Combination Immune Checkpoint Blockade Enhances IL-2 and CD107a Production from HIV-Specific T Cells Ex Vivo in People Living with HIV on Antiretroviral Therapy.

In people with HIV (PWH) on antiretroviral therapy (ART), immune dysfunction persists, including elevated expression of immune checkpoint (IC) proteins on total and HIV-specific T cells. Reversing immune exhaustion is one strategy to enhance the elimination of HIV-infected cells that persist in PWH on ART. We aimed to evaluate whether blocking CTL-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell Ig domain and mucin domain 3 (TIM-3), T cell Ig and ITIM domain (TIGIT) and lymphocyte activation gene-3 (LAG-3) alone or in combination would enhance HIV-specific CD4+ and CD8+ T cell function ex vivo. Intracellular cytokine staining was performed using human PBMCs from PWH on ART (n = 11) and expression of CD107a, IFN-γ, TNF-α, and IL-2 was quantified with HIV peptides and Abs to IC. We found the following: 1) IC blockade enhanced the induction of CD107a and IL-2 but not IFN-γ and TNF-α in response to Gag and Nef peptides; 2) the induction of CD107a and IL-2 was greatest with multiple combinations of two Abs; and 3) Abs to LAG-3, CTLA-4, and TIGIT in combinations showed synergistic induction of IL-2 in HIV-specific CD8+ and CD107a and IL-2 production in HIV-specific CD4+ and CD8+ T cells. These results demonstrate that the combination of Abs to LAG-3, CTLA-4, or TIGIT can increase the frequency of cells expressing CD107a and IL-2 that associated with cytotoxicity and survival of HIV-specific CD4+ and CD8+ T cells in PWH on ART. These combinations should be further explored for an HIV cure.

Relationship between CD4 T cell turnover, cellular differentiation and HIV persistence during ART.

The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p<0.001). TEM had the fastest replacement rates, were most highly enriched for intDNA and caRNA, and contained the most clonal proviral expansion. Clonal proviruses detected in less mature subpopulations were more expanded in TEM, suggesting that they were maintained through cell differentiation. Earlier ART initiation was associated with lower levels of intDNA, caRNA and fractional replacement rates. In conclusion, circulating integrated HIV proviruses appear to be maintained both by slow turnover of immature CD4 subpopulations, and by clonal expansion as well as cell differentiation into effector cells with faster replacement rates.

Diverse effects of interferon alpha on the establishment and reversal of HIV latency.

HIV latency is the major barrier to a cure for people living with HIV (PLWH) on antiretroviral therapy (ART) because the virus persists in long-lived non-proliferating and proliferating latently infected CD4+ T cells. Latently infected CD4+ T cells do not express viral proteins and are therefore not visible to immune mediated clearance. Therefore, identifying interventions that can reverse latency and also enhance immune mediated clearance is of high interest. Interferons (IFNs) have multiple immune enhancing effects and can inhibit HIV replication in activated CD4+ T cells. However, the effects of IFNs on the establishment and reversal of HIV latency is not understood. Using an in vitro model of latency, we demonstrated that plasmacytoid dendritic cells (pDC) inhibit the establishment of HIV latency through secretion of type I IFNα, IFNß and IFNω but not IFNε or type III IFNλ1 and IFNλ3. However, once latency was established, IFNα but no other IFNs were able to efficiently reverse latency in both an in vitro model of latency and CD4+ T cells collected from PLWH on suppressive ART. Binding of IFNα to its receptor expressed on primary CD4+ T cells did not induce activation of the canonical or non-canonical NFκB pathway but did induce phosphorylation of STAT1, 3 and 5 proteins. STAT5 has been previously demonstrated to bind to the HIV long terminal repeat and activate HIV transcription. We demonstrate diverse effects of interferons on HIV latency with type I IFNα; inhibiting the establishment of latency but also reversing HIV latency once latency is established.

Combination Immune Checkpoint Blockade to Reverse HIV Latency.

In people living with HIV on antiretroviral therapy, HIV latency is the major barrier to a cure. HIV persists preferentially in CD4+ T cells expressing multiple immune checkpoint (IC) molecules, including programmed death (PD)-1, T cell Ig and mucin domain-containing protein 3 (TIM-3), lymphocyte associated gene 3 (LAG-3), and T cell immunoreceptor with Ig and ITIM domains (TIGIT). We aimed to determine whether these and other IC molecules have a functional role in maintaining HIV latency and whether blocking IC molecules with Abs reverses HIV latency. Using an in vitro model that establishes latency in both nonproliferating and proliferating human CD4+ T cells, we show that proliferating cells express multiple IC molecules at high levels. Latent infection was enriched in proliferating cells expressing PD-1. In contrast, nonproliferating cells expressed IC molecules at significantly lower levels, but latent infection was enriched in cells expressing PD-1, TIM-3, CTL-associated protein 4 (CTLA-4), or B and T lymphocyte attenuator (BTLA). In the presence of an additional T cell-activating stimulus, staphylococcal enterotoxin B, Abs to CTLA-4 and PD-1 reversed HIV latency in proliferating and nonproliferating CD4+ T cells, respectively. In the absence of staphylococcal enterotoxin B, only the combination of Abs to PD-1, CTLA-4, TIM-3, and TIGIT reversed latency. The potency of latency reversal was significantly higher following combination IC blockade compared with other latency-reversing agents, including vorinostat and bryostatin. Combination IC blockade should be further explored as a strategy to reverse HIV latency.

CXCR4-Using HIV Strains Predominate in Naive and Central Memory CD4+ T Cells in People Living with HIV on Antiretroviral Therapy: Implications for How Latency Is Established and Maintained.

HIV can persist in people living with HIV (PLWH) on antiretroviral therapy (ART) in multiple CD4+ T cell subsets, including naive cells, central memory (CM) cells, transitional (TM) cells, and effector memory (EM) cells. Since these cells express different levels of the viral coreceptors CXCR4 and CCR5 on their surface, we sought to determine whether the HIV envelope protein (Env) was genotypically and phenotypically different between CD4+ T cell subsets isolated from PLWH on suppressive ART (n = 8). Single genome amplification for the HIV env gene was performed on genomic DNA extracts from different CD4+ T cell subsets. We detected CXCR4-using (X4) strains in five of the eight participants studied, and in these participants, the prevalence of X4 strains was higher in naive CD4+ T cells than in the memory subsets. Conversely, R5 strains were mostly found in the TM and EM populations. Identical sets of env sequences, consistent with clonal expansion of some infected cells, were more frequent in EM cells. These expanded identical sequences could also be detected in multiple CD4+ T cell subsets, suggesting that infected cells can undergo T cell differentiation. These identical sequences largely encoded intact and functional Env proteins. Our results are consistent with a model in which X4 HIV strains infect and potentially establish latency in naive and CM CD4+ T cells through direct infection, in addition to maintenance of the reservoir through differentiation and proliferation of infected cells.IMPORTANCE In people living with HIV (PLWH) on suppressive ART, latent HIV can be found in a diverse range of CD4+ T cells, including quiescent naive and central memory cells that are typically difficult to infect in vitro It is currently unclear how latency is established in these cells in vivo We show that in CD4+ T cells from PLWH on suppressive ART, the use of the coreceptor CXCR4 was prevalent among viruses amplified from naive and central memory CD4+ T cells. Furthermore, we found that expanded numbers of identical viral sequences were most common in the effector memory population, and these identical sequences were also found in multiple different CD4+ T cell subsets. Our results help to shed light on how a range of CD4+ T cell subsets come to harbor HIV DNA, which is one of the major barriers to eradicating the virus from PLWH.

Human Immunodeficiency Virus (HIV)-Infected CCR6+ Rectal CD4+ T Cells and HIV Persistence On Antiretroviral Therapy.

BACKGROUND: Identifying where human immunodeficiency virus (HIV) persists in people living with HIV and receiving antiretroviral therapy is critical to develop cure strategies. We assessed the relationship of HIV persistence to expression of chemokine receptors and their chemokines in blood (n = 48) and in rectal (n = 20) and lymph node (LN; n = 8) tissue collected from people living with HIV who were receiving suppressive antiretroviral therapy. METHODS: Cell-associated integrated HIV DNA, unspliced HIV RNA, and chemokine messenger RNA were quantified by quantitative polymerase chain reaction. Chemokine receptor expression on CD4+ T cells was determined using flow cytometry. RESULTS: Integrated HIV DNA levels in CD4+ T cells, CCR6+CXCR3+ memory CD4+ T-cell frequency, and CCL20 expression (ligand for CCR6) were highest in rectal tissue, where HIV-infected CCR6+ T cells accounted for nearly all infected cells (median, 89.7%). Conversely in LN tissue, CCR6+ T cells were infrequent, and there was a statistically significant association of cell-associated HIV DNA and RNA with CCL19, CCL21, and CXCL13 chemokines. CONCLUSIONS: HIV-infected CCR6+ CD4+ T cells accounted for the majority of infected cells in rectal tissue. The different relationships between HIV persistence and T-cell subsets and chemokines in rectal and LN tissue suggest that different tissue-specific strategies may be required to eliminate HIV persistence and that assessment of biomarkers for HIV persistence may not be generalizable between blood and other tissues.

Myeloid Dendritic Cells Induce HIV Latency in Proliferating CD4+ T Cells.

HIV latency occurs predominantly in long-lived resting CD4+ T cells; however, latent infection also occurs in T cell subsets, including proliferating CD4+ T cells. We compared the establishment and maintenance of latent infection in nonproliferating and proliferating human CD4+ T cells cocultured with syngeneic myeloid dendritic cells (mDC). Resting CD4+ T cells were labeled with the proliferation dye eFluor 670 and cultured alone or with mDC, plasmacytoid dendritic cells, or monocytes in the presence of staphylococcal enterotoxin B (SEB). Cells were cultured for 24 h and infected with CCR5-tropic enhanced GFP (EGFP) reporter HIV. Five days postinfection, nonproductively infected EGFP- CD4+ T cells that were either nonproliferating (eFluor 670hi) or proliferating (eFluor 670lo) were sorted and cultured for an additional 7 d (day 12) with IL-7 and antiretrovirals. At day 5 postinfection, sorted, nonproductively infected T cells were stimulated with anti-CD3/CD28, and induced expression of EGFP was measured to determine the frequency of latent infection. Integrated HIV in these cells was confirmed using quantitative PCR. By these criteria, latent infection was detected at day 5 and 12 in proliferating T cells cocultured with mDC and monocytes but not plasmacytoid dendritic cells, where CD4+ T cells at day 12 were poor. At day 5 postinfection, nonproliferating T cells expressing SEB-specific TCR Vß-17 were enriched in latent infection compared with non-SEB-specific TCR Vß-8.1. Together, these data show that both nonproliferating and proliferating CD4+ T cells can harbor latent infection during SEB-stimulated T cell proliferation and that the establishment of HIV latency in nonproliferating T cells is linked to expression of specific TCR that respond to SEB.

The Pathway To Establishing HIV Latency Is Critical to How Latency Is Maintained and Reversed.

HIV infection requires lifelong antiretroviral therapy because of the persistence of latently infected CD4+ T cells. The induction of virus expression from latently infected cells occurs following T cell receptor (TCR) activation, but not all latently infected cells respond to TCR stimulation. We compared two models of latently infected cells using an enhanced green fluorescent protein (EGFP) reporter virus to infect CCL19-treated resting CD4+ (rCD4+) T cells (preactivation latency) or activated CD4+ T cells that returned to a resting state (postactivation latency). We isolated latently infected cells by sorting for EGFP-negative (EGFP-) cells after infection. These cells were cultured with antivirals and stimulated with anti-CD3/anti-CD28, mitogens, and latency-reversing agents (LRAs) and cocultured with monocytes and anti-CD3. Spontaneous EGFP expression was more frequent in postactivation than in preactivation latency. Stimulation of latently infected cells with monocytes/anti-CD3 resulted in an increase in EGFP expression compared to that for unstimulated controls using the preactivation latency model but led to a reduction in EGFP expression in the postactivation latency model. The reduced EGFP expression was not associated with reductions in the levels of viral DNA or T cell proliferation but depended on direct contact between monocytes and T cells. Monocytes added to the postactivation latency model during the establishment of latency reduced spontaneous virus expression, suggesting that monocyte-T cell interactions at an early time point postinfection can maintain HIV latency. This direct comparison of pre- and postactivation latency suggests that effective strategies needed to reverse latency will depend on how latency is established.IMPORTANCE One strategy being evaluated to eliminate latently infected cells that persist in HIV-infected individuals on antiretroviral therapy (ART) is to activate HIV expression or production with the goal of inducing virus-mediated cytolysis or immune-mediated clearance of infected cells. The gold standard for the activation of latent virus is T cell receptor stimulation with anti-CD3/anti-CD28. However, this stimulus activates only a small proportion of latently infected cells. We show clear differences in the responses of latently infected cells to activating stimuli based on how latent infection is established, an observation that may potentially explain the persistence of noninduced intact proviruses in HIV-infected individuals on ART.

Identifying Cytomegalovirus Complications Using the Quantiferon-CMV Assay After Allogeneic Hematopoietic Stem Cell Transplantation.

Background: A simple test to identify recovery of CMV-specific T-cell immunity following hematopoietic stem cell transplantation (HSCT) could assist clinicians in managing CMV-related complications. Methods: In an observational, multicenter, prospective study of 94 HSCT recipients we evaluated CMV-specific T-cell immunity at baseline, 3, 6, 9, and 12 months after transplant using the Quantiferon-CMV, an enzyme-linked immunosorbent spot assay (ELISpot), and intracellular cytokine staining. Results: At 3 months after HSCT, participants who developed CMV disease (n = 8) compared with CMV reactivation (n = 26) or spontaneous viral control (n = 25) had significantly lower CD8+ T-cell production of interferon-γ (IFN-γ) in response to CMV antigens measured by Quantiferon-CMV (P = .0008). An indeterminate Quantiferon-CMV result had a positive predictive value of 83% and a negative predictive value of 98% for identifying participants at risk of further CMV reactivation. Participants experiencing CMV reactivation compared with patients without CMV reactivation had a reduced proportion of polyfunctional (IFN-γ+/tumor necrosis factor α-positive) CD4+ and CD8+ T cells and a higher proportion of interleukin 2-secreting cells (P = .01 and P = .002, respectively). Conclusions: Quantifying CMV-specific T-cell immunity after HSCT can identify participants at increased risk of clinically relevant CMV-related outcomes.

Human Immunodeficiency Virus Persistence and T-Cell Activation in Blood, Rectal, and Lymph Node Tissue in Human Immunodeficiency Virus-Infected Individuals Receiving Suppressive Antiretroviral Therapy.

Background: Immune activation and inflammation remain elevated in human immunodeficiency virus (HIV)-infected individuals receiving antiretroviral therapy (ART) and may contribute to HIV persistence. Methods: Using flow cytometry expression of CD38, HLA-DR and PD-1 were measured in blood (n = 48), lymph node (LN; n = 9), and rectal tissue (n = 17) from virally suppressed individuals. Total and integrated HIV DNA, 2-LTR circles, and cell-associated unspliced HIV RNA were quantified. Results: CD4+ T cells from rectal tissue had a higher frequency of integrated HIV DNA compared with blood (4.26 fold-change in DNA; 95% confidence interval [CI] = 2.61-7.00; P < .001) and LN (2.32 fold-change in DNA; 95% CI = 1.22-4.41; P = .01). In rectal tissue, there were positive associations between integrated HIV DNA with PD-1+ CD4+ T-cells (1.44 fold-change in integrated HIV DNA per 10-unit increase in PD-1+ CD4+ T cells; 95% CI = 1.01-2.05; P = .045) and CD38+HLA-DR+ CD8+ T cells (1.40 fold-change in integrated HIV DNA per 1-unit increase in CD38+HLA-DR+ CD8+ T cells; 95% CI = 1.05-1.86; P = .02). Both associations were independent of current and nadir CD4+ T-cell counts. Conclusions: During ART, rectal tissue is an important reservoir for HIV persistence with a high frequency of activated CD4+ and CD8+ T cells. PD-1 may represent a marker of HIV persistence in rectal tissue.

Cytomegalovirus Reactivation Is Associated with Increased Risk of Late-Onset Invasive Fungal Disease after Allogeneic Hematopoietic Stem Cell Transplantation: A Multicenter Study in the Current Era of Viral Load Monitoring.

Opportunistic infections such as cytomegalovirus (CMV) reactivation and invasive fungal disease (IFD) cause significant morbidity and mortality to recipients of hematopoietic stem cell transplant (HSCT). We aimed to characterize the risk and relationship of CMV reactivation post-HSCT to IFD in the current era of CMV viral load monitoring using highly sensitive plasma DNA. A multicenter, retrospective, cohort study was conducted of consecutive patients undergoing allogeneic HSCT from January 2006 to December 2010 in Melbourne, Australia. CMV reactivation was defined as detection of plasma CMV DNA ≥ 546 IU/mL or development of CMV disease. IFD was classified in accordance with current international consensus guidelines. Of the 419 study participants, the median age was 44 years (IQR, 34 to 54), and CMV reactivation occurred in 106 participants (25%) at a median time of 56 days (IQR, 45 to 79). Thirty-eight participants (9.1%) were identified with 41 cases of IFD (n = 22 proven, n = 8 probable, n = 11 possible) at a median time of 76 days (IQR, 24 to 344). The incidence of IFD was higher in participants with CMV reactivation compared with no CMV reactivation (15% versus 7%, P = .012). In a multivariate analysis CMV reactivation remained an independent risk factor for IFD (hazard ratio, 3.7; 95% CI, 1.6 to 8.5; P = .002). The cumulative incidence of all IFD in patients with and without CMV reactivation using a competing risk regression was a hazard ratio of 2.2 (95% CI, 1.2 to 4.1; P = .017) and for late-onset IFD was a hazard ratio of 3.95 (95% CI, 1.7 to 9; P = .001). The median time to IFD onset was longer in participants with than without CMV reactivation (184 versus 37 days, P = .03). The peak viral load, detection of any level of viremia, and experiencing more than 1 episode of CMV reactivation were not associated with development of IFD. CMV reactivation in HSCT recipients in the post-transplant period is associated with an increased risk of developing late-onset IFD. Further research is warranted to understand the interaction between these 2 important infectious complications.

HIV integration sites in latently infected cell lines: evidence of ongoing replication.

BACKGROUND: Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. RESULTS: We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. CONCLUSION: Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.

HIV integration and the establishment of latency in CCL19-treated resting CD4(+) T cells require activation of NF-κB.

BACKGROUND: Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4(+) T cells. We previously reported that HIV latency could be established in resting CD4(+) T cells in the presence of the chemokine CCL19. To define how CCL19 facilitated the establishment of latent HIV infection, the role of chemokine receptor signalling was explored. RESULTS: In resting CD4(+) T cells, CCL19 induced phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt), nuclear factor kappa B (NF-κB), extracellular-signal-regulated kinase (ERK) and p38. Inhibition of the phosphoinositol-3-kinase (PI3K) and Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/ERK signalling pathways inhibited HIV integration, without significant reduction in HIV nuclear entry (measured by Alu-LTR and 2-LTR circle qPCR respectively). Inhibiting activation of MEK1/ERK1/2, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1) and NF-κB, but not p38, also inhibited HIV integration. We also show that HIV integrases interact with Pin1 in CCL19-treated CD4(+) T cells and inhibition of JNK markedly reduced this interaction, suggesting that CCL19 treatment provided sufficient signals to protect HIV integrase from degradation via the proteasome pathway. Infection of CCL19-treated resting CD4(+) T cells with mutant strains of HIV, lacking NF-κB binding sites in the HIV long terminal repeat (LTR) compared to infection with wild type virus, led to a significant reduction in integration by up to 40-fold (range 1-115.4, p = 0.03). This was in contrast to only a modest reduction of 5-fold (range 1.7-11, p > 0.05) in fully activated CD4(+) T cells infected with the same mutants. Finally, we demonstrated significant differences in integration sites following HIV infection of unactivated, CCL19-treated, and fully activated CD4(+) T cells. CONCLUSIONS: HIV integration in CCL19-treated resting CD4(+) T cells depends on NF-κB signalling and increases the stability of HIV integrase, which allow subsequent integration and establishment of latency. These findings have implications for strategies needed to prevent the establishment, and potentially reverse, latent infection.

Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy.

UNLABELLED: Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells. TRIAL REGISTRATION: ClinicalTrials.gov NCT01365065.

The role of antigen presenting cells in the induction of HIV-1 latency in resting CD4(+) T-cells.

BACKGROUND: Combination antiretroviral therapy (cART) is able to control HIV-1 viral replication, however long-lived latent infection in resting memory CD4(+) T-cells persist. The mechanisms for establishment and maintenance of latent infection in resting memory CD4(+) T-cells remain unclear. Previously we have shown that HIV-1 infection of resting CD4(+) T-cells co-cultured with CD11c(+) myeloid dendritic cells (mDC) produced a population of non-proliferating T-cells with latent infection. Here we asked whether different antigen presenting cells (APC), including subpopulations of DC and monocytes, were able to induce post-integration latent infection in resting CD4(+) T-cells, and examined potential cell interactions that may be involved using RNA-seq. RESULTS: mDC (CD1c(+)), SLAN(+) DC and CD14(+) monocytes were most efficient in stimulating proliferation of CD4(+) T-cells during syngeneic culture and in generating post-integration latent infection in non-proliferating CD4(+) T-cells following HIV-1 infection of APC-T cell co-cultures. In comparison, plasmacytoid DC (pDC) and B-cells did not induce latent infection in APC-T-cell co-cultures. We compared the RNA expression profiles of APC subpopulations that could and could not induce latency in non-proliferating CD4(+) T-cells. Gene expression analysis, comparing the CD1c(+) mDC, SLAN(+) DC and CD14(+) monocyte subpopulations to pDC identified 53 upregulated genes that encode proteins expressed on the plasma membrane that could signal to CD4(+) T-cells via cell-cell interactions (32 genes), immune checkpoints (IC) (5 genes), T-cell activation (9 genes), regulation of apoptosis (5 genes), antigen presentation (1 gene) and through unknown ligands (1 gene). CONCLUSIONS: APC subpopulations from the myeloid lineage, specifically mDC subpopulations and CD14(+) monocytes, were able to efficiently induce post-integration HIV-1 latency in non-proliferating CD4(+) T-cells in vitro. Inhibition of key pathways involved in mDC-T-cell interactions and HIV-1 latency may provide novel targets to eliminate HIV-1 latency.

Myeloid dendritic cells induce HIV-1 latency in non-proliferating CD4+ T cells.

Latently infected resting CD4(+) T cells are a major barrier to HIV cure. Understanding how latency is established, maintained and reversed is critical to identifying novel strategies to eliminate latently infected cells. We demonstrate here that co-culture of resting CD4(+) T cells and syngeneic myeloid dendritic cells (mDC) can dramatically increase the frequency of HIV DNA integration and latent HIV infection in non-proliferating memory, but not naïve, CD4(+) T cells. Latency was eliminated when cell-to-cell contact was prevented in the mDC-T cell co-cultures and reduced when clustering was minimised in the mDC-T cell co-cultures. Supernatants from infected mDC-T cell co-cultures did not facilitate the establishment of latency, consistent with cell-cell contact and not a soluble factor being critical for mediating latent infection of resting CD4(+) T cells. Gene expression in non-proliferating CD4(+) T cells, enriched for latent infection, showed significant changes in the expression of genes involved in cellular activation and interferon regulated pathways, including the down-regulation of genes controlling both NF-κB and cell cycle. We conclude that mDC play a key role in the establishment of HIV latency in resting memory CD4(+) T cells, which is predominantly mediated through signalling during DC-T cell contact.

Human immunodeficiency virus (HIV)-1 integration sites in viral latency.

The persistence of human immunodeficiency virus type 1 (HIV-1) in latent reservoirs is a major barrier to HIV cure. Reservoir establishment depends on low viral expression that may be related to provirus integration sites (IS). In vitro, in cell lines and primary T cells, latency is associated with specific IS through reduced viral expression mediated by transcriptional interference by host cellular promoters, reverse orientation, and the presence of specific epigenetic modifiers. In primary T cell models of latency, specific IS are associated with intracellular viral antigen expression that is not directly related to cell activation. In contrast, in patient CD4+ T cells, there is enrichment for IS in genes controlling cell cycle and survival and in some clonally expanded T cell subpopulations. Multiple insertion sites within some specific genes may suggest that integrated HIV can increase the host's T cell survival.

Identification of lineage relationships and novel markers of blood and skin human dendritic cells.

The lineage relationships and fate of human dendritic cells (DCs) have significance for a number of diseases including HIV where both blood and tissue DCs may be infected. We used gene expression profiling of human monocyte and DC subpopulations sorted directly from blood and skin to define the lineage relationships. We also compared these with monocyte-derived DCs (MDDCs) and MUTZ3 Langerhans cells (LCs) to investigate their relevance as model skin DCs. Hierarchical clustering analysis showed that myeloid DCs clustered according to anatomical origin rather than putative lineage. Plasmacytoid DCs formed the most discrete cluster, but ex vivo myeloid cells formed separate clusters of cells both in blood and in skin. Separate and specific DC populations could be determined within skin, and the proportion of CD14(+) dermal DCs (DDCs) was reduced and CD1a(+) DDCs increased during culture, suggesting conversion to CD1a(+)-expressing cells in situ. This is consistent with origin of the CD1a(+) DDCs from a local precursor rather than directly from circulating blood DCs or monocyte precursors. Consistent with their use as model skin DCs, the in vitro-derived MDDC and MUTZ3 LC populations grouped within the skin DC cluster. MDDCs clustered most closely to CD14(+) DDCs; furthermore, common unique patterns of C-type lectin receptor expression were identified between these two cell types. MUTZ3 LCs, however, did not cluster closely with ex vivo-derived LCs. We identified differential expression of novel genes in monocyte and DC subsets including genes related to DC surface receptors (including C-type lectin receptors, TLRs, and galectins).

Targeting antigen to bone marrow stromal cell-2 expressed by conventional and plasmacytoid dendritic cells elicits efficient antigen presentation.

Bone marrow stromal cell-2 (BST-2) has major roles in viral tethering and modulation of interferon production. Here we investigate BST-2 as a receptor for the delivery of antigen to dendritic cells (DCs). We show that BST-2 is expressed by a panel of mouse and human DC subsets, particularly under inflammatory conditions. The outcome of delivering antigen to BST-2 expressed by steady state and activated plasmacytoid DC (pDC) or conventional CD8(+) and CD8(-) DCs was determined. T-cell responses were measured for both MHC class I (MHCI) and MHC class II (MHCII) antigen presentation pathways in vitro. Delivering antigen via BST-2 was compared with that via receptors DEC205 or Siglec-H. We show that despite a higher antigen load and faster receptor internalisation, when antigen is delivered to steady state or activated pDC via BST-2, BST-2-targeted activated conventional DCs present antigen more efficiently. Relative to DEC205, BST-2 was inferior in its capacity to deliver antigen to the MHCI cross-presentation pathway. In contrast, BST-2 was superior to Siglec-H at initiating either MHCI or MHCII antigen presentation. In summary, BST-2 is a useful receptor to target with antigen, given its broad expression pattern and ability to access both MHCI and MHCII presentation pathways with relative efficiency.

Tissue dendritic cells as portals for HIV entry.

Dendritic cells (DCs) are found at the portals of pathogen entry such as the mucosal surfaces of the respiratory, gastrointestinal and genital tracts where they represent the first line of contact between the immune system and the foreign invaders. They are found throughout the body in multiple subsets where they express unique combinations of C-type lectin receptors to best aid them in detection of pathogens associated with their anatomical location. DCs are important in the establishment in HIV infection for two reasons. Firstly, they are one of the first cells to encounter the virus, and the specific interaction that occurs between these cells and HIV is critical to HIV establishing a foothold infection. Secondly and most importantly, HIV is able to efficiently transfer the virus to its primary target cell, the CD4(+) T lymphocyte, in which it replicates explosively. Infection of CD4(+) T lymphocytes via DCs is far more efficient than direct infection. This review surveys the various DCs subsets found within the human sexual mucosa and their interactions with HIV. Mechanisms of HIV uptake are discussed as well as how the virus then traffics through the DC and is transferred to T cells. Until recently, most research has focussed on vaginal transmission despite the increased transmission rate associated with anal intercourse. Here, we also discuss recent advances in our understanding of HIV transmission in the colon.