MAdCAM costimulation through Integrin-α4ß7 promotes HIV replication.

Human gut-associated lymphoid tissues (GALT) play a key role in the acute phase of HIV infection. The propensity of HIV to replicate in these tissues, however, is not fully understood. Access and migration of naive and memory CD4+ T cells to these sites is mediated by interactions between integrin α4ß7, expressed on CD4+ T cells, and MAdCAM, expressed on high endothelial venules. We report here that MAdCAM delivers a potent costimulatory signal to naive and memory CD4+ T cells following ligation with α4ß7. Such costimulation promotes high levels of HIV replication. An anti-α4ß7 mAb that prevents mucosal transmission of SIV blocks MAdCAM signaling through α4ß7 and MAdCAM-dependent viral replication. MAdCAM costimulation of memory CD4+ T cells is sufficient to drive cellular proliferation and the upregulation of CCR5, while naive CD4+ T cells require both MAdCAM and retinoic acid to achieve the same response. The pairing of MAdCAM and retinoic acid is unique to the GALT, leading us to propose that HIV replication in these sites is facilitated by MAdCAM-α4ß7 interactions. Moreover, complete inhibition of MAdCAM signaling by an anti-α4ß7 mAb, an analog of the clinically approved therapeutic vedolizumab, highlights the potential of such agents to control acute HIV infection.

Neutralizing and Targeting Properties of a New Set of α4ß7-Specific Antibodies Are Influenced by Their Isotype.

The homing of lymphocytes to the mucosa is mainly controlled by α4ß7 integrin, and it is amplified during gut chronic inflammation, as occurs with HIV and/or inflammatory bowel diseases. We designed and applied an improved immunization strategy based on an innovative selection process to isolate new α4ß7 lymphocyte-specific monoclonal antibodies that are able to prevent their migration into inflamed gut tissues and/or to counteract HIV infection in vitro. First, 5 monoclonal antibodies (1 IgA, 1 IgM, and 4 IgGs) were selected based on their capacity to recognize α4 or ß7 homodimers and α4ß7 heterodimers in transfected human cells. Their ability to block gp120/α4ß7 or MAdCAM-1/α4ß7 interactions was then measured in vitro with human T and B lymphocytes. In vitro, the anti-α4ß7 IgA isotype was found to have the highest affinity for the α4ß7 heterodimer, and it significantly reduced HIV replication in retinoic acid-treated α4ß7 CD4 human T cells. This α4ß7-specific IgA also displayed a high avidity for human and mouse α4ß7 lymphocytes in both mouse and human inflammatory colitis tissues. These new antibodies, and in particular those with mucosa-targeting isotypes such as IgA, could therefore be potential novel therapeutic tools for treating HIV and inflammatory bowel disease.

Sustained virologic control in SIV+ macaques after antiretroviral and α4ß7 antibody therapy.

Antiretroviral drug therapy (ART) effectively suppresses replication of both the immunodeficiency viruses, human (HIV) and simian (SIV); however, virus rebounds soon after ART is withdrawn. SIV-infected monkeys were treated with a 90-day course of ART initiated at 5 weeks post infection followed at 9 weeks post infection by infusions of a primatized monoclonal antibody against the α4ß7 integrin administered every 3 weeks until week 32. These animals subsequently maintained low to undetectable viral loads and normal CD4+ T cell counts in plasma and gastrointestinal tissues for more than 9 months, even after all treatment was withdrawn. This combination therapy allows macaques to effectively control viremia and reconstitute their immune systems without a need for further therapy.

HIV-1 gp120: A Target for Therapeutics and Vaccine Design.

Although extraordinary progress has been made in the treatment and prevention of HIV infection, the AIDS pandemic continues to rage globally with 2.1 million infections and 1.6 million AIDS-related deaths reported in 2013. Until an effective vaccine is developed, new strategies for treatment and prevention are needed. Regarding the prevention of HIV infection, a major focus of prevention research in general and vaccine research in particular involves the interaction of the HIV-1 envelope protein gp120 with cell-surface receptors, with the hope that a greater understanding of these interactions will lead to the development of novel strategies aimed at preventing and even treating HIV-1 infection. Particular attention has been directed toward gaining a more precise understanding of the early events in transmission focusing on that critical window of time when HIV first establishes infection in the host. Here we describe some of the recent findings involving HIV-1 envelope interactions with cell surface receptors that are relevant to transmission and which may represent new opportunities to develop strategies to prevent HIV infection.

The HIV-1 envelope protein gp120 impairs B cell proliferation by inducing TGF-ß1 production and FcRL4 expression.

The humoral immune response after acute infection with HIV-1 is delayed and ineffective. The HIV-1 envelope protein gp120 binds to and signals through integrin α4ß7 on T cells. We found that gp120 also bound to and signaled through α4ß7 on naive B cells, which resulted in an abortive proliferative response. In primary B cells, signaling by gp120 through α4ß7 resulted in increased expression of the immunosuppressive cytokine TGF-ß1 and FcRL4, an inhibitory receptor expressed on B cells. Coculture of B cells with HIV-1-infected autologous CD4(+) T cells also increased the expression of FcRL4 by B cells. Our findings indicated that in addition to mediating chronic activation of the immune system, viral proteins contributed directly to HIV-1-associated B cell dysfunction. Our studies identify a mechanism whereby the virus may subvert the early HIV-1-specific humoral immune response.

The genotype of early-transmitting HIV gp120s promotes α (4) ß(7)-reactivity, revealing α (4) ß(7) +/CD4+ T cells as key targets in mucosal transmission.

Mucosal transmission of HIV is inefficient. The virus must breach physical barriers before it infects mucosal CD4+ T cells. Low-level viral replication occurs initially in mucosal CD4+ T cells, but within days high-level replication occurs in Peyer's patches, the gut lamina propria and mesenteric lymph nodes. Understanding the early events in HIV transmission may provide valuable information relevant to the development of an HIV vaccine. The viral quasispecies in a donor contracts through a genetic bottleneck in the recipient, such that, in low-risk settings, infection is frequently established by a single founder virus. Early-transmitting viruses in subtypes A and C mucosal transmission tend to encode gp120s with reduced numbers of N-linked glycosylation sites at specific positions throughout the V1-V4 domains, relative to typical chronically replicating isolates in the donor quasispecies. The transmission advantage gained by the absence of these N-linked glycosylation sites is unknown. Using primary α4ß7/CD4+ T cells and a flow-cytometry based steady-state binding assay we show that the removal of transmission-associated N-linked glycosylation sites results in large increases in the specific reactivity of gp120 for integrin-α4ß7. High-affinity for integrin α4ß7, although not found in many gp120s, was observed in early-transmitting gp120s that we analyzed. Increased α4ß7 affinity is mediated by sequences encoded in gp120 V1/V2. α4ß7-reactivity was also influenced by N-linked glycosylation sites located in C3/V4. These results suggest that the genetic bottleneck that occurs after transmission may frequently involve a relative requirement for the productive infection of α4ß7+/CD4+ T cells. Early-transmitting gp120s were further distinguished by their dependence on avidity-effects to interact with CD4, suggesting that these gp120s bear unusual structural features not present in many well-characterized gp120s derived from chronically replicating viruses. Understanding the structural features that characterize early-transmitting gp120s may aid in the design of an effective gp120-based subunit vaccine.

The integrin alpha4beta7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1.

Both activated and resting CD4(+) T cells in mucosal tissues play important roles in the earliest phases of infection after sexual transmission of HIV-1, a process that is inefficient. HIV-1 gp120 binds to integrin alpha(4)beta(7) (alpha(4)beta(7)), the gut mucosal homing receptor. We find that alpha(4)beta(7)(high) CD4(+) T cells are more susceptible to productive infection than are alpha(4)beta(7)(low-neg) CD4(+) T cells in part because this cellular subset is enriched with metabolically active CD4(+) T cells. alpha(4)beta(7)(high) CD4(+) T cells are CCR5(high) and CXCR4(low); on these cells, alpha(4)beta(7) appears in a complex with CD4. The specific affinity of gp120 for alpha(4)beta(7) provides a mechanism for HIV-1 to target activated cells that are critical for efficient virus propagation and dissemination following sexual transmission.

Identification of two new synthetic histone deacetylase inhibitors that modulate globin gene expression in erythroid cells from healthy donors and patients with thalassemia.

We have identified two new histone deacetylase (HDAC) inhibitors (9 and 24) capable of inducing the expression of gamma-globin and/or beta-globin promoter-driven reporter genes in a synthetic model of Hb switch. Both compounds also increased, with different mechanisms, the gamma/(gamma+beta) ratio expressed in vitro by normal human erythroblasts. Compound 9 increased the levels of gamma-globin mRNA and the gamma/(gamma+beta) ratio (both by 2-fold). Compound 24 increased by 3-fold the level of gamma-globin and decreased by 2-fold that of beta-globin mRNA, increasing the gamma/(gamma+beta) ratio by 6-fold, and raising (by 50%) the cell HbF content. Both compounds raised the acetylation state of histone H4 in primary cells, an indication that their activity was mediated through HDAC inhibition. Compounds 9 and 24 were also tested as gamma/(gamma+beta) mRNA inducers in erythroblasts obtained from patients with beta(0) thalassemia. Progenitor cells from patients with beta(0) thalassemia generated in vitro morphologically normal proerythroblasts that, unlike normal cells, failed to mature in the presence of EPO and expressed low beta-globin levels but 10 times higher-than-normal levels of the alpha hemoglobin-stabilizing protein (AHSP) mRNA. Both compounds ameliorated the impaired in vitro maturation in beta(0) thalassemic erythroblasts, decreasing AHSP expression to normal levels. In the case of two patients (of five analyzed), the improved erythroblast maturation was associated with detectable increases in the gamma/(gamma+beta) mRNA ratio. The low toxicity exerted by compounds 9 and 24 in all of the assays investigated suggests that these new HDAC inhibitors should be considered for personalized therapy of selected patients with beta(0) thalassemia.

Interleukin-3 and erythropoietin cooperate in the regulation of the expression of erythroid-specific transcription factors during erythroid differentiation.

OBJECTIVE: To characterize how interleukin-3 and erythropoietin regulate cell fate by modulating the expression of lineage-specific transcription factors. METHODS: This study analyzed mRNA and protein levels, gene transcription rates, and mRNA and protein stabilities of erythroid-specific transcription factors in lineage-restricted cells derived from the 32D cell line cultured either in interleukin-3 or erythropoietin. RESULTS: Erythroid 32D subclones expressed levels of Idl, Gata-2, and Scl comparable and levels of Eklf and Gata-1 higher than those expressed by myeloid subclones. While maintained in interleukin-3, erythroid cells remained immature despite their high expression of Gata-1, Gata-2, Scl, Eklf, and Idl. Switching the erythroid cells to erythropoietin induced cell maturation (within 48 hours) and reduced expression of Gata-2 and Idl (in 24 hours) but did not alter the expression of Gata-1. The effects of interleukin-3 were mostly mediated by increases in transcription rates (Scl and Gata-2), and that of erythropoietin was apparently due to increased mRNA and protein (Gata-1, Scl, and Eklf) stability. In particular, erythropoietin increased the stability of the processed and transcriptionally more active form of GATA-1 protein. CONCLUSIONS: These results suggest that interleukin-3 and erythropoietin cooperate to establish the lineage-specific transcription factor milieu of erythroid cells: interleukin-3 regulates mainly gene transcription and erythropoietin consistently increases mRNA and protein stability.