Dynamic shift from CD85j/ILT-2 to NKG2D NK receptor expression pattern on human decidual NK during the first trimester of pregnancy

During the first trimester of human pregnancy, Natural Killer (NK) cells of the maternal uterine mucosa (e.g. decidua) have a unique phenotype and are involved in crucial physiological processes during pregnancy. We investigated whether modifications of the NK receptor repertoire occur during the first trimester of pregnancy. We found significantly decreased expression of KIR2DL1/S1 and KIR2DL2/L3/S2 receptors, NKp30 and NKp44 activatory receptors, and the CD85j (ILT-2) inhibitory receptor. We also observed significantly increased expression of the NKG2D activatory receptor at the decidual NK cell surface. By flow cytometry, we further highlighted an evolution of NK subsets between 8 and 12 weeks of gestation, with a shift from the KIR2DL1/S1⁺/KIR2DL2/L3/S2⁺ subset towards the double negative subset, coupled with a decrease of the CD85j⁺/NKG2D⁻ subset in favour of the CD85j⁻/NKG2D⁺ subset. Furthermore, cell surface expression of NK receptor ligands, including CD85j and NKG2D ligands, has been characterized by flow cytometry on decidual immune CD14⁺ and CD3⁺ cells. HLA-G, the high affinity ligand of CD85j, was detected on both cell types. In contrast, NKG2D ligands ULBP-2 ULBP-3 and MICA/B were not expressed on CD14⁺ and CD3⁺ cells, however a variable expression of ULBP-1 was observed. The ligand expression of KIR2DL1/S1 and KIR2DL2/L3/S2 was also analyzed: the HLA-C molecule was expressed at a low level on some CD14⁺ cells whereas it was not detected on CD3⁺ cell surface. NK receptor ligands are known to be also expressed on the invading placental trophoblast cells. Thus, the phenotypic evolutions of decidual NK cells described in this present study may preserve their activation/inhibition balance during the first trimester of pregnancy.     

Dendritic cells are less susceptible to human immunodeficiency virus type 2 (HIV-2) infection than to HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) has been documented in vivo and may be an important contributor to HIV-1 transmission and pathogenesis. HIV-1-specific CD4⁺ T cells respond to HIV antigens presented by HIV-1-infected DCs and in this process become infected, thereby providing a mechanism through which HIV-1-specific CD4⁺ T cells could become preferentially infected in vivo. HIV-2 disease is attenuated with respect to HIV-1 disease, and host immune responses are thought to be contributory. Here we investigated the susceptibility of primary myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to infection by HIV-2. We found that neither CCR5-tropic primary HIV-2 isolates nor a lab-adapted CXCR4-tropic HIV-2 strain could efficiently infect mDCs or pDCs, though these viruses could infect primary CD4⁺ T cells in vitro. HIV-2-exposed mDCs were also incapable of transferring virus to autologous CD4⁺ T cells. Despite this, we found that HIV-2-specific CD4⁺ T cells contained more viral DNA than memory CD4⁺ T cells of other specificities in vivo. These data suggest that either infection of DCs is not an important contributor to infection of HIV-2-specific CD4⁺ T cells in vivo or that infection of DCs by HIV-2 occurs at a level that is undetectable in vitro. The frequent carriage of HIV-2 DNA within HIV-2-specific CD4⁺ T cells, however, does not appear to be incompatible with preserved numbers and functionality of HIV-2-specific CD4⁺ T cells in vivo, suggesting that additional mechanisms contribute to maintenance of HIV-2-specific CD4⁺ T-cell help in vivo.     

Ligand-independent exhaustion of killer immunoglobulin-like receptor-positive CD8+ T cells in human immunodeficiency virus type 1 infection

Virus-specific CD8⁺ T cells play a central role in the control of viral infections, including human immunodeficiency virus type 1 (HIV-1) infection. However, despite the presence of strong and broad HIV-specific CD8⁺ T-cell responses in chronic HIV-1 infection, these cells progressively lose critical effector functions and fail to clear the infection. Mounting evidence suggests that the upregulation of several inhibitory regulatory receptors on the surface of CD8⁺ T cells during HIV-1 infection may contribute directly to the impairment of T-cell function. Here, we investigated the role of killer immunoglobulin receptors (KIR), which are expressed on NK cells and on CD8⁺ T cells, in regulating CD8⁺ T-cell function in HIV-1 infection. KIR expression was progressively upregulated on CD8⁺ T cells during HIV-1 infection and correlated with the level of viral replication. Expression of KIR was associated with a profound inhibition of cytokine secretion, degranulation, proliferation, and activation by CD8⁺ T cells following stimulation with T-cell receptor (TCR)-dependent stimuli. In contrast, KIR⁺ CD8⁺ T cells responded potently to TCR-independent stimulation, demonstrating that these cells are functionally competent. KIR-associated suppression of CD8⁺ T-cell function was independent of ligand engagement, suggesting that these regulatory receptors may constitutively repress TCR activation. This ligand-independent repression of TCR activation of KIR⁺ CD8⁺ T cells may represent a significant barrier to therapeutic interventions aimed at improving the quality of the HIV-specific CD8⁺ T-cell response in infected individuals.     

Role of human immunodeficiency virus (HIV)-specific T-cell immunity in control of dual HIV-1 and HIV-2 infection

Progressive immune dysfunction and AIDS develop in most cases of human immunodeficiency virus type 1 (HIV-1) infection but in only 25 to 30% of persons with HIV-2 infection. However, the natural history and immunologic responses of individuals with dual HIV-1 and HIV-2 infection are largely undefined. Based on our previous findings, we hypothesized that among patients with dual infection the control of HIV-1 is associated with the ability to respond to HIV-2 Gag epitopes and to maintain HIV-specific CD4⁺ T-cell responses. To test this, we compared the HIV-specific ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay responses of 19 dually infected individuals to those of persons infected with HIV-1 or HIV-2 only. Further, we assessed the functional profile of HIV Gag-specific CD4⁺ and CD8⁺ T cells from nine HIV dually infected patients by using a multicolor intracellular cytokine staining assay. As determined by ELISPOT assay, the magnitude and frequency of IFN-γ-secreting T-cell responses to gene products of HIV-1 were higher than those to gene products of HIV-2 (2.64 versus 1.53 log₁₀ IFN-γ spot-forming cells/10⁶ cells [90% versus 63%, respectively].) Further, HIV-1 Env-, Gag-, and Nef- and HIV-2 Gag-specific responses were common; HIV-2 Nef-specific responses were rare. HIV-specific CD4⁺ T helper responses were detected in nine of nine dually infected subjects, with the majority of these T cells producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and, to a lesser extent, interleukin-2. The HIV-1 plasma viral load was inversely correlated with HIV-2 Gag-specific IFN-γ-/TNF-α-secreting CD4⁺ and HIV-2 Gag-specific IFN-γ-secreting CD8⁺ T cells. In conclusion, the T-cell memory responses associated with containment of single HIV-1 and HIV-2 infection play a similar significant role in the immune control of dual HIV-1 and HIV-2 infection.     

Influenza Vaccine Induces Intracellular Immune Memory of Human NK Cells

Influenza vaccines elicit antigen-specific antibodies and immune memory to protect humans from infection with drift variants. However, what supports or limits vaccine efficacy and duration is unclear. Here, we vaccinated healthy volunteers with annual vaccine formulations and investigated the dynamics of T cell, natural killer (NK) cell and antibody responses upon restimulation with heterologous or homologous influenza virus strains. Influenza vaccines induced potential memory NK cells with increased antigen-specific recall IFN-γ responses during the first 6 months. In the absence of significant changes in other NK cell markers (CD45RO, NKp44, CXCR6, CD57, NKG2C, CCR7, CD62L and CD27), influenza vaccines induced memory NK cells with the distinct feature of intracellular NKp46 expression. Indeed, surface NKp46 was internalized, and the dynamic increase in NKp46(intracellular)⁺CD56^dim NK cells positively correlated with increased IFN-γ production to influenza virus restimulation after vaccination. In addition, anti-NKp46 antibodies blocked IFN-γ responses. These findings provide insights into a novel mechanism underlying vaccine-induced immunity and NK-related diseases, which may help to design persisting and universal vaccines in the future.     

Strong Ability of Nef-Specific CD4+ Cytotoxic T Cells To Suppress Human Immunodeficiency Virus Type 1 (HIV-1) Replication in HIV-1-Infected CD4+ T Cells and Macrophages

A restricted number of studies have shown that human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic CD4⁺ T cells are present in HIV-1-infected individuals. However, the roles of this type of CD4⁺ T cell in the immune responses against an HIV-1 infection remain unclear. In this study, we identified novel Nef epitope-specific HLA-DRB1*0803-restricted cytotoxic CD4⁺ T cells. The CD4⁺ T-cell clones specific for Nef187-203 showed strong gamma interferon production after having been stimulated with autologous B-lymphoblastoid cells infected with recombinant vaccinia virus expressing Nef or pulsed with heat-inactivated virus particles, indicating the presentation of the epitope antigen through both exogenous and endogenous major histocompatibility complex class II processing pathways. Nef187-203-specific CD4⁺ T-cell clones exhibited strong cytotoxic activity against both HIV-1-infected macrophages and CD4⁺ T cells from an HLA-DRB1*0803⁺ donor. In addition, these Nef-specific cytotoxic CD4⁺ T-cell clones exhibited strong ability to suppress HIV-1 replication in both macrophages and CD4⁺ T cells in vitro. Nef187-203-specific cytotoxic CD4⁺ T cells were detected in cultures of peptide-stimulated peripheral blood mononuclear cells (PBMCs) and in ex vivo PBMCs from 40% and 20% of DRB1*0803⁺ donors, respectively. These results suggest that HIV-1-specific CD4⁺ T cells may directly control HIV-1 infection in vivo by suppressing virus replication in HIV-1 natural host cells.Human immunodeficiency virus (HIV)-specific CD8⁺ cytotoxic T cells (CTLs) play a central role in the control of HIV type 1 (HIV-1) during acute and chronic phases of an HIV-1 infection (5, 29, 34). However, HIV-1 escapes from the immune surveillance of CD8⁺ CTLs by mechanisms such as mutations of immunodominant CTL epitopes and downregulation of major histocompatibility complex class I (MHC-I) molecules on the infected cells (9, 11, 12, 49). Therefore, most HIV-1-infected patients without highly active antiretroviral therapy (HAART) develop AIDS eventually.HIV-1-specific CD4⁺ T cells also play an important role in host immune responses against HIV-1 infections. An inverse association of CD4⁺ T-cell responses with viral load in chronically HIV-1-infected patients was documented in a series of earlier studies (8, 36, 39, 41, 48), although the causal relationship between them still remains unclear (23). Classically, CD4⁺ T cells help the expansion of CD8⁺ CTLs by producing growth factors such as interleukin-2 (IL-2) or by their CD40 ligand interaction with antigen-processing cells and CD8⁺ CTLs. In addition, CD4⁺ T cells provide activation of macrophages, which can professionally maintain CD8⁺ T-cell memory (17). On the other hand, the direct ability of virus-specific cytotoxic CD4⁺ T cells (CD4⁺ CTLs) to kill target cells has been widely observed in human virus infections such as those by human cytomegalovirus, Epstein-Barr virus (EBV), hepatitis B virus, Dengue virus, and HIV-1 (2, 4, 10, 19, 30, 31, 38, 50). Furthermore, one study showed that mouse CD4⁺ T cells specific for lymphocytic choriomeningitis virus have cytotoxic activity in vivo (25). These results, taken together, indicate that a subset of effector CD4⁺ T cells develops cytolytic activity in response to virus infections.HIV-1-specific CD4⁺ CTLs were found to be prevalent in HIV-1 infections, as Gag-specific cytotoxic CD4⁺ T cells were detected directly ex vivo among peripheral blood mononuclear cells (PBMCs) from an HIV-1-infected long-term nonprogressor (31). Other studies showed that up to 50% of the CD4⁺ T cells in some HIV-1-infected donors can exhibit a clear cytolytic potential, in contrast to the fact that healthy individuals display few of these cells (3, 4). These studies indicate the real existence of CD4⁺ CTLs in HIV-1 infections.The roles of CD4⁺ CTLs in the control of an HIV-1 infection have not been widely explored. It is known that Gag-specific CD4⁺ CTLs can suppress HIV-1 replication in a human T-cell leukemia virus type 1-immortalized CD4⁺ T-cell line (31). However, the functions of CD4⁺ T cells specific for other HIV-1 antigens remain unclear. On the other hand, the abilities of CD4⁺ CTLs to suppress HIV-1 replication in infected macrophages and CD4⁺ T cells may be different, as in the case of CD8⁺ CTLs for HIV-1-infected macrophages (17). In this study, we identified Nef-specific CD4⁺ T cells and investigated their ability to kill HIV-1 R5 virus-infected macrophages and HIV-1 X4 virus-infected CD4⁺ T cells and to suppress HIV-1 replication in the infected macrophages and CD4⁺ T cells. The results obtained in the present study show for the first time the ability of HIV-1-specific CD4⁺ CTLs to suppress HIV-1 replication in natural host cells, i.e., macrophages and CD4⁺ T cells.     

Preferential infection of dendritic cells during human immunodeficiency virus type 1 infection of blood leukocytes

Human immunodeficiency virus type 1 (HIV-1) transmission by the parenteral route is similar to mucosal transmission in the predominance of virus using the CCR5 coreceptor (R5 virus), but it is unclear whether blood dendritic cells (DCs), monocytes, or T cells are the cells initially infected. We used ex vivo HIV-1 infection of sorted blood mononuclear cells to model the in vivo infection of blood leukocytes. Using quantitative real-time PCR to detect full-length HIV-1 DNA, both sorted CD11c⁺ myeloid and CD11c⁻ plasmacytoid DCs were more frequently infected than other blood mononuclear cells, including CD16⁺ or CD14⁺ monocytes or resting CD4⁺ T cells. There was a strong correlation between CCR5 coreceptor use and preferential DC infection across a range of HIV-1 isolates. After infection of unsorted blood mononuclear cells, HIV-1 was initially detected in the CD11c⁺ DCs and later in other leukocytes, including clustering DCs and activated T cells. DC infection with R5 virus was productive, as shown by efficient transmission to CD4⁺ T cells in coculture. Blood DCs infected with HIV-1 in vitro and cultured alone expressed only low levels of multiply spliced HIV-1 RNA unless cocultured with CD4⁺ T cells. Early selective infection of immature blood DCs by R5 virus and upregulation of viral expression during DC-T-cell interaction and transmission provide a potential pathway for R5 selection following parenteral transmission.     

Endogenous Production of β-Chemokines by CD4+, but Not CD8+, T-Cell Clones Correlates with the Clinical State of Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and May Be Responsible for Blocking Infection with NonSyncytium-Inducing HIV-1 In Vitro

Recent studies have demonstrated that the β-chemokines RANTES, MIP-1α, and MIP-1β suppress human immunodeficiency virus type 1 (HIV-1) replication in vitro and may play an important role in protecting exposed but uninfected individuals from HIV-1 infection. However, levels of β-chemokines in AIDS patients are comparable to and can exceed levels in nonprogressing individuals, indicating that global β-chemokine production may have little effect on HIV-1 disease progression. We sought to clarify the role of β-chemokines in nonprogressors and AIDS patients by examination of β-chemokine production and HIV-1 infection in patient T-lymphocyte clones established by herpesvirus saimiri immortalization. Both CD4⁺ and CD8⁺ clones were established, and they resembled primary T cells in their phenotypes and expression of activated T-cell markers. CD4⁺ T-cell clones from all patients had normal levels of mRNA-encoding CCR5, a coreceptor for non-syncytium-inducing (NSI) HIV-1. CD4⁺ clones from nonprogressors and CD8⁺ clones from AIDS patients secreted high levels of RANTES, MIP1α, and MIP-1β. In contrast, CD4⁺ clones from AIDS patients produced no RANTES and little or no MIP-1α or MIP-1β. The infection of CD4⁺ clones with the NSI HIV-1 strain ADA revealed an inverse correlation to β-chemokine production; clones from nonprogressors were poorly susceptible to ADA replication, but clones from AIDS patients were highly infectable. The resistance to ADA infection in CD4⁺ clones from nonprogressors could be partially reversed by treatment with anti-β-chemokine antibodies. These results indicate that CD4⁺ cells can be protected against NSI-HIV-1 infection in culture through endogenously produced factors, including β-chemokines, and that β-chemokine production by CD4⁺, but not CD8⁺, T cells may constitute one mechanism of disease-free survival for HIV-1-infected individuals.     

Generation of a novel regulatory NK cell subset from peripheral blood CD34+ progenitors promoted by membrane-bound IL-15

Background

NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated.

Methodology/Principal Findings

Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of “NK-ireg” cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34⁺ PB-HP. Finally, a small subset of NKp46⁺ HLA-G⁺ IL-10⁺ is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells.

Conclusions/Significance

In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56⁺ CD16⁺ NKp30⁺ NKp44⁺ NKp46⁺ CD94⁺ CD69⁺ CCR7⁺) generated from specific pSTAT6⁺ GATA3⁺ precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells.     

Costimulation of Naive CD8+ Lymphocytes Induces CD4 Expression and Allows Human Immunodeficiency Virus Type 1 Infection

Human immunodeficiency virus type 1 (HIV-1) infection requires cell surface expression of CD4. Costimulation of CD8⁺/CD4⁻ T lymphocytes by anti-CD3 and anti-CD28 antibodies or by allogeneic dendritic cells induced expression of CD4 and rendered these CD8 cells susceptible to HIV-1 infection. Naive CD45RA⁺ cells responded with greater expression of CD4 than did CD45RO⁺ cells. CD8⁺ lymphocytes derived from fetal or newborn sources exhibited a greater tendency to express CD4, consistent with their naive states. This mechanism of infection suggests HIV-induced perturbation of the CD8 arm of the immune response and could explain the generally rapid disease progression seen in HIV-infected children.     

Immunogenic Profiling in Mice of a HIV/AIDS Vaccine Candidate (MVA-B) Expressing Four HIV-1 Antigens and Potentiation by Specific Gene Deletions

Background

The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B), that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs) with immunoregulatory function.

Methodology/Principal Findings

In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R), known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1β, respectively (referred as MVA-B ΔA41L/ΔB16R). A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B ΔA41L/ΔB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4⁺ and CD8⁺ T cells, with the CD8⁺ T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B ΔA41L/ΔB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4⁺ and CD8⁺ T-cell immune responses. HIV-1-specific CD4⁺ T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8⁺ T-cell responses, MVA-B ΔA41L/ΔB16R induced more GPN-specific CD8⁺ T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env.

Conclusions/Significance

These findings revealed that MVA-B and MVA-B ΔA41L/ΔB16R induced in mice robust, polyfunctional and durable T-cell responses to HIV-1 antigens, but the double deletion mutant showed enhanced magnitude and quality of HIV-1 adaptive and memory responses. Our observations are relevant in the immune evaluation of MVA-B and on improvements of MVA vectors as HIV-1 vaccines.     

Naïve and Memory CD4 T Cells Differ in Their Susceptibilities to Human Immunodeficiency Virus Type 1 Infection following CD28 Costimulation: Implications for Transmission and Pathogenesis

In vitro evidence suggests that memory CD4⁺ cells are preferentially infected by human immunodeficiency virus type 1 (HIV-1), yet studies of HIV-1-infected individuals have failed to detect preferential memory cell depletion. To explore this paradox, we stimulated CD45RA⁺ CD4⁺ (naïve) and CD45RO⁺ CD4⁺ (memory) cells with antibodies to CD3 and CD28 and infected them with either CCR5-dependent (R5) or CXCR4-dependent (X4) HIV-1 isolates. Naïve CD4⁺ cells supported less X4 HIV replication than their memory counterparts. However, naïve cells were susceptible to R5 viral infection, while memory cells remained resistant to infection and viral replication. As with the unseparated cells, mixing the naïve and memory cells prior to infection resulted in cells resistant to R5 infection and highly susceptible to X4 infection. While both naïve and memory CD4⁺ subsets downregulated CCR5 expression in response to CD28 costimulation, only the memory cells produced high levels of the β-chemokines RANTES, MIP-1α, and MIP-1β upon stimulation. Neutralization of these β-chemokines rendered memory CD4⁺ cells highly sensitive to infection with R5 HIV-1 isolates, indicating that downregulation of CCR5 is not sufficient to mediate complete protection from CCR5 strains of HIV-1. These results indicate that susceptibility to R5 HIV-1 isolates is determined not only by the level of CCR5 expression but also by the balance of CCR5 expression and β-chemokine production. Furthermore, our results suggest a model of HIV-1 transmission and pathogenesis in which naïve rather than memory CD4⁺ T cells serve as the targets for early rounds of HIV-1 replication.     

Regulatory T Cell Suppression of Gag-Specific CD8+ T Cell Polyfunctional Response After Therapeutic Vaccination of HIV-1-Infected Patients on ART

We tested the hypothesis that therapeutic vaccination against HIV-1 can increase the frequency and suppressive function of regulatory, CD4⁺ T cells (Treg), thereby masking enhancement of HIV-1-specific CD8⁺ T cell response. HIV-1-infected subjects on antiretroviral therapy (N = 17) enrolled in a phase I therapeutic vaccine trial received 2 doses of autologous dendritic cells (DC) loaded with HIV-1 peptides. The frequency of CD4⁺CD25^hiFOXP3⁺ Treg in blood was determined prior to and after vaccination in subjects and normal controls. Polyfunctional CD8⁺ T cell responses were determined pre- and post-vaccine (N = 7) for 5 immune mediators after in vitro stimulation with Gag peptide, staphylococcal enterotoxin B (SEB), or medium alone. Total vaccine response (post-vaccine–pre-vaccine) was compared in the Treg(+) and Treg-depleted (Treg-) sets. After vaccination, 12/17 subjects showed a trend of increased Treg frequency (P = 0.06) from 0.74% to 1.2%. The increased frequency did not correlate with CD8⁺ T cell vaccine response by enzyme linked immunosorbent assay for interferon γ production. Although there was no significant change in CD8⁺ T cell polyfunctional response after vaccination, Treg depletion increased the polyfunctionality of the total vaccine response (P = 0.029), with a >2-fold increase in the percentage of CD8⁺ T cells producing multiple immune mediators. In contrast, depletion of Treg did not enhance polyfunctional T cell response to SEB, implying specificity of suppression to HIV-1 Gag. Therapeutic immunization with a DC-based vaccine against HIV-1 caused a modest increase in Treg frequency and a significant increase in HIV-1-specific, Treg suppressive function. The Treg suppressive effect masked an increase in the vaccine-induced anti-HIV-1-specific polyfunctional response. The role of Treg should be considered in immunotherapeutic trials of HIV-1 infection.     

CD4+ T Cell-Derived IL-2 Signals during Early Priming Advances Primary CD8+ T Cell Responses

Stimulating naïve CD8⁺ T cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. In this study, we show that the activation and differentiation of CD8⁺ T cells require IL-2 provided by activated CD4⁺ T cells at the initial priming stage within 0–2.5 hours after stimulation. This critical IL-2 signal from CD4⁺ cells is mediated through the IL-2Rβγ of CD8⁺ cells, which is independent of IL-2Rα. The activation of IL-2 signaling advances the restriction point of the cell cycle, and thereby expedites the entry of antigen-stimulated CD8⁺ T-cell into the S phase. Besides promoting cell proliferation, IL-2 stimulation increases the amount of IFNγ and granzyme B produced by CD8⁺ T cells. Furthermore, IL-2 at priming enhances the ability of P14 effector cells generated by antigen activation to eradicate B16.gp33 tumors in vivo. Therefore, our studies demonstrate that a full CD8⁺ T-cell response is elicited by a critical temporal function of IL-2 released from CD4⁺ T cells, providing mechanistic insights into the regulation of CD8⁺ T cell activation and differentiation.     

Engagement of the CD4 Receptor Affects the Redistribution of Lck to the Immunological Synapse in Primary T Cells: Implications for T-Cell Activation during Human Immunodeficiency Virus Type 1 Infection

Understanding the molecular mechanisms underlying dysregulated immune responses in human immunodeficiency virus type 1 (HIV-1) infection is crucial for the control of HIV/AIDS. Despite the postulate that HIV envelope glycoprotein gp120-CD4 interactions lead to impaired T-cell responses, the precise mechanisms underlying such association are not clear. To address this, we analyzed Lck and F-actin redistribution into the immunological synapse in stimulated human primary CD4⁺ T cells from HIV-1-infected donors. Similar experiments were performed with CD4⁺ T cells from HIV-uninfected donors, which were exposed to anti-CD4 domain 1 antibodies, as an in vitro model of gp120-CD4 interactions, or aldithriol-inactivated HIV-1 virions before stimulation. CD4⁺ T cells from HIV-infected patients exhibited a two- to threefold inhibition of both Lck and F-actin recruitment into the synapse, compared to cells from uninfected donors. Interestingly, defective recruitment of Lck was ameliorated following suppressive highly active antiretroviral therapy. Engagement of the CD4 receptor on T cells from HIV-uninfected donors before anti-CD3/CD28 stimulation led to similar defects. Furthermore, the redistribution of Lck into lipid rafts was abrogated by CD4 preengagement. Our results suggest that the engagement of CD4 by HIV gp120 prior to T-cell receptor stimulation leads to dysregulation of early signaling events and could consequently play an important role in impaired CD4⁺ T-cell function.     

IL-2 Mediates CD4+ T Cell Help in the Breakdown of Memory-Like CD8+ T Cell Tolerance under Lymphopenic Conditions

Background

Lymphopenia results in the proliferation and differentiation of naïve T cells into memory-like cells in the apparent absence of antigenic stimulation. This response, at least in part due to a greater availability of cytokines, is thought to promote anti-self responses. Although potentially autoreactive memory-like CD8⁺ T cells generated in a lymphopenic environment are subject to the mechanisms of peripheral tolerance, they can induce autoimmunity in the presence of antigen-specific memory-like CD4⁺ T helper cells.

Methodology/Principal Findings

Here, we studied the mechanisms underlying CD4 help under lymphopenic conditions in transgenic mice expressing a model antigen in the beta cells of the pancreas. Surprisingly, we found that the self-reactivity mediated by the cooperation of memory-like CD8⁺ and CD4⁺ T cells was not abrogated by CD40L blockade. In contrast, treatment with anti-IL-2 antibodies inhibited the onset of autoimmunity. IL-2 neutralization prevented the CD4-mediated differentiation of memory-like CD8⁺ T cells into pathogenic effectors in response to self-antigen cross-presentation. Furthermore, in the absence of helper cells, induction of IL-2 signaling by an IL-2 immune complex was sufficient to promote memory-like CD8⁺ T cell self-reactivity.

Conclusions/Significance

IL-2 mediates the cooperation of memory-like CD4⁺ and CD8⁺ T cells in the breakdown of cross-tolerance, resulting in effector cytotoxic T lymphocyte differentiation and the induction of autoimmune disease.     

Determinant in Human Immunodeficiency Virus Type 1 for Efficient Replication under Cytokine-Induced CD4+ T-Helper 1 (Th1)- and Th2-Type Conditions

Cytokines are potent stimuli for CD4⁺-T-cell differentiation. Among them, interleukin-12 (IL-12) and IL-4 induce naive CD4⁺ T cells to become T-helper 1 (Th1) or Th2 cells, respectively. In this study we found that macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains replicated more efficiently in IL-12-induced Th1-type cultures derived from normal CD4⁺ T cells than did T-cell-line-tropic (T-tropic) strains. In contrast, T-tropic strains preferentially infected IL-4-induced Th2-type cultures derived from the same donor CD4⁺ T cells. Additional studies using chimeric viruses demonstrated that the V3 region of HIV-1 gp120 was the principal determinant for efficiency of replication. Cell fusion analysis showed that cells expressing envelope protein from a T-tropic strain effectively fused with IL-4-induced Th2-type culture cells. Flow cytometric analysis showed that the level of CCR5 expression was higher on IL-12-induced Th1-type culture cells, whereas CXCR4 was highly expressed on IL-4-induced Th2-type culture cells, although a low level of CXCR4 expression was observed on IL-12-induced Th1-type culture cells. These results indicate that HIV-1 isolates exhibit differences in the ability to infect CD4⁺-T-cell subsets such as Th1 or Th2 cells and that this difference may partly correlate with the expression of particular chemokine receptors on these cells. The findings suggest that immunological conditions are one of the factors responsible for inducing selection of HIV-1 strains.     

Role of Natural Killer Cells in a Cohort of Elite Suppressors: Low Frequency of the Protective KIR3DS1 Allele and Limited Inhibition of Human Immunodeficiency Virus Type 1 Replication In Vitro

Natural killer (NK) cells are associated with the innate immune response and are important in many viral infections. Recent studies indicate that NK cells can control human immunodeficiency virus type 1 (HIV-1) replication. We studied the effect of NK cells on HIV-1 replication in a subpopulation of HIV-1-infected individuals termed elite suppressors (ES) or elite controllers. These patients maintain a clinically undetectable viral load without treatment and thus provide a fascinating cohort in which to study the immunological response to HIV-1. Using an autologous system, we analyzed the effects of NK cells and CD8⁺ T cells on viral replication in CD4⁺ T lymphoblasts. Although we had postulated that NK cells of ES would be highly effective at controlling viral replication, we found that NK cells from some, but not all, ES were capable of inhibiting replication in the presence of interleukin-2, and the inhibition was less robust than that mediated by CD8⁺ T cells. Additionally, we examined whether particular alleles of the KIR receptors, specifically KIR3DS1 and KIR3DL1, or allele-ligand combinations correlated with the control of HIV-1 replication by NK cells and whether any specific KIR alleles were overrepresented in ES. Our ES cohort did not differ from the general population with respect to the frequency of individual KIR. However, of the eight ES studied, the four exhibiting the most NK cell-mediated control of viral replication also had the fewest activating KIR and were haplotype A. Thus, the strong NK cell-mediated inhibition of viral replication is not necessary for the immunological control of HIV-1 in all ES.A small subset of untreated, human immunodeficiency virus type 1 (HIV-1)-infected individuals referred to as elite suppressors (ES) control viremia to levels that are undetectable by ultrasensitive commercial assays while maintaining high CD4⁺ T-cell counts (13, 37). While defective virus has been shown to account for the control of virus in some patients, examining multiple host factors in ES with replication-competent virus (9) already has provided critical information on the immune response to HIV-1 and may yield important insights into future therapies and vaccine development.Research on ES suggests that CD8⁺ T cells play a crucial role in an effective response to HIV-1. CD8⁺ T cells from ES are capable of controlling viral replication in autologous CD4⁺ T cells significantly better than CD8⁺ T cells from progressors (36), and only the former proliferate (29) and secrete multiple cytokines (8) in response to HIV-1 antigens. Furthermore, certain class I HLA alleles, such as HLA-B*27 and HLA-B*57, which appear to be important in the cytotoxic T-lymphocyte (CTL) response, are overrepresented in ES (15, 19, 21, 30, 32). A second, less well studied cytotoxic cell also may play a role in the control of HIV-1. Natural killer (NK) cells are part of the innate immune system and are an important component of the host response to many viral infections. They act on target cells via cytokine release and cytolysis in response to the integration of signals from inhibitory and activating receptors.The striking propensity of HIV-1 to evolve rapidly in response to immunologic or pharmacologic pressure suggests that the virus has the capability to evade the NK cell response, and indeed selection for evasive measures seems to have occurred. The virus-induced downregulation of HLA-A and -B molecules on infected cells provides some protection against the CTL response; at the same time, however, HLA-C molecules are not downregulated upon infection (12). NK cell interaction with HLA-C can inhibit NK cytotoxic effects, and thus the retention of HLA-C on infected cells can provide some protection against the NK cell response. Additionally, a variety of alterations in NK cell function have been observed during HIV-1 infection. NK cells of patients with chronic HIV-1 have altered phenotypes and effector capabilities: NK cells from viremic patients have an increased expression of inhibitory receptors, and there is an expansion of the defective CD56⁻ NK cells compared to the levels in patients on highly active antiretroviral therapy or in ES (7, 27). These changes may be due to alterations in the cytokine environment during infection, which can affect the activation of the NK cells (39); they also may be due to direct interactions between HIV-1 gene products and the NK cells (20). Although the precise cause is unknown, the result is the development of defective NK cells that express an altered receptor and NK cell marker phenotype.Studies specifically examining a role for NK cells in the response to HIV-1 have yielded conflicting results. During acute HIV-1 infection, the NK cell population is activated and expands, particularly the cytotoxic CD56^dim population (2, 3). This activation declines in the chronic phase, and at least one study suggests that the drop in the viral load (VL) of patients during acute infection occurs before the CD8⁺ T-cell response is fully activated; this could be attributed to the effect of NK cells (2). At the same time, the study of exposed, uninfected individuals shows a correlation between resistance to acquiring HIV-1 infection and NK cell activation levels, cytokine release, and cytotoxicity to NK cell-sensitive cell lines (33, 38). Additionally, a recent whole-genome association study identified three single-nucleotide polymorphisms that appear to be important for the host control of HIV-1 (16). Two of these may have an impact on NK cell function, one that is associated with HLA-B*57 and a second that correlates with higher HLA-C mRNA expression. Taken together, such data suggest that NK cells are important for preventing HIV-1 infection and/or reducing the magnitude of viral replication in acute infection, thereby contributing to the ability of ES to control viremia.In this study, we provide the first characterization of NK cells in patients who naturally control HIV-1 infection. Considering that the effectiveness of CD8⁺ T cells against viral replication is well documented, we directly compared the effect of NK cells to that of CD8⁺ T cells from ES on viral replication to put the effect of NK cells in perspective. We studied the NK cell response by measuring the change in p24 production when autologous effector cell populations were coincubated with infected CD4⁺ lymphoblasts with and without the addition of interleukin-2 (IL-2). Additionally, we examined the killer immunoglobulin-like receptors (KIR) and KIR ligand genotype of ES patients to determine whether any KIR are overrepresented in ES and whether KIR-ligand combinations correlated with the HIV-1 inhibitory activity of the NK cells from specific patients. Previous studies have identified correlations between the expression of certain KIR and progression to AIDS in chronic progressors (25, 26); however, a connection between KIR, KIR ligands, and the control of HIV-1 has yet to be identified in ES. The results of these studies significantly advance the understanding of the nature of NK cells and of their potential role in reducing HIV-1 replication.     

In Vitro Priming Recapitulates In Vivo HIV-1 Specific T Cell Responses,Revealing Rapid Loss of Virus Reactive CD4+ T Cells in Acute HIV-1 Infection

Background

The requirements for priming of HIV-specific T cell responses initially seen in infected individuals remain to be defined. Activation of T cell responses in lymph nodes requires cell-cell contact between T cells and DCs, which can give concurrent activation of T cells and HIV transmission.

Methodology

The study aim was to establish whether DCs pulsed with HIV-1 could prime HIV-specific T cell responses and to characterize these responses. Both infectious and aldrithiol-2 inactivated noninfectious HIV-1 were compared to establish efficiencies in priming and the type of responses elicited.

Findings

Our findings show that both infectious and inactivated HIV-1 pulsed DCs can prime HIV-specific responses from naïve T cells. Responses included several CD4⁺ and CD8⁺ T cell epitopes shown to be recognized in vivo by acutely and chronically infected individuals and some CD4⁺ T cell epitopes not identified previously. Follow up studies of acute and recent HIV infected samples revealed that these latter epitopes are among the earliest recognized in vivo, but the responses are lost rapidly, presumably through activation-induced general CD4⁺ T cell depletion which renders the newly activated HIV-specific CD4⁺ T cells prime targets for elimination.

Conclusion

Our studies highlight the ability of DCs to efficiently prime naïve T cells and induce a broad repertoire of HIV-specific responses and also provide valuable insights to the pathogenesis of HIV-1 infection in vivo.     

Heterosexual and Homosexual Partners Practising Unprotected Sex May Develop Allogeneic Immunity and to a Lesser Extent Tolerance

Background

Epidemiological studies suggest that allogeneic immunity may inhibit HIV-1 transmission from mother to baby and is less frequent in multiparous than uniparous women. Alloimmune responses may also be elicited during unprotected heterosexual intercourse, which is associated ex vivo with resistance to HIV infection.

Methodology/Principal Findings

The investigation was carried out in well-defined heterosexual and homosexual monogamous partners, practising unprotected sex and a heterosexual cohort practising protected sex. Allogeneic CD4⁺ and CD8⁺ T cell proliferative responses were elicited by stimulating PBMC with the partners'' irradiated monocytes and compared with 3^rd party unrelated monocytes, using the CFSE method. Significant increase in allogeneic proliferative responses was found in the CD4⁺ and CD8⁺ T cells to the partners'' irradiated monocytes, as compared with 3^rd party unrelated monocytes (p≤0.001). However, a significant decrease in proliferative responses, especially of CD8⁺ T cells to the partners'' compared with 3^rd party monocytes was consistent with tolerization, in both the heterosexual and homosexual partners (p<0.01). Examination of CD4⁺CD25⁺FoxP3⁺ regulatory T cells by flow cytometry revealed a significantly greater proportion of these cells in the homosexual than heterosexual partners practising unprotected sex (p<0.05). Ex vivo studies of infectivity of PBMC with HIV-1 showed significantly greater inhibition of infectivity of PBMC from heterosexual subjects practising unprotected compared with those practising protected sex (p = 0.02).

Conclusions/Significance

Both heterosexual and homosexual monogamous partners practising unprotected sex develop allogeneic CD4⁺ and CD8⁺ T cell proliferative responses to the partners'' unmatched cells and a minority may be tolerized. However, a greater proportion of homosexual rather than heterosexual partners developed CD4⁺CD25FoxP3⁺ regulatory T cells. These results, in addition to finding greater inhibition of HIV-1 infectivity in PBMC ex vivo in heterosexual partners practising unprotected, compared with those practising protected sex, suggest that allogeneic immunity may play a significant role in the immuno-pathogenesis of HIV-1 infection.