Extending Remission and Reversing New-Onset Type 1 Diabetes by Targeted Ablation of Autoreactive T Cells.

Preserving endogenous insulin production is clinically advantageous and remains a vital unmet challenge in the treatment and reversal of type 1 diabetes. Although broad immunosuppression has had limited success in prolonging the so-called remission period, it comes at the cost of compromising beneficial immunity. Here, we used a novel strategy to specifically deplete the activated diabetogenic T cells that drive pathogenesis while preserving not only endogenous insulin production but also protective immunity. Effector T (Teff) cells, such as diabetogenic T cells, are naturally poised on the edge of apoptosis because of activation-induced DNA damage that stresses the p53 regulation of the cell cycle. We have found that using small molecular inhibitors that further potentiate p53 while inhibiting the G2/M cell cycle checkpoint control drives apoptosis of activated T cells in vivo. When delivered at the onset of disease, these inhibitors significantly reduce diabetogenic Teff cells, prolong remission, preserve functional islets, and protect islet allografts while leaving naive, memory, and regulatory T-cell populations functionally untouched. Thus, the targeted manipulation of p53 and cell cycle checkpoints represents a new therapeutic modality for the preservation of islet ß-cells in new-onset type 1 diabetes or after islet transplant.

Manipulating DNA damage-response signaling for the treatment of immune-mediated diseases.

Antigen-activated lymphocytes undergo extraordinarily rapid cell division in the course of immune responses. We hypothesized that this unique aspect of lymphocyte biology leads to unusual genomic stress in recently antigen-activated lymphocytes and that targeted manipulation of DNA damage-response (DDR) signaling pathways would allow for selective therapeutic targeting of pathological T cells in disease contexts. Consistent with these hypotheses, we found that activated mouse and human T cells display a pronounced DDR in vitro and in vivo. Upon screening a variety of small-molecule compounds, we found that potentiation of p53 (via inhibition of MDM2) or impairment of cell cycle checkpoints (via inhibition of CHK1/2 or WEE1) led to the selective elimination of activated, pathological T cells in vivo. The combination of these strategies [which we termed "p53 potentiation with checkpoint abrogation" (PPCA)] displayed therapeutic benefits in preclinical disease models of hemophagocytic lymphohistiocytosis and multiple sclerosis, which are driven by foreign antigens or self-antigens, respectively. PPCA therapy targeted pathological T cells but did not compromise naive, regulatory, or quiescent memory T-cell pools, and had a modest nonimmune toxicity profile. Thus, PPCA is a therapeutic modality for selective, antigen-specific immune modulation with significant translational potential for diverse immune-mediated diseases.

Roles of natural killer cells in antiviral immunity.

Natural killer (NK) cells are important in immune defense against virus infections. This is predominantly considered a function of rapid, innate NK-cell killing of virus-infected cells. However, NK cells also prime other immune cells through the release of interferon gamma (IFN-γ) and other cytokines. Additionally, NK cells share features with long-lived adaptive immune cells and can impact disease pathogenesis through the inhibition of adaptive immune responses by virus-specific T and B cells. The relative contributions of these diverse and conflicting functions of NK cells in humans are poorly defined and likely context-dependent, thereby complicating the development of therapeutic interventions. Here we focus on the contributions of NK cells to disease in diverse virus infections germane to human health.

Eliminating encephalitogenic T cells without undermining protective immunity.

The current clinical approach for treating autoimmune diseases is to broadly blunt immune responses as a means of preventing autoimmune pathology. Among the major side effects of this strategy are depressed beneficial immunity and increased rates of infections and tumors. Using the experimental autoimmune encephalomyelitis model for human multiple sclerosis, we report a novel alternative approach for purging autoreactive T cells that spares beneficial immunity. The moderate and temporally limited use of etoposide, a topoisomerase inhibitor, to eliminate encephalitogenic T cells significantly reduces the onset and severity of experimental autoimmune encephalomyelitis, dampens cytokine production and overall pathology, while dramatically limiting the off-target effects on naive and memory adaptive immunity. Etoposide-treated mice show no or significantly ameliorated pathology with reduced antigenic spread, yet have normal T cell and T-dependent B cell responses to de novo antigenic challenges as well as unimpaired memory T cell responses to viral rechallenge. Thus, etoposide therapy can selectively ablate effector T cells and limit pathology in an animal model of autoimmunity while sparing protective immune responses. This strategy could lead to novel approaches for the treatment of autoimmune diseases with both enhanced efficacy and decreased treatment-associated morbidities.

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.

The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands.

The programmed death (PD)-1 molecule and its ligands (PD-L1 and PD-L2), negative regulatory members of the B7 family, play an important role in peripheral tolerance. Previous studies have demonstrated that PD-1 is up-regulated on T cells following TCR-mediated activation; however, little is known regarding PD-1 and Ag-independent, cytokine-induced T cell activation. The common gamma-chain (gamma c) cytokines IL-2, IL-7, IL-15, and IL-21, which play an important role in peripheral T cell expansion and survival, were found to up-regulate PD-1 and, with the exception of IL-21, PD-L1 on purified T cells in vitro. This effect was most prominent on memory T cells. Furthermore, these cytokines induced, indirectly, the expression of PD-L1 and PD-L2 on monocytes/macrophages in PBMC. The in vivo correlate of these observations was confirmed on PBMC isolated from HIV-infected individuals receiving IL-2 immunotherapy. Exposure of gamma c cytokine pretreated T cells to PD-1 ligand-IgG had no effect on STAT5 activation, T cell proliferation, or survival driven by gamma c cytokines. However, PD-1 ligand-IgG dramatically inhibited anti-CD3/CD28-driven proliferation and Lck activation. Furthermore, following restimulation with anti-CD3/CD28, cytokine secretion by both gamma c cytokine and anti-CD3/CD28 pretreated T cells was suppressed. These data suggest that gamma c cytokine-induced PD-1 does not interfere with cytokine-driven peripheral T cell expansion/survival, but may act to suppress certain effector functions of cytokine-stimulated cells upon TCR engagement, thereby minimizing immune-mediated damage to the host.

HIV-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells.

Infection with human immunodeficiency virus 1 (HIV-1) results in the dissemination of virus to gut-associated lymphoid tissue. Subsequently, HIV-1 mediates massive depletion of gut CD4+ T cells, which contributes to HIV-1-induced immune dysfunction. The migration of lymphocytes to gut-associated lymphoid tissue is mediated by integrin alpha4beta7. We demonstrate here that the HIV-1 envelope protein gp120 bound to an activated form of alpha4beta7. This interaction was mediated by a tripeptide in the V2 loop of gp120, a peptide motif that mimics structures presented by the natural ligands of alpha4beta7. On CD4+ T cells, engagement of alpha4beta7 by gp120 resulted in rapid activation of LFA-1, the central integrin involved in the establishment of virological synapses, which facilitate efficient cell-to-cell spreading of HIV-1.

Structural basis for the interaction between focal adhesion kinase and CD4.

Focal adhesion kinase (FAK) and CD4 fulfil vital functions in cellular signal transduction: FAK is a central component in integrin signalling, whereas CD4 plays essential roles in the immune defence. In T lymphocytes, FAK and CD4 localise to the same signalling complexes after stimulation by either the human immunodeficiency virus (HIV) gp120 glycoprotein or an antigen, suggesting the concerted action of FAK and CD4 in these cells. Using crystallography and microcalorimetry, we here show that the focal adhesion targeting (FAT) domain of FAK binds specifically to the CD4 endocytosis motif in vitro. This FAT-CD4 complex is structurally and thermodynamically similar to the one FAT forms with paxillin LD motifs. The CD4 binding site on FAT presents the same features as the established CD4 binding site on the HIV-1 Nef protein. The binding of FAT to CD4 is incompatible with the binding of Lck to CD4. We further show that HIV-1 gp120 triggers the association of CD4 with FAK in T cells, under conditions that are known to dissociate Lck from CD4. Our results suggest that the FAK-CD4 complex represents an alternative route for eliciting T-cell-specific signals and that it links gp120 engagement to distinctive T-cell signalling during HIV infection. In infected cells, HIV-1 Nef may displace FAK from CD4 to protect the cells from apoptosis.

CD25+ regulatory T cells isolated from HIV-infected individuals suppress the cytolytic and nonlytic antiviral activity of HIV-specific CD8+ T cells in vitro.

HIV infection is characterized by CD4(+) T cell depletion and progressive immune dysfunction; particularly impacted are HIV-specific T cell responses. An important component of immune-mediated control of HIV replication, killing of infected cells, appears to be impaired, in part due to poor cytolytic activity of HIV-specific cytotoxic T cells (CTL). In vitro, several functions of HIV-specific T cells, such as cytokine production, can be enhanced by the depletion of the immunosuppressive CD25(+) FoxP3(+) CD4(+) regulatory (Treg) cell subset. However, the effect of CD25(+) Treg cells on virus-specific cytolytic activity in the context of HIV or any human viral infection has not been investigated. The present study demonstrates that CD25(+) Treg cells isolated from the peripheral blood of HIV-infected subjects significantly suppress HIV Gag-specific cytolytic activity in vitro. In addition, CD25(+) Treg cells suppress effector function (coexpression of TNF-alpha and IFN-gamma) of HIV-specific CD8(+) T cells that proliferate in response to HIV antigen. Finally, the secretion of HIV-inhibitory CC-chemokines by HIV-specific and nonspecific CD8(+) T cells is significantly reduced in the presence of CD25(+) Treg cells. These data suggest that CD25(+) Treg-mediated suppression of the antiviral activity of HIV-specific CD8(+) T cells could impact the ability of HIV-infected individuals to control HIV replication in vivo.

Suppression of HIV-specific T cell activity by lymph node CD25+ regulatory T cells from HIV-infected individuals.

CD25(+) CD4(+) FoxP3(+) regulatory T (Treg) cells isolated from the peripheral blood of asymptomatic HIV-infected individuals have been demonstrated to significantly suppress HIV-specific immune responses in vitro. CD25(+) Treg cell suppressor activity in the peripheral blood seems to diminish with progression of HIV disease, and it has been suggested that loss of Treg cells contributes to aberrant immune activation and disease progression. However, phenotypic studies suggest that Treg cells may migrate to, and be maintained or even expanded in, tissue sites of HIV replication. Currently, it is not known whether tissue-associated Treg cells maintain suppressive activity in the context of HIV infection, particularly in individuals with advanced disease. The present study demonstrates that CD25(+) Treg cells isolated from lymph nodes and peripheral blood of HIV(+) subjects, even those with high viral loads and/or low CD4(+) T cell counts, maintain potent suppressive activity against HIV-specific cytolytic T cell function. This activity was better in lymph node as compared with peripheral blood, particularly in patients with high levels of plasma viremia. In addition, the expression of certain CD25(+) Treg-associated markers on CD4(+) T cells isolated from lymph nodes differed significantly from those on CD4(+) T cell subsets isolated from the peripheral blood. These data suggest that CD25(+) Treg cell-mediated suppression of HIV-specific responses continues throughout the course of HIV disease and, because of their particularly potent suppression of HIV-specific CTL activity in lymphoid tissue, may considerably impact the ability to control HIV replication in vivo.