Karen P. McKinnon

Differential Roles for CCR5 Expression on Donor T Cells during Graft-versus-Host Disease Based on Pretransplant Conditioning

The coordinated expression of chemokines and receptors may be important in the directed migration of alloreactive T cells during graft-vs-host disease (GVHD). Recent work demonstrated in a murine model that transfer of CCR5-deficient (CCR5−/−) donor cells to nonconditioned haploidentical recipients resulted in reduced donor cell infiltration in liver and lymphoid tissues compared with transfer of CCR5+/+ cells. To investigate the function of CCR5 during GVHD in conditioned transplant recipients, we transferred CCR5−/− or wild-type C57BL/6 (B6) T cells to lethally irradiated B6D2 recipients. Unexpectedly, we found an earlier time to onset and a worsening of GVHD using CCR5−/− T cells, which was associated with significant increases in the accumulation of alloreactive CD4+ and CD8+ T cells in liver and lung. Conversely, the transfer of CCR5−/− donor cells to nonirradiated recipients led to reduced infiltration of target organs, confirming previous studies and demonstrating that the role of CCR5 on donor T cells is dependent on conditioning of recipients. Expression of proinflammatory chemokines in target tissues was dependent on conditioning of recipients, such that CXCL10 and CXCL11 were most highly expressed in tissues of irradiated recipients during the first week post-transplant. CCR5−/− T cells were shown to have enhanced migration to CXCL10, and blocking this ligand in vivo improved survival in irradiated recipients receiving CCR5−/− T cells. Our data indicate that the effects of inhibiting CCR5/ligand interaction on donor T cells during GVHD differ depending on conditioning of recipients, a finding with potentially important clinical significance.

CD200 is induced by ERK and is a potential therapeutic target in melanoma

Immune-mediated antitumor responses occur in patients with metastatic melanoma (MM), and therapies designed to augment such responses are clinically beneficial. Despite the immunogenicity of melanoma, immunomodulatory therapies fail in the majority of patients with MM. An inability of DCs to sufficiently activate effector cells may, in part, underlie this failure of the antitumor response seen in most patients. In this work, we show that mutation of N-RAS or B-RAF, signature genetic lesions present in most MMs, potently induced the expression of cell-surface CD200, a repressor of DC function. Employing 2 independent, genome-wide microarray analyses, we identified CD200 as a highly dynamic, downstream target of RAS/RAF/MEK/ERK activation in melanoma. CD200 protein was similarly overexpressed in human melanoma cell lines and primary tumors. CD200 mRNA expression correlated with progression and was higher in melanoma than in other solid tumors or acute leukemia. Melanoma cell lines expressing endogenous CD200 repressed primary T cell activation by DCs, while knockdown of CD200 by shRNA abrogated this immunosuppressive effect. These data indicate that in addition to its effects on growth, survival, and motility, ERK activation in MM attenuates a host antitumor immune response, implicating CD200 and its interaction with the CD200 receptor as a potential therapeutic target for MM.

The SecA2 secretion factor of Mycobacterium tuberculosis promotes growth in macrophages and inhibits the host immune response.

ABSTRACT The SecA protein is present in all bacteria, and it is a central component of the general Sec-dependent protein export pathway. An unusual property of Mycobacterium tuberculosis is the presence of two SecA proteins: SecA1, the essential “housekeeping” SecA, and SecA2, the accessory secretion factor. Here, we report that a ΔsecA2 mutant of M. tuberculosis was defective for growth in the early stages of low-dose aerosol infection of C57BL/6 mice, a time during which the bacillus is primarily replicating in macrophages. Consistent with this in vivo phenotype, we found that the ΔsecA2 mutant was defective for growth in macrophages from C57BL/6 mice. The ΔsecA2 mutant was also attenuated for growth in macrophages from phox−/− mice and from NOS2−/− mice. These mice are defective in the reactive oxygen intermediate (ROI)-generating phagocyte oxidase and the reactive nitrogen intermediate (RNI)-generating inducible nitric oxide synthase, respectively. This indicated a role for SecA2 in the intracellular growth of M. tuberculosis that is independent of protecting against these ROIs or RNIs. Macrophages infected with the ΔsecA2 mutant produced higher levels of tumor necrosis factor alpha, interleukin-6, RNI, and gamma interferon-induced major histocompatibility complex class II. This demonstrated a function for M. tuberculosis SecA2 in suppressing macrophage immune responses, which could explain the role of SecA2 in intracellular growth. Our results provide another example of a relationship between M. tuberculosis virulence and inhibition of the host immune response.

Regulation and Specificity of MHC2TA Promoter Usage in Human Primary T Lymphocytes and Cell Line

Although activated human T cells express MHC class II antigens, the regulation of these antigens in T cells is poorly understood. This study focuses on the control of the MHC2TA gene in these cells. MHC2TA encodes the transcriptional master regulator of MHC class II, the class II trans-activator (CIITA). It has at least three distinct promoters (PI, PIII, and PIV), each active in an overlapping subset of cell types and directing a slightly different product. This report used highly purified blood T cells prepared by negative immunoselection to analyze CIITA. Real-time PCR analysis indicates that resting T cells do not express detectable CIITA transcript, while activated T cells express the PIII CIITA form. Transient transfection of activated blood T cells using wild-type and mutant PIII promoter-reporter constructs shows that two promoter elements, activation response element-1 (ARE-1) and ARE-2, are important for PIII function. cAMP response element binding protein, a known activator of gene expression in activated T cells, activates PIII in primary T cells. However, an intact ARE-2 site is not required for this activation, indicating that cAMP response element binding protein does not activate via this site. EMSAs indicate that an activating transcription factor/cAMP response element binding protein/cAMP response element modulator family member, but not phosphorylated cAMP response element binding protein-1, binds to ARE-2. ARE-2 also forms a complex with an unidentified protein. The ARE-2 binding protein is constitutively expressed in a DR+ T cell line, reflecting differences between the DR+ cell line and primary blood lymphocytes. These results show that MHC2TA PIII is induced in activated T lymphocytes, and that the induced binding of ARE-2 is a crucial step in this process.

A Novel Viral System for Generating Antigen-Specific T Cells

Dendritic cell (DC)-based vaccines are increasingly used for the treatment of patients with malignancies. Although these vaccines are typically safe, consistent and lasting generation of tumor-specific immunity has been rarely demonstrated. Improved methods for delivering tumor Ags to DCs and approaches for overcoming tolerance or immune suppression to self-Ags are critical for improving immunotherapy. Viral vectors may address both of these issues, as they can be used to deliver intact tumor Ags to DCs, and have been shown to inhibit the suppression mediated by CD4+CD25+ regulatory T cells. We have evaluated the potential use of Venezuelan equine encephalitis virus replicon particles (VRPs) for in vitro Ag delivery to human monocyte-derived DCs. VRPs efficiently transduced immature human DCs in vitro, with ∼50% of immature DCs expressing a vector-driven Ag at 12 h postinfection. VRP infection of immature DCs was superior to TNF-α treatment at inducing phenotypic maturation of DCs, and was comparable to LPS stimulation. Additionally, VRP-infected DC cultures secreted substantial amounts of the proinflammatory cytokines IL-6, TNF-α, and IFN-α. Finally, DCs transduced with a VRP encoding the influenza matrix protein (FMP) stimulated 50% greater expansion of FMP-specific CD8+ CTL when compared with TNF-α-matured DCs pulsed with an HLA-A*0201-restricted FMP peptide. Thus, VRPs can be used to deliver Ags to DCs resulting in potent stimulation of Ag-specific CTL. These findings provide the rationale for future studies evaluating the efficacy of VRP-transduced DCs for tumor immunotherapy.

Transfection of Macrophage Inflammatory Protein 1α into B16 F10 Melanoma Cells Inhibits Growth of Pulmonary Metastases But Not Subcutaneous Tumors

Macrophage inflammatory protein 1α (MIP-1α), a CC chemokine, is a chemoattractant for T cells and immature dendritic cells. Plasmacytoma cells expressing MIP-1α generate a cytotoxic T cell response without affecting tumor growth. To understand this discrepancy, we compared a local tumor model with a metastatic one using MIP-1α-transfected B16 F10 melanoma cells. Clonal idiosyncrasies were controlled by selecting three lipotransfected tumor clones and two pcDNA vector transfected control clones with equivalent in vitro proliferative capacities. No significant differences were seen between the MIP-1α-producing and control melanoma cells after s.c. injection in the hind leg. All animals had a leg diameter of 10 cm in 18.5–21.5 days. However, after i.v. injection the number of pulmonary foci was significantly reduced in the MIP-1α-producing clones. Injection of 106 control transfected cells resulted in a median of 98.5 tumor foci in 2 wk, whereas the injection of the MIP-1α-producing clones resulted in 89.5, 26.5, and 0 foci. The number of metastatic foci was inversely proportional to the amount of MIP-1α produced by the clone in vitro. Flow cytometry showed a significant increase in CD8+ cells in lungs of mice with MIP-1α-transfected tumors 3 days after injection. This increase was not maintained 10 days later despite continued production of MIP-1α. The protection offered by transfection with MIP-1α was significantly impaired in β2-microglobulin−/− mice. Our findings suggest that MIP-1α is effective in preventing the initiation of metastasis, but not at sustaining an effective antitumor response.

Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy

Immunotherapy holds tremendous promise for improving cancer treatment1. Administering radiotherapy with immunotherapy has been shown to improve immune responses and can elicit an “abscopal effect”2. Unfortunately, response rates for this strategy remain low3. Herein, we report an improved cancer immunotherapy approach that utilizes antigen-capturing nanoparticles (AC-NPs). We engineered several AC-NPs formulations and demonstrated that the set of protein antigens captured by each AC-NP formulation is dependent upon NP surface properties. We showed that AC-NPs deliver tumor specific proteins to antigen-presenting cells and significantly improve the efficacy of αPD-1 treatment using the B16F10 melanoma model, generating up to 20% cure rate as compared to 0% without AC-NPs. Mechanistic studies revealed that AC-NPs induced an expansion of CD8+ cytotoxic T cells and increased both CD4+/Treg and CD8+/Treg ratios. Our work presents a novel strategy for improving cancer immunotherapy with nanotechnology.

Dendritic cells can be rapidly expanded ex vivo and safely administered in patients with metastatic breast cancer

PurposeImmunotherapy using either dendritic cells (DCs) or expanded cytotoxic T cells (CTLs) has received increased interest in the treatment of specific malignancies including metastatic breast cancer (MBC). DCs can be generated ex vivo from monocytes or CD34+ precursors. The ability to expand and safely administer CD34-derived DCs in patients with MBC that have received prior cytotoxic chemotherapy has not been evaluated.MethodsWe enrolled ten patients with MBC that had received prior chemotherapy for the treatment of metastatic disease on a phase I/II trial designed to test the safety and feasibility of administering ex vivo expanded DCs from CD34+ progenitor cells.ResultsUsing a cocktail of multiple different cytokines, we could expand DCs 19-fold compared to the initial CD34-selected product, which allowed the administration of as many as six vaccine treatments per patient. Patients received three to six injections i.v. of DCs pulsed with either the wild type GP2 epitope from the HER-2/neu protein or an altered peptide ligand, isoleucine to leucine (I2L). Toxicity was mild, with no patients demonstrating grade III toxicity during the treatment. Two patients with subcutaneous disease had a partial response to therapy, while IFN-γ-producing CD8+ T cells could be found in two other patients during treatment.ConclusionsThis approach is safe and effective in generating a significant quantity of DCs from CD34-precursors.

CC chemokine receptor 8 potentiates donor Treg survival and is critical for the prevention of murine graft-versus-host disease.

The infusion of donor regulatory T cells (Tregs) has been used to prevent acute graft-versus-host disease (GVHD) in mice and has shown promise in phase 1 clinical trials. Previous work suggested that early Treg migration into lymphoid tissue was important for GVHD prevention. However, it is unclear how and where Tregs function longitudinally to affect GVHD. To better understand their mechanism of action, we studied 2 Treg-associated chemokine receptors in murine stem cell transplant models. CC chemokine receptor (CCR) 4 was dispensable for donor Treg function in the transplant setting. Donor Tregs lacking CCR8 (CCR8(-/-)), however, were severely impaired in their ability to prevent lethal GVHD because of increased cell death. By itself, CCR8 stimulation was unable to rescue Tregs from apoptosis. Instead, CCR8 potentiated Treg survival by promoting critical interactions with dendritic cells. In vivo, donor bone marrow-derived CD11c(+) antigen-presenting cells (APCs) were important for promoting donor Treg maintenance after transplant. In contrast, host CD11c(+) APCs appeared to be dispensable for early activation and expansion of donor Tregs. Collectively, our data indicate that a sustained donor Treg presence is critical for their beneficial properties, and that their survival depends on CCR8 and donor but not host CD11c(+) APCs.

Cutting Edge: Rho Activation and Actin Polarization Are Dependent on Plexin-A1 in Dendritic Cells

We recently identified expression of the semaphorin receptor, plexin-A1, in dendritic cells (DCs); however, its function in these cells remains to be elucidated. To investigate function and maximize physiological relevance, we devised a retroviral approach to ablate plexin-A1 gene expression using small hairpin RNA (shRNA) in primary bone marrow-derived DCs. We show that plexin-A1 localizes within the cytoplasm of immature DCs, becomes membrane-associated, and is enriched at the immune synapse in mature DCs. Reducing plexin-A1 expression with shRNA greatly reduced actin polarization as well as Rho activation without affecting Rac or Cdc42 activation. A Rho inhibitor, C3, also reduced actin polarization. These changes were accompanied by the near-ablation of T cell activation. We propose a mechanism of adaptive immune regulation in which plexin-A1 controls Rho activation and actin cytoskeletal rearrangements in DCs that is associated with enhanced DC-T cell interactions.