The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity.

T cell activation is subject to tight regulation to avoid inappropriate responses to self antigens. Here we show that genetic deficiency in the ubiquitin ligase Peli1 caused hyperactivation of T cells and rendered T cells refractory to suppression by regulatory T cells and transforming growth factor-ß (TGF-ß). As a result, Peli1-deficient mice spontaneously developed autoimmunity characterized by multiorgan inflammation and autoantibody production. Peli1 deficiency resulted in the nuclear accumulation of c-Rel, a member of the NF-κB family of transcription factors with pivotal roles in T cell activation. Peli1 negatively regulated c-Rel by mediating its Lys48 (K48) ubiquitination. Our results identify Peli1 as a critical factor in the maintenance of peripheral T cell tolerance and demonstrate a previously unknown mechanism of c-Rel regulation.

Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer.

Antigen specific T cell migration to sites of infection or cancer is critical for an effective immune response. In mouse models of cancer, the number of lymphocytes reaching the tumor is typically only a few hundred, yet technology capable of imaging these cells using bioluminescence has yet to be achieved. A combination of codon optimization, removal of cryptic splice sites and retroviral modification was used to engineer an enhanced firefly luciferase (ffLuc) vector. Compared with ffLuc, T cells expressing our construct generated >100 times more light, permitting detection of as few as three cells implanted s.c. while maintaining long term coexpression of a reporter gene (Thy1.1). Expression of enhanced ffLuc in mouse T cells permitted the tracking of <3 x 10(4) adoptively transferred T cells infiltrating sites of vaccination and preestablished tumors. Penetration of light through deep tissues, including the liver and spleen, was also observed. Finally, we were able to enumerate infiltrating mouse lymphocytes constituting <0.3% of total tumor cellularity, representing a significant improvement over standard methods of quantitation including flow cytometry.

Genetic Engineering of T Cells to Target HERV-K, an Ancient Retrovirus on Melanoma.

PURPOSE: The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) expressed on melanoma but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T-cell surface, such that they target tumors in advanced stages of melanoma. EXPERIMENTAL DESIGN: Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immunohistochemical (IHC) analysis. HERV-K env-specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env-specific CAR(+) T cells were expanded ex vivo on activating and propagating cells (AaPC) and characterized for CAR expression and specificity. This includes evaluating the HERV-K-specific CAR(+) T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model. RESULTS: We detected HERV-K env protein on melanoma but not in normal tissues. After electroporation of T cells and selection on HERV-K(+) AaPC, more than 95% of genetically modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env(+) tumor targets in an antigen-specific manner. Even though there is apparent shedding of this TAA from tumor cells that can be recognized by HERV-K env-specific CAR(+) T cells, we observed a significant antitumor effect. CONCLUSIONS: Adoptive cellular immunotherapy with HERV-K env-specific CAR(+) T cells represents a clinically appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors.

Activating and propagating polyclonal gamma delta T cells with broad specificity for malignancies.

PURPOSE: To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved. EXPERIMENTAL DESIGN: Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing. RESULTS: γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1(neg)Vδ2(neg) subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1(neg)Vδ2(neg)>Vδ2) of survival of mice with ovarian cancer xenografts. CONCLUSIONS: Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans.

Treating brain tumor-initiating cells using a combination of myxoma virus and rapamycin.

BACKGROUND: Intratumoral heterogeneity in glioblastoma multiforme (GBM) poses a significant barrier to therapy in certain subpopulation such as the tumor-initiating cell population, being shown to be refractory to conventional therapies. Oncolytic virotherapy has the potential to target multiple compartments within the tumor and thus circumvent some of the barriers facing conventional therapies. In this study, we investigate the oncolytic potential of myxoma virus (MYXV) alone and in combination with rapamycin in vitro and in vivo using human brain tumor-initiating cells (BTICs). METHODS: We cultured fresh GBM specimens as neurospheres and assayed their growth characteristics in vivo. We then tested the susceptibility of BTICs to MYXV infection with or without rapamycin in vitro and assessed viral biodistribution/survival in vivo in orthotopic xenografts. RESULTS: The cultured neurospheres were found to retain stem cell markers in vivo, and they closely resembled human infiltrative GBM. In this study we determined that (i) all patient-derived BTICs tested, including those resistant to temozolomide, were susceptible to MYXV replication and killing in vitro; (ii) MYXV replicated within BTICs in vivo, and intratumoral administration of MYXV significantly prolonged survival of BTIC-bearing mice; (iii) combination therapy with MYXV and rapamycin improved antitumor activity, even in mice bearing "advanced" BTIC tumors; (iv) MYXV treatment decreased expression of stem cell markers in vitro and in vivo. CONCLUSIONS: Our study suggests that MYXV in combination with rapamycin infects and kills both the BTICs and the differentiated compartments of GBM and may be an effective treatment even in TMZ-resistant patients.

A central role for RAF→MEK→ERK signaling in the genesis of pancreatic ductal adenocarcinoma.

UNLABELLED: KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDA) but remains an intractable pharmacologic target. Consequently, defining RAS effector pathway(s) required for PDA initiation and maintenance is critical to improve treatment of this disease. Here, we show that expression of BRAF(V600E), but not PIK3CA(H1047R), in the mouse pancreas leads to pancreatic intraepithelial neoplasia (PanIN) lesions. Moreover, concomitant expression of BRAF(V600E) and TP53(R270H) result in lethal PDA. We tested pharmacologic inhibitors of RAS effectors against multiple human PDA cell lines. Mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) inhibition was highly effective both in vivo and in vitro and was synergistic with AKT inhibition in most cell lines tested. We show that RAF→MEK→ERK signaling is central to the initiation and maintenance of PDA and to rational combination strategies in this disease. These results emphasize the value of leveraging multiple complementary experimental systems to prioritize pathways for effective intervention strategies in PDA. SIGNIFICANCE: PDA is diffi cult to treat, in large part, due to recurrent mutations in the KRAS gene. Here, we defi ne rational treatment approaches for the disease achievable today with existing drug combinations by thorough genetic and pharmacologic dissection of the major KRAS effector pathways, RAF→MEK→ERK and phosphoinositide 3'-kinase (PI3'K)→AKT.

Antitumor activity mediated by CpG: the route of administration is critical.

Unmethylated CpG oligodeoxynucleotides (CpG) are synthetic toll-like receptor 9 agonists that activate innate immune cells and which have been tested as an immune therapy in a number of cancer clinical trials. Although some antitumor immune responses have been reported, so far the majority of studies have failed to show significant clinical responses to CpG. Here we showed that the route of administration is critical to the antitumor activity of CpG. Although intravenous (i.v.) injection of CpG was capable of inducing the activation and expansion of tumor antigen-specific T cells, most of these activated T cells failed to migrate to tumor sites. By contrast, intratumoral (i.t.) injection of CpG led to extensive tumor infiltration of antigen-specific T cells and subsequent tumor suppression. We further showed that very high levels of inflammatory chemokines [regulated upon activation, normal T-cell expressed, and secreted (RANTES), interferon-inducible protein-10 (IP-10), monocyte chemoattractant protein-1, monocyte chemotactic protein (MCP5), macrophage inflammatory proteins (MIP1α, and MIP1ß)] were induced in the tumor microenvironment after i.t. CpG injection, compared with administration by the i.v. route. It is interesting to note that, in vivo depletion of plasmacytoid dendritic cells greatly reduced the levels of chemokines induced; also, T-cell accumulation and antitumor effect were impaired. We also showed that i.t. but not i.v. CpG injection induced a broad antigen-specific T-cell response against tumor-derived antigens. Collectively, our data provides evidence that the route of CpG administration is a critical factor in mediating antitumor activity. By inducing localized inflammatory signals at tumor sites, i.t. CpG effectively promotes the migration, activation and function of immune cells, ultimately leading to improved tumor control.

Imaging adoptive cell transfer based cancer immunotherapy.

The prospect of mobilizing the power of the immune system in the fight against cancer has fascinated generations of immunologists. After decades of sporadic successes scattered amongst frequent failures, the field of tumor immunology is now poised to take advantage of a greatly improved understanding of the molecular and cellular mechanisms of immune tolerance. Further enhancing the prospects of cancer immunotherapy are technological advantages that have resulted in sophisticated diagnostic and immune monitoring approaches. Amongst these approaches, molecular imaging has the potential to play a transformational role in the development of highly efficient immune therapies. This brief review outlines recent significant steps in this direction, and discusses strategies to incorporate molecular imaging in the design of the next generation of experimental and clinical cell-based immune interventions against cancer.

Transduction of tumor-specific T cells with CXCR2 chemokine receptor improves migration to tumor and antitumor immune responses.

PURPOSE: One of the most important rate-limiting steps in adoptive cell transfer (ACT) is the inefficient migration of T cells to tumors. Because melanomas specifically express the chemokines CXCL1 and CXCL8 that are known to facilitate the CXCR2-dependent migration by monocytes, our aim is to evaluate whether introduction of the CXCR2 gene into tumor-specific T cells could further improve the effectiveness of ACT by enhancing T-cell migration to tumor. EXPERIMENTAL DESIGN: In this study, we used transgenic pmel-1 T cells, which recognize gp100 in the context of H-2Db, that were transduced with luciferase gene to monitor the migration of transferred T cells in vivo. To visualize luciferase-expressing T cells within a tumor, a nonpigmented tumor is required. Therefore, we used the MC38 tumor model, which naturally expresses CXCL1. RESULTS: Mice bearing MC38/gp100 tumor cells treated with CXCR2/luciferase-transduced pmel-1 T cells showed enhanced tumor regression and survival compared with mice receiving control luciferase-transduced pmel-1 T cells. We also observed preferential accumulation of CXCR2-expressing pmel-1 T cells in the tumor sites of these mice using bioluminescence imaging. A similar enhancement in tumor regression and survival was observed when CXCR2-transduced pmel-1 T cells were transferred into mice bearing CXCL1-transduced B16 tumors compared with mice treated with control pmel-1 T cells. CONCLUSIONS: These results implicate that the introduction of the CXCR2 gene into tumor-specific T cells can enhance their localization to tumors and improve antitumor immune responses. This strategy may ultimately enable personalization of cancer therapies based on chemokine expression by tumors.

A role for the MHC class I-like Mill molecules in nutrient metabolism and wound healing.

MHC class I family members serve multiple functions beyond antigen presentation. We provide insight into the structure, expression and function of the Mill subfamily. This family includes two surface glycoproteins, Mill1 and Mill2. Protein sequences for Mill1 and Mill2 are most highly related to the NKG2D ligands, MICA and MICB, but neither of them bound to NKG2D. Computer-based protein modelling indicated that hereditary haemochromatosis protein (HFE), a molecule involved in iron uptake, was most similar. Mill1 and Mill2 were observed on cycling thymocytes, proliferating smooth muscle cells and fibroblasts. Using soluble Mill proteins, we found evidence for a soluble ligand in serum. Like HFE, the Mill family may be involved in nutrient metabolism. Skin was one of the only three organs found to express transcripts for both Mill1 and Mill2. Addition of antibodies specific for Mill2 to wounded skin enhanced healing. Our results suggest a role for the Mill proteins in cellular metabolism, with possible therapeutic significance.

Immunosynapse formation coincides with rapid activation of NK cells by syngeneic T cells and correlates with clustering of MHC class I.

T cells cultured for 3 h with antigen-presenting cells (APCs) stimulated syngeneic IL-2-activated NK cells as measured via a standard chromium-release assay. Discrete caps containing both TCR and MHC-I had formed on the surface of these activated T cells. When conjugates were formed between NK cells and these activated T cells, >80% of the contact sites were in the MHC-I(dim) region outside the TCR-MHC-I cap. Stimulation with phorbol myristate acetate plus Ionomycin, which bypasses the need for cell surface events during activation, did not induce either cap formation or NK cell activation. Further, the addition of the protein transport inhibitor Brefeldin A did not block activation of NK cells. MHC-I is the major inhibitory ligand recognized by NK cells. One possible mechanism for the activation of NK cells by TCR-MHC-I-capped T cells is that aggregation of MHC-I into one region leaves the remaining T cell surface denuded of ligands for NK-inhibitory receptors. As a test of this hypothesis, we aggregated MHC-I on T cells with plate-bound anti-MHC-I mAb. This treatment conferred upon the T cells the capacity to activate NK cells, suggesting that MHC-I clustering could contribute to the observed phenomenon.

Survival versus neglect: redefining thymocyte subsets based on expression of NKG2D ligand(s) and MHC class I.

BALB/c thymocytes can be divided into three distinct subsets according to the expression of a ligand for the NK activation receptor NKG2D (NKG2D-L) and the expression of MHC class I (MHC-I). The first subset (MHC-Imid/NKG2D-Lhigh or "N+") is predominately CD4+CD8+ double positive (DP), comprises approximately 35% of thymocytes in a 6-8-week-old adult and contains uncommitted cells that have neither undergone selection nor are committed to death by neglect. The second subset (MHC-Ilow/NKG2D-Llow or "M-"), also mostly DP cells, comprises approximately 50% of thymocytes and consists of cells committed to death by apoptosis, likely due to neglect. By contrast, the third subset (MHC-Ihigh/NKG2D-Llow or "M+") is largely single positive (SP), represents approximately 15% of thymocytes and mostly contains more mature cells that have undergone successful positive selection. The major advantage of the analysis is that it splits DP cells into two subpopulations, one committed to death by apoptosis and the other subjected to selection. The analysis also suggests that NKG2D-L may play a role in thymocyte development.

Expression cloning and function of the rat NK activating and inhibitory receptors NKR-P1A and -P1B.

We have characterized the rat NK receptors NKR-P1A and -P1B. A cDNA library was constructed from the rat NK cell line, RNK-16. Using the pMX retroviral cloning system, the library was expressed in the human NK cell line, YTSeco, and cells staining with the anti-rat mAb 10/78 identified, FACS sorted and cloned. Two genes, corresponding to rat NK receptors NKR-P1A and -P1B, were identified. YTSeco clones expressing either NKR-P1A or -P1B were functionally tested using (51)Cr-release redirected lysis assays and calcium flux experiments. This demonstrated that NKR-P1A functions as an activation receptor, as previously shown, and that NKR-P1B functions as an inhibitory receptor, as predicted by the presence of an immunoreceptor tyrosine-based inhibition motif. Although annotated as NKR-P1A specific, we found that mAb 10/78 stained YTSeco clones expressing NKR-P1A or -P1B equally well, as did the mAb 3.2.3 used for the original cloning of rat NKR-P1A.

Activated, but not resting, T cells can be recognized and killed by syngeneic NK cells.

We demonstrate that IL-2-activated NK cells or lymphokine-activated killer cells recognize and kill syngeneic CD4(+) and CD8(+) T cells that have been activated by APCs. Induction with APC required TCR-specific Ag, and lysis was perforin mediated. Brefeldin A, which disrupts protein transport, inhibited the sensitivity induced by activation. In BALB/c, expression of NKG2D ligands correlated with lysis and could be inhibited by brefeldin A. As well, addition of anti-NKG2D mAb to a killing assay completely abrogated lysis. Transduction of mouse NKG2D into a human NK cell line, YTSeco, conferred upon it the ability to kill activated BALB/c T cells, indicating that NKG2D is necessary for recognition. Our data provide a basis for studying a role for NK cells in T cell regulation.