Ronald L. Rabin

HIV-specific CD8+ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors

It is unclear why immunological control of HIV replication is incomplete in most infected individuals. We examined here the CD8+ T cell response to HIV-infected CD4+ T cells in rare patients with immunological control of HIV. Although high frequencies of HIV-specific CD8+ T cells were present in nonprogressors and progressors, only those of nonprogressors maintained a high proliferative capacity. This proliferation was coupled to increases in perforin expression. These results indicated that nonprogressors were differentiated by increased proliferative capacity of HIV-specific CD8+ T cells linked to enhanced effector function. In addition, the relative absence of these functions in progressors may represent a mechanism by which HIV avoids immunological control.

Mycobacterium tuberculosis Triggers Host Type I IFN Signaling To Regulate IL-1β Production in Human Macrophages

Mycobacterium tuberculosis is a virulent intracellular pathogen that survives in macrophages even in the presence of an intact adaptive immune response. Type I IFNs have been shown to exacerbate tuberculosis in mice and to be associated with disease progression in infected humans. Nevertheless, the mechanisms by which type I IFNs regulate the host response to M. tuberculosis infection are poorly understood. In this study, we show that M. tuberculosis induces an IFN-related gene expression signature in infected primary human macrophages, which is dependent on host type I IFN signaling as well as the mycobacterial virulence factor, region of difference-1. We further demonstrate that type I IFNs selectively limit the production of IL-1β, a critical mediator of immunity to M. tuberculosis. This regulation occurs at the level of IL1B mRNA expression, rather than caspase-1 activation or autocrine IL-1 amplification and appears to be preferentially used by virulent mycobacteria since avirulent M. bovis bacillus Calmette-Guérin (BCG) fails to trigger significant expression of type I IFNs or release of mature IL-1β protein. The latter property is associated with decreased caspase-1–dependent IL-1β maturation in the BCG-infected macrophages. Interestingly, human monocytes in contrast to macrophages produce comparable levels of IL-1β in response to either M. tuberculosis or BCG. Taken together, these findings demonstrate that virulent and avirulent mycobacteria employ distinct pathways for regulating IL-1β production in human macrophages and reveal that in the case of M. tuberculosis infection the induction of type I IFNs is a major mechanism used for this purpose.

Alpha and Lambda Interferon Together Mediate Suppression of CD4 T Cells Induced by Respiratory Syncytial Virus

ABSTRACT The mechanism by which respiratory syncytial virus (RSV) suppresses T-cell proliferation to itself and other antigens is poorly understood. We used monocyte-derived dendritic cells (MDDC) and CD4 T cells and measured [3H]thymidine incorporation to determine the factors responsible for RSV-induced T-cell suppression. These two cell types were sufficient for RSV-induced suppression of T-cell proliferation in response to cytomegalovirus or Staphylococcus enterotoxin B. Suppressive activity was transferable with supernatants from RSV-infected MDDC and was not due to transfer of live virus or RSV F (fusion) protein. Supernatants from RSV-infected MDDC, but not MDDC exposed to UV-killed RSV or mock conditions, contained alpha interferon (IFN-α; median, 43 pg/ml) and IFN-λ (approximately 1 to 20 ng/ml). Neutralization of IFN-α with monoclonal antibody (MAb) against one of its receptor chains, IFNAR2, or of IFN-λ with MAb against either of its receptor chains, IFN-λR1 (interleukin 28R [IL-28R]) or IL-10R2, had a modest effect. In contrast, blocking the two receptors together markedly reduced or completely blocked the RSV-induced suppression of CD4 T-cell proliferation. Defining the mechanism of RSV-induced suppression may guide vaccine design and provide insight into previously uncharacterized human T-cell responses and activities of interferons.

Interferon-α is the primary plasma type-I IFN in HIV-1 infection and correlates with immune activation and disease markers.

Type-I interferon (IFN-I) has been increasingly implicated in HIV-1 pathogenesis. Various studies have shown elevated IFN-I and an IFN-I-induced gene and protein expression signature in HIV-1 infection, yet the elevated IFN-I species has not been conclusively identified, its source remains obscure and its role in driving HIV-1 pathogenesis is controversial. We assessed IFN-I species in plasma by ELISAs and bioassay, and we investigated potential sources of IFN-I in blood and lymph node tissue by qRT-PCR. Furthermore, we measured the effect of therapeutic administration of IFNα in HCV-infected subjects to model the effect of IFNα on chronic immune activation. IFN-I bioactivity was significantly increased in plasma of untreated HIV-1-infected subjects relative to uninfected subjects (p = 0.012), and IFNα was the predominant IFN-I subtype correlating with IFN-I bioactivity (r = 0.658, p<0.001). IFNα was not detectable in plasma of subjects receiving anti-retroviral therapy. Elevated expression of IFNα mRNA was limited to lymph node tissue cells, suggesting that peripheral blood leukocytes are not a major source of IFNα in untreated chronic HIV-1 infection. Plasma IFN-I levels correlated inversely with CD4 T cell count (p = 0.003) and positively with levels of plasma HIV-1 RNA and CD38 expression on CD8 T cells (p = 0.009). In hepatitis C virus-infected subjects, treatment with IFN-I and ribavirin increased expression of CD38 on CD8 T cells (p = 0.003). These studies identify IFNα derived from lymph nodes, rather than blood leukocytes, as a possible source of the IFN-I signature that contributes to immune activation in HIV-1 infection.

Characterization of subsets of CD4+ memory T cells reveals early branched pathways of T cell differentiation in humans

The pathways for differentiation of human CD4+ T cells into functionally distinct subsets of memory cells in vivo are unknown. The identification of these subsets and pathways has clear implications for the design of vaccines and immune-targeted therapies. Here, we show that populations of apparently naïve CD4+ T cells express the chemokine receptors CXCR3 or CCR4 and demonstrate patterns of gene expression and functional responses characteristic of memory cells. The proliferation history and T cell receptor repertoire of these chemokine-receptor+ cells suggest that they are very early memory CD4+ T cells that have “rested down” before acquiring the phenotypes described for “central” or “effector” memory T cells. In addition, the chemokine-receptor+ “naïve” populations contain Th1 and Th2 cells, respectively, demonstrating that Th1/Th2 differentiation can occur very early in vivo in the absence of markers conventionally associated with memory cells. We localized ligands for CXCR3 and CCR4 to separate foci in T cell zones of tonsil, suggesting that the chemokine-receptor+ subsets may be recruited and contribute to segregated, polarized microenvironments within lymphoid organs. Importantly, our data suggest that CD4+ T cells do not differentiate according to a simple schema from naïve → CD45RO+ noneffector/central memory → effector/effector memory cells. Rather, developmental pathways branch early on to yield effector/memory populations that are highly heterogeneous and multifunctional and have the potential to become stable resting cells.

Respiratory syncytial virus interferon antagonist NS1 protein suppresses and skews the human T lymphocyte response

We recently demonstrated that the respiratory syncytial virus (RSV) NS1 protein, an antagonist of host type I interferon (IFN-I) production and signaling, has a suppressive effect on the maturation of human dendritic cells (DC) that was only partly dependent on released IFN-I. Here we investigated whether NS1 affects the ability of DC to activate CD8+ and CD4+ T cells. Human DC were infected with RSV deletion mutants lacking the NS1 and/or NS2 genes and assayed for the ability to activate autologous T cells in vitro, which were analyzed by multi-color flow cytometry. Deletion of the NS1, but not NS2, protein resulted in three major effects: (i) an increased activation and proliferation of CD8+ T cells that express CD103, a tissue homing integrin that directs CD8+ T cells to mucosal epithelial cells of the respiratory tract and triggers cytolytic activity; (ii) an increased activation and proliferation of Th17 cells, which have recently been shown to have anti-viral effects and also indirectly attract neutrophils; and (iii) decreased activation of IL-4-producing CD4+ T cells - which are associated with enhanced RSV disease - and reduced proliferation of total CD4+ T cells. Except for total CD4+ T cell proliferation, none of the T cell effects appeared to be due to increased IFN-I signaling. In the infected DC, deletion of the NS1 and NS2 genes strongly up-regulated the expression of cytokines and other molecules involved in DC maturation. This was partly IFN-I-independent, and thus might account for the T cell effects. Taken together, these data demonstrate that the NS1 protein suppresses proliferation and activation of two of the protective cell populations (CD103+ CD8+ T cells and Th17 cells), and promotes proliferation and activation of Th2 cells that can enhance RSV disease.

HIV Envelope Induces Virus Expression from Resting CD4+ T Cells Isolated from HIV-Infected Individuals in the Absence of Markers of Cellular Activation or Apoptosis

Resting CD4+ T cells containing integrated HIV provirus constitute one of the long-lived cellular reservoirs of HIV in vivo. This cellular reservoir of HIV had been thought to be quiescent with regard to virus replication based on the premise that HIV production in T cells is inexorably linked to cellular activation as determined by classical activation markers. The transition of T cells within this HIV reservoir from a resting state to an activated HIV-producing state is believed to be associated with a shorten life span due to susceptibility to activation-associated apoptosis. Evidence is mounting, however, that HIV production may occur in T cells that have not undergone classic T cell activation. HIV encodes several proteins, including envelope and Nef, which trigger a variety of signaling pathways associated with cellular activation, thereby facilitating HIV replication in nondividing cells. The present study demonstrates that production of infectious virus from resting CD4+ T cells isolated from HIV-infected individuals can be induced following exposure of these cells to HIV-1 recombinant (oligomeric gp140) envelope protein. Envelope-mediated induction of HIV expression occurs in the presence of reverse transcriptase inhibitors and is not associated with markers of classic T cell activation, proliferation, or apoptosis. The ability of HIV envelope to induce virus replication in HIV-infected resting CD4+ T cells without triggering apoptosis provides a mechanism for the virus itself to directly participate in the maintenance of HIV production from this cellular reservoir.

Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3

Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza virus type 3 (HPIV3) are common, important respiratory pathogens, but HRSV has a substantially greater impact with regard to acute disease, long-term effects on airway function, and frequency of re-infection. It has been reported to strongly interfere with the functioning of dendritic cells (DC). We compared HRSV to HMPV and HPIV3 with regard to their effects on human monocyte-derived immature DC (IDC). Side-by-side analysis distinguished between common effects versus those specific to individual viruses. The use of GFP-expressing viruses yielded clear identification of robustly infected cells and provided the means to distinguish between direct effects of robust viral gene expression versus bystander effects. All three viruses infected inefficiently based on GFP expression, with considerable donor-to donor-variability. The GFP-negative cells exhibited low, abortive levels of viral RNA synthesis. The three viruses induced low-to-moderate levels of DC maturation and cytokine/chemokine responses, increasing slightly in the order HRSV, HMPV, and HPIV3. Infection at the individual cell level was relatively benign, such that in general GFP-positive cells were neither more nor less able to mature compared to GFP-negative bystanders, and cells were responsive to a secondary treatment with lipopolysaccharide, indicating that the ability to mature was not impaired. However, there was a single exception, namely that HPIV3 down-regulated CD38 expression at the RNA level. Maturation by these viruses was anti-apoptotic. Inefficient infection of IDC and sub-optimal maturation might result in reduced immune responses, but these effects would be common to all three viruses rather than specific to HRSV.

Effects of human respiratory syncytial virus, metapneumovirus, parainfluenza virus 3 and influenza virus on CD4+ T cell activation by dendritic cells.

Background Human respiratory syncytial virus (HRSV), and to a lesser extent human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), re-infect symptomatically throughout life without antigenic change, suggestive of incomplete immunity. One causative factor is thought to be viral interference with dendritic cell (DC)-mediated stimulation of CD4+ T cells. Methodology, Principal Findings We infected human monocyte-derived DC with purified HRSV, HMPV, HPIV3, or influenza A virus (IAV) and compared their ability to induce activation and proliferation of autologous CD4+ T cells in vitro. IAV was included because symptomatic re-infection without antigenic change is less frequent, suggesting that immune protection is more complete and durable. We examined virus-specific memory responses and superantigen-induced responses by multiparameter flow cytometry. Live virus was more stimulatory than inactivated virus in inducing DC-mediated proliferation of virus-specific memory CD4+ T cells, suggesting a lack of strong suppression by live virus. There were trends of increasing proliferation in the order: HMPV<HRSV<HPIV3<IAV, and greater production of interferon-γ and tumor necrosis factor-α by proliferating cells in response to IAV, but differences were not significant. Exposure of DC to HRSV, HPIV3, or IAV reduced CD4+ T cell proliferation in response to secondary stimulus with superantigen, but the effect was transitory and greatest for IAV. T cell cytokine production was similar, with no evidence of Th2 or Th17 skewing. Conclusions, Significance Understanding the basis for the ability of HRSV in particular to symptomatically re-infect without significant antigenic change is of considerable interest. The present results show that these common respiratory viruses are similar in their ability to induce DC to activate CD4+ T cells. Thus, the results do not support the common model in which viral suppression of CD4+ T cell activation and proliferation by HRSV, HMPV, and HPIV3 is a major factor in the difference in re-infectability compared to IAV.

CCR2 Identifies a Stable Population of Human Effector Memory CD4+ T Cells Equipped for Rapid Recall Response

Because T cells act primarily through short-distance interactions, homing receptors can identify colocalizing cells that serve common functions. Expression patterns for multiple chemokine receptors on CD4+ T cells from human blood suggested a hierarchy of receptors that are induced and accumulate during effector/memory cell differentiation. We characterized CD4+CD45RO+ T cells based on expression of two of these receptors, CCR5 and CCR2, the principal subsets being CCR5−CCR2− (∼70%), CCR5+CCR2− (∼25%), and CCR5+CCR2+ (∼5%). Relationships among expression of CCR5 and CCR2 and CD62L, and the subsets’ proliferation histories, suggested a pathway of progressive effector/memory differentiation from the CCR5−CCR2− to CCR5+CCR2− to CCR5+CCR2+ cells. Sensitivity and rapidity of TCR-mediated activation, TCR signaling, and effector cytokine production by the subsets were consistent with such a pathway. The subsets also showed increasing responsiveness to IL-7, and the CCR5+CCR2+ cells were CD127bright and invariably showed the greatest response to tetanus toxoid. CCR5+CCR2+ cells also expressed the largest repertoire of chemokine receptors and migrated to the greatest number of chemokines. By contrast, the CCR5+CCR2− cells had the greatest percentages of regulatory T cells, activated/cycling cells, and CMV-reactive cells, and were most susceptible to apoptosis. Our results indicate that increasing memory cell differentiation can be uncoupled from susceptibility to death, and is associated with an increase in chemokine responsiveness, suggesting that vaccination (or infection) can produce a stable population of effector-capable memory cells that are highly enriched in the CCR5+CCR2+ subset and ideally equipped for rapid recall responses in tissue.