B7-H1 Influences the Accumulation of Virus-Specific Tissue Resident Memory T Cells in the Central Nervous System.

Therapies that target the PD-1/B7-H1 axis have revolutionized cancer treatment, yet precise knowledge of how this pathway provides benefit continues to evolve. Here, we report a novel role for the immune checkpoint ligand B7-H1 in the accumulation of tissue-resident memory CD8+ T-cells (TRM). After intracranial infection, Theiler's murine encephalomyelitis virus (TMEV) generates TRM that are maintained in the central nervous system (CNS) tissues of B7-H1WT animals. Although no differences in acute T-cell responses between B7-H1WT and B7-H1KO are observed, at long-term periods post-infection the maintenance of CD8+ TRM is diminished in B7-H1KO animals. This is accompanied by redistribution of the resident CD8+ population from primarily CD103+ TRM to a diminished population of TRM and a preponderance of non-specified PD-1+ CD103- CD8+ T-cells. T-cell transfer studies demonstrate that host B7-H1 is necessary for maintaining TRM and limiting accumulation of PD-1+ CD103- CD8+ T-cells. The lack of host B7-H1 results in compromised control of a heterologous virus re-challenge demonstrating a functional defect in TRM mediated virus control. This study reveals a new role for B7-H1 in TRM and pro-inflammatory PD-1+ CD103- CD8+ T-cell accumulation in the CNS and gives insight for using B7-H1/PD-1 blockade in modulating long-term T-cell protection.

Enhancing the Tumor Selectivity of a Picornavirus Virotherapy Promotes Tumor Regression and the Accumulation of Infiltrating CD8+ T Cells.

Picornaviruses have emerged as promising cancer therapies due to their ability to drive cytotoxic cellular immune responses and for promoting oncolysis. These properties include preferential replication in tumor cells, the induction of strong innate and adaptive immune responses, and the ease with which their genomes can be manipulated. We have developed Theiler's murine encephalomyelitis virus (TMEV) as an immunotherapy vector that promotes strong adaptive immune responses to tumor antigens embedded within its genome. To further explore its usefulness as cancer therapy, we investigated whether direct intratumoral delivery of TMEV could promote tumor regression. We generated several picornavirus hybrids using substrains of TMEV that have unique immunopathologic characteristics, despite their extensive sequence homology. These hybrids exhibit a unique propensity to infect and replicate in melanoma. We have identified GD7-KS1, a virus that is particularly effective at replicating and infecting B16 melanoma in vitro and provides benefit as an oncolytic therapy in vivo after intratumoral injection. In addition, this virus promotes the mobilization and accumulation of CD8(+) T cells within treated tumors. Altogether, these findings demonstrate that picornavirus substrains can be used to rationally design virus hybrids that promote antitumor responses and add to the known strategies identified by us and others to further enhance the therapeutic potential of vectors used to treat cancer.

Widespread Non-Canonical Epigenetic Modifications in MMTV-NeuT Breast Cancer.

Breast tumors in (FVB × BALB-NeuT) F1 mice have characteristic loss of chromosome 4 and sporadic loss or gain of other chromosomes. We employed the Illumina GoldenGate genotyping platform to quantitate loss of heterozygosity (LOH) across the genome of primary tumors, revealing strong biases favoring chromosome 4 alleles from the FVB parent. While allelic bias was not observed on other chromosomes, many tumors showed concerted LOH (C-LOH) of all alleles of one or the other parent on sporadic chromosomes, a pattern consistent with cytogenetic observations. Surprisingly, comparison of LOH in tumor samples relative to normal unaffected tissues from these animals revealed significant variegated (stochastic) deviations from heterozygosity (V-LOH) in every tumor genome. Sequence analysis showed expected changes in the allelic frequency of single nucleotide polymorphisms (SNPs) in cases of C-LOH. However, no evidence of LOH due to mutations, small deletions, or gene conversion at the affected SNPs or surrounding DNA was found at loci with V-LOH. Postulating an epigenetic mechanism contributing to V-LOH, we tested whether methylation of template DNA impacts allele detection efficiency using synthetic oligonucleotide templates in an assay mimicking the GoldenGate genotyping format. Methylated templates were systematically over-scored, suggesting that the observed patterns of V-LOH may represent extensive epigenetic DNA modifications across the tumor genomes. As most of the SNPs queried do not contain standard (CpG) methylation targets, we propose that widespread, non-canonical DNA modifications occur during Her2/neuT-driven tumorigenesis.

Detection of constant domain of human T cell antigen receptor alpha-chain via novel monoclonal antibody 7F18.

The αß T cell antigen receptor (TCR) endows T lymphocytes with immune specificity and controls their effector functions. Each person possesses a vast repertoire of TCRs that is generated by the well-studied processes of somatic recombination and thymic selection. While many antibodies specific for TCRß variable domains are available, antibodies specific for human TCRα are rare. We now report a novel monoclonal antibody, 7F18, which binds to human TCRα constant region, with specificity for a denatured epitope that can be visualized by SDS-PAGE followed by Western blot. Both immature and mature TCR α-chain products can be visualized, making 7F18 potentially applicable to various biochemical assays of multiprotein complex assembly and maturation. This new monoclonal antibody provides a tool that can potentially facilitate the biochemical analysis of comprehensive populations of human αß TCR complexes that need not be limited to small subsets of the repertoire.

A CD8 T-cell epitope variant enhances immune targeting to a recombinant picornavirus vaccine antigen.

Recombinant virus vaccines are often less effective due to immunodominant responses against endogenous vector antigens. However, the use of small RNA virus vectors provides an opportunity to limit host exposure to endogenous virus antigens and focus immune responses on the desired vaccine antigen. Using the Daniel's strain of Theiler's murine encephalomyelitis virus, we have identified strategies to modulate responses to endogenous viral proteins by manipulating the host CD8+ T-cell repertoire prior to infection or through the use of mutations introduced into the virus genome. Both of these approaches enhance responses to vaccine antigens introduced into the picornavirus. However, the use of mutant immunodominant epitopes provides an opportunity for enhancing vaccine responses without further manipulation of the host. Using this strategy, we demonstrate that modification of the consensus MHC class I anchor residue within the virus genome can promote enhanced immunity to foreign antigens and self-antigens embedded in the virus genome.

An elite controller of picornavirus infection targets an epitope that is resistant to immune escape.

The emergence of novel viral pathogens can lead to devastating consequences in the infected population. However, on occasion, rare hyper-responsive elite controllers are able to mount a protective primary response to infection and clear the new pathogen. Factors distinguishing elite controllers from other members of the population are not completely understood. We have been using Theiler's murine encephalomyelitis as a model of primary infection in mice and clearance of the virus is limited to one MHC genotype capable of generating a protective response to a single viral peptide VP2121-130. The genetics of host susceptibility to TMEV, a natural mouse pathogen, has been studied extensively and non-protective CD8 responses to other peptides have been documented, however, little is known why the protective response to infection focuses on the VP2121-130 peptide. To study this question, we have generated TMEV mutants that encode for mutations within the VP2121-130 peptide. We find that very few of mutants are able to assemble and infect in vitro. These mutations are not related to virus RNA structure since non-coding mutations do not interfere with assembly. In the rare event when functional VP2121-130 mutant viruses did emerge, they were attenuated to some level or retained the ability to develop an immune response to the wild-type VP2121-130 sequence, demonstrating that the virus is incapable of escaping the protective response. These findings advance our understanding of how characteristics of the host immune response and an infectious agent can interact to lead to the appearance of rare super controllers in a population. Furthermore, the immutable nature of the viral antigen highlights the importance of choosing appropriate vaccine antigens and has implications for the development of agents that are able to generate protective CD8 T-cell responses.

The epitope integration site for vaccine antigens determines virus control while maintaining efficacy in an engineered cancer vaccine.

Picornaviruses have been developed as potential therapies for gene delivery and vaccination. One drawback to their use is the potential for recombination and viral persistence. Therefore, the engineering strategies used must take into account the possibility for virus escape. We have developed Theiler's murine encephalomyelitis virus (TMEV) as a potential vaccine vector for use in immunotherapy. This study shows that insertion of a vaccine epitope at a unique site within the TMEV leader protein can dramatically increase the type I interferon (IFN) response to infection and promote rapid viral clearance. This live virus vaccine maintains its ability to drive antigen-specific CD8(+) T-cell responses to a model antigen as well as to the weakly immunogenic tumor antigen Her2/neu. Furthermore, the epitope integration site does not affect the efficacy of this vaccine as cancer immunotherapy for treating models of melanoma and breast cancer as demonstrated by delayed tumor outgrowth and increased survival in animals implanted with these tumors. These findings show that an attenuated virus retaining limited ability to replicate nonetheless can effectively mobilize CD8(+) cellular immunity and will be important for the design of picornavirus vectors used as immunotherapy in clinical settings.

Nonequivalence of classical MHC class I loci in ability to direct effective antiviral immunity.

Structural diversity in the peptide binding sites of the redundant classical MHC antigen presenting molecules is strongly selected in humans and mice. Although the encoded antigen presenting molecules overlap in antigen presenting function, differences in polymorphism at the MHC I A, B and C loci in humans and higher primates indicate these loci are not functionally equivalent. The structural basis of these differences is not known. We hypothesize that classical class I loci differ in their ability to direct effective immunity against intracellular pathogens. Using a picornavirus infection model and chimeric H-2 transgenes, we examined locus specific functional determinants distinguishing the ability of class I sister genes to direct effective anti viral immunity. Whereas, parental FVB and transgenic FVB mice expressing the H-2K(b) gene are highly susceptible to persisting Theiler's virus infection within the CNS and subsequent demyelination, mice expressing the D(b) transgene clear the virus and are protected from demyelination. Remarkably, animals expressing a chimeric transgene, comprised primarily of K(b) but encoding the peptide binding domain of D(b), develop a robust anti viral CTL response yet fail to clear virus and develop significant demyelination. Differences in expression of the chimeric K(b)α1α2D(b) gene (low) and D(b) (high) in the CNS of infected mice mirror expression levels of their endogenous H-2(q) counterparts in FVB mice. These findings demonstrate that locus specific elements other than those specifying peptide binding and T cell receptor interaction can determine ability to clear virus infection. This finding provides a basis for understanding locus-specific differences in MHC polymorphism, characterized best in human populations.

Theiler's murine encephalomyelitis virus as a vaccine candidate for immunotherapy.

The induction of sterilizing T-cell responses to tumors is a major goal in the development of T-cell vaccines for treating cancer. Although specific components of anti-viral CD8+ immunity are well characterized, we still lack the ability to mimic viral CD8+ T-cell responses in therapeutic settings for treating cancers. Infection with the picornavirus Theiler's murine encephalomyelitis virus (TMEV) induces a strong sterilizing CD8+ T-cell response. In the absence of sterilizing immunity, the virus causes a persistent infection. We capitalized on the ability of TMEV to induce strong cellular immunity even under conditions of immune deficiency by modifying the virus to evaluate its potential as a T-cell vaccine. The introduction of defined CD8+ T-cell epitopes into the leader sequence of the TMEV genome generates an attenuated vaccine strain that can efficiently drive CD8+ T-cell responses to the targeted antigen. This virus activates T-cells in a manner that is capable of inducing targeted tissue damage and glucose dysregulation in an adoptive T-cell transfer model of diabetes mellitus. As a therapeutic vaccine for the treatment of established melanoma, epitope-modified TMEV can induce strong cytotoxic T-cell responses and promote infiltration of the T-cells into established tumors, ultimately leading to a delay in tumor growth and improved survival of vaccinated animals. We propose that epitope-modified TMEV is an excellent candidate for further development as a human T-cell vaccine for use in immunotherapy.

The pathogen recognition receptor NOD2 regulates human FOXP3+ T cell survival.

The expression of pathogen recognition receptors in human FOXP3+ T regulatory cells is established, yet the function of these receptors is currently obscure. In the process of studying the function of both peripheral and lamina propria FOXP3+ lymphocytes in patients with the human inflammatory bowel disease Crohn's disease, we observed a clear deficiency in the quantity of FOXP3+ lymphocytes in patients with disease-associated polymorphisms in the pathogen recognition receptor gene NOD2. Subsequently, we determined that the NOD2 ligand, muramyl dipeptide (MDP), activates NF-kappaB in primary human FOXP3+ T cells. This activation is functionally relevant, as MDP-stimulated human FOXP3+ T cells are protected from death receptor Fas-mediated apoptosis. Importantly, apoptosis protection was not evident in MDP-stimulated FOXP3+ T cells isolated from a patient with the disease-associated polymorphism. Thus, we propose that one function of pathogen recognition receptors in human T regulatory cells is the protection against death receptor-mediated apoptosis in a Fas ligand-rich environment, such as that of the inflamed intestinal subepithelial space.

T-bet expression by dendritic cells is required for the repolarization of allergic airway inflammation.

By cross-linking B7-DC on dendritic cells (DC) the human IgM antibody (B7-DC XAb) shifts polarized immune responses from Th2 to Th1 in an antigen-specific manner. The molecular determinants governing the ability of DC to reprogram the polarity of T cell recall responses are not yet known. In addition to the expected role of T-bet expressed by T cells in regulating Th1 responses, we find using in vitro assays and an established in vivo model of allergic airway inflammation that T-bet expression by DC is also required for the polarity shift promoted by B7-DC XAb. T-bet expression by both T cells and DC is critically important for B7-DC XAb-induced down-regulation of IL-4, up-regulation of IFN-gamma and suppression of allergic airway inflammation. Moreover, retroviral reconstitution of T-bet expression in T-bet-deficient DC rescued their ability to modulate both naive and memory T-cell responses from Th2 to Th1. Our observations further our understanding of the critical mediators controlling the ability of DC to modify the responses of previously activated T cells and reveal the interesting use of the same transcription factor to regulate the inductive phenotype of DC and the inducible phenotype of T cells.

Reprogrammed FoxP3+ T regulatory cells become IL-17+ antigen-specific autoimmune effectors in vitro and in vivo.

Lymphocyte differentiation from naive CD4(+) T cells into mature Th1, Th2, Th17, or T regulatory cell (Treg) phenotypes has been considered end stage in character. In this study, we demonstrate that dendritic cells (DCs) activated with a novel immune modulator B7-DC XAb (DC(XAb)) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DC(XAb) interaction is Ag-specific, IL-6-dependent, and results in the functional reprogramming of the mature T cell phenotype. The reprogrammed Tregs cease to express IL-10 and TGFbeta, fail to suppress T cell responses, and gain the ability to produce IFN-gamma, IL-17, and TNF-alpha. The ability of IL-6(+) DC(XAb) and the inability of IL-6(-/-) DC(XAb) vaccines to protect animals from lethal melanoma suggest that exogenously modulated DC can reprogram host Tregs. In support of this hypothesis and as a test for Ag specificity, transfer of DC(XAb) into RIP-OVA mice causes a break in immune tolerance, inducing diabetes. Conversely, adoptive transfer of reprogrammed Tregs but not similarly treated CD25(-) T cells into naive RIP-OVA mice is also sufficient to cause autoimmune diabetes. Yet, treatment of normal mice with B7-DC XAb fails to elicit generalized autoimmunity. The finding that mature Tregs can be reprogrammed into competent effector cells provides new insights into the plasticity of T cell lineage, underscores the importance of DC-T cell interaction in balancing immunity with tolerance, points to Tregs as a reservoir of autoimmune effectors, and defines a new approach for breaking tolerance to self Ags as a strategy for cancer immunotherapy.

FOXP3 regulates TLR10 expression in human T regulatory cells.

Although functionally relevant TLRs can be expressed on human T regulatory (Treg) cells, little is known about the transcriptional control of their expression. We hypothesized that the transcription factor forkhead box P3 (FOXP3) regulates the expression of TLR family members in human Treg cells. Using primary human T cells and a reporter assay in Jurkat T cell lines, we dissected the regulation of TLR10, a TLR highly expressed in human Treg cells. We determined that TLR10 was expressed in human Treg cells through quantitative PCR, Western blotting, and flow cytometry. DNA binding of FOXP3 to a suspected cis-regulatory region in proximity to the transcription start site of TLR10 was established through EMSA and chromatin immunoprecipitation. Transcriptional control of TLR10 by FOXP3 was determined through luciferase reporter assays in Jurkat T cell lines. Relevance of FOXP3 to TLR10 gene transcription in primary T cells was established through the transfection of primary CD4(+)CD25(-)FOXP3(-) T cells with a FOXP3 expression vector, which resulted in prompt production of TLR10 mRNA. Enhanced expression of TLR10 protein in primary Treg cells was induced in a calcium-dependent fashion through TCR activation. The suspected promotional cooperation between FOXP3 and NF-AT was established in the abolition of the luciferase signal upon transfection of a mutant FOXP3 devoid of NF-AT-binding activity. These results suggest that human Treg cells express TLR10, and this expression is regulated through a cooperative complex of FOXP3 and NF-AT.

CD28 ligation costimulates cell death but not maturation of double-positive thymocytes due to defective ERK MAPK signaling.

The differentiation of double-positive (DP) CD4(+)CD8(+) thymocytes to single-positive CD4(+) or CD8(+) T cells is regulated by signals that are initiated by coengagement of the Ag (TCR) and costimulatory receptors. CD28 costimulatory receptors, which augment differentiation and antiapoptotic responses in mature T lymphocytes, have been reported to stimulate both differentiation and apoptotic responses in TCR-activated DP thymocytes. We have used artificial APCs that express ligands for TCR and CD28 to show that CD28 signals increase expression of CD69, Bim, and cell death in TCR-activated DP thymocytes but do not costimulate DP thymocytes to initiate the differentiation program. The lack of a differentiation response is not due to defects in CD28-initiated TCR proximal signaling events but by a selective defect in the activation of ERK MAPK. To characterize signals needed to initiate the death response, a mutational analysis was performed on the CD28 cytoplasmic domain. Although mutation of all of CD28 cytoplasmic domain signaling motifs blocks cell death, the presence of any single motif is able to signal a death response. Thus, there is functional redundancy in the CD28 cytoplasmic domain signaling motifs that initiate the thymocyte death response. In contrast, immobilized Abs can initiate differentiation responses and cell death in DP thymocytes. However, because Ab-mediated differentiation occurs through CD28 receptors with no cytoplasmic domain, the response may be mediated by increased adhesion to immobilized anti-TCR Abs.

Ly9 (CD229)-deficient mice exhibit T cell defects yet do not share several phenotypic characteristics associated with SLAM- and SAP-deficient mice.

Signaling lymphocyte activation molecule (SLAM) family receptors are critically involved in modulating innate and adaptive immune responses. Several SLAM family receptors have been shown to interact with the adaptor molecule SAP; however, subsequent intracellular signaling is poorly defined. Notably, mutations in SLAM-associated protein (SAP) lead to X-linked lymphoproliferative disease, a rare but fatal immunodeficiency. Although the SLAM family member Ly9 (CD229) is known to interact with SAP, the functions of this receptor have remained elusive. Therefore, we have generated Ly9-/- mice and compared their phenotype with that of SLAM-/- and SAP-/- mice. We report that Ly9-/- T cells exhibit a mild Th2 defect associated with reduced IL-4 production after stimulation with anti-TCR and anti-CD28 in vitro. This defect is similar in magnitude to the previously reported Th2 defect in SLAM-/- mice but is more subtle than that observed in SAP-/- mice. In contrast to SLAM-/- and SAP-/- mice, T cells from Ly9-/- mice proliferate poorly and produce little IL-2 after suboptimal stimulation with anti-CD3 in vitro. We have also found that Ly9-/- macrophages exhibit no defects in cytokine production or bacterial killing as was observed in SLAM-/- macrophages. Additionally, Ly9-/- mice differ from SAP-/- mice in that they foster normal development of NKT cells and mount appropriate T and B cell responses to lymphocytic choriomeningitis virus. We have identified significant phenotypic differences between Ly-9-/- mice as compared with both SLAM-/- and SAP-/- mice. Although Ly9, SLAM, and SAP play a common role in promoting Th2 polarization, Ly-9 is uniquely involved in enhancing T cell activation.

Increased thymic output in HIV-negative patients after antiretroviral therapy.

OBJECTIVE: To determine the effects of antiretroviral therapy on thymic output independent of HIV infection. METHODS: Thymic output was evaluated by quantifying signal joint T-cell receptor (TCR) recombination excision circles in peripheral blood lymphocytes from HIV-negative patients undergoing prophylactic antiretroviral therapy. Additionally, effects of the HIV protease inhibitor nelfinavir were assessed in vivo on TCR-induced death of murine double-positive thymocytes. RESULTS: Five out of seven HIV-negative patients undergoing prophylactic antiretroviral therapy exhibited a dramatic increase (1-3 log10) in recent thymic emigrants containing signal joint TCR recombination excision circles while their peripheral T cell compartments remained relatively unaffected. None of the patients developed subsequent HIV infections. Interestingly, nelfinavir did not have significant effects on TCR-induced apoptosis of murine thymocytes in vivo. CONCLUSION: Antiretroviral therapy augments thymic output independent of HIV. Furthermore, nelfinavir does not dramatically affect TCR-induced thymocyte death in mice, thus central tolerance remains intact.