Murine CD4+ T cell responses are inhibited by cytotoxic T cell-mediated killing of dendritic cells and are restored by antigen transfer

Cytotoxic T lymphocytes (CTL) provide protection against pathogens and tumors. In addition, experiments in mouse models have shown that CTL can also kill antigen-presenting dendritic cells (DC), reducing their ability to activate primary and secondary CD8(+) T cell responses. In contrast, the effects of CTL-mediated killing on CD4(+) T cell responses have not been fully investigated. Here we use adoptive transfer of TCR transgenic T cells and DC immunization to show that specific CTL significantly inhibited CD4(+) T cell proliferation induced by DC loaded with peptide or low concentrations of protein antigen. In contrast, CTL had little effect on CD4(+) T cell proliferation induced by DC loaded with high protein concentrations or expressing antigen endogenously, even if these DC were efficiently killed and failed to accumulate in the lymph node (LN). Residual CD4(+) T cell proliferation was due to the transfer of antigen from carrier DC to host APC, and predominantly involved skin DC populations. Importantly, the proliferating CD4(+) T cells also developed into IFN-γ producing memory cells, a property normally requiring direct presentation by activated DC. Thus, CTL-mediated DC killing can inhibit CD4(+) T cell proliferation, with the extent of inhibition being determined by the form and amount of antigen used to load DC. In the presence of high antigen concentrations, antigen transfer to host DC enables the generation of CD4(+) T cell responses regardless of DC killing, and suggests mechanisms whereby CD4(+) T cell responses can be amplified.     

Neuropeptide signaling activates dendritic cell-mediated type 1 immune responses through neurokinin-2 receptor

Neurokinin A (NKA), a neurotransmitter distributed in the central and peripheral nervous system, strictly controls vital responses, such as airway contraction, by intracellular signaling through neurokinin-2 receptor (NK2R). However, the function of NKA-NK2R signaling on involvement in immune responses is less-well defined. We demonstrate that NK2R-mediated neuropeptide signaling activates dendritic cell (DC)-mediated type 1 immune responses. IFN-γ stimulation significantly induced NK2R mRNA and remarkably enhanced surface protein expression levels of bone marrow-derived DCs. In addition, the DC-mediated NKA production level was significantly elevated after IFN-γ stimulation in vivo and in vitro. We found that NKA treatment induced type 1 IFN mRNA expressions in DCs. Transduction of NK2R into DCs augmented the expression level of surface MHC class II and promoted Ag-specific IL-2 production by CD4(+) T cells after NKA stimulation. Furthermore, blockade of NK2R by an antagonist significantly suppressed IFN-γ production by both CD4(+) T and CD8(+) T cells stimulated with the Ag-loaded DCs. Finally, we confirmed that stimulation with IFN-γ or TLR3 ligand (polyinosinic-polycytidylic acid) significantly induced both NK2R mRNA and surface protein expression of human PBMC-derived DCs, as well as enhanced human TAC1 mRNA, which encodes NKA and Substance P. Thus, these findings indicate that NK2R-dependent neuropeptide signaling regulates Ag-specific T cell responses via activation of DC function, suggesting that the NKA-NK2R cascade would be a promising target in chronic inflammation caused by excessive type 1-dominant immunity.     

Model for studying virus attachment. II. Binding of biotinylated human T cell leukemia virus type I to human blood mononuclear cells potential targets for human T cell leukemia virus type I infection.

Purified human T cell leukemia virus type I (HTLV-I) was biotinylated and used to study its attachment to human PBMC. The use of biotinylated HTLV-I (biot-HTLV-I) in conjunction with mouse mAb specific for selected cell-surface molecules and flow cytometric analysis allowed us to positively identify virus-binding cells among a heterogeneous blood mononuclear cell population. Biot-HTLV-I efficiently bound not only to T cells, but also to B cells and monocytes. Preincubation of monocytes with excess of unlabeled HTLV-I significantly reduced the attachment of biot-HTLV-I. HTLV-I not only bound to, but also infected, B cells, as suggested by: i) in situ hybridization of a 35S-labeled full length HTLV-I DNA probe with EBV-transformed B cells, previously cocultured with HTLV-I-producing (G11MJ) T cells, and ii) hybridization of the same nick-translated 32P-labeled DNA probe with blotted DNA from similar HTLV-I-infected EBV-transformed B cells. HTLV-I infection did not affect the ability of B cells to secrete IgG. These findings suggest that HTLV-I cannot only infect cells of the T lineage, but can also infect B cells.     

A milk protein lactoferrin enhances human T cell leukemia virus type I and suppresses HIV-1 infection

Human T cell leukemia virus type I (HTLV-I) and HIV-1, causative agents of adult T cell leukemia/lymphoma and AIDS, respectively, are transmitted vertically via breast milk. Here we demonstrate that lactoferrin, a milk protein that has a variety of antimicrobial and immunomodulatory activities, facilitates replication of HTLV-I in lymphocytes derived from asymptomatic HTLV-I carriers and transmission to cord blood lymphocytes in vitro. Transient expression assays revealed that lactoferrin can transactivate HTLV-I long terminal repeat promoter. In contrast, lactoferrin inhibits HIV-1 replication, at least in part, at the level of viral fusion/entry. These results suggest that lactoferrin may have different effects on vertical transmission of the two milk-borne retroviruses.     

The Toxoplasma gondii bradyzoite antigens BAG1 and MAG1 induce early humoral and cell-mediated immune responses upon human infection

Infection of humans by Toxoplasma gondii leads to an acute systemic phase, in which tachyzoites disseminate throughout the body, followed by a chronic phase characterized by the presence of tissue cysts, containing bradyzoites, in brain, heart and skeletal muscles. This work focused on studying the antigenic regions of bradyzoite-specific proteins involved in human B- and T-cell responses. To this aim, we constructed a phage-display library of DNA fragments derived from the bradyzoite-specific genes BAG1, MAG1, SAG2D, SAG4, BSR4, LDH2, ENO1 and p-ATPase. Challenge of the bradyzoite library with sera of infected individuals led to the identification of antigenic regions within BAG1 and MAG1 gene products. Analysis of the humoral and lymphoproliferative responses to recombinant antigens demonstrated that the BAG1 fragment induced T-cell proliferation in 34% of T. gondii-exposed individuals, while 50% of them had specific IgG. In the same subjects, the MAG1 fragment was recognized by T cells from 17% of the exposed donors and by antibodies from 73% of them. A detailed analysis of the antibody response against BAG1 and MAG1 antigen fragments demonstrated that the immune response against bradyzoites occurs early after infection in humans. Finally, we provide evidence that the T-cell response against BAG1 is associated with the production of interferon-gamma, suggesting that bradyzoite antigens should be considered in the design of potential vaccines in humans.     

Vehicles for genetic vaccines against human immunodeficiency virus: induction of T cell-mediated immune responses

Success of a candidate vaccine against human immunodeficiency virus (HIV) depends on the type, site, strength, longevity and specificity of the immune responses it induces. The specificity of a vaccine is determined by the HIV-derived immunogens it employs in its formulation. Central to the other features is a correct and efficient delivery of the immunogens to the relevant cells of the immune system, which leads to orchestrated actions of millions of cells of several types and functions at multiple sites in the body. Thus, for elicitation of cytotoxic T lymphocyte responses, immunogens have to be delivered to the so called 'professional' antigen-presenting cells in a way that leads to a specific activation and expansion of na?ve or precursor T cells, subsequent maturation of effector functions and, importantly, generation of a potent immunological memory. Many aspects of theseprocesses are currently unknown. However, it is very likely that the immunogenicities of genetic vaccines, i.e. vaccines delivering genes coding for immunogens rather than purified possibly adjuvanted proteins or peptides themselves, are in great part determined by the choice of vaccine vehicles and route of administration. In addition, vaccine immunogenicities can be augmented semi-rationally by immunogen engineering and co-delivering immunomodulatory molecules, and empirically by combining different vehicles expressing the same immunogen in heterologous prime-boost protocols. In any case, a successful vaccination strategy against HIV as well as other chronic viral infections has to elicit better immune responses than the natural infections do.     

Virus-specific interleukin-17-producing CD4+ T cells are detectable in early human immunodeficiency virus type 1 infection

T(H)-17 cells have been shown to play a role in bacterial defense, acute inflammation, and autoimmunity. We examined the role of interleukin 17 (IL-17) production in human immunodeficiency virus type 1 (HIV-1) infection. Both HIV-1- and cytomegalovirus (CMV)-specific IL-17-producing CD4(+) T cells were detectable in early HIV-1 infection but were reduced to nondetectable levels in chronic and nonprogressive HIV-1 infection. IL-17-producing CMV-specific cells were not detected in blood from HIV-1-uninfected normal volunteers. Virus-specific T(H)-17 cells could coexpress other cytokines and could express CCR4 or CXCR3. Although the etiology of these cells has yet to be established, we propose that microbial translocation may induce them.     

T cell responses to human endogenous retroviruses in HIV-1 infection

Human endogenous retroviruses (HERVs) are remnants of ancient infectious agents that have integrated into the human genome. Under normal circumstances, HERVs are functionally defective or controlled by host factors. In HIV-1-infected individuals, intracellular defense mechanisms are compromised. We hypothesized that HIV-1 infection would remove or alter controls on HERV activity. Expression of HERV could potentially stimulate a T cell response to HERV antigens, and in regions of HIV-1/HERV similarity, these T cells could be cross-reactive. We determined that the levels of HERV production in HIV-1-positive individuals exceed those of HIV-1-negative controls. To investigate the impact of HERV activity on specific immunity, we examined T cell responses to HERV peptides in 29 HIV-1-positive and 13 HIV-1-negative study participants. We report T cell responses to peptides derived from regions of HERV detected by ELISPOT analysis in the HIV-1-positive study participants. We show an inverse correlation between anti-HERV T cell responses and HIV-1 plasma viral load. In HIV-1-positive individuals, we demonstrate that HERV-specific T cells are capable of killing cells presenting their cognate peptide. These data indicate that HIV-1 infection leads to HERV expression and stimulation of a HERV-specific CD8+ T cell response. HERV-specific CD8+ T cells have characteristics consistent with an important role in the response to HIV-1 infection: a phenotype similar to that of T cells responding to an effectively controlled virus (cytomegalovirus), an inverse correlation with HIV-1 plasma viral load, and the ability to lyse cells presenting their target peptide. These characteristics suggest that elicitation of anti-HERV-specific immune responses is a novel approach to immunotherapeutic vaccination. As endogenous retroviral sequences are fixed in the human genome, they provide a stable target, and HERV-specific T cells could recognize a cell infected by any HIV-1 viral variant. HERV-specific immunity is an important new avenue for investigation in HIV-1 pathogenesis and vaccine design.     

Antibodies to the envelope glycoprotein of human T cell leukemia virus type 1 robustly activate cell-mediated cytotoxic responses and directly neutralize viral infectivity at multiple steps of the entry process

Infection of human cells by human T cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response, and therefore, represent attractive components for a HTLV-1 vaccine. To begin to explore the immunological properties of potential envelope-based subunit vaccine candidates, we have used a soluble recombinant surface glycoprotein (gp46, SU) fused to the Fc region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice. The recombinant SU protein is highly immunogenic and induces high titer Ab responses, facilitating selection of hybridomas that secrete mAbs targeting SU. Many of these mAbs recognize envelope displayed on the surface of HTLV-1-infected cells and virions and several of the mAbs robustly antagonize envelope-mediated membrane fusion and neutralize pseudovirus infectivity. The most potently neutralizing mAbs recognize the N-terminal receptor-binding domain of SU, though there is considerable variation in neutralizing proficiency of the receptor-binding domain-targeted mAbs. By contrast, Abs targeting the C-terminal domain of SU tend to lack robust neutralizing activity. Importantly, we find that both neutralizing and poorly neutralizing Abs strongly stimulate neutrophil-mediated cytotoxic responses to HTLV-1-infected cells. Our data demonstrate that recombinant forms of SU possess immunological features that are of significant utility to subunit vaccine design.     

Endemic impact of human T cell leukemia virus type 1 screening in bone allografts

Allograft bone is a widely used as a convenient tool for reconstructing massive bone defects in orthopedic surgery. However, allografts are associated with the risk of viral disease transmission. One of the viruses transmitted in this manner is human T-lymphotropic virus type 1 (HTLV-1), which is found worldwide but is unevenly distributed. The southwestern parts of Japan are a highly endemic for HTLV-1. We investigated the HTLV-1 seroprevalence in candidate allograft donors at the regional bone bank in Kagoshima, Japan during its first 5 years of service. Between 2008 and 2012, we collected 282 femoral heads at the Kagoshima regional bone bank from living donors with osteoarthritis of the hip joint. Among the 282 candidate donors, 32 donors (11.3 %) were seropositive for anti-HTLV-1 antibody; notably, this prevalence is higher than that reported for blood donors in this area. Additionally, to determine if HTLV-1 genes are detectable after processing, we examined the bone marrow of the femoral heads from seropositive donors by conducting PCR assays. Our results confirm the existence of viral genes following the heat treatment processing of the femoral heads. Therefore, it is important to inactivate a virus completely by heat-treatment. Together, our findings highlight the importance of HTLV-1 screening at bone banks, particularly in HTLV-1-endemic areas such as southwest Japan.     

Analysis of potential phosphorylation sites in human T cell leukemia virus type 1 Tax

The human T cell leukemia virus type 1 (HTLV-1) Tax is a phosphoprotein, however, the contribution of phosphorylation to Tax activity is unknown. Previous studies have shown that phosphorylation of Tax occurs on serine residue(s), within one tryptic fragment, in response to 4-phorbol-12-myristate-13-acetate, in both mouse and human cells. Studies were conducted in multiple cell lines to identify the specific phosphorylated serines as a prelude to functional analysis. The phosphorylation pattern of Tax was found to be different in 293T and COS-7 cells in comparison with MT-4 and Px-1 cells. However, one tryptic fragment remained consistent in comigration analyses among all cell lines. Using selected Tax serine mutants a tryptic fragment containing a serine at residue 113 believed to be the site of phosphorylation of Tax did not comigrate with the common phosphorylated tryptic fragment. Analysis of selected Tax mutants for ability totrans-activate the cytomegalovirus promoter demonstrated mutation of serine 77 to alanine reducedtrans-activation by 90% compared to wild-type Tax. However, examination of the phosphorylation pattern of the serine 77 mutant demonstrated that it is not the site of phosphorylation. These studies demonstrate the importance of using relevant cell lines to characterize the role of phosphorylation in protein function.     

The human T cell immune response to Epstein-Barr virus

The Epstein-Barr virus (EBV) is a gamma-herpes virus which establishes latent, life-long infection in more than 95% of the human adult population. Despite its growth transforming capacity, most carriers control EBV associated malignacies efficiently and remain free of EBV+ tumors. Though EBV is controlled by a potent immune response, this virus uses latency to persist in vivo. This review summarizes work which has been done to characterize T cell responses to EBV. The CD8 T cell responses are rather well characterized and have been shown by several groups to be highly focused towards early lytic antigens. Much less is known about CD4 T cell epitopes, due to the small size of the CD4 compartment. However, recent data indicate a control of lytic and latent cycles of EBV by specific CD4+ T cells. A clear understanding of the T cell response to EBV is important with a view to developing immunotherapies for the virus and its related malignancies.     

B cells drive early T cell autoimmunity in vivo prior to dendritic cell-mediated autoantigen presentation

Both B cells and dendritic cells (DCs) have been implicated as autoantigen-presenting cells in the activation of self-reactive T cells. However, most self-proteins are ubiquitously and/or developmentally expressed, making it difficult to determine the source and the exposure of autoantigens to APCs in a controlled manner. In this study, we have used an Ig transgenic mouse model to examine the mechanisms by which B cells and other APCs acquire and present lupus autoantigens in vivo. Targeting a lupus autoantigen, the small nuclear ribonucleoprotein particle D protein, to the BCR activates autoreactive T cells in the periphery. Our in vivo studies demonstrate that autoantigen-specific B cells, when present in the repertoire, are the first subset of APCs to capture and present self-proteins for activating T cells. Thereafter, DCs acquire self-Ag and become effective APCs for stimulating the same subsets of autoreactive T cells. This mechanism provides one explanation of how early steps in autoimmunity can focus responses, via BCR, at a small group of self-proteins among the total milieu of intracellular self-proteins. Subsequently, DCs and other professional APCs may then amplify and perpetuate the autoimmune T cell response.     

Human T cells recovered from human/Balb radiation chimeras are hypersensitive to human immunodeficiency virus type 1 infection.

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated by virus-encoded regulatory proteins, as well as by a variety of cellular factors. Productive infection of human T lymphocytes by HIV-1 is dependent upon the activation status of the target cells. In general, short-term mitogenic stimulation of CD4 T cells is used to enhance infection of peripheral blood mononuclear cells (PBMC) in vitro. Recently, we demonstrated that adoptive transfer of human PBMC into lethally irradiated BALB/c mice, radioprotected with severe combined immunodeficiency (SCID) mouse bone marrow, leads to marked T-cell activation and proliferation. In the present study, we investigated the effect of such xenoactivation of human T cells on their susceptibility to HIV-1 infection. Human cells that were recovered from human/Balb radiation chimeras supported efficient replication of laboratory strains of HIV-1, as well as of HIV-1 clinical isolates. The multiplicity of infection required to attain effective virus replication in the recovered xenoactivated human cells was 10- to 100-fold lower than that needed for infection of short- or long-term phytohemagglutinin (PHA)-stimulated blasts or of various T-cell lines. Analysis of human cell surface activation markers has indicated that xenoactivation in the mouse, in contrast to in vitro stimulation with PHA, is associated with a marked downregulation of CD25 (interleukin 2 receptor). Our results demonstrate that human cells recovered from human/Balb radiation chimeras, which are hypersensitive to HIV-1 infection, differ from in vitro-stimulated cells in their activation status. Therefore, this system could be used to study host factors that participate in HIV-1 infection and replication in vitro and in vivo.     

Conferral of enhanced natural killer cell function by KIR3DS1 in early human immunodeficiency virus type 1 infection

A flurry of recent reports on the role of activating and inhibitory forms of the killer cell immunoglobulin-like receptors (KIR) in natural killer (NK) cell activity against human immunodeficiency virus type 1 (HIV-1) have yielded widely divergent results. The role of the activating NK receptor encoded by the KIR3DS1 allele and its putative ligands, members of the HLA class I Bw4Ile80 cluster, in early HIV-1 disease is controversial. We selected 60 treatment-naïve adults for study from the OPTIONS cohort of individuals with early HIV-1 infection in San Francisco. We performed NK cell functional assays measuring gamma interferon (IFN-γ) and CD107a expression by NK cells in the unstimulated state and after stimulation by the major histocompatibility complex class I-deficient 721.221 B-lymphoblastoid cell line. In addition, we measured CD38 expression (a T-cell activation marker) on T and NK cells. Persons who have at least one copy of the KIR3DS1 gene had higher IFN-γ and CD107a expression in the unstimulated state compared to those who do not possess this gene. After stimulation, both groups experienced a large induction of IFN-γ and CD107a, with KIR3DS1 carriers achieving a greater amount of IFN-γ expression. Differences in effector activity correlating with KIR3DS1 were not attributable to joint carriage of HLA Bw4Ile80 and KIR3DS1. We detected a partial but not complete dependence of KIR3DS1 on the members of B*58 supertype (B*57 and B*58) leading to higher NK cell function. Possessing KIR3DS1 was associated with lower expression of CD38 on both CD8⁺ T and NK cells and with a loss or weakening of the known strong associations between CD8⁺ T-cell expression of CD38 mean fluorescence intensity and the HIV-1 viral load. We observed that possessing KIR3DS1 was associated with higher NK cell effector functions in early HIV-1 disease, despite the absence of HLA Bw4Ile80, a putative ligand of KIR3DS1. Carriage of KIR3DS1 was associated with diminished CD8⁺ T-cell activation, as determined by expression of CD38, and a disruption of the traditional relationship between viral load and activation in HIV-1 disease, which may lead to better clinical outcomes for these individuals.NK cell function is regulated by a family of receptors encoded by the killer cell immunoglobulin-like receptor (KIR) genes (18, 33). Within the KIR family, certain genes encode inhibitory receptors that recognize HLA class I ligands (i.e., HLA-Bw4 or HLA-C), whereas other KIR genes encode activating receptors which are not completely known. Studies on the role of KIRs in human immunodeficiency virus (HIV) disease have focused on the activating receptor encoded by the KIR3DS1 allele. However, recent genetic association and functional studies of KIR and HIV disease have yielded widely disparate results on the role of KIR3DS1 and its putative ligands, a subset of HLA class I-B alleles referred to as Bw4Ile80. The Bw4Ile80 cluster is a subset of HLA-B alleles that bear an isoleucine at position 80 in the α-1 helix, on the rim of the peptide-binding cleft. The inhibitory receptors encoded by KIR3DL1 alleles, which are highly related in the extracellular domains to the activating receptor encoded by KIR3DS1, specifically recognize HLA-Bw4 ligands (5). Because of this similarity, KIR3DS1 has been assumed to also recognize Bw4Ile80 ligands. In 2002, Martin et al. reported that HIV-infected individuals in the Multicenter AIDS Cohort Study possessing the KIR3DS1 allele demonstrated significantly delayed progression to AIDS, provided that the individuals also expressed a Bw4Ile80 allele (20).In 2005, Gaudieri et al. reported on the association of the entire KIR gene cluster and HLA class I in HIV disease progression in an Australian HIV cohort (8, 9). These authors observed a trend toward slowed CD4⁺ T-cell percent loss among those who carried both Bw4Ile80 and KIR3DS1 (8). However, this trend was not statistically significant, and Gaudieri et al. simultaneously observed an acceleration of time to AIDS (1987 definition) among joint KIR3DS1 and Bw4Ile80 carriers. In 2006, Qi et al. published a follow-up report from the Multicenter AIDS Cohort Study cohort documenting an association between the coexpression of KIR3DS1 and Bw4Ile80 and enhanced protection against certain opportunistic infections in HIV-infected individuals (26), an effect partially attributed to very modest differences in viral load. In 2007, our group observed that KIR3DS1 gene carriage was associated with higher CD4⁺ T-cell counts and hence protection against HIV type 1 (HIV-1) progression in early disease (4); however, we observed that this effect was not attributable to differences in the viral load and further was independent of Bw4Ile80. In other words, our analyses suggested that the KIR3DS1 and Bw4Ile80 genes were each associated with protection against HIV disease but via different mechanisms.Until recently, it was not clear if KIR3DS1 was expressed on the surface of NK cells; however, two recent reports have conclusively established that KIR3DS1 is expressed on NK cells (6, 24) and that expression is dose dependent, with higher expression for homozygotes. These studies also demonstrated that KIR3DS1 recognizes neither HLA-Bw4 nor HLA-Bw6 ligands, at least when these major histocompatibility complex (MHC) class I molecules are expressed on Epstein-Barr virus-transformed B-lymphoblastoid cell lines. Similarly, an independent study by another group reported that KIR3DS1 fails to bind to soluble Bw4Ile80 tetrameric complexes (10). In contrast, Alter et al. have presented results from in vitro cytotoxicity assays suggesting that target cells possessing HLA-Bw4Ile80 are better targets for NK cells possessing KIR3DS1 (1); however, no evidence was provided to confirm a physical interaction between the KIR3DS1 and HLA-Bw4 proteins.Here, we present a study of the NK cell phenotype and function in 60 treatment-naïve, recently HIV-1-infected persons with defined HLA-B and KIR3DS1/KIR3DL1 allotypes. We also measured the expression of CD38 on NK cells and CD8⁺ T cells, a widely used marker of disease progression and virulence in HIV research and a marker of immune activation. The expression of CD38, as measured by flow cytometry, is known to be elevated on CD8⁺ T cells in HIV disease, reaching steady-state levels in early HIV-1 infection (7), and predicts disease progression independently of the viral load (19). The individuals studied were selected from our recent genetic association study of KIR and HLA among 255 recently HIV-1-infected persons (4), in which KIR3DS1 carriage alone was associated with higher CD4⁺ T-cell counts, despite the absence of a difference in the viral loads. On the basis of these clinical findings, we performed this study to determine whether persons who carried the KIR3DS1 gene had enhanced NK cell phenotypic and functional profiles and if these profiles were further enhanced by carriage of the putative KIR3DS1 ligands encoded by HLA-Bw4Ile80 alleles. Flow cytometry-based detection of KIR3DS1 has been hampered by the absence of a monoclonal antibody that can bind to KIR3DS1 specifically and not cross-react with the related KIR3DL1 proteins (25). Hence, we used genotypic KIR assignments for our analyses rather than flow cytometry-based methods.     

Differential susceptibility to human immunodeficiency virus type 1 infection of myeloid and plasmacytoid dendritic cells

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) plays an important role in HIV-1 transmission and pathogenesis. Here, we studied the susceptibility of ex vivo-isolated CD11c+ myeloid DCs (MDCs) and CD123+ plasmacytoid DCs (PDCs) to HIV-1 infection and the function of these cells early after infection. Both DC subsets were susceptible to CCR5- and CXCR4-using HIV-1 isolates (BaL and IIIB, respectively). However, MDCs were more susceptible to HIV-1(BaL) infection than donor-matched PDCs. In addition, HIV-1(BaL) infected MDCs more efficiently than HIV-1(IIIB), whereas PDCs were equally susceptible to both isolates. While exposure to HIV-1 alone resulted in only weak maturation of DCs, Toll-like receptor 7/8 ligation induced full maturation in both infected and uninfected DCs. Maturation did not increase HIV-1 replication in infected DCs, and infected DCs retained their ability to produce tumor necrosis factor alpha after stimulation. Both HIV-1 isolates induced alpha interferon production exclusively in PDCs, irrespective of productive infection. In conclusion, PDCs and MDCs were susceptible to HIV-1 infection, but neither displayed functional defects as a consequence of infection. The difference in susceptibility of PDCs and MDCs to HIV-1 may have implications for HIV-1 transmission and DC-mediated transfer of HIV-1 to T cells.     

Lactoferrin prevents dendritic cell-mediated human immunodeficiency virus type 1 transmission by blocking the DC-SIGN--gp120 interaction

One of the cell types first encountered by human immunodeficiency virus type 1 (HIV-1) following sexual transmission are dendritic cells (DC). DC capture HIV-1 through C-type lectin receptors, of which the best studied example is DC-SIGN, which mediates HIV-1 internalization. DC can keep the virus infectious for several days and are able to transmit HIV-1 to CD4(+) T cells. We tested proteins from milk and serum for their ability to block DC-mediated HIV-1 transmission, of which bovine lactoferrin (bLF) is the most potent inhibitor. bLF binds strongly to DC-SIGN, thus preventing virus capture and subsequent transmission. Interestingly, bLF is a much more efficient inhibitor of transmission than human lactoferrin. Since bLF is nontoxic and easy to purify in large quantities, it is an interesting candidate microbicide against HIV-1. Another advantage of bLF is its ability to block HIV-1 replication in T cells. DC-mediated capture of a bLF-resistant HIV-1 variant that was selected during long-term culturing in T cells could still be blocked by bLF. This underscores the usefulness of bLF as a microbicide drug to prevent HIV-1 transmission.     

Reduced frequency, diversity, and function of human T cell leukemia virus type 1-specific CD8+ T cell in adult T cell leukemia patients

Human T cell lymphotropic virus type 1 (HTLV-1)-specific CTL are thought to be immune effectors that reduce the risk of adult T cell leukemia (ATL). However, in vivo conditions of anti-HTLV-1 CTL before and after ATL development have yet to be determined. To characterize anti-HTLV-1 CTL in asymptomatic HTLV-1 carriers (AC) and ATL patients, we analyzed the frequency and diversity of HTLV-1-specific CD8+ T cells in PBMC of 35 AC and 32 ATL patients using 16 distinct epitopes of HTLV-1 Tax or Env/HLA tetramers along with intracellular cytolytic effector molecules (IFN-gamma, perforin, and granzyme B). Overall frequency of subjects possessing Tax-specific CD8+ T cells was significantly lower in ATL than AC (53 vs 90%; p = 0.001), whereas the difference in Env-specific CD8+ T cells was not statistically significant. AC possessed Tax11-19/HLA-A*0201-specific tetramer+ cells by 90% and Tax301-309/HLA-A*2402-specific tetramer+ cells by 92%. Some AC recognized more than one epitope. In contrast, ATL recognized only Tax11-19 with HLA-A*0201 and Tax301-309 with HLA-A*2402 at frequencies of 30 and 55%. There were also significant differences in percentage of cells binding Tax11-19/HLA-A*0201 and Tax301-309/HLA-A*2402 tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-gamma in response to Tax. In contrast, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC. Frequency of Tax-specific CD8+ T cells in AC was related to proviral load in HLA-A*0201. These results suggest that decreased frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be one of the risks of ATL development.