have been identified that contribute to immune suppression. Recently, it was revealed that aberrant or sustained type 1 interferon (IFN‐I) production or signaling is a central contributor to immune suppression elicited during persistent viral infection. In this issue, Honke et al. [Eur. J. Immunol. 2016. 46: 372–380] identify that IFN‐I signaling promotes an immune suppressive state during persistent lymphocytic choriomeningitis virus infection by inhibiting enforced virus replication in CD169⁺ macrophages. The authors demonstrate that mice infected with a persistent strain of lymphocytic choriomeningitis virus have blunted humoral immune responses to a superinfecting vesicular stomatitis virus infection. The absence of virus replication in CD169⁺ macrophages was not due to antiviral CD8⁺ T cell‐mediated killing of CD169⁺ macrophages, but required sustained IFN‐I responses. In turn, reduction in vesicular stomatitis virus replication in CD169⁺ macrophages resulted in a reduction in antigen production, which is necessary for generating optimal humoral responses. This study highlights a novel mechanism by which IFN‐I signaling promotes an immune suppressive state during persistent viral infection.     

Upregulation of CD4+CD25+ T lymphocyte by adenovirus-mediated gene transfer of CTLA4Ig fusion protein in experimental autoimmune myocarditis

Objective: To explore the effects of adenovirus vector-mediated gene transfer of CTLA4Ig fusion protein on CD4⁺CD25⁺ T cells in experimental autoimmune myocarditis (EAM).

Methods: EAM was induced by porcine cardiac myosin as previously described. Adenovirus vector-mediated CTLA4Ig gene was administrated intravenously in EAM rats on days 1, 4 and 7, with EGFP as control. On day 21, myocardium histopathology was examined and CD4⁺CD25⁺ T cells were isolated. Proliferation and suppression assays were used to evaluate the suppressive capacity of CD4⁺CD25⁺ T cells in vitro. Relative mRNA level of Foxp3 and TGF-β was determined by quantitative real-time RT-PCR; expression of CTLA-4, B7-1 and B7-2 protein was compared with Western blot in CD4⁺CD25⁺ T_regs.

Results: Severe inflammatory lesions were observed in the hearts of EGFP-treated EAM rats and the untreated ones, while Ad–CMV–CTLA4Ig alleviated the myocarditis histologically. Adenovirus vector-mediated CTLA4Ig gene transfer up-regulated the proportion of CD4⁺CD25⁺ T_regs significantly. T cell proliferation was greatly inhibited in the CTLA4Ig group compared with the untreated and EGFP-treated groups in vitro. CTLA-4 and B7-2 proteins were down-regulated in the CTLA4Ig group, Foxp3 and TGF-β mRNA was up-regulated significantly by CTLA4Ig treatment.

Conclusions: Adenovirus vector-mediated CTLA4Ig gene transfer alleviated inflammation in EAM, one of the potential mechanisms is up-regulation of CD4⁺CD25⁺ T_regs.     

Down-regulation of CXCR4 expression by SDF-KDEL in CD34 hematopoietic stem cells: An anti-human immunodeficiency virus strategy

CXCR4 plays an essential role as the first discovered coreceptor for the entry of T cell tropic isolates of HIV-1. Blocking the surface expression of this receptor may be a potential strategy to prevent HIV-1 infection. A lentiviral vector, pLenti6/V5-S-K, expressing a SDF-KDEL fusion protein was constructed and a replication-incompetent lentiviral stock was produced. The lentiviral stock was transduced into CD34⁺ hHSC and the transient expression of the recombinant protein, SDF-1, was assayed using indirect immunofluorescence. The surface expression of CXCR4 in CD34⁺ hHSC pretreated with different amounts of recombinant lentiviral vectors was detected by flow cytometric analysis. A marked down-regulation of CXCR4 expression in the cells transduced with recombinant lentiviral vectors pLenti6/V5-S-K was observed by flow cytometry with PE-conjugated anti-human CXCR4 monoclonal antibodies which showed the percentages of the inhibition effects of CXCR4-SDF-1 mediated syncytium formation are presented by concentration. P24 antigen levels of cell culture supernatants were detected on the 4th, 7th, and 10th day, with 10³ TCID50 HIV-1 infected CD34⁺ hHSC to evaluate the inhibitory effect of pLenti6/V5-S-K transduction on HIV-1 infection. The cells transfected with pLenti6/V5-S-K had a significant reduction of HIV-1 DP27 infection compared to controls (P < 0.05).     

Infants with late breast milk acquisition of HIV-1 generate interferon-gamma responses more rapidly than infants with early peripartum acquisition

Infants infected with HIV-1 after the first month of life have a lower viral set-point and slower disease progression than infants infected before 1 month. We investigated the kinetics of HIV-1-specific CD8⁺ T lymphocyte secretion of interferon (IFN)-γ in infants infected before 1 month of life compared with those infected between months 1 and 12 (late infection). HIV-1 infection was assessed at birth and at months 1, 3, 6, 9 and 12 and timing of infection was determined by HIV-1 gag DNA from dried blood spots and verified by plasma HIV-1 RNA levels. HIV-1 peptide-specific IFN-γ responses were measured by enzyme-linked immunospot at months 1, 3, 6, 9 and 12. Timing of development of IFN-γ responses was compared using the log–rank test and Kaplan–Meier survival curves. Infants infected late developed HIV-1-specific CD8⁺ T cell responses 2·8 months sooner than infants infected peripartum: 2·3 versus 5·1 months after HIV-1 infection (n = 52, P = 0·04). Late-infected infants had more focused epitope recognition than early-infected infants (median 1 versus 2 peptides, P = 0·03); however, there were no differences in the strength of IFN-γ responses. In infants infected with HIV-1 after the first month of life, emergence of HIV-1-specific CD8⁺ IFN-γ responses is coincident with the decline in viral load, nearly identical to what is observed in adults and more rapid than in early-infected infants.     

Recombinant vector-induced HIV/SIV-specific CD4 T lymphocyte responses in rhesus monkeys

The recently reported modest success of the RV144 Thai trial vaccine regimen in preventing HIV-1 acquisition has focused interest on the potential contribution to that protection of vaccine-elicited CD4⁺ T cell responses. We evaluated the induction of virus-specific CD4⁺ T cell responses in rhesus monkeys using a series of diverse vaccine vectors. We assessed both the magnitudes and functional profiles of the antigen-specific CD4⁺ T cells by measuring cytokine production, memory differentiation, and the expression of mucosal homing molecules. We found that DNA prime/recombinant MVA boost immunizations induced particularly high-frequency virus-specific CD4⁺ T cell responses with polyfunctional repertoires, and these responses were partially preserved following SHIV-89.6P challenge. The majority of the vaccine-elicited CD4⁺ T cells were CD28⁺ memory T cells that expressed low levels of β7. Neither the magnitudes nor the functional profiles of the virus-specific CD4⁺ T cells generated by vaccination were associated with a preservation of CD4⁺ T cells or control of viral replication following SHIV-89.6P challenge. Interestingly, monkeys primed with recombinant Ad5 immunogens showed a dramatic expansion of both the magnitude and polyfunctionality of the vaccine-elicited CD4⁺ T cell responses following envelope protein boost. These results demonstrate that vaccine strategies that include recombinant MVA or recombinant Ad5 vectors can elicit robust CD4⁺ T cell responses.     

IDO expressing fibroblasts promote the expansion of antigen specific regulatory T cells

Regulatory CD4⁺CD25⁺Foxp3⁺ T cells (Tregs) can be induced and expanded by dendritic cells (DCs) in the presence of the enzyme indoleamine 2,3-dioxygenase (IDO). Here we report that a possible alternative to DCs are IDO expressing dermal fibroblasts (DFs), which are easier to isolate and sustain in culture compared to DCs. When mouse splenocytes were co-cultured with IDO expressing DFs, a significant increase in frequency and the number of Tregs was found compared to those of control group (13.16% ± 1.8 vs. 5.53% ± 1.2, p < 0.05). Despite observing a higher total number of dead CD4⁺ cells in the IDO group, there was a more abundant live CD4⁺CD25⁺ subpopulation in this group. Further analysis reveales that these CD4⁺ CD25⁺ cells have the capacity to expand in the presence of IDO expressing DFs. Greater number of CTLA-4⁺ cells and high expression of TGF-β and IL-10 were found in CD4⁺ cells of the IDO group compared to those of the controls. This finding confirmed a suppressive functionality of the expanded Tregs. Furthermore, CD4⁺ CD25⁺ cells isolated from the IDO group showed an alloantigen specific suppressive effect in a mixed lymphocyte reaction assay. These results confirm that IDO expressing dermal fibroblasts can expand a population of suppressive antigen specific Tregs. In conclusion, IDO expressing dermal fibroblasts have the capacity to stimulate the expansion of a subset of Tregs which can be used to generate antigen-specific immune tolerance.     

Human Immunodeficiency Virus Type 1 Replication,Immune Activation, and Circulating CytotoxicT Cells Against Uninfected CD4+ T Cells

Cytotoxic T lymphocytes (CTL) that kill uninfected activated CD4⁺ T cells can be induced in vitro by stimulating CD8⁺ T cells with activated autologous CD4⁺ T cells. Similar CTL have been detected in circulating T cells from human immunodeficiency virus type 1 (HIV)-infected individuals. To define the in vivo correlates of this CTL activity, we studied plasma -2 microglobulin and HIV RNA levels, T-lymphocyte subset counts, and expression of CD28 on CD8⁺ T cells concurrently with circulating CTL activity against uninfected CD4⁺ T cells in 75 HIV-infected individuals at different stages of disease progression. Mean values of each parameter were compared in subsets of this group of 75 segregated on the basis of this CTL activity. The group with CTL against uninfected activated CD4⁺ T lymphocytes had more CD8⁺ T cells, a higher percentage of CD28^– CD8⁺ T cells, and higher plasma levels of HIV RNA and -2 microglobulin. CTL against uninfected activated CD4⁺ T cells were predominantly CD28^– and in HIV-infected individuals were associated with immunological or virological evidence of progressive disease. In HIV infection, circulating CTL activity against uninfected activated CD4⁺ T lymphocytes is associated with immune activation, CD8⁺ T cell expansion, accumulation of CD28^– CD8⁺ T cells, and inadequate suppression of HIV replication.