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The CD4 receptor, by stabilizing TCR-MHC II interactions, plays a central role in adaptive immunity. It also serves as the HIV docking receptor. The HIV gp120 envelope protein binds directly to CD4. This interaction is a prerequisite for viral entry. gp120 also binds to ⍺4β7, an integrin that is expressed on a subset of memory CD4+ T cells. HIV tropisms for CD4+ T cells and gut tissues are central features of HIV pathogenesis. We report that CD4 binds directly to ⍺4β7 in a dynamic way, consistent with a cis regulatory interaction. The molecular details of this interaction are related to the way in which gp120 interacts with both receptors. Like MAdCAM-1 and VCAM-1, two recognized ligands of ⍺4β7, the binding interface on CD4 includes 2 sites (1° and accessory), distributed across its two N-terminal IgSF domains (D1 and D2). The 1° site includes a sequence in the G β-strand of CD4 D2, KIDIV, that binds directly to ⍺4β7. This pentapeptide sequence occurs infrequently in eukaryotic proteins. However, a closely related and conserved sequence, KLDIV, appears in the V2 domain of gp120. KLDIV mediates gp120-⍺4β7 binding. The accessory ⍺4β7 binding site on CD4 includes Phe43. The Phe43 aromatic ring protrudes outward from one edge of a loop connecting the C'C" strands of CD4 D1. Phe43 is a principal contact for HIV gp120. It interacts with conserved residues in the recessed CD4 binding pocket. Substitution of Phe43 abrogates CD4 binding to both gp120 and ⍺4β7. As such, the interactions of gp120 with both CD4 and ⍺4β7 reflect elements of their interactions with each other. These findings indicate that gp120 specificities for CD4 and ⍺4β7 are interrelated and suggest that selective pressures which produced a CD4 tropic virus that replicates in gut tissues are linked to a dynamic interaction between these two receptors. Author summary: Integrins modulate cell trafficking and provide costimulatory signals to leukocytes. An increasing number have also been shown to interact in cis with immune receptors. Formation of these multi-receptor complexes is dynamic and typically serves a regulatory function. We report a cis interaction between ⍺4β7, the gut-homing integrin and CD4, a receptor that plays a central role in the adaptive immune response. CD4-⍺4β7 interactions share features with MAdCAM-1 and VCAM-1, two natural ligands that engage ⍺4β7 in trans. All three receptors engage ⍺4β7 through two discreet sites distributed across their two N-terminal IgSF domains. Specific features of the two ⍺4β7 binding sites on CD4 are related to HIV pathogenesis in a unique and informative way. HIV gp120 binds to both CD4 and ⍺4β7. A key finding of this report is that specific elements of gp120-CD4 and gp120-⍺4β7 interactions reflect the interaction of these two receptors with each other. We conclude that the selective pressures that resulted in a virus which infects and depletes gut CD4+ T cells are linked to CD4-⍺4β7 cis interactions. [ABSTRACT FROM AUTHOR]

The CD4 receptor, by stabilizing TCR-MHC II interactions, plays a central role in adaptive immunity. It also serves as the HIV docking receptor. The HIV gp120 envelope protein binds directly to CD4. This interaction is a prerequisite for viral entry. gp120 also binds to ⍺4β7, an integrin that is expressed on a subset of memory CD4+ T cells. HIV tropisms for CD4+ T cells and gut tissues are central features of HIV pathogenesis. We report that CD4 binds directly to ⍺4β7 in a dynamic way, consistent with a cis regulatory interaction. The molecular details of this interaction are related to the way in which gp120 interacts with both receptors. Like MAdCAM-1 and VCAM-1, two recognized ligands of ⍺4β7, the binding interface on CD4 includes 2 sites (1° and accessory), distributed across its two N-terminal IgSF domains (D1 and D2). The 1° site includes a sequence in the G β-strand of CD4 D2, KIDIV, that binds directly to ⍺4β7. This pentapeptide sequence occurs infrequently in eukaryotic proteins. However, a closely related and conserved sequence, KLDIV, appears in the V2 domain of gp120. KLDIV mediates gp120-⍺4β7 binding. The accessory ⍺4β7 binding site on CD4 includes Phe43. The Phe43 aromatic ring protrudes outward from one edge of a loop connecting the C'C" strands of CD4 D1. Phe43 is a principal contact for HIV gp120. It interacts with conserved residues in the recessed CD4 binding pocket. Substitution of Phe43 abrogates CD4 binding to both gp120 and ⍺4β7. As such, the interactions of gp120 with both CD4 and ⍺4β7 reflect elements of their interactions with each other. These findings indicate that gp120 specificities for CD4 and ⍺4β7 are interrelated and suggest that selective pressures which produced a CD4 tropic virus that replicates in gut tissues are linked to a dynamic interaction between these two receptors. Author summary: Integrins modulate cell trafficking and provide costimulatory signals to leukocytes. An increasing number have also been shown to interact in cis with immune receptors. Formation of these multi-receptor complexes is dynamic and typically serves a regulatory function. We report a cis interaction between ⍺4β7, the gut-homing integrin and CD4, a receptor that plays a central role in the adaptive immune response. CD4-⍺4β7 interactions share features with MAdCAM-1 and VCAM-1, two natural ligands that engage ⍺4β7 in trans. All three receptors engage ⍺4β7 through two discreet sites distributed across their two N-terminal IgSF domains. Specific features of the two ⍺4β7 binding sites on CD4 are related to HIV pathogenesis in a unique and informative way. HIV gp120 binds to both CD4 and ⍺4β7. A key finding of this report is that specific elements of gp120-CD4 and gp120-⍺4β7 interactions reflect the interaction of these two receptors with each other. We conclude that the selective pressures that resulted in a virus which infects and depletes gut CD4+ T cells are linked to CD4-⍺4β7 cis interactions. [ABSTRACT FROM AUTHOR]

CD4+ tissue resident memory T cells (TRMs) are implicated in the formation of persistent HIV reservoirs that are established during the very early stages of infection. The tissue-specific factors that direct T cells to establish tissue residency are not well defined, nor are the factors that establish viral latency. We report that costimulation via MAdCAM-1 and retinoic acid (RA), two constituents of gut tissues, together with TGF-β, promote the differentiation of CD4+ T cells into a distinct subset α4β7+CD69+CD103+ TRM -like cells. Among the costimulatory ligands we evaluated, MAdCAM-1 was unique in its capacity to upregulate both CCR5 and CCR9. MAdCAM-1 costimulation rendered cells susceptible to HIV infection. Differentiation of TRM -like cells was reduced by MAdCAM-1 antagonists developed to treat inflammatory bowel diseases. These finding provide a framework to better understand the contribution of CD4+ TRMs to persistent viral reservoirs and HIV pathogenesis. Author summary: Although antiretroviral drugs strongly suppress viral replication in HIV infected subjects, viral reservoirs persist. To achieve a cure, these reservoirs need to be eliminated. However, the location and identity of persistently infected cells are not well understood. Tissue resident memory CD4+ T cells (TRMs) are a cell type that may play an important role. These cells reside in tissues for extended periods of time and circulate infrequently. To increase our understanding of the role of TRMs in HIV reservoirs this study identifies MAdCAM-1 and RA as two key components of the gut immune system involved in the generation of TRMs. Treatment of primary CD4+ T cells with MAdCAM-1 and RA renders cells susceptible to HIV infection and primes them to adopt a TRM -like phenotype. Addition of a TGF-β, an anti-proliferative cytokine, induces them to adopt a more complete TRM -like phenotype. This study provides a new approach toward studying the persistent reservoirs that are a barrier to an HIV cure. [ABSTRACT FROM AUTHOR]

A novel in vitro culture system using variable concentrations of biotin/streptavidin to label red blood cells (RBCs) that allows for the simultaneous comparison of growth rates in Plasmodium falciparum malaria parasite in four heterogeneous target RBC populations is described. Donor RBCs containing both P. falciparum‐infected RBCs and non‐infected RBCs at 0.5% parasitemia were first labeled with 7‐hydroxy‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) succinimidyl ester (DDAO‐SE) followed by co‐culture with a mixture of equal numbers of four differentially biotin/streptavidin labeled RBC populations. After two to three schizogonic growth cycles, co‐cultures were harvested and stained with streptavidin‐phycoerythrin (SA‐PE) followed by staining of parasite‐infected RBCs with nucleic acid fluorochrome SYBR Green I. To demonstrate the application of this method, some target RBC populations that had sialic acid residues removed using neuraminidase treatment were mixed with RBC populations without enzymatic treatment and incubated with donor parasitized RBCs strain W2 (sialic acid‐dependent) or 3D7 (sialic acid‐independent). Significant less susceptibility to malaria parasite invasion was obtained with enzyme‐treated RBC populations when compared with non‐treated RBCs in blood samples from the same individual when using malaria parasite strain W2, whereas no difference in percent parasitemias was noted following infection with malaria parasite strain 3D7. This novel malaria culture method is cheap and provides increased sensitivity for direct comparison of parasite growth over time of any of the four RBC populations under identical conditions and eliminates the experimental bias due to contaminated donor RBCs. The application of biotin‐labeled RBCs will therefore provide a better understanding of invasion phenotype‐specific host–parasite interactions and the extent of complex malaria invasion mechanism. © 2019 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Wide-spread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-α and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake., Competing Interests: Competing interests: The authors declare they have no competing interests. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health