CCL2: a Chemokine Potentially Promoting Early Seeding of the Latent HIV Reservoir.

HIV infects long-lived CD4 memory T cells, establishing a latent viral reservoir that necessitates lifelong antiretroviral therapy (ART). How this reservoir is formed so quickly after infection remains unclear. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, leading to recruitment of cells expressing the CCR2 receptor, including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find (i) treatment of humanized mice with anti-CCL2 antibodies during early HIV infection decreases reservoir seeding and preserves CCR2/5+ cells and (ii) CCR2/5+ cells from the blood of HIV-infected individuals on long-term ART contain significantly more integrated provirus than CCR2/5-negative memory or naive cells. Together, these studies support a model where the host's innate inflammatory response to HIV infection, including CCL2 production, leads to the recruitment of CCR2/5+ central memory CD4 T cells to zones of virus-associated inflammation, likely contributing to rapid formation of the latent HIV reservoir. IMPORTANCE There are currently over 35 million people living with HIV worldwide, and we still have no vaccine or scalable cure. One of the difficulties with HIV is its ability to rapidly establish a viral reservoir in lymphoid tissues that allows it to elude antivirals and the immune system. Thus, it is important to understand how HIV accomplishes this so we can develop preventive strategies. Our current results show that an early inflammatory response to HIV infection includes production of the chemokine CCL2, which recruits a unique subset of CCR2/5+ CD4+ T cells that become infected and form a significant reservoir for latent infection. Furthermore, we show that blockade of CCL2 in humanized mice significantly reduces persistent HIV infection. This information is relevant to the development of therapeutics to prevent and/or treat chronic HIV infections.

Cell-Extrinsic Priming Increases Permissiveness of CD4+ T Cells to Human Immunodeficiency Virus Infection by Increasing C-C Chemokine Receptor Type 5 Co-receptor Expression and Cellular Activation Status.

The chemokine receptor CCR5 is expressed on multiple cell types, including macrophages, dendritic cells, and T cells, and is the major co-receptor used during HIV transmission. Using a standard αCD3/CD28 in vitro stimulation protocol to render CD4+ T cells from PBMCs permissive to HIV infection, we discovered that the percentage of CCR5+ T cells was significantly elevated in CD4+ T cells when stimulated in the presence of peripheral blood mononuclear cells (PBMCs) as compared to when stimulated as purified CD4+ T cells. This indicated that environmental factors unique to the T-PBMCs condition affect surface expression of CCR5 on CD4+ T cells. Conditioned media from αCD3/CD28-stimulated PBMCs induced CCR5 expression in cultures of unstimulated cells. Cytokine profile analysis of these media suggests IL-12 as an inducer of CCR5 expression. Mass cytometric analysis showed that stimulated T-PBMCs exhibited a uniquely activated phenotype compared to T-Pure. In line with increased CCR5 expression and activation status in stimulated T-PBMCs, CD4+ T cells from these cultures were more susceptible to infection by CCR5-tropic HIV-1 as compared with T-Pure cells. These results suggest that in order to increase ex vivo infection rates of blood-derived CD4+ T cells, standard stimulation protocols used in HIV infection studies should implement T-PBMCs or purified CD4+ T cells should be supplemented with IL-12.

Hyaluronic acid is a negative regulator of mucosal fibroblast-mediated enhancement of HIV infection.

The majority of HIV infections are established through the genital or rectal mucosa. Fibroblasts are abundant in these tissues, and although not susceptible to infection, can potently enhance HIV infection of CD4+ T cells. Hyaluronic acid (HA) is a major component of the extracellular matrix of fibroblasts, and its levels are influenced by the inflammatory state of the tissue. Since inflammation is known to facilitate HIV sexual transmission, we investigated the role of HA in genital mucosal fibroblast-mediated enhancement of HIV infection. Depletion of HA by CRISPR-Cas9 in primary foreskin fibroblasts augmented the ability of the fibroblasts to increase HIV infection of CD4+ T cells. This amplified enhancement required direct contact between the fibroblasts and CD4+ T cells, and could be attributed to both increased rates of trans-infection and the increased ability of HA-deficient fibroblasts to push CD4+ T cells into a state of higher permissivity to infection. This HIV-permissive state was characterized by differential expression of genes associated with regulation of cell metabolism and death. Our results suggest that conditions resulting in diminished cell-surface HA on fibroblasts, such as genital inflammation, can promote HIV transmission by conditioning CD4+ T cells toward a state more vulnerable to infection by HIV.

Evaluating a New Class of AKT/mTOR Activators for HIV Latency Reversing Activity Ex Vivo and In Vivo.

An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to SIV-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined.IMPORTANCE If combined with immune therapeutics, latency reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8+ T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing next generation LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.

Attacking Latent HIV with convertibleCAR-T Cells, a Highly Adaptable Killing Platform.

Current approaches to reducing the latent HIV reservoir entail first reactivating virus-containing cells to become visible to the immune system. A critical second step is killing these cells to reduce reservoir size. Endogenous cytotoxic T-lymphocytes (CTLs) may not be adequate because of cellular exhaustion and the evolution of CTL-resistant viruses. We have designed a universal CAR-T cell platform based on CTLs engineered to bind a variety of broadly neutralizing anti-HIV antibodies. We show that this platform, convertibleCAR-T cells, effectively kills HIV-infected, but not uninfected, CD4 T cells from blood, tonsil, or spleen and only when armed with anti-HIV antibodies. convertibleCAR-T cells also kill within 48 h more than half of the inducible reservoir found in blood of HIV-infected individuals on antiretroviral therapy. The modularity of convertibleCAR-T cell system, which allows multiplexing with several anti-HIV antibodies yielding greater breadth and control, makes it a promising tool for attacking the latent HIV reservoir.

Distinct mechanisms regulate IL1B gene transcription in lymphoid CD4 T cells and monocytes.

Interleukin 1ß is a pro-inflammatory cytokine important for both normal immune responses and chronic inflammatory diseases. The regulation of the 31 kDa proIL-1ß precursor coded by the IL1B gene has been extensively studied in myeloid cells, but not in lymphoid-derived CD4 T cells. Surprisingly, we found that some CD4 T cell subsets express higher levels of proIL-1ß than unstimulated monocytes, despite relatively low IL1B mRNA levels. We observed a significant increase in IL1B transcription and translation in CD4 T cells upon ex vivo CD3/CD28 activation, and a similar elevation in the CCR5+ effector memory population compared to CCR5- T cells in vivo. The rapid and vigorous increase in IL1B gene transcription for stimulated monocytes has previously been associated with the presence of Spi-1/PU.1 (Spi1), a myeloid-lineage transcription factor, pre-bound to the promoter. In the case of CD4 T cells, this increase occurred despite the lack of detectable Spi1 at the IL1B promoter. Additionally, we found altered epigenetic regulation of the IL1B locus in CD3/CD28-activated CD4 T cells. Unlike monocytes, activated CD4 T cells possess bivalent H3K4me3+/H3K27me3+ nucleosome marks at the IL1B promoter, reflecting low transcriptional activity. These results support a model in which the IL1B gene in CD4 T cells is transcribed from a low-activity bivalent promoter independent of Spi1. Accumulated cytoplasmic proIL-1ß may ultimately be cleaved to mature 17 kDa bioactive IL-1ß, regulating T cell polarization and pathogenic chronic inflammation.

Silencing of high-affinity insulin-reactive B lymphocytes by anergy and impact of the NOD genetic background in mice.

AIMS/HYPOTHESIS: Previous studies have demonstrated that high-affinity insulin-binding B cells (IBCs) silenced by anergy in healthy humans lose their anergy in islet autoantibody-positive individuals with recent-onset type 1 diabetes, and in autoantibody-negative first-degree relatives carrying certain risk alleles. Here we explore the hypothesis that IBCs are found in the immune periphery of disease-resistant C57BL/6-H2g7 mice, where, as in healthy humans, they are anergic, but that in disease-prone genetic backgrounds (NOD) they become activated and migrate to the pancreas and pancreatic lymph nodes, where they participate in the development of type 1 diabetes. METHODS: We compared the status of high-affinity IBCs in disease-resistant VH125.C57BL/6-H2g7 and disease-prone VH125.NOD mice. RESULTS: Consistent with findings in healthy humans, high-affinity IBCs reach the periphery in disease-resistant mice and are anergic, as indicated by a reduced expression of membrane IgM, unresponsiveness to antigen and failure to become activated or accumulate in the pancreatic lymph nodes or pancreas. In NOD mice, high-affinity IBCs reach the periphery early in life and increase in number prior to the onset of hyperglycaemia. These cells are not anergic; they become activated, produce autoantibodies and accumulate in the pancreas and pancreatic lymph nodes prior to disease development. CONCLUSIONS/INTERPRETATION: These findings are consistent with genetic determination of the escape of high-affinity IBCs from anergy and their early contribution to the development of type 1 diabetes.

Neutrophil transfer of miR-223 to lung epithelial cells dampens acute lung injury in mice.

Intercellular transfer of microRNAs can mediate communication between critical effector cells. We hypothesized that transfer of neutrophil-derived microRNAs to pulmonary epithelial cells could alter mucosal gene expression during acute lung injury. Pulmonary-epithelial microRNA profiling during coculture of alveolar epithelial cells with polymorphonuclear neutrophils (PMNs) revealed a selective increase in lung epithelial cell expression of microRNA-223 (miR-223). Analysis of PMN-derived supernatants showed activation-dependent release of miR-223 and subsequent transfer to alveolar epithelial cells during coculture in vitro or after ventilator-induced acute lung injury in mice. Genetic studies indicated that miR-223 deficiency was associated with severe lung inflammation, whereas pulmonary overexpression of miR-223 in mice resulted in protection during acute lung injury induced by mechanical ventilation or by infection with Staphylococcus aureus Studies of putative miR-223 gene targets implicated repression of poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) in the miR-223-dependent attenuation of lung inflammation. Together, these findings suggest that intercellular transfer of miR-223 from neutrophils to pulmonary epithelial cells may dampen acute lung injury through repression of PARP-1.

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function.

B cells reactive with a specific antigen usually occur at a frequency of <0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

B Cell Receptor Affinity for Insulin Dictates Autoantigen Acquisition and B Cell Functionality in Autoimmune Diabetes.

B cells have been strongly implicated in the development of human type 1 diabetes and are required for disease in the NOD mouse model. These functions are dependent on B cell antigen receptor (BCR) specificity and expression of MHC, implicating linked autoantigen recognition and presentation to effector T cells. BCR-antigen affinity requirements for participation in disease are unclear. We hypothesized that BCR affinity for the autoantigen insulin differentially affects lymphocyte functionality, including tolerance modality and the ability to acquire and become activated in the diabetogenic environment. Using combined transgenic and retrogenic heavy and light chain to create multiple insulin-binding BCRs, we demonstrate that affinity for insulin is a critical determinant of the function of these autoreactive cells. We show that both BCR affinity for insulin and genetic background affect tolerance induction in immature B cells. We also find new evidence that may explain the enigmatic ability of B cells expressing 125 anti-insulin BCR to support development of TID in NOD mice despite a reported affinity beneath requirements for binding insulin at in vivo concentrations. We report that when expressed as an antigen receptor the affinity of 125 is much higher than determined by measurements of the soluble form. Finally, we show that in vivo acquisition of insulin requires both sufficient BCR affinity and permissive host/tissue environment. We propose that a confluence of BCR affinity, pancreas environment, and B cell tolerance-regulating genes in the NOD animal allows acquisition of insulin and autoimmunity.

Loss of anergic B cells in prediabetic and new-onset type 1 diabetic patients.

Although dogma predicts that under normal circumstances, potentially offensive autoreactive cells are silenced by mechanisms of immune tolerance, islet antigen-reactive B lymphocytes are known to play a crucial role in the development of autoimmunity in type 1 diabetes (T1D). Thus, participation of these cells in T1D may reflect escape from silencing mechanisms. Consistent with this concept, we found that in healthy subjects, high-affinity insulin-binding B cells occur exclusively in the anergic naive IgD(+), IgM(-) B-cell (BND) compartment. Antigen receptors expressed by these cells are polyreactive and have N-region additions, Vh usage, and charged complementarity-determining region 3 consistent with autoreactivity. Consistent with a potential early role in autoimmunity, these high-affinity insulin-binding B cells are absent from the anergic compartment of some first-degree relatives and all prediabetic and new-onset (<1 year) T1D patients tested, but return to normal levels in individuals diabetic for >1 year. Interestingly, these changes were correlated by transient loss of the entire BND compartment. These findings suggest that environmental events such as infection or injury may, by disrupting B-cell anergy, dispose individuals toward autoimmunity, the precise nature of which is specified by genetic risk factors, such as HLA alleles.

B lymphocyte antigen receptor signaling: initiation, amplification, and regulation.

B lymphocytes and their differentiated daughters are charged with responding to the myriad pathogens in our environment and production of protective antibodies. A sample of the protective antibody produced by each clone is utilized as a component of the cell's antigen receptor (BCR). Transmembrane signals generated upon antigen binding to this receptor provide the primary directive for the cell's subsequent response. In this report, we discuss recent progress and current controversy regarding B cell receptor signal initiation, transduction and regulation.

STING/MPYS mediates host defense against Listeria monocytogenes infection by regulating Ly6C(hi) monocyte migration.

MPYS (also known as STING, MITA, and TMEM173) is a type I IFN stimulator that is essential for host defense against DNA virus infection and appears important in defense against certain bacteria. The in vivo significance and mechanisms by which MPYS mediates host defense against nonviral pathogens are unknown. Using an MPYS-deficient mouse (Tmem173()), we determined that, distinct from the IFNAR(-/-) mice, MPYS deficiency leads to increased bacterial burden in the liver upon Listeria monocytogenes infection. The increase was correlated with the diminished MCP-1 and MCP-3 chemokine production and decreased blood and liver Ly6C(hi) monocyte frequency. We further demonstrate that MPYS-deficient Ly6C(hi) monocytes are intrinsically defective in migration to the liver. Lastly, adoptive transfer of wild-type Ly6C(hi) monocyte into MPYS-deficient mice decreases their liver bacterial burden. Our findings reveal a novel in vivo function of MPYS that is distinct from its role in activating type I IFN production.

COPD is associated with production of autoantibodies to a broad spectrum of self-antigens, correlative with disease phenotype.

The role of autoimmune pathology in development and progression of chronic obstructive pulmonary disease (COPD) is becoming increasingly appreciated. In this study, we identified serum autoantibody reactivities associated with chronic bronchitis or emphysema, as well as systemic autoimmunity and associated lung disease. Using autoantigen array analysis, we demonstrated that COPD patients produce autoantibodies reactive to a broad spectrum of self-antigens. Further, the level and reactivities of these antibodies, or autoantibody profile, correlated with disease phenotype. Patients with emphysema produced autoantibodies of higher titer and reactive to an increased number of array antigens. Strikingly, the autoantibody reactivities observed in emphysema were increased over those detected in rheumatoid arthritis patients, and included similar reactivities to those associated with lupus. These findings raise the possibility that autoantibody profiles may be used to determine COPD risk, as well as provide a diagnostic and prognostic tool. They shed light on the heterogeneity of autoantibody reactivities associated with COPD phenotype and could be of use in the personalization of medical treatment, including determining and monitoring therapeutic interventions.

B cell depletion therapy exacerbates murine primary biliary cirrhosis.

UNLABELLED: Primary biliary cirrhosis (PBC) is considered a model autoimmune disease due to the clinical homogeneity of patients and the classic hallmark of antimitochondrial antibodies (AMAs). Indeed, the presence of AMAs represents the most highly directed and specific autoantibody in autoimmune diseases. However, the contribution of B cells to the pathogenesis of PBC is unclear. Therefore, although AMAs appear to interact with the biliary cell apotope and contribute to biliary pathology, there is no correlation of disease severity and titer of AMAs. The recent development of well-characterized monoclonal antibodies specific for the B cell populations, anti-CD20 and anti-CD79, and the development of a well-defined xenobiotic-induced model of autoimmune cholangitis prompted us to use these reagents and the model to address the contribution of B cells in the pathogenesis of murine PBC. Prior to the induction of autoimmune cholangitis, mice were treated with either anti-CD20, anti-CD79, or isotype-matched control monoclonal antibody and followed for B cell development, the appearance of AMAs, liver pathology, and cytokine production. Results of the studies reported herein show that the in vivo depletion of B cells using either anti-CD20 or anti-CD79 led to the development of a more severe form of cholangitis than observed in control mice, which is in contrast with results from several other autoimmune models that have documented an important therapeutic role of B cell-specific depletion. Anti-CD20/CD79-treated mice had increased liver T cell infiltrates and higher levels of proinflammatory cytokines. CONCLUSION: Our results reflect a novel disease-protective role of B cells in PBC and suggest that B cell depletion therapy in humans with PBC should be approached with caution.