HIV-1-Specific Chimeric Antigen Receptor T Cells Fail To Recognize and Eliminate the Follicular Dendritic Cell HIV Reservoir In Vitro.

The major obstacle to a cure for HIV infection is the persistence of replication-competent viral reservoirs during antiretroviral therapy. HIV-specific chimeric antigen receptor (CAR) T cells have been developed to target latently infected CD4+ T cells that express virus either spontaneously or after intentional latency reversal. Whether HIV-specific CAR-T cells can recognize and eliminate the follicular dendritic cell (FDC) reservoir of HIV-bound immune complexes (ICs) is unknown. We created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a CAR construct that enables the expression of CD4 (domains 1 and 2) and the carbohydrate recognition domain of mannose binding lectin (MBL) to target native HIV Env (CD4-MBL CAR). We assessed CAR-T cell cytotoxicity using a carboxyfluorescein succinimidyl ester (CFSE) release assay and evaluated CAR-T cell activation through interferon gamma (IFN-γ) production and CD107a membrane accumulation by flow cytometry. CD4-MBL CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4+ T cells but were ineffective at targeting FDC bearing HIV-ICs. CD4-MBL CAR-T cells were unresponsive to cell-free HIV or concentrated, immobilized HIV-ICs in cell-free experiments. Blocking intercellular adhesion molecule-1 (ICAM-1) inhibited the cytolytic response of CD4-MBL CAR-T cells to Env-expressing cell lines and HIV-infected CD4+ T cells, suggesting that factors such as adhesion molecules are necessary for the stabilization of the CAR-Env interaction to elicit a cytotoxic response. Thus, CD4-MBL CAR-T cells are unable to eliminate the FDC-associated HIV reservoir, and alternative strategies to eradicate this reservoir must be sought.IMPORTANCE Efforts to cure HIV infection have focused primarily on the elimination of latently infected CD4+ T cells. Few studies have addressed the unique reservoir of infectious HIV that exists on follicular dendritic cells (FDCs), persists in vivo during antiretroviral therapy, and likely contributes to viral rebound upon cessation of antiretroviral therapy. We assessed the efficacy of a novel HIV-specific chimeric antigen receptor (CAR) T cell to target both HIV-infected CD4+ T cells and the FDC reservoir in vitro Although CAR-T cells eliminated CD4+ T cells that express HIV, they did not respond to or eliminate FDC bound to HIV. These findings reveal a fundamental limitation to CAR-T cell therapy to eradicate HIV.

Alpha-1-antitrypsin interacts with gp41 to block HIV-1 entry into CD4+ T lymphocytes.

BACKGROUND: Study of a clinic case reveals that alpha-1-antitrypsin (AAT) deficiency is related to CD4+ T cell count decline and AIDS progression, suggesting that AAT might be an endogenous inhibitor of HIV/AIDS. Previous study shows that AAT inhibits HIV-1 replication in infected host cells and the C-terminus fragment of AAT, VIRIP, interferes with HIV-1 infection. However, it is still unclear whether and how intact AAT inhibits HIV-1 infection. It is also unknown what the mechanism of AAT is and which critical step(s) are involved. RESULTS: In the present study, the C-terminus of AAT (C) was synthesized. C terminus-truncated AAT (ΔAAT) was also prepared by digesting AAT with metalloproteinase. Primary CD4+ T cells were then co-cultured with HIV-1 with the presence or absence of AAT/C/ΔAAT to detect cis-infection of HIV-1. The interaction between AAT/C/ΔAAT and gp120/gp41 was also measured. Meanwhile, HIV-1 reverse transcriptase activity and viral DNA integration were also detected in these lymphocytes. The results demonstrated that AAT and C, not ΔAAT, inhibited HIV-1 entry by directly interacting with gp41. Meanwhile, AAT, C and ΔAAT could not directly interfere with the steps of viral RNA reverse transcription and viral DNA integration. CONCLUSION: AAT inhibits HIV-1 entry by directly interacting with gp41 through its C-terminus and thereby inhibits HIV-1 infection.

HIV replication in CD4+ T lymphocytes in the presence and absence of follicular dendritic cells: inhibition of replication mediated by α-1-antitrypsin through altered IκBα ubiquitination.

Follicular dendritic cells (FDCs) increase HIV replication and virus production in lymphocytes by increasing the activation of NF-κB in infected cells. Because α-1-antitrypsin (AAT) decreases HIV replication in PBMCs and monocytic cells and decreases NF-κB activity, we postulated that AAT might also block FDC-mediated HIV replication. Primary CD4(+) T cells were infected with HIV and cultured with FDCs or their supernatant with or without AAT, and ensuing viral RNA and p24 production were monitored. NF-κB activation in the infected cells was also assessed. Virus production was increased in the presence of FDC supernatant, but the addition of AAT at concentrations >0.5 mg/ml inhibited virus replication. AAT blocked the nuclear translocation of NF-κB p50/p65 despite an unexpected elevation in associated phosphorylated and ubiquitinated IκBα (Ub-IκBα). In the presence of AAT, degradation of cytoplasmic IκBα was dramatically inhibited compared with control cultures. AAT did not inhibit the proteasome; however, it altered the pattern of ubiquitination of IκBα. AAT decreased IκBα polyubiquitination linked through ubiquitin lysine residue 48 and increased ubiquitination linked through lysine residue 63. Moreover, lysine reside 63-linked Ub-IκBα degradation was substantially slower than lysine residue 48-linked Ub-IκBα in the presence of AAT, correlating altered ubiquitination with a prolonged IκBα t(1/2). Because AAT is naturally occurring and available clinically, examination of its use as an inhibitory agent in HIV-infected subjects may be informative and lead to the development of similar agents that inhibit HIV replication using a novel mechanism.

The evolution of HIV: inferences using phylogenetics.

Molecular phylogenetics has revolutionized the study of not only evolution but also disparate fields such as genomics, bioinformatics, epidemiology, ecology, microbiology, molecular biology and biochemistry. Particularly significant are its achievements in population genetics as a result of the development of coalescent theory, which have contributed to more accurate model-based parameter estimation and explicit hypothesis testing. The study of the evolution of many microorganisms, and HIV in particular, have benefited from these new methodologies. HIV is well suited for such sophisticated population analyses because of its large population sizes, short generation times, high substitution rates and relatively small genomes. All these factors make HIV an ideal and fascinating model to study molecular evolution in real time. Here we review the significant advances made in HIV evolution through the application of phylogenetic approaches. We first examine the relative roles of mutation and recombination on the molecular evolution of HIV and its adaptive response to drug therapy and tissue allocation. We then review some of the fundamental questions in HIV evolution in relation to its origin and diversification and describe some of the insights gained using phylogenies. Finally, we show how phylogenetic analysis has advanced our knowledge of HIV dynamics (i.e., phylodynamics).

Follicular dendritic cells and human immunodeficiency virus type 1 transcription in CD4+ T cells.

HIV replication occurs throughout the natural course of infection in secondary lymphoid tissues and in particular within the germinal centers (GCs), where follicular dendritic cells (FDCs) are adjacent to CD4(+) T cells. Because FDCs provide signaling that increases lymphocyte activation, we postulated that FDCs could increase human immunodeficiency virus (HIV) replication. We cultured HIV-infected CD4(+) T cells alone or with FDCs and measured subsequent virus expression using HIV-p24 production and reverse transcription-PCR analyses. When cultured with FDCs, infected CD4(+) T cells produced almost fourfold more HIV than when cultured alone, and the rate of virus transcription was doubled. Both FDCs and their supernatant increased HIV transcription and resulted in nuclear translocation of NF-kappaB and phosphorylated c-Jun in infected cells. FDCs produced soluble tumor necrosis factor alpha (TNF-alpha) ex vivo, and the addition of a blocking soluble TNF receptor ablated FDC-mediated HIV transcription. Furthermore, TNF-alpha was found highly expressed within GCs, and ex vivo GC CD4(+) T cells supported greater levels of HIV-1 replication than other CD4(+) T cells. These data indicated that FDCs increase HIV transcription and production by a soluble TNF-alpha-mediated mechanism. This FDC-mediated effect may account, at least in part, for the presence of persistent HIV replication in GCs. Therefore, in addition to providing an important reservoir of infectious virus, FDCs increase HIV production, contributing to a tissue microenvironment that is highly conducive to HIV transmission and expression.

Characterization of the follicular dendritic cell reservoir of human immunodeficiency virus type 1.

Throughout the natural course of human immunodeficiency virus (HIV) infection, follicular dendritic cells (FDCs) trap and retain large quantities of particle-associated HIV RNA in the follicles of secondary lymphoid tissue. We have previously found that murine FDCs in vivo could maintain trapped virus particles in an infectious state for at least 9 months. Here we sought to determine whether human FDCs serve as an HIV reservoir, based on the criteria that virus therein must be replication competent, genetically diverse, and archival in nature. We tested our hypothesis using postmortem cells and tissues obtained from three HIV-infected subjects and antemortem blood samples obtained from one of these subjects. Replication competence was determined using coculture, while genetic diversity and the archival nature of virus were established using phylogenetic and population genetics methods. We found that FDC-trapped virus was replication competent and demonstrated greater genetic diversity than that of virus found in most other tissues and cells. Antiretrovirus-resistant variants that were not present elsewhere were also detected on FDCs. Furthermore, genetic similarity was observed between FDC-trapped HIV and viral species recovered from peripheral blood mononuclear cells obtained 21 and 22 months antemortem, but was not present in samples obtained 4 and 18 months prior to the patient's death, indicating that FDCs can archive HIV. These data indicate that FDCs represent a significant reservoir of infectious and diverse HIV, thereby providing a mechanism for viral persistence for months to years.

Multivariate profiling of African green monkey and rhesus macaque T lymphocytes.

The complexity of immune responses limits the usefulness of univariate methods in answering complex immunology questions. To demonstrate the utility of a multivariate approach, we employ such approach to compare T cells of African green monkeys (AGMs) and rhesus macaques (RMs). Among the most prominent distinguishing features we found were lower CD3 and higher CD28 surface expression in AGMs compared to RMs. After in vitro stimulation, a larger proportion of AGM T cells secreted cytokines, especially those producing more than one cytokine (i.e. multifunctional cells). To find out whether multifunctional responses associate with protection in other species, we compared T cells of cynomolgus macaques (CMs) infected with wild-type Simian Immunodeficiency Virus (SIV) to those of CMs infected (vaccinated) with a replication-defective virus. Wild-type SIV infection in macaques leads to simian Acquired Immunodeficiency Syndrome (AIDS), which does not happen in animals previously vaccinated with a replication-defective virus. Interestingly, after in vitro stimulation, multifunctional cells were more abundant among T cells of vaccinated CMs. Our results propose T-cell multifunctionality as a potentially useful marker of immunity, although additional verification is needed. Finally, we hope our multivariate model and its associated validation methods will inform future studies in the field of immunology.

Proteomic and phototoxic characterization of melanolipofuscin: correlation to disease and model for its origin.

PURPOSE: Melanolipofuscin (MLF) is a complex granule, exhibiting properties of both melanosomes and lipofuscin (LF) granules, which accumulates in retinal pigment epithelial (RPE) cells and may contribute to the etiology of age-related macular degeneration (AMD). MLF accumulation has been reported by Feeney-Burns to more closely reflect the onset of AMD than the accumulation of lipofuscin. In an effort to assess the possible contribution MLF may have to the onset of AMD, we analyzed the phototoxicity and protein composition of MLF and compared those results to that of LF. METHODS: Specifically, we observed the accumulation of MLF in human RPE from different decades of life, and assessed the phototoxicity of these granules. We also employed fluorescence spectroscopy, atomic force microscopy, transmission and scanning electron microscopy and proteomic analysis to examine the composition of MLF granules in an effort to ascertain their origin. RESULTS: Our results show that MLF granules are phototoxic and their accumulation more closely reflects the onset of AMD than does LF accumulation. Our compositional analysis of MLF has shown that while these granules contain some similarities to LF granules, MLF is substantially different. Of significant interest is the finding that MLF, in contrast to LF, does not contain photoreceptor-specific proteins, suggesting that MLF may not originate from the phagocytosis of photoreceptor outer segments. Instead the presence of RPE- and melanosome-specific proteins would suggest that MLF accumulates as a result of the melanosomal autophagocytosis of RPE cells. CONCLUSIONS: Our results provide significant insight into understanding the formation and toxicity of MLF and suggest a possible contribution to the etiology of retinal diseases.

Examining the proteins of functional retinal lipofuscin using proteomic analysis as a guide for understanding its origin.

PURPOSE: To elucidate the origins of biologically active retinal lipofuscin (RLF) by examining its protein composition. METHODS: Total protein and total lipid were extracted and quantified. Proteins in this lipoprotein granule were identified by limited-scale proteomic analysis using both two-dimensional (2D) gel electrophoresis and SDS-PAGE coupled with MALDI-QqToF MSMS and automated LCMSMS, respectively. RESULTS: RLF granules were 44% protein and 50% lipid. Proteomic analyses identified 41 constituent proteins. Hydrophobic proteins and several proteins specific to photoreceptors, including rhodopsin, that have not previously been reported, were identified. Extensive protein modification, especially oxidative damage, was observed. CONCLUSIONS: Proteins identified support the model that RLF accumulates in RPE cells as a result of the buildup of undigested material from the phagocytosis of photoreceptor outer segments. Perhaps oxidative damage renders some of these proteins indigestible and thus leads to the accumulation of RLF granules.

Low-density lipoprotein receptor-related protein 1 mediates α1-antitrypsin internalization in CD4+ T lymphocytes.

In α1-antitrypsin-deficient HIV patients, an accelerated decline of CD4(+) T cell numbers is observed, suggesting that α1-antitrypsin is a potential endogenous HIV inhibitor. In infected T lymphocytes, α1-antitrypsin potently blocks NF-κB activation and HIV-1 replication by directly interacting with IκBα in the cytosol, thereby altering its ubiquitination pattern. However, the mechanism of α1-antitrypsin entry into the cytosol, where IκBα locates, remains unclear. In the present study, we investigated the mechanism of α1-antitrypsin internalization in CD4(+) T cells. Thus, primary CD4(+) T cells were infected with HIV-1 and then incubated with α1-antitrypsin to detect its internalization. We found that CD4(+) T cells internalized α1-antitrypsin through a clathrin-dependent endocytosis process. Next, intracellular α1-antitrypsin exerted the inhibitory effect on NF-κB activation and HIV-1 replication. On primary CD4(+) T cells, α1-antitrypsin interacted with low-density lipoprotein receptor-related protein 1 to initiate the internalization. Inside CD4(+) T lymphocytes, α1-antitrypsin was transported from the endosome to the lysosome and then released into the cytosol, where it is possible for α1-antitrypsin to directly interact with IκBα. These results together suggest that α1-antitrypsin internalization is a clathrin-dependent and low-density lipoprotein receptor-related protein 1-mediated endocytosis process. Internalized α1-antitrypsin is transported through the endosome-lysosome-cytosol routine to interact with cytosolic IκBα and block NF-κB activation and HIV-1 replication.

Cumulative mechanisms of lymphoid tissue fibrosis and T cell depletion in HIV-1 and SIV infections.

The hallmark of HIV-1 and SIV infections is CD4(+) T cell depletion. Both direct cell killing and indirect mechanisms related to immune activation have been suggested to cause the depletion of T cells. We have now identified a mechanism by which immune activation-induced fibrosis of lymphoid tissues leads to depletion of naive T cells in HIV-1 infected patients and SIV-infected rhesus macaques. The T regulatory cell response to immune activation increased procollagen production and subsequent deposition as fibrils via the TGF-ß1 signaling pathway and chitinase 3-like-1 activity in fibroblasts in lymphoid tissues from patients infected with HIV-1. Collagen deposition restricted T cell access to the survival factor IL-7 on the fibroblastic reticular cell (FRC) network, resulting in apoptosis and depletion of T cells, which, in turn, removed a major source of lymphotoxin-ß, a survival factor for FRCs during SIV infection in rhesus macaques. The resulting loss of FRCs and the loss of IL-7 produced by FRCs may thus perpetuate a vicious cycle of depletion of T cells and the FRC network. Because this process is cumulative, early treatment and antifibrotic therapies may offer approaches to moderate T cell depletion and improve immune reconstitution during HIV-1 infection.

Follicular dendritic cell contributions to HIV pathogenesis.

Early after infection, large quantities of HIV are trapped on follicular dendritic cells (FDCs) thus establishing a potent reservoir of infectious virus adjacent to highly susceptible CD4-bearing T lymphocytes. Throughout much of the disease course, active HIV infection is largely confined to sites surrounding FDCs suggesting that this microenvironment is highly conducive to infection. FDCs maintain HIV infectivity and trapped virus can cause infection even in the presence of neutralizing antibody. FDCs also contribute signaling to the germinal center microenvironment that appears to increase HIV infection and replication. This article discusses these FDC contributions to HIV pathogenesis.

Follicular dendritic cell regulation of CXCR4-mediated germinal center CD4 T cell migration.

Follicular dendritic cells (FDCs) up-regulate the chemokine receptor CXCR4 on CD4 T cells, and a major subpopulation of germinal center (GC) T cells (CD4(+)CD57(+)), which are adjacent to FDCs in vivo, expresses high levels of CXCR4. We therefore reasoned that GC T cells would actively migrate to stromal cell-derived factor-1 (CXCL12), the CXCR4 ligand, and tested this using Transwell migration assays with GC T cells and other CD4 T cells (CD57(-)) that expressed much lower levels of CXCR4. Unexpectedly, GC T cells were virtually nonresponsive to CXCL12, whereas CD57(-)CD4 T cells migrated efficiently despite reduced CXCR4 expression. In contrast, GC T cells efficiently migrated to B cell chemoattractant-1/CXCL13 and FDC supernatant, which contained CXCL13 produced by FDCs. Importantly, GC T cell nonresponsiveness to CXCL12 correlated with high ex vivo expression of regulator of G protein signaling (RGS), RGS13 and RGS16, mRNA and expression of protein in vivo. Furthermore, FDCs up-regulated both RGS13 and RGS16 mRNA expression in non-GC T cells, resulting in their impaired migration to CXCL12. Finally, GC T cells down-regulated RGS13 and RGS16 expression in the absence of FDCs and regained migratory competence to CXCL12. Although GC T cells express high levels of CXCR4, signaling through this receptor appears to be specifically inhibited by FDC-mediated expression of RGS13 and RGS16. Thus, FDCs appear to directly affect GC T cell migration within lymphoid follicles.

Follicular dendritic cells and the persistence of HIV infectivity: the role of antibodies and Fcgamma receptors.

Large quantities of HIV are found trapped on the surface of follicular dendritic cells (FDCs), and virus persists on these cells until they ultimately die. We recently found that FDCs maintain HIV infectivity for long periods in vivo and in vitro. Because FDCs trap Ags (and virus) in the form of immune complexes and are rich in FcgammaRs, we reasoned that Ab and FcgammaRs may be required for FDC-mediated maintenance of HIV infectivity. To investigate this hypothesis, HIV immune complexes were formed in vitro and incubated for increasing times with or without FDCs, after which the remaining infectious virus was determined by HIV-p24 production in rescue cultures. FDCs maintained HIV infectivity in vitro in a dose-dependent manner but required the presence of specific Ab for this activity regardless of whether laboratory-adapted or primary X4 and R5 isolates were tested. In addition, Abs against either virally or host-encoded proteins on the virion permitted FDC-mediated maintenance of HIV infectivity. We found that the addition of FDCs to HIV immune complexes at the onset of culture gave optimal maintenance of infectivity. Moreover, blocking FDC-FcgammaRs or killing the FDCs dramatically reduced their ability to preserve virus infectivity. Finally, FDCs appeared to decrease the spontaneous release of HIV-1 gp120, suggesting that FDC-virus interactions stabilize the virus particle, thus contributing to the maintenance of infectivity. Therefore, optimal maintenance of HIV infectivity requires both Ab against particle-associated determinants and FDC-FcgammaRs.

Follicular dendritic cell-mediated up-regulation of CXCR4 expression on CD4 T cells and HIV pathogenesis.

Follicular dendritic cells (FDCs) represent a major reservoir of HIV, and active infection occurs surrounding these cells, suggesting that this microenvironment is highly conducive to virus transmission. Because CD4 T cells around FDCs in germinal centers express the HIV coreceptor, CXCR4, whereas CD4 lymphocytes in many other sites do not, it prompted the hypothesis that FDCs may increase CXCR4 expression on CD4 T cells, thereby facilitating infection. To test this, HIV receptor/coreceptor expression was determined on CD4 T cells cultured with or without FDCs, and its consequence to infection was assessed by measuring virus binding and entry. FDCs had little effect on CCR5 or CD4 expression but increased CXCR4 expression on CD4 T cells. FDC-mediated up-regulation of CXCR4 on CD4 T cells occurred by 24 h and was sustained for at least 96 h in vitro, and FDC-CD4 T cell contact was necessary. Importantly, increased CXCR4 expression directly correlated with increased binding and entry of HIV-1 X4 isolates. Furthermore, CD4(+)CD57(+) germinal center T cells expressed high levels of CXCR4 and supported enhanced entry of X4 HIV compared with other CD4 T cells from the same tissue. Thus, in addition to serving as a reservoir of infectious virus, FDCs render surrounding germinal center T cells highly susceptible to infection with X4 isolates of HIV-1.