Tet2 deletion in CD4+ T cells disrupts Th1 lineage commitment in memory cells and enhances T follicular helper cell recall responses to viral rechallenge.

Following viral clearance, antigen-specific CD4+ T cells contract and form a pool of distinct Th1 and Tfh memory cells that possess unique epigenetic programs, allowing them to rapidly recall their specific effector functions upon rechallenge. DNA methylation programing mediated by the methylcytosine dioxygenase Tet2 contributes to balancing Th1 and Tfh cell differentiation during acute viral infection; however, the role of Tet2 in CD4+ T cell memory formation and recall is unclear. Using adoptive transfer models of antigen-specific wild type and Tet2 knockout CD4+ T cells, we find that Tet2 is required for full commitment of CD4+ T cells to the Th1 lineage and that in the absence of Tet2, memory cells preferentially recall a Tfh like phenotype with enhanced expansion upon secondary challenge. These findings demonstrate an important role for Tet2 in enforcing lineage commitment and programing proliferation potential, and highlight the potential of targeting epigenetic programing to enhance adaptive immune responses.

Microbiota promotes systemic T-cell survival through suppression of an apoptotic factor.

Symbiotic microbes impact the severity of a variety of diseases through regulation of T-cell development. However, little is known regarding the molecular mechanisms by which this is accomplished. Here we report that a secreted factor, Erdr1, is regulated by the microbiota to control T-cell apoptosis. Erdr1 expression was identified by transcriptome analysis to be elevated in splenic T cells from germfree and antibiotic-treated mice. Suppression of Erdr1 depends on detection of circulating microbial products by Toll-like receptors on T cells, and this regulation is conserved in human T cells. Erdr1 was found to function as an autocrine factor to induce apoptosis through caspase 3. Consistent with elevated levels of Erdr1, germfree mice have increased splenic T-cell apoptosis. RNA sequencing of Erdr1-overexpressing cells identified the up-regulation of genes involved in Fas-mediated cell death, and Erdr1 fails to induce apoptosis in Fas-deficient cells. Importantly, forced changes in Erdr1 expression levels dictate the survival of auto-reactive T cells and the clinical outcome of neuro-inflammatory autoimmune disease. Cellular survival is a fundamental feature regulating appropriate immune responses. We have identified a mechanism whereby the host integrates signals from the microbiota to control T-cell apoptosis, making regulation of Erdr1 a potential therapeutic target for autoimmune disease.

Performance of Anti-Carbamylated Protein Antibody Testing in the Routine Evaluation of Rheumatoid Arthritis from a Single Center.

BACKGROUND: Detection of anticyclic citrullinated peptide antibodies (anti-CCP) and rheumatoid factors (RF) in sera support the diagnosis of rheumatoid arthritis (RA); however, these markers are not detected in about 20% of RA patients. More recently, antibodies against carbamylated proteins (anti-CarP) have emerged with implications for preclinical RA diagnosis. The objective of this study was to assess the clinical performance of anti-CarP and correlate with disease severity in routine clinical practice. METHODS: Retrospective chart review of 331 subjects submitted for RA panel serology: 136 clinically defined RA-positive and 195 RA-negative patients. Fifty additional individuals were recruited for healthy controls. Patients' sera were tested for anti-CCP, anti-CarP, and RF antibodies. Clinical performance characteristics were evaluated for anti-CarP individually and in combination with anti-CCP and RF. Documented erosions and synovitis were correlated with anti-CarP positivity. RESULTS: Anti-CarP had a clinical sensitivity and specificity of 27% and 94%, respectively, for established RA. This sensitivity was lower than anti-CCP (79%) and RF (85%). The specificity of anti-CarP was similar to anti-CCP (93%) and higher than RF (69%). Anti-CarP in combination with anti-CCP and RF increased specificity (100%) but decreased sensitivity (21%). There was no correlation of anti-CarP positivity with presence of bone erosions; however, there was an increase in anti-CarP positivity among patients with synovitis. CONCLUSIONS: Anti-CarP demonstrates high specificity in diagnosis of established RA but lacks clinical sensitivity. In combination, anti-CarP does not improve clinical performance of anti-CCP and RF but may be useful in anti-CCP negative patients and in identifying patients with more active disease.

Comparison of three methods for the detection of antibodies against muscle-specific kinase.

OBJECTIVES: To compare 3 different methods for the detection of antibodies against muscle-specific kinase (MuSK). METHODS: MuSK antibody testing was performed in 237 serum samples by enzyme-linked immunosorbent assay (ELISA) and fixed cell-based assay (f-CBA-IFA). One hundred and forty-eight (148) of the sera had previously been tested by RIA during clinical testing: 47 MuSK antibody positive and 101 MuSK antibody negative. Of the MuSK RIA negative antibodies, 46 tested positive for other neural antibodies. Additionally, 89 sera were subsequently tested by all three methods: 70 healthy controls and 19 sera positive for other neural antibodies. RESULTS: Qualitative inter-assay agreement based on tiered RIA values was 100% for results of 1.00 nmol/L or greater by both methods; 81% and 94% for results between 0.21 and 0.99 nmol/L by ELISA and f-CBA-IFA, respectively; and 0% for results of 0.04-0.20 nmol/L by both methods. Negative results showed 100% agreement between RIA and both ELISA and f-CBA-IFA (n = 55). None of the controls positive for other neural autoantibodies or healthy controls were positive in any assay. CONCLUSION: Overall, excellent agreement was observed between the 3 methods used to detect antibodies against MuSK. Both the f-CBA-IFA and ELISA performed comparably to RIA and exhibited excellent overall accuracy for MuSK IgG detection, with the f-CBA-IFA demonstrating higher agreement between positive samples with the RIA than the ELISA without identifying false positives in the control samples. Advantages of non-radioactive methods for the detection of MuSK antibodies include reduced handling and disposal of hazardous materials, potential for automation and the reagents having a longer shelf-life, reducing costs associated with both workflow and lot validations. Thus, commercially available ELISA and transfected cell-based assays are viable alternatives to the traditional radioactive assay used for serologic determination of MuSK IgG.

Reactivation of latent HIV-1 in central memory CD4⁺ T cells through TLR-1/2 stimulation.

BACKGROUND: Toll-like receptors (TLRs) are crucial for recognition of pathogen-associated molecular patterns by cells of the innate immune system. TLRs are present and functional in CD4⁺ T cells. Memory CD4⁺ T cells, predominantly central memory cells (TCM), constitute the main reservoir of latent HIV-1. However, how TLR ligands affect the quiescence of latent HIV within central memory CD4⁺ T cells has not been studied. RESULTS: We evaluated the ability of a broad panel of TLR agonists to reactivate latent HIV-1. The TLR-1/2 agonist Pam3CSK4 leads to viral reactivation of quiescent HIV in a model of latency based on cultured TCM and in resting CD4⁺ T cells isolated from aviremic patients. In addition, we investigated the signaling pathway associated with Pam3CSK4 involved in HIV-1 reactivation. We show that the transcription factors NFκB, NFAT and AP-1 cooperate to induce viral reactivation downstream of TLR-1/2 stimulation. Furthermore, increasing levels of cyclin T1 is not required for TLR-mediated viral reactivation, but induction of viral expression requires activated pTEFb. Finally, Pam3CSK4 reactivates latent HIV-1 in the absence of T cell activation or proliferation, in contrast to antigen stimulation. CONCLUSIONS: Our findings suggest that the signaling through TLR-1/2 pathway via Pam3CSK4 or other reagents should be explored as an anti-latency strategy either alone or in combination with other anti-latency drugs.

Presence of anti-gp210 or anti-sp100 antibodies in AMA-positive patients may help support a diagnosis of primary biliary cholangitis.

BACKGROUND: Anti-mitochondrial antibody (AMA) positivity is not always associated with primary biliary cholangitis (PBC). We aimed to determine the additional value of anti-sp100 or anti-gp210 antibody in AMA-positive patients for PBC. METHODS: Patients (n = 190) and healthy donors (n = 50) were evaluated for AMA, anti-gp210 and anti-sp100 antibodies by ELISA. Antibody frequencies in cohorts and performance characteristics in some patients categorized as 'definitive-', 'probable-', and 'no PBC' were determined following review of their charts. RESULTS: Of the patients (n = 190), 38.4% were AMA-positive (n = 73) and 61.6% AMA-negative (n = 117). Frequency of anti-sp100 or anti-gp210 antibody was 17.8%, 2.6%, and 0% in AMA-positive, AMA-negative and healthy controls, respectively. Clinical data was available for 63 of 73 AMA-positive patients with 28.6%, 22.2%, and 49.2% categorized as definite, probable, and no PBC, respectively. Patients with definite PBC had higher mean levels of AMA and frequencies of sp100 or gp210 antibody compared to other groups. Sensitivities were low (anti-sp100: 18.8% and anti-gp210: 16.7%) with specificities above 98.0% for both. CONCLUSION: AMA-positive patients positive for anti-sp100 or anti-gp210 antibody were more likely to have a diagnosis of definite or probable PBC than those with AMA alone. Use of all tests is likely to improve characterization of patients at-risk for PBC.

Tet2 coordinates with Foxo1 and Runx1 to balance T follicular helper cell and T helper 1 cell differentiation.

In response to various types of infection, naïve CD4+ T cells differentiate into diverse helper T cell subsets; however, the epigenetic programs that regulate differentiation in response to viral infection remain poorly understood. Demethylation of CpG dinucleotides by Tet methylcytosine dioxygenases is a key component of epigenetic programing that promotes specific gene expression, cellular differentiation, and function. We report that following viral infection, Tet2-deficient CD4+ T cells preferentially differentiate into highly functional germinal center T follicular helper (TFH) cells that provide enhanced help for B cells. Using genome-wide DNA methylation and transcription factor binding analyses, we find that Tet2 coordinates with multiple transcription factors, including Foxo1 and Runx1, to mediate the demethylation and expression of target genes, including genes encoding repressors of TFH differentiation. Our findings establish Tet2 as an important regulator of TFH cell differentiation and reveal pathways that could be targeted to enhance immune responses against infectious disease.

Activation of the Anti-Oxidative Stress Response Reactivates Latent HIV-1 Through the Mitochondrial Antiviral Signaling Protein Isoform MiniMAVS.

The mitochondrial antiviral signaling protein (MAVS) is part of the cell's innate immune mechanism of defense. MAVS mRNA is bicistronic and can give rise to a full length-MAVS and a shorter isoform termed miniMAVS. In response to viral infections, viral RNA can be sensed by the cytosolic RNA sensors retinoic acid-inducible gene I (RIG-I) and/or melanoma differentiation-associated protein 5 (MDA5) and activate NF-κB through interaction with MAVS. MAVS can also sense cellular stress and activate an anti-oxidative stress (AOS) response through the activation of NF-κB. Because NF-κB is a main cellular transcription factor for HIV-1, we wanted to address what role MAVS plays in HIV-1 reactivation from latency in CD4 T cells. Our results indicate that RIG-I agonists required full length-MAVS whereas the AOS response induced by Dynasore through its catechol group can reactivate latent HIV-1 in a MAVS dependent manner through miniMAVS isoform. Furthermore, we uncover that PKC agonists, a class of latency-reversing agents, induce an AOS response in CD4 T cells and require miniMAVS to fully reactivate latent HIV-1. Our results indicate that the AOS response, through miniMAVS, can induce HIV-1 transcription in response to cellular stress and targeting this pathway adds to the repertoire of approaches to reactivate latent HIV-1 in 'shock-and-kill' strategies.

Targeting Cellular and Tissue HIV Reservoirs With Toll-Like Receptor Agonists.

The elimination of both cellular and tissue latent reservoirs is a challenge toward a successful HIV cure. "Shock and Kill" are among the therapeutic strategies that have been more extensively studied to target these reservoirs. These strategies are aimed toward the reactivation of the latent reservoir using a latency-reversal agent (LRA) with the subsequent killing of the reactivated cell either by the cytotoxic arm of the immune system, including NK and CD8 T cells, or by viral cytopathic mechanisms. Numerous LRAs are currently being investigated in vitro, ex vivo as well as in vivo for their ability to reactivate and reduce latent reservoirs. Among those, several toll-like receptor (TLR) agonists have been shown to reactivate latent HIV. In humans, there are 10 TLRs that recognize different pathogen-associated molecular patterns. TLRs are present in several cell types, including CD4 T cells, the cell compartment that harbors the majority of the latent reservoir. Besides their ability to reactivate latent HIV, TLR agonists also increase immune activation and promote an antiviral response. These combined properties make TLR agonists unique among the different LRAs characterized to date. Additionally, some of these agonists have shown promise toward finding an HIV cure in animal models. When in combination with broadly neutralizing antibodies, TLR-7 agonists have shown to impact the SIV latent reservoir and delay viral rebound. Moreover, there are FDA-approved TLR agonists that are currently being investigated for cancer therapy and other diseases. All these has prompted clinical trials using TLR agonists either alone or in combination toward HIV eradication approaches. In this review, we provide an extensive characterization of the state-of-the-art of the use of TLR agonists toward HIV eradication strategies and the mechanism behind how TLR agonists target both cellular and tissue HIV reservoirs.

Dual TLR2 and TLR7 agonists as HIV latency-reversing agents.

The presence of a reservoir of latently infected cells in HIV-infected patients is a major barrier towards finding a cure. One active cure strategy is to find latency-reversing agents that induce viral reactivation, thus leading to immune cell recognition and elimination of latently infected cells, known as the shock-and-kill strategy. Therefore, the identification of molecules that reactivate latent HIV and increase immune activation has the potential to further these strategies into the clinic. Here, we characterized synthetic molecules composed of a TLR2 and a TLR7 agonist (dual TLR2/7 agonists) as latency-reversing agents and compared their activity with that of the TLR2 agonist Pam2CSK4 and the TLR7 agonist GS-9620. We found that these dual TLR2/7 agonists reactivate latency by 2 complementary mechanisms. The TLR2 component reactivates HIV by inducing NF-κB activation in memory CD4+ T cells, while the TLR7 component induces the secretion of TNF-α by monocytes and plasmacytoid dendritic cells, promoting viral reactivation in CD4+ T cells. Furthermore, the TLR2 component induces the secretion of IL-22, which promotes an antiviral state and blocks HIV infection in CD4+ T cells. Our study provides insight into the use of these agonists as a multipronged approach targeting eradication of latent HIV.

Mycobacterium tuberculosis reactivates latent HIV-1 in T cells in vitro.

Following proviral integration into the host cell genome and establishment of a latent state, the human immunodeficiency virus type 1 (HIV-1) can reenter a productive life cycle in response to various stimuli. HIV-1 reactivation occurs when transcription factors, such as nuclear factor-κB (NF-κB), nuclear factor of activated T cells (NFAT), and activator protein -1 (AP-1), bind cognate sites within the long terminal repeat (LTR) region of the HIV-1 provirus to promote transcription. Interestingly, pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) can reactivate latent HIV-1 through activation of the transcription factor NF-κB. Some PRRs are expressed on central memory CD4+ T cells (TCM), which in HIV-1 patients constitute the main reservoir of latent HIV-1. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), interacts with PRRs through membrane components. However, the ability of Mtb to reactivate latent HIV-1 has not been extensively studied. Here we show that phosphatidylinositol mannoside 6 (PIM6), a component of the Mtb membrane, in addition to whole bacteria in co-culture, can reactivate HIV-1 in a primary TCM cell model of latency. Using a JLAT model of HIV-1 latency, we found this interaction to be mediated through Toll-like receptor-2 (TLR-2). Thus, we describe a mechanism by which Mtb can exacerbate HIV-1 infection. We hypothesize that chronic Mtb infection can drive HIV-1 reactivation. The phenomenon described here could explain, in part, the poor prognosis that characterizes HIV-1/Mtb co-infection.

Benzotriazoles Reactivate Latent HIV-1 through Inactivation of STAT5 SUMOylation.

The presence of latent HIV-1 in infected individuals represents a major barrier preventing viral eradication. For that reason, reactivation of latent viruses in the presence of antiretroviral regimens has been proposed as a therapeutic strategy to achieve remission. We screened for small molecules and identified several benzotriazole derivatives with the ability to reactivate latent HIV-1. In the presence of IL-2, benzotriazoles reactivated and reduced the latent reservoir in primary cells, and, remarkably, viral reactivation was achieved without inducing cell proliferation, T cell activation, or cytokine release. Mechanistic studies showed that benzotriazoles block SUMOylation of phosphorylated STAT5, increasing STAT5's activity and occupancy of the HIV-1 LTR. Our results identify benzotriazoles as latency reversing agents and STAT5 signaling and SUMOylation as targets for HIV-1 eradication strategies. These compounds represent a different direction in the search for "shock and kill" therapies.

Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus.

HIV-1 latently infected cells in vivo can be found in extremely low frequencies. Therefore, in vitro cell culture models have been used extensively for the study of HIV-1 latency. Often, these in vitro systems utilize defective viruses. Defective viruses allow for synchronized infections and circumvent the use of antiretrovirals. In addition, replication-defective viruses cause minimal cytopathicity because they fail to spread and usually do not encode env or accessory genes. On the other hand, replication-competent viruses encode all or most viral genes and better recapitulate the nuances of the viral replication cycle. The study of latency with replication-competent viruses requires the use of antiretroviral drugs in culture, and this mirrors the use of antiretroviral treatment (ART) in vivo. We describe a model that utilizes cultured central memory CD4(+) T cells and replication-competent HIV-1. This method generates latently infected cells that can be reactivated using latency reversing agents in the presence of antiretroviral drugs. We also describe a method for the removal of productively infected cells prior to viral reactivation, which takes advantage of the downregulation of CD4 by HIV-1, and the use of a GFP-encoding virus for increased throughput.