Canine peripheral non-conventional TCRαß+ CD4-CD8α- double-negative T cells show T helper 2-like and regulatory properties.

The dog is an important companion animal and also serves as model species for human diseases. Given the central role of T cells in immune responses, a basic understanding of canine conventional T cell receptor (TCR)αß+ T cells, comprising CD4+ single-positive (sp) T helper (Th) and CD8α+ sp cytotoxic T cell subsets, is available. However, characterization of canine non-conventional TCRαß+ CD4+CD8α+ double-positive (dp) and TCRαß+ CD4-CD8α- double-negative (dn) T cells is limited. In this study, we performed a comprehensive analysis of canine dp and dn T cells in comparison with their conventional counterparts. TCRαß+ T cells from peripheral blood of healthy dogs were sorted according to their CD4/CD8α phenotype into four populations (i.e. CD4+ sp, CD8α+ sp, dp, and dn) and selected surface markers, transcription factors and effector molecules were analyzed ex vivo and after in vitro stimulation by RT-qPCR. Novel characteristics of canine dp T cells were identified, expanding the previously characterized Th1-like phenotype to Th17-like and Th2-like properties. Overall, mRNA expression of various Th cell-associated cytokines (i.e. IFNG, IL17A, IL4, IL13) in dp T cells upon stimulation highlights their versatile immunological potential. Furthermore, we demonstrated that the CD4-CD8α- dn phenotype is stable during in vitro stimulation. Strikingly, dn T cells were found to express highest mRNA levels of type 2 effector cytokines (IL4, IL5, and IL13) upon stimulation. Their strong ability to produce IL-4 was confirmed at the protein level. Upon stimulation, the percentage of IL-4-producing cells was even higher in the non-conventional dn than in the conventional CD4+ sp population. Constitutive transcription of IL1RL1 (encoding IL-33Rα) further supports Th2-like properties within the dn T cell population. These data point to a role of dn T cells in type 2 immunity. In addition, the high potential of dn T cells to transcribe the gene encoding the co-inhibitory receptor CTLA-4 and to produce the inhibitory cytokine IL-10 indicates putative immunosuppressive capacity of this population. In summary, this study reveals important novel aspects of canine non-conventional T cells providing the basis for further studies on their effector and/or regulatory functions to elucidate their role in health and disease.

Human CD4-binding site antibody elicited by polyvalent DNA prime-protein boost vaccine neutralizes cross-clade tier-2-HIV strains.

The vaccine elicitation of HIV tier-2-neutralization antibodies has been a challenge. Here, we report the isolation and characterization of a CD4-binding site (CD4bs) specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent DNA prime-protein boost HIV vaccine. HmAb64 is derived from heavy chain variable germline gene IGHV1-18 and light chain germline gene IGKV1-39. It has a third heavy chain complementarity-determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 20 (10%), including tier-2 strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 in complex with a CNE40 SOSIP trimer revealed details of its recognition; HmAb64 uses both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4bs. This study demonstrates that a gp120-based vaccine can elicit antibodies capable of tier 2-HIV neutralization.

A new mode for the diagnosis of angioimmunoblastic T-cell lymphoma.

In order to explore a new mode for the diagnosis of angioimmunoblastic T-cell lymphoma (AITL), 31 cases of AITL and 28 cases of peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) were used as the study subjects. Identifying T follicular helper (TFH) cells with CD4, CD10, Bcl-6, and PD-1, identifying proliferative B cells with CD20 and EZH2, identifying proliferative follicular dendritic cells (FDCs) with CD21 and CD23, and analyzing the value of TFH/B/FDC proliferation and immunolocalization in the diagnosis of AITL. (1) Outside the inherent lymphoid follicles, simultaneous proliferation of TFH/B/FDC (a new diagnostic mode) were observed in AITL [83.87%; 26/31], with their immunolocalizations in the same site [83.87%; 26/31], while this phenomenon was not observed in 28 cases of PTCL-NOS (P<0.05). (2) The sensitivity and specificity of using this new mode to diagnose AITL were both high (83.87%, 100%), which was superior to CD2 (100%, 0%), CD3 (100%, 0%), CD4 (100%, 32.14%), CD5 (100%, 25%), CD10 (61.9%, 100%), Bcl-6 (42.86%, 100%), PD-1 (83.87%, 96.43%), and its Youden Index (0.84) was the highest. The areas under the curve (AUC) of CD10, Bcl-6, PD-1, and new mode to diagnosis AITL were 0.81, 0.71, 0.90, and 0.92, respectively, while the new mode had the highest AUC. The simultaneous proliferation of TFH/B/FDC cells outside the inherent lymphoid follicles can be used to assist in the diagnosis of AITL, and the simultaneous spatiotemporal proliferation of TFH/B/FDC cells is a specific immunomorphology of AITL.

Identification and functional analyses of CD4-1+ cells in grass carp (Ctenopharyngodon idella).

In mammals, CD4 is found to be expressed on T cells and innate immune cells, however, teleost cells bearing CD4 have not been well identified and characterized. In this study, we identified two different CD4-1+ cell subsets in grass carp (Ctenopharyngodon idella): CD4-1+ lymphocytes (Lym) and CD4-1+ myeloid cells (Mye), both of which had the highest proportions in the head kidney. The mRNA expression analysis showed that CD4-1, CD4-2, TCRß, CD3γ/δ, and LCK1 are highly expressed in CD4-1+ Lym and also expressed in CD4-1+ Mye. Furthermore, we found that CD4-1+ Lym have a Lym morphology and highly express T-cell cytokines, suggesting that they are CD4+ T cells equivalent to mammalian Th cells. On the other hand, CD4-1+ Mye were found to have a morphology of macrophage and highly express macrophage marker gene MCSFR, indicating that they are macrophages. In addition, functional analysis revealed that CD4-1+ Mye possess phagocytic ability and great antigen-processing ability. Taken together, our study sheds further light on the composition and function of CD4+ cells in teleost fish.

[Clinical Characteristics of CD4-CD56+ Blastic Plasmacytoid Dendritic Cell Neoplasm].

OBJECTIVE: To explore the clinical manifestations, pathological features, immunophenotype, as well as diagnosis, treatment and prognosis of patients with CD4-CD56+ blastic plasmacytoid dendritic cell neoplasm (BPDCN), in order to further understand the rare disease. METHODS: The clinical data, laboratory examinations and treatment regimens of two patients with CD4-CD56+ BPDCN in the First Affiliated Hospital of Wannan Medical College were retrospectively analyzed. RESULTS: The two patients were both elderly males with tumor involved in skin, bone marrow, lymph nodes, etc. Immunohistochemical results of skin lesions showed that both CD56 and CD123 were positive, while CD4, CD34, TdT, CD3, CD20, MPO and EBER were negative. Flow cytometry of bone marrow demonstrated that CD56, CD123, and CD304 were all positive, while specific immune markers of myeloid and lymphoid were negative. Two patients were initially very sensitive to acute lymphoblastic leukemia or lymphomatoid chemotherapy regimens, but prone to rapid relapse. The overall survival of both patients was 36 months and 4 months, respectively. CONCLUSION: CD4-CD56+ BPDCN is very rare and easily misdiagnosed as other hematological tumors with poor prognosis. Acute lymphoblastic leukemia or lymphomatoid therapy should be used first to improve the poor prognosis.

Stoichiometry for entry and binding properties of the Env protein of R5 T cell-tropic HIV-1 and its evolutionary variant of macrophage-tropic HIV-1.

Human immunodeficiency virus type 1 typically requires a high density of CD4 for efficient entry as a mechanism to target CD4+ T cells (T-tropic), with CCR5 being used most often as the coreceptor. When target T cells are limiting, the virus can evolve to infect cells with a low density of CD4 such as macrophages (M-tropic). The entry phenotype is known to be encoded in the viral Env protein on the surface of the virus particle. Using data showing a dose response for infectivity based on CD4 surface density, we built a model consistent with T-tropic viruses requiring multiple CD4 molecules to mediate infection, whereas M-tropic viruses can infect cells using a single CD4 receptor molecule interaction. We also found that T-tropic viruses bound to the surface of cells with a low density of CD4 are released more slowly than M-tropic viruses which we modeled to be due to multiple interactions of the T-tropic virus with multiple CD4 molecules to allow the initial stable binding. Finally, we found that some M-tropic Env proteins, as the gp120 subunit, possess an enhanced affinity for CD4 compared with their T-tropic pair, indicating that the evolution of macrophage tropism can be reflected both in the closed Env trimer conformation on the virion surface and, in some cases, also in the open confirmation of gp120 Env. Collectively, these studies reveal differences in the stoichiometry of interaction of T-tropic and M-tropic viruses with CD4 and start to identify the basis of binding differences at the biochemical level. IMPORTANCE: Human immunodeficiency virus type 1 normally targets CD4+ T cells for viral replication. When T cells are limiting, the virus can evolve to infect myeloid cells. The evolutionary step involves a change from requiring a high surface density of CD4 for entry to being able to infect cells with a low density of CD4, as is found on myeloid lineage cells such as macrophage and microglia. Viruses able to infect macrophages efficiently are most often found in the CNS late in the disease course, and such viruses may contribute to neurocognitive impairment. Here, we examine the CD4 binding properties of the viral Env protein to explore these two different entry phenotypes.

High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4.

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods may have important implications in drug development and discovery. Here, we present the first approach that employs computational high-throughput virtual screening and molecular docking to search for small molecules in chemical libraries that function as mechano-regulators of the stability of human cluster of differentiation 4, receptor of HIV-1. Using single-molecule force spectroscopy, we prove that these small molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, that is, mechano-regulation, paving the way toward mechanopharmacology.

Inactivation of cell-free HIV-1 by designing potent peptides based on mutations in the CD4 binding site.

Human immunodeficiency virus type 1 (HIV-1) is a major global health problem, with over 38 million people infected worldwide. Current anti-HIV-1 drugs are limited in their ability to prevent the virus from replicating inside host cells, making them less effective as preventive measures. In contrast, viral inhibitors that inactivate the virus before it can bind to a host cell have great potential as drugs. In this study, we aimed to design mutant peptides that could block the interaction between gp120 and the CD4 receptor on host cells, thus preventing HIV-1 infection. We designed a 20-amino-acid peptide that mimicked the amino acids of the CD4 binding site and docked it to gp120. Molecular dynamics simulations were performed to calculate the energy of MMPBSA (Poisson-Boltzmann Surface Area) for each residue of the peptide, and unfavorable energy residues were identified as potential mutation points. Using MAESTRO (Multi AgEnt STability pRedictiOn), we measured ΔΔG (change in the change in Gibbs free energy) for mutations and generated a library of 240 mutated peptides using OSPREY software. The peptides were then screened for allergenicity and binding affinity. Finally, molecular dynamics simulations (via GROMACS 2020.2) and control docking (via HADDOCK 2.4) were used to evaluate the ability of four selected peptides to inhibit HIV-1 infection. Three peptides, P3 (AHRQIRQWFLTRGPNRSLWQ), P4 (VHRQIRQWFLTRGPNRSLWQ), and P9 (AHRQIRQMFLTRGPNRSLWQ), showed practical and potential as HIV inhibitors, based on their binding affinity and ability to inhibit infection. These peptides have the ability to inactivate the virus before it can bind to a host cell, thus representing a promising approach to HIV-1 prevention. Our findings suggest that mutant peptides designed to block the interaction between gp120 and the CD4 receptor have potential as HIV-1 inhibitors. These peptides could be used as preventive measures against HIV-1 transmission, and further research is needed to evaluate their safety and efficacy in clinical settings.

Idiotopes of antibodies against HIV-1 CD4-induced epitope shared with those against microorganisms.

Induction of antibodies (Abs) against the conformational CD4-induced (CD4i) epitope is frequent in HIV-1 infection. However, the mechanism of development of anti-CD4i Abs is unclear. We used anti-idiotypic (aID) monoclonal Abs (mAbs) of anti-CD4i mAbs to isolate anti-CD4i mAbs from infected subjects and track the causative antigens. One anti-aID mAb sorted from infected subjects by aID mAbs had the characteristics of anti-CD4i Abs, including IGHV1-69 usage and ability to bind to HIV-1 Env enhanced by sCD4. Critical amino acid sequences for the binding of six anti-aID mAbs, with shared idiotope to anti-CD4i mAbs, were analysed by phage display. The identified amino acid sequences showed similarity to proteins from human microbiota and infectious agents. Peptides synthesized from Caudoviricetes sp and Vibrio vulnificus based on the identified sequences were reactive to most anti-aID and some anti-CD4i mAbs. These results suggest that anti-CD4i Abs may evolve from B cells primed by microorganisms.

Potent latency reversal by Tat RNA-containing nanoparticle enables multi-omic analysis of the HIV-1 reservoir.

The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells' transcriptome and proteome profiles.

HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes.

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD41-4. Although high-resolution structures of Env in a complex with the soluble domains of CD4 have been determined, the binding process is less understood in native membranes5-13. Here we used cryo-electron tomography to monitor Env-CD4 interactions at the membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing the opposing membranes closer together. Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound to one, two and finally three CD4 molecules, after which Env adopted an open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which probably has consequences for antibody-mediated immune responses and vaccine immunogen design.

Intermediate conformations of CD4-bound HIV-1 Env heterotrimers.

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120 and gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops1-4 and in fully saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops3-9. To investigate changes resulting from substoichiometric CD4 binding, we solved single-particle cryo-electron microscopy (cryo-EM) structures of soluble, native-like heterotrimeric Envs bound to one or two CD4 molecules. Most of the Env trimers bound to one CD4 adopted the closed, prefusion Env state, with a minority exhibiting a heterogeneous partially open Env conformation. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state10 that showed gp120 outward rotation but maintained the prefusion three-stranded gp120 bridging sheet with only partial V1V2 displacement and V3 exposure. We conclude that most of the engagements with one CD4 molecule were insufficient to stimulate CD4-induced conformational changes, whereas binding two CD4 molecules led to Env opening in CD4-bound protomers only. The substoichiometric CD4-bound soluble Env heterotrimer structures resembled counterparts derived from a cryo-electron tomography study of complexes between virion-bound Envs and membrane-anchored CD4 (ref. 11), validating their physiological relevance. Together, these results illuminate intermediate conformations of HIV-1 Env and illustrate its structural plasticity.

TScan-II: A genome-scale platform for the de novo identification of CD4+ T cell epitopes.

CD4+ T cells play fundamental roles in orchestrating immune responses and tissue homeostasis. However, our inability to associate peptide human leukocyte antigen class-II (HLA-II) complexes with their cognate T cell receptors (TCRs) in an unbiased manner has hampered our understanding of CD4+ T cell function and role in pathologies. Here, we introduce TScan-II, a highly sensitive genome-scale CD4+ antigen discovery platform. This platform seamlessly integrates the endogenous HLA-II antigen-processing machinery in synthetic antigen-presenting cells and TCR signaling in T cells, enabling the simultaneous screening of multiple HLAs and TCRs. Leveraging genome-scale human, virome, and epitope mutagenesis libraries, TScan-II facilitates de novo antigen discovery and deep exploration of TCR specificity. We demonstrate TScan-II's potential for basic and translational research by identifying a non-canonical antigen for a cancer-reactive CD4+ T cell clone. Additionally, we identified two antigens for clonally expanded CD4+ T cells in Sjögren's disease, which bind distinct HLAs and are expressed in HLA-II-positive ductal cells within affected salivary glands.

Single cell and spatial transcriptomics analysis of kidney double negative T lymphocytes in normal and ischemic mouse kidneys.

T cells are important in the pathogenesis of acute kidney injury (AKI), and TCR+CD4-CD8- (double negative-DN) are T cells that have regulatory properties. However, there is limited information on DN T cells compared to traditional CD4+ and CD8+ cells. To elucidate the molecular signature and spatial dynamics of DN T cells during AKI, we performed single-cell RNA sequencing (scRNA-seq) on sorted murine DN, CD4+, and CD8+ cells combined with spatial transcriptomic profiling of normal and post AKI mouse kidneys. scRNA-seq revealed distinct transcriptional profiles for DN, CD4+, and CD8+ T cells of mouse kidneys with enrichment of Kcnq5, Klrb1c, Fcer1g, and Klre1 expression in DN T cells compared to CD4+ and CD8+ T cells in normal kidney tissue. We validated the expression of these four genes in mouse kidney DN, CD4+ and CD8+ T cells using RT-PCR and Kcnq5, Klrb1, and Fcer1g genes with the NIH human kidney precision medicine project (KPMP). Spatial transcriptomics in normal and ischemic mouse kidney tissue showed a localized cluster of T cells in the outer medulla expressing DN T cell genes including Fcer1g. These results provide a template for future studies in DN T as well as CD4+ and CD8+ cells in normal and diseased kidneys.

V3 tip determinants of susceptibility to inhibition by CD4-mimetic compounds in natural clade A human immunodeficiency virus (HIV-1) envelope glycoproteins.

IMPORTANCE: CD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.

Drug hypersensitivity and eosinophilia: The decisive role of p-i stimulation.

Eosinophilia is a common finding in drug hypersensitivity reactions (DHR). Its cause is unclear, as neither antigen/allergen-driven inflammation nor clonal expansion is involved. Most delayed-DHRs are due to p-i (pharmacologic interaction of drugs with immune receptors). These are off-target activities of drugs with immune receptors that result in various types of T-cell stimulation, some of which involve excessive IL-5 production. Functional and phenotypic studies of T-cell clones and their TCR-transfected hybridoma cell lines revealed that some p-i-induced drug stimulations occur without CD4/ CD8 co-receptor engagement. The CD4/CD8 co-receptors link Lck (lymphocyte-specific protein tyrosine kinase) and LAT (linker for activation of T cells) to the TCR. Alteration of Lck or LAT can result in a TCR signalosome with enhanced IL-5 production. Thus, if a more affine TCR-[drug/peptide/HLA] interaction allows bypassing the CD4 co-receptor, a modified Lck/LAT activation may lead to a TCR signalosome with elevated IL-5 production. This "IL-5-TCR-signalosome" hypothesis could also explain eosinophilia in superantigen or allo-stimulation (graft-versus-host disease), in which evasion of CD4/CD8 co-receptors has also been described. It may open new therapeutic possibilities in certain eosinophilic diseases by directly targeting the IL-5-TCR signalosome.

HIV-1 virological synapse formation enhances infection spread by dysregulating Aurora Kinase B.

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity.

Soluble CD4 effectively prevents excessive TLR activation of resident macrophages in the onset of sepsis.

T lymphopenia, occurring in the early phase of sepsis in response to systemic inflammation, is commonly associated with morbidity and mortality of septic infections. We have previously shown that a sufficient number of T cells is required to constrain Toll-like receptors (TLRs) mediated hyperinflammation. However, the underlying mechanisms remains unsolved. Herein, we unveil that CD4+ T cells engage with MHC II of macrophages to downregulate TLR pro-inflammatory signaling. We show further that the direct contact between CD4 molecule of CD4+ T cells or the ectodomain of CD4 (soluble CD4, sCD4), and MHC II of resident macrophages is necessary and sufficient to prevent TLR4 overactivation in LPS and cecal ligation puncture (CLP) sepsis. sCD4 serum concentrations increase after the onset of LPS sepsis, suggesting its compensatory inhibitive effects on hyperinflammation. sCD4 engagement enables the cytoplasmic domain of MHC II to recruit and activate STING and SHP2, which inhibits IRAK1/Erk and TRAF6/NF-κB activation required for TLR4 inflammation. Furthermore, sCD4 subverts pro-inflammatory plasma membrane anchorage of TLR4 by disruption of MHC II-TLR4 raft domains that promotes MHC II endocytosis. Finally, sCD4/MHCII reversal signaling specifically interferes with TLR4 but not TNFR hyperinflammation, and independent of the inhibitive signaling of CD40 ligand of CD4+ cells on macrophages. Therefore, a sufficient amount of soluble CD4 protein can prevent excessive inflammatory activation of macrophages via alternation of MHC II-TLR signaling complex, that might benefit for a new paradigm of preventive treatment of sepsis.

Piperidine CD4-Mimetic Compounds Expose Vulnerable Env Epitopes Sensitizing HIV-1-Infected Cells to ADCC.

The ability of the HIV-1 accessory proteins Nef and Vpu to decrease CD4 levels contributes to the protection of infected cells from antibody-dependent cellular cytotoxicity (ADCC) by preventing the exposure of Env vulnerable epitopes. Small-molecule CD4 mimetics (CD4mc) based on the indane and piperidine scaffolds such as (+)-BNM-III-170 and (S)-MCG-IV-210 sensitize HIV-1-infected cells to ADCC by exposing CD4-induced (CD4i) epitopes recognized by non-neutralizing antibodies that are abundantly present in plasma from people living with HIV. Here, we characterize a new family of CD4mc, (S)-MCG-IV-210 derivatives, based on the piperidine scaffold which engages the gp120 within the Phe43 cavity by targeting the highly conserved Asp368 Env residue. We utilized structure-based approaches and developed a series of piperidine analogs with improved activity to inhibit the infection of difficult-to-neutralize tier-2 viruses and sensitize infected cells to ADCC mediated by HIV+ plasma. Moreover, the new analogs formed an H-bond with the α-carboxylic acid group of Asp368, opening a new avenue to enlarge the breadth of this family of anti-Env small molecules. Overall, the new structural and biological attributes of these molecules make them good candidates for strategies aimed at the elimination of HIV-1-infected cells.