Scientific Resources

Natural T regulatory cells (nTregs) play a key role in inducing and maintaining immunological tolerance. Cell-based therapy using purified nTregs is under consideration for several conditions,but procedures employed to date have resulted in cell populations that are contaminated with cytokine secreting effector cells. We have established a method for isolation and ex vivo expansion of human nTregs from healthy blood donors for cellular therapy aimed at preventing allograft rejection in organ transplants. The Robosep instrument was used for initial nTreg isolation and rapamycin was included in the expansion phase of cell cultures. The resulting cell population exhibited a stable CD4(+)CD25(++bright)Foxp3(+) phenotype,had potent functional ability to suppress CD4(+)CD25(negative) T cells without evidence of conversion to effector T cells including TH17 cells,and manifested little to no production of pro-inflammatory cytokines upon in vitro stimulation. Boolean gating analysis of cytokine-expressing cells by flow cytometry for 32 possible profile end points revealed that 96% of expanded nTregs did not express any cytokine. From a single buffy coat,approximately 80 million pure nTregs were harvested after expansion under cGMP conditions; these cell numbers are adequate for infusion of approximately one million cells kg�?�¹ for cell therapy in clinical trials. View Publication

CD4(+)Vβ5(+) peripheral T cells in C57BL/6 mice respond to encounter with a peripherally expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process,cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes,driving surface expression of novel TCRs. Although postrevision CD4(+)Vβ5(-)TCRβ(+) T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire,it is unknown whether they respond to homeostatic and antigenic stimuli and thus may benefit the host. We demonstrate in this study that postrevision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ,and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly,postrevision cells do not proliferate in response to the tolerizing superantigen,implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of postrevision cells is not driven by the transgene-encoded receptor. Postrevision cells proliferate extensively to commensal bacterial Ags and can generate I-A(b)-restricted responses to Ag by producing IFN-γ following Listeria monocytogenes challenge. These data show that rescued postrevision T cells are responsive to homeostatic signals and recognize self- and foreign peptides in the context of self-MHC and are thus useful to the host. View Publication

Cell-based therapies against HIV/AIDS have been gaining increased interest. Natural killer (NK) cells are a key component of the innate immune system with the ability to kill diverse tumor cells and virus-infected cells. While NK cells have been shown to play an important role in the control of HIV-1 replication,their functional activities are often compromised in HIV-1-infected individuals. We have previously demonstrated the derivation of NK cells from human embryonic stem cells (hESCs) with the ability to potently kill multiple types of tumor cells both in vitro and in vivo. We now demonstrate the derivation of functional NK cells from human induced pluripotent stem cells (iPSCs). More importantly,both hESC- and iPSC-derived NK cells are able to inhibit HIV-1 NL4-3 infection of CEM-GFP cells. Additional studies using HIV-1-infected human primary CD4(+) T cells illustrated that hESC- and iPSC-derived NK cells suppress HIV-1 infection by at least three distinct cellular mechanisms: killing of infected targets through direct lysis,antibody-dependent cellular cytotoxicity,and production of chemokines and cytokines. Our results establish the potential to utilize hESC- and iPSC-derived NK cells to better understand anti-HIV-1 immunity and provide a novel cellular immunotherapeutic approach to treat HIV/AIDS. View Publication

The viral infection of higher vertebrates elicits potent innate and adaptive host immunity. However,an excessive or inappropriate immune response also may lead to host pathology that often is more severe than the direct effects of viral replication. Therefore,several mechanisms exist that regulate the magnitude and class of the immune response. Here,we have examined the potential involvement of regulatory T (Treg) cells in limiting pathology induced by influenza A virus (IAV) infection. Using lymphocyte-deficient mice as hosts,we showed that Treg cell reconstitution resulted in a significant delay in weight loss and prolonged survival following infection. The adoptively transferred Treg cells did not affect the high rate of IAV replication in the lungs of lymphocyte-deficient hosts,and therefore their disease-ameliorating effect was mediated through the suppression of innate immune pathology. Mechanistically,Treg cells reduced the accumulation and altered the distribution of monocytes/macrophages in the lungs of IAV-infected hosts. This reduction in lung monocytosis was associated with a specific delay in monocyte chemotactic protein-2 (MCP-2) induction in the infected lungs. Nevertheless,Treg cells failed to prevent the eventual development of severe disease in lymphocyte-deficient hosts,which likely was caused by the ongoing IAV replication. Indeed,using T-cell-deficient mice,which mounted a T-cell-independent B cell response to IAV,we further showed that the combination of virus-neutralizing antibodies and transferred Treg cells led to the complete prevention of clinical disease following IAV infection. Taken together,these results suggested that innate immune pathology and virus-induced pathology are the two main contributors to pathogenesis during IAV infection. View Publication

The early events that determine the decision between lymphocyte tolerance and activation are not well-understood. Using a model of systemic self-antigen recognition by CD4(+) T cells,we show,using single-cell biochemical analyses,that tolerance is characterized by transient signaling events downstream of T-cell receptor engagement in the mammalian target of rapamycin (mTOR) and NF-κB pathways. Parallel studies done by live cell imaging show that the key difference between tolerance and activation is the duration of the T cell-antigen presenting cell (APC) interaction,as revealed by stable T-cell immobilization on antigen encounter. Brief T cell-APC interactions result in tolerance,and prolonged interactions are associated with activation and the development of effector cells. These studies show that the duration of T cell-APC interactions and magnitude of associated TCR-mediated signaling are key determinants of lymphocyte tolerance vs. activation. View Publication

The active vitamin A metabolite retinoic acid (RA) imprints gut-homing specificity on lymphocytes upon activation by inducing the expression of α4β7 integrin and CCR9. RA receptor (RAR) activation is essential for their expression,whereas retinoid X receptor (RXR) activation is not essential for α4β7 expression. However,it remains unclear whether RXR activation affects the RA-dependent CCR9 expression on T cells and their gut homing. The major physiological RA,all-trans-RA,binds to RAR but not to RXR at physiological concentrations. Cell-surface CCR9 expression was often induced on a limited population of murine naive CD4(+) T cells by all-trans-RA or the RAR agonist Am80 alone upon CD3/CD28-mediated activation in vitro,but it was markedly enhanced by adding the RXR agonist PA024 or the RXR-binding environmental chemicals tributyltin and triphenyltin. Accordingly,CD4(+) T cells treated with the combination of all-trans-RA and tributyltin migrated into the small intestine upon adoptive transfer much more efficiently than did those treated with all-trans-RA alone. Furthermore,naive TCR transgenic CD4(+) T cells transferred into wild-type recipients migrated into the small intestinal lamina propria following i.p. injection of Ag,and the migration was enhanced by i.p. injection of PA024. We also show that PA024 markedly enhanced the all-trans-RA-induced CCR9 expression on naturally occurring naive-like regulatory T cells upon activation,resulting in the expression of high levels of α4β7,CCR9,and Foxp3. These results suggest that RXR activation enhances the RAR-dependent expression of CCR9 on T cells and their homing capacity to the small intestine. View Publication

Persistent pulmonary infection with Cryptococcus neoformans in C57BL/6 mice results in chronic inflammation that is characterized by an injurious Th2 immune response. In this study,we performed a comparative analysis of cryptococcal infection in wild-type versus CD40-deficient mice (in a C57BL/6 genetic background) to define two important roles of CD40 in the modulation of fungal clearance as well as Th2-mediated immunopathology. First,CD40 promoted microanatomic containment of the organism within the lung tissue. This protective effect was associated with: i) a late reduction in fungal burden within the lung; ii) a late accumulation of lung leukocytes,including macrophages,CD4+ T cells,and CD8+ T cells; iii) both early and late production of tumor necrosis factor-α and interferon-γ by lung leukocytes; and iv) early IFN-γ production at the site of T cell priming in the regional lymph nodes. In the absence of CD40,systemic cryptococcal dissemination was increased,and mice died of central nervous system infection. Second,CD40 promoted pathological changes in the airways,including intraluminal mucus production and subepithelial collagen deposition,but did not alter eosinophil recruitment or the alternative activation of lung macrophages. Collectively,these results demonstrate that CD40 helps limit progressive cryptococcal growth in the lung and protects against lethal central nervous system dissemination. CD40 also promotes some,but not all,elements of Th2-mediated immunopathology in response to persistent fungal infection in the lung. View Publication

Improving effector T-cell functions is highly desirable for preventive or therapeutic interventions of diverse diseases. Signal transducer and activator of transcription 3 (Stat3) in the myeloid compartment constrains Th1-type immunity,dampening natural and induced antitumor immune responses. We have recently developed an in vivo small interfering RNA (siRNA) delivery platform by conjugating a Toll-like receptor 9 agonist with siRNA that efficiently targets myeloid and B cells. Here,we show that either CpG triggering combined with the genetic Stat3 ablation in myeloid/B cell compartments or administration of the CpG-Stat3siRNA drastically augments effector functions of adoptively transferred CD8+ T cells. Specifically,we show that both approaches are capable of increasing dendritic cell and CD8(+) T-cell engagement in tumor-draining lymph nodes. Furthermore,both approaches can significantly activate the transferred CD8(+) T cells in vivo,upregulating effector molecules such as perforin,granzyme B,and IFN-γ. Intravital multiphoton microscopy reveals that Stat3 silencing combined with CpG triggering greatly increases killing activity and tumor infiltration of transferred T cells. These results suggest the use of CpG-Stat3siRNA,and possibly other Stat3 inhibitors,as a potent adjuvant to improve T-cell therapies. View Publication

Two critical concerns in clinical cord blood transplantation are the initial time to engraftment and the subsequent restoration of immune function. These studies measured the impact of progenitor cell dose on both the pace and strength of hematopoietic reconstitution by transplanting nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-gamma-null (NSγ) mice with lineage-depleted aldehyde dehydrogenase-bright CD34(+) human cord blood progenitors. The progress of each transplant was monitored over an extended time course by repeatedly analyzing the peripheral blood for human hematopoietic cells. In vivo human hematopoietic development was complete. After long-term transplantation assays (≥ 19 weeks),human T-cell development was documented within multiple tissues in 16 of 32 NSγ mice. Human T-cell differentiation was active within NSγ thymuses,as documented by the presence of CD4(+) CD8(+) T-cell progenitors as well as T-cell receptor excision circles. It is important to note that although myeloid and B-cell engraftment was detected as early as 4 weeks after transplantation,human T-cell development was exclusively late onset. High progenitor cell doses were associated with a robust human hematopoietic chimerism that accelerated both initial time to engraftment and subsequent T-cell development. At lower progenitor cell doses,the chimerism was weak and the human hematopoietic lineage development was frequently incomplete. View Publication

Progressive multifocal leukoencephalopathy (PML) is an often fatal demyelinating disease caused by lytic infection of oligodendrocytes with JC virus (JCV). The development of PML in non-immunosuppressed individuals is a growing concern with reports of mortality in patients treated with mAb therapies. JCV can persist in the kidneys,lymphoid tissue and bone marrow. JCV gene expression is restricted by non-coding viral regulatory region sequence variation and cellular transcription factors. Because JCV latency has been associated with cells undergoing haematopoietic development,transcription factors previously reported as lymphoid specific may regulate JCV gene expression. This study demonstrates that one such transcription factor,Spi-B,binds to sequences present in the JCV promoter/enhancer and may affect early virus gene expression in cells obtained from human brain tissue. We identified four potential Spi-B-binding sites present in the promoter/enhancer elements of JCV sequences from PML variants and the non-pathogenic archetype. Spi-B sites present in the promoter/enhancers of PML variants alone bound protein expressed in JCV susceptible brain and lymphoid-derived cell lines by electromobility shift assays. Expression of exogenous Spi-B in semi- and non-permissive cells increased early viral gene expression. Strikingly,mutation of the Spi-B core in a binding site unique to the Mad-4 variant was sufficient to abrogate viral activity in progenitor-derived astrocytes. These results suggest that Spi-B could regulate JCV gene expression in susceptible cells,and may play an important role in JCV activity in the immune and nervous systems. View Publication

The contribution of mammary epithelial cells (MEC) to human immunodeficiency virus type 1 (HIV-1) in breast milk remains largely unknown. While breast milk contains CD4(+) cells throughout the breast-feeding period,it is not known whether MEC directly support HIV-1 infection or facilitate infection of CD4(+) cells in the breast compartment. This study evaluated primary human MEC for direct infection with HIV-1 and for indirect transfer of infection to CD4(+) target cells. Primary human MEC were isolated and assessed for expression of HIV-1 receptors. MEC were exposed to CCR5-,CXCR4- and dual-tropic strains of HIV-1 and evaluated for viral reverse transcription and integration and productive viral infection. MEC were also tested for the ability to transfer HIV to CD4(+) target cells and to activate resting CD4(+) T cells. Our results demonstrate that MEC express HIV-1 receptor proteins CD4,CCR5,CXCR4,and galactosyl ceramide (GalCer). While no evidence for direct infection of MEC was found,HIV-1 virions were observed in MEC endosomal compartments. Coculture of HIV-exposed MEC resulted in productive infection of activated CD4(+) T cells. In addition,MEC secretions increased HIV-1 replication and proliferation of infected target cells. Overall,our results indicate that MEC are capable of endosomal uptake of HIV-1 and can facilitate virus infection and replication in CD4(+) target cells. These findings suggest that MEC may serve as a viral reservoir for HIV-1 and may enhance infection of CD4(+) T lymphocytes in vivo. View Publication

BACKGROUND: Therapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects,including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments,with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However,current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover,FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific,whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these limitations,we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs,and maintained the capacity to suppress conventional T cell responses directed against tyrosinase,as well as bystander T cell responses. Using this methodology in a model tumor system,murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging. CONCLUSIONS/SIGNIFICANCE: These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy. View Publication