Haplotype-specific chromatin looping reveals genetic interactions of regulatory regions modulating gene expression in 8p23.1.

A major goal of genetics research is to elucidate mechanisms explaining how genetic variation contributes to phenotypic variation. The genetic variants identified in genome-wide association studies (GWASs) generally explain only a small proportion of heritability of phenotypic traits, the so-called missing heritability problem. Recent evidence suggests that additional common variants beyond lead GWAS variants contribute to phenotypic variation; however, their mechanistic underpinnings generally remain unexplored. Herein, we undertake a study of haplotype-specific mechanisms of gene regulation at 8p23.1 in the human genome, a region associated with a number of complex diseases. The FAM167A-BLK locus in this region has been consistently found in the genome-wide association studies (GWASs) of systemic lupus erythematosus (SLE) in all major ancestries. Our haplotype-specific chromatin interaction (Hi-C) experiments, allele-specific enhancer activity measurements, genetic analyses, and epigenome editing experiments revealed that: 1) haplotype-specific long-range chromatin interactions are prevalent in 8p23.1; 2) BLK promoter and cis-regulatory elements cooperatively interact with haplotype-specificity; 3) genetic variants at distal regulatory elements are allele-specific modifiers of the promoter variants at FAM167A-BLK; 4) the BLK promoter interacts with and, as an enhancer-like promoter, regulates FAM167A expression and 5) local allele-specific enhancer activities are influenced by global haplotype structure due to chromatin looping. Although systemic lupus erythematosus causal variants at the FAM167A-BLK locus are thought to reside in the BLK promoter region, our results reveal that genetic variants at distal regulatory elements modulate promoter activity, changing BLK and FAM167A gene expression and disease risk. Our results suggest that global haplotype-specific 3-dimensional chromatin looping architecture has a strong influence on local allelic BLK and FAM167A gene expression, providing mechanistic details for how regional variants controlling the BLK promoter may influence disease risk.

IL-4 and IL-15 promotion of virtual memory CD8+ T cells is determined by genetic background.

Virtual memory (VM) CD8+ T cells are present in unimmunized mice, yet possess T-cell receptors specific for foreign antigens. To date, VM cells have only been characterized in C57BL/6 mice. Here, we assessed the cytokine requirements for VM cells in C57BL/6 and BALB/c mice. As reported previously, VM cells in C57BL/6 mice rely mostly on IL-15 and marginally on IL-4. In stark contrast, VM cells in BALB/c mice rely substantially on IL-4 and marginally on IL-15. Further, NKT cells are the likely source of IL-4, because CD1d-deficient mice on a BALB/c background have significantly fewer VM cells. Notably, this NKT/IL-4 axis contributes to appropriate effector and memory T-cell responses to infection in BALB/c mice, but not in C57BL/6 mice. However, the effects of IL-4 are manifest prior to, rather than during, infection. Thus, cytokine-mediated control of the precursor population affects the development of virus-specific CD8+ T-cell memory. Depending upon the genetic background, different cytokines encountered before infection may influence the subsequent ability to mount primary and memory anti-viral CD8+ T-cell responses.

Protecting and rescuing the effectors: roles of differentiation and survival in the control of memory T cell development.

Vaccines, arguably the single most important intervention in improving human health, have exploited the phenomenon of immunological memory. The elicitation of memory T cells is often an essential part of successful long-lived protective immunity. Our understanding of T cell memory has been greatly aided by the development of TCR Tg mice and MHC tetrameric staining reagents that have allowed the precise tracking of antigen-specific T cell responses. Indeed, following acute infection or immunization, naïve T cells undergo a massive expansion culminating in the generation of a robust effector T cell population. This peak effector response is relatively short-lived and, while most effector T cells die by apoptosis, some remain and develop into memory cells. Although the molecular mechanisms underlying this cell fate decision remain incompletely defined, substantial progress has been made, particularly with regards to CD8(+) T cells. For example, the effector CD8(+) T cells generated during a response are heterogeneous, consisting of cells with more or less potential to develop into full-fledged memory cells. Development of CD8(+) T cell memory is regulated by the transcriptional programs that control the differentiation and survival of effector T cells. While the type of antigenic stimulation and level of inflammation control effector CD8(+) T cell differentiation, availability of cytokines and their ability to control expression and function of Bcl-2 family members governs their survival. These distinct differentiation and survival programs may allow for finer therapeutic intervention to control both the quality and quantity of CD8(+) T cell memory. Effector to memory transition of CD4(+) T cells is less well characterized than CD8(+) T cells, emerging details will be discussed. This review will focus on the recent progress made in our understanding of the mechanisms underlying the development of T cell memory with an emphasis on factors controlling survival of effector T cells.

Bcl-2 allows effector and memory CD8+ T cells to tolerate higher expression of Bim.

As acute infections resolve, most effector CD8(+) T cells die, whereas some persist and become memory T cells. Recent work showed that subsets of effector CD8(+) T cells, identified by reciprocal expression of killer cell lectin-like receptor G1 (KLRG1) and CD127, have different lifespans. Similar to previous reports, we found that effector CD8(+) T cells reported to have a longer lifespan (i.e., KLRG1(low)CD127(high)) have increased levels of Bcl-2 compared with their shorter-lived KLRG1(high)CD127(low) counterparts. Surprisingly, we found that these effector KLRG1(low)CD127(high) CD8(+) T cells also had increased levels of Bim compared with KLRG1(high)CD127(low) cells. Similar effects were observed in memory cells, in which CD8(+) central memory T cells expressed higher levels of Bim and Bcl-2 than did CD8(+) effector memory T cells. Using both pharmacologic and genetic approaches, we found that survival of both subsets of effector and memory CD8(+) T cells required Bcl-2 to combat the proapoptotic activity of Bim. Interestingly, inhibition or absence of Bcl-2 led to significantly decreased expression of Bim in surviving effector and memory T cells. In addition, manipulation of Bcl-2 levels by IL-7 or IL-15 also affected expression of Bim in effector CD8(+) T cells. Finally, we found that Bim levels were significantly increased in effector CD8(+) T cells lacking Bax and Bak. Together, these data indicate that cells having the highest levels of Bim are selected against during contraction of the response and that Bcl-2 determines the level of Bim that effector and memory T cells can tolerate.

Distinct roles of Cdc42 in thymopoiesis and effector and memory T cell differentiation.

Cdc42 of the Rho GTPase family has been implicated in cell actin organization, proliferation, survival, and migration but its physiological role is likely cell-type specific. By a T cell-specific deletion of Cdc42 in mouse, we have recently shown that Cdc42 maintains naïve T cell homeostasis through promoting cell survival and suppressing T cell activation. Here we have further investigated the involvement of Cdc42 in multiple stages of T cell differentiation. We found that in Cdc42(-/-) thymus, positive selection of CD4(+)CD8(+) double-positive thymocytes was defective, CD4(+) and CD8(+) single-positive thymocytes were impaired in migration and showed an increase in cell apoptosis triggered by anti-CD3/-CD28 antibodies, and thymocytes were hyporesponsive to anti-CD3/-CD28-induced cell proliferation and hyperresponsive to anti-CD3/-CD28-stimulated MAP kinase activation. At the periphery, Cdc42-deficient naive T cells displayed an impaired actin polymerization and TCR clustering during the formation of mature immunological synapse, and showed an enhanced differentiation to Th1 and CD8(+) effector and memory cells in vitro and in vivo. Finally, Cdc42(-/-) mice exhibited exacerbated liver damage in an induced autoimmune disease model. Collectively, these data establish that Cdc42 is critically involved in thymopoiesis and plays a restrictive role in effector and memory T cell differentiation and autoimmunity.

A major role for Bim in regulatory T cell homeostasis.

We have previously shown that regulatory T cells (Treg) accumulate dramatically in aged animals and negatively impact the ability to control persistent infection. However, the mechanisms underlying the age-dependent accrual of Treg remain unclear. In this study, we show that Treg accumulation with age is progressive and likely not the result of increased thymic output, increased peripheral proliferation, or from enhanced peripheral conversion. Instead, we found that Treg from aged mice are more resistant to apoptosis than Treg from young mice. Although Treg from aged mice had increased expression of functional IL-7Rα, we found that IL-7R signaling was not required for maintenance of Treg in vivo. Notably, aged Treg exhibit decreased expression of the proapoptotic molecule Bim compared with Treg from young mice. Furthermore, in the absence of Bim, Treg accumulate rapidly, accounting for >25% of the CD4(+) T cell compartment by 6 mo of age. Additionally, accumulation of Treg in Bim-deficient mice occurred after the cells left the transitional recent thymic emigrant compartment. Mechanistically, we show that IL-2 drives preferential proliferation and accumulation of Bim(lo) Treg. Collectively, our data suggest that chronic stimulation by IL-2 leads to preferential expansion of Treg having low expression of Bim, which favors their survival and accumulation in aged hosts.

Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis.

T-cell homeostasis is essential for normal functioning of the immune system. IL-7 receptor (IL-7R) and T-cell receptor (TCR) signaling are pivotal for T-cell homeostatic regulation. The detailed mechanisms regulating T-cell homeostasis and how IL-7R and TCR signaling are coordinated are largely unknown. Here we demonstrate that T cell-specific deletion of cell-division cycle 42 (Cdc42) GTPase causes a profound loss of mature T cells. Deletion of Cdc42 leads to a markedly increased expression of growth factor independence-1 (Gfi-1) and represses expression of IL-7Rα. In the absence of Cdc42, aberrant ERK1/2 MAP kinase activity results in enhanced, TCR-mediated T-cell proliferation. In vivo reconstitution of effector-binding-defective Cdc42 mutants and the effector p21 protein-activated kinase 1 (PAK1) into Cdc42-deficient T cells showed that PAK1 is both necessary and sufficient for Cdc42-regulated T-cell homeostasis. Thus, T-cell homeostasis is maintained through a concerted regulation of Gfi-1-IL-7R-controlled cytokine responsiveness and ERK-mediated TCR signaling strength by the Cdc42-PAK1 signaling axis.

Immune responses to coiled coil supramolecular biomaterials.

Self-assembly has been increasingly utilized in recent years to create peptide-based biomaterials for 3D cell culture, tissue engineering, and regenerative medicine, but the molecular determinants of these materials' immunogenicity have remained largely unexplored. In this study, a set of molecules that self-assembled through coiled coil oligomerization was designed and synthesized, and immune responses against them were investigated in mice. Experimental groups spanned a range of oligomerization behaviors and included a peptide from the coiled coil region of mouse fibrin that did not form supramolecular structures, an engineered version of this peptide that formed coiled coil bundles, and a peptide-PEG-peptide triblock bioconjugate that formed coiled coil multimers and supramolecular aggregates. In mice, the native peptide and engineered peptide did not produce any detectable antibody response, and none of the materials elicited detectable peptide-specific T cell responses, as evidenced by the absence of IL-2 and interferon-gamma in cultures of peptide-challenged splenocytes or draining lymph node cells. However, specific antibody responses were elevated in mice injected with the multimerizing peptide-PEG-peptide. Minimal changes in secondary structure were observed between the engineered peptide and the triblock peptide-PEG-peptide, making it possible that the triblock's multimerization was responsible for this antibody response.

STAT5 is critical to maintain effector CD8+ T cell responses.

During an immune response, most effector T cells die, whereas some are maintained and become memory T cells. Factors controlling the survival of effector CD4(+) and CD8(+) T cells remain unclear. In this study, we assessed the role of IL-7, IL-15, and their common signal transducer, STAT5, in maintaining effector CD4(+) and CD8(+) T cell responses. Following viral infection, IL-15 was required to maintain a subpopulation of effector CD8(+) T cells expressing high levels of killer cell lectin-like receptor subfamily G, member 1 (KLRG1), and lower levels of CD127, whereas IL-7 and IL-15 acted together to maintain KLRG1(low)CD127(high) CD8(+) effector T cells. In contrast, effector CD4(+) T cell numbers were not affected by the individual or combined loss of IL-15 and IL-7. Both IL-7 and IL-15 drove phosphorylation of STAT5 within effector CD4(+) and CD8(+) T cells. When STAT5 was deleted during the course of infection, both KLRG1(high)CD127(low) and KLRG1(low)CD127(high) CD8(+) T cells were lost, although effector CD4(+) T cell populations were maintained. Furthermore, STAT5 was required to maintain expression of Bcl-2 in effector CD8(+), but not CD4(+), T cells. Finally, IL-7 and IL-15 required STAT5 to induce Bcl-2 expression and to maintain effector CD8(+) T cells. Together, these data demonstrate that IL-7 and IL-15 signaling converge on STAT5 to maintain effector CD8(+) T cell responses.

Contracting the 'mus cells'--does down-sizing suit us for diving into the memory pool?

Maintenance of T-cell homeostasis is critical for normal functioning of the immune system. After thymocyte selection, T cells enter the peripheral lymphoid organs, where they are maintained as naive cells. Transient disruption of homeostasis occurs when naive T cells undergo antigen-driven expansion and acquire effector functions. Effector T cells then either undergo apoptosis (i.e. contraction at the population level) or survive to become memory cells. This apoptotic process is crucial: it resets T-cell homeostasis, promotes protective immunity, and limits autoimmunity. Although initial studies using in vitro models supported a role for death receptor signaling, more recent in vivo studies have implicated Bcl-2 family members as being critical for the culling of T-cell responses. While several Bcl-2 family members likely contribute to T-cell contraction, the pro-apoptotic molecule Bim and its anti-apoptotic antagonist Bcl-2 are essential regulators of the process. This review discusses the progress made in our understanding of the mechanisms underlying contraction of T-cell responses and how some cells avoid this cell death and become memory T cells.

CD8 T cell-initiated vascular endothelial growth factor expression promotes central nervous system vascular permeability under neuroinflammatory conditions.

Dysregulation of the blood-brain barrier (BBB) is a hallmark feature of numerous neurologic disorders as diverse as multiple sclerosis, stroke, epilepsy, viral hemorrhagic fevers, cerebral malaria, and acute hemorrhagic leukoencephalitis. CD8 T cells are one immune cell type that have been implicated in promoting vascular permeability in these conditions. Our laboratory has created a murine model of CD8 T cell-mediated CNS vascular permeability using a variation of the Theiler's murine encephalomyelitis virus system traditionally used to study multiple sclerosis. Previously, we demonstrated that CD8 T cells have the capacity to initiate astrocyte activation, cerebral endothelial cell tight junction protein alterations and CNS vascular permeability through a perforin-dependent process. To address the downstream mechanism by which CD8 T cells promote BBB dysregulation, in this study, we assess the role of vascular endothelial growth factor (VEGF) expression in this model. We demonstrate that neuronal expression of VEGF is significantly upregulated prior to, and coinciding with, CNS vascular permeability. Phosphorylation of fetal liver kinase-1 is significantly increased early in this process indicating activation of this receptor. Specific inhibition of neuropilin-1 significantly reduced CNS vascular permeability and fetal liver kinase-1 activation, and preserved levels of the cerebral endothelial cell tight junction protein occludin. Our data demonstrate that CD8 T cells initiate neuronal expression of VEGF in the CNS under neuroinflammatory conditions, and that VEGF may be a viable therapeutic target in neurologic disease characterized by inflammation-induced BBB disruption.

Gamma interferon signaling in macrophage lineage cells regulates central nervous system inflammation and chemokine production.

Intracranial (i.c.) infection of mice with lymphocytic choriomeningitis virus (LCMV) results in anorexic weight loss, mediated by T cells and gamma interferon (IFN-gamma). Here, we assessed the role of CD4(+) T cells and IFN-gamma on immune cell recruitment and proinflammatory cytokine/chemokine production in the central nervous system (CNS) after i.c. LCMV infection. We found that T-cell-depleted mice had decreased recruitment of hematopoietic cells to the CNS and diminished levels of IFN-gamma, CCL2 (MCP-1), CCL3 (MIP-1alpha), and CCL5 (RANTES) in the cerebrospinal fluid (CSF). Mice deficient in IFN-gamma had decreased CSF levels of CCL3, CCL5, and CXCL10 (IP-10), and decreased activation of both resident CNS and infiltrating antigen-presenting cells (APCs). The effects of IFN-gamma signaling on macrophage lineage cells was assessed using transgenic mice, called "macrophages insensitive to interferon gamma" (MIIG) mice, that express a dominant-negative IFN-gamma receptor under the control of the CD68 promoter. MIIG mice had decreased levels of CCL2, CCL3, CCL5, and CXCL10 compared to controls despite having normal numbers of LCMV-specific CD4(+) T cells in the CNS. MIIG mice also had decreased recruitment of infiltrating macrophages and decreased activation of both resident CNS and infiltrating APCs. Finally, MIIG mice were significantly protected from LCMV-induced anorexia and weight loss. Thus, these data suggest that CD4(+) T-cell production of IFN-gamma promotes signaling in macrophage lineage cells, which control (i) the production of proinflammatory cytokines and chemokines, (ii) the recruitment of macrophages to the CNS, (iii) the activation of resident CNS and infiltrating APC populations, and (iv) anorexic weight loss.

Bim/Bcl-2 balance is critical for maintaining naive and memory T cell homeostasis.

We examined the role of the antiapoptotic molecule Bcl-2 in combating the proapoptotic molecule Bim in control of naive and memory T cell homeostasis using Bcl-2(-/-) mice that were additionally deficient in one or both alleles of Bim. Naive T cells were significantly decreased in Bim(+/-)Bcl-2(-/-) mice, but were largely restored in Bim(-/-)Bcl-2(-/-) mice. Similarly, a synthetic Bcl-2 inhibitor killed wild-type, but not Bim(-/-), T cells. Further, T cells from Bim(+/-)Bcl-2(-/-) mice died rapidly ex vivo and were refractory to cytokine-driven survival in vitro. In vivo, naive CD8(+) T cells required Bcl-2 to combat Bim to maintain peripheral survival, whereas naive CD4(+) T cells did not. In contrast, Bim(+/-)Bcl-2(-/-) mice generated relatively normal numbers of memory T cells after lymphocytic choriomeningitis virus infection. Accumulation of memory T cells in Bim(+/-)Bcl-2(-/-) mice was likely caused by their increased proliferative renewal because of the lymphopenic environment of the mice. Collectively, these data demonstrate a critical role for a balance between Bim and Bcl-2 in controlling homeostasis of naive and memory T cells.

Cutting Edge: Limiting amounts of IL-7 do not control contraction of CD4+ T cell responses.

During the acute T cell response most effector T cells die while some survive and become memory T cells. Selective expression of CD127 (IL-7Ralpha) on effector T cells has been proposed to engender their survival into the memory pool. We assessed the role of IL-7 in effector T cell survival using MHC class II tetramers to track a CD4+ T cell response following infection with a recombinant vaccinia virus (rVV-2W1S). Exogenous IL-7 prevented the contraction of the 2W1S-specific CD4+ T cell response after rVV-2W1S infection. IL-7 increased proliferation of, and Bcl-2 expression within, 2W1S-specific T cells; the latter was required for IL-7-driven prevention of contraction. Conversely, in vivo neutralization of IL-7 or Bcl-2 did not exacerbate the contraction of 2W1S-specific CD4+ T cells. These data suggest that IL-7 administration may enhance the survival of effector T cells but that IL-7 is not the limiting factor during normal contraction of the response.

An adenoviral vector for probing promoter activity in primary immune cells.

Functional analysis of the DNA regulatory regions that control gene expression has largely been performed through transient transfection of promoter-reporter constructs into transformed cells. However, transformed cells are often poor models of primary cells. To directly analyze DNA regulatory regions in primary cells, we generated a novel adenoviral luciferase reporter vector, pShuttle-luciferase-GFP (pSLUG) that contains a promoterless luciferase cassette (with an upstream cloning site) for probing promoter activity, and a GFP expression cassette that allows for the identification of transduced cells. Recombinant adenoviruses generated from this vector can transduce a wide range of primary immune cells with high efficiency, including human macrophages, dendritic cells and T cells; and mouse T cells transgenic for the coxsackie and adenoviral receptor (CAR). In primary T cells, we show inducible nuclear factor of activated T cells (NF-AT) activity using a recombinant pSLUG adenovirus containing a consensus NF-AT promoter. We further show inducible IL-12/23 p40 promoter activity in primary macrophages and dendritic cells using a recombinant pSLUG adenovirus containing the proximal human IL-12/23 p40 promoter. The pSLUG system promises to be a powerful tool for the analysis of DNA regulatory regions in diverse types of primary immune cells.

Identification of molecular targets for immunotherapy of patients with head and neck squamous cell carcinoma.

To identify molecular targets for immunotherapy of head and neck squamous cell carcinoma (HNSCC) patients, we analyzed gene expression profile in matched tumor (HN) and normal fibroblast (FB) cell lines established from a HNSCC patient using microarray technique followed by real-time RT-PCR. Screening against a series of established normal and malignant cell lines followed by screening against a panel of normal human tissues led to the identification of 7 genes (AREG, CDH3, KLK10, NmU, SLPI, ANAX3 and MAL2), which were over-expressed at least 10-fold in tumors over any of the normal tissues. We determined the expression of mRNA encoding these genes against a panel of 15 HNSCC primary tumor samples. Relative expression of these genes was at least 20-fold. Expression of AREG, CDH3, KLK10, NmU and SLPI at the protein level was determined by immunohistochemistry in seven supraglottic laryngeal cancer specimens. All five proteins were expressed in these tumor samples with high intensity. We conclude that these molecules are potential targets for immunotherapy of HNSCC patients.

Recombinant vaccinia virus expressing IL-2 generates effective anti-tumor responses in an orthotopic murine model of head and neck carcinoma.

We evaluated the efficacy of a replication-competent, attenuated recombinant vaccinia virus (rvv) expressing IL-2 as a tumor vaccine in an immunocompetent murine model of head and neck squamous cell carcinoma. We implanted oral tumors by injection of tumor cells (SCC VII/SF) into the floor of the mouth of the syngeneic C3H/HeJ mice. Previous studies with this model suggested the presence of tumor-induced immune suppression. To circumvent the immune suppression we subcutaneously (s.c.) immunized the mice with irradiated, rvv-infected tumor cells prior to or along with intratumoral (i.t.) vaccination. A single s.c. vaccination invoked tumor-specific T cell proliferation and cytotoxicity. Mice treated by this protocol survived longer compared to those treated with i.t. vaccination alone. Tumor growth was significantly inhibited (P < 0.0002) and tumor regression was observed in all mice. The numbers of CD4(+) and CD8(+) lymphocytes as well as macrophages in the tumor beds and in tumor-draining lymph nodes were significantly increased in the mice treated by s.c. plus i.t. vaccination compared to s.c. or i.t. vaccination alone. These results suggested that s.c. vaccination along with i.t. vaccination increased antitumoral immunity and that CD4(+), CD8(+) lymphocytes as well as macrophages may play an important role in this antitumoral immunity.