Kavita M. Dhodapkar

Antitumor Monoclonal Antibodies Enhance Cross-Presentation of Cellular Antigens and the Generation of Myeloma-specific Killer T Cells by Dendritic Cells

The mechanism of antitumor effect of monoclonal antibodies (mAbs) is not fully understood. Here we show that coating myeloma cells with anti–syndecan-1 antibody promotes cross-presentation of cellular antigens by dendritic cells (DCs) to autologous T cells from healthy donors. The tumor cells treated with anti–syndecan-1 or isotype-matched control antibody were fed to HLA-mismatched monocyte-derived immature DCs. Tumor cell–loaded mature DCs induced a strong CD8+ T cell response that was specific for the cancer-testis (C-T) antigens expressed in the tumor. The CD8+ T cells killed peptide-pulsed targets, as well as myeloma tumor cells. Importantly, mAbs-coated tumor-loaded DCs were consistently superior to DCs loaded with peptides or dying cells for eliciting tumor-specific killer T cells. This enhanced cross-presentation was not due to enhanced tumor cell uptake or to DC maturation. When mixtures of NY-Eso-1-positive and -negative myeloma cells were captured by DCs, the anti–syndecan-1 antibody had to be on the NY-Eso-1-positive cells to elicit NY-Eso-1–specific response. Cross-presentation was inhibited by pretreatment of DCs with Fcγ receptor blocking antibodies. Targeting of mAb-coated tumors to DCs may contribute to the efficacy of tumor-reactive mAb and offers a new strategy for immunotherapy.

A Reversible Defect in Natural Killer T Cell Function Characterizes the Progression of Premalignant to Malignant Multiple Myeloma

We studied the function of antitumor T and natural killer T (NKT) cells from the blood and tumor bed in 23 patients with premalignant gammopathy, nonprogressive myeloma, or progressive multiple myeloma. We show that antitumor killer T cells can be detected in patients with both progressive or nonprogressive myeloma. Vα24+Vβ11+ invariant NKT cells are detectable in the blood and tumor bed of all cohorts. However, freshly isolated NKT cells from both the blood and tumor bed of patients with progressive disease, but not nonprogressive myeloma or premalignant gammopathy, have a marked deficiency of ligand-dependent interferon-γ production. This functional defect can be overcome in vitro using dendritic cells pulsed with the NKT ligand, α-galactosylceramide (α-GalCer). Fresh myeloma cells express CD1d, and can be efficiently killed by autologous NKT cells. We hypothesize that presentation of tumor derived glycolipids by myeloma cells leads to NKT dysfunction in vivo. These data demonstrate that clinical progression in patients with monoclonal gammopathies is associated with an acquired but potentially reversible defect in NKT cell function and support the possibility that these innate lymphocytes play a role in controlling the malignant growth of this incurable B cell tumor in patients.

Combination Therapy with Anti–CTLA-4 and Anti–PD-1 Leads to Distinct Immunologic Changes In Vivo

Combination therapy concurrently targeting PD-1 and CTLA-4 immune checkpoints leads to remarkable antitumor effects. Although both PD-1 and CTLA-4 dampen the T cell activation, the in vivo effects of these drugs in humans remain to be clearly defined. To better understand biologic effects of therapy, we analyzed blood/tumor tissue from 45 patients undergoing single or combination immune checkpoint blockade. We show that blockade of CTLA-4, PD-1, or combination of the two leads to distinct genomic and functional signatures in vivo in purified human T cells and monocytes. Therapy-induced changes are more prominent in T cells than in monocytes and involve largely nonoverlapping changes in coding genes, including alternatively spliced transcripts and noncoding RNAs. Pathway analysis revealed that CTLA-4 blockade induces a proliferative signature predominantly in a subset of transitional memory T cells, whereas PD-1 blockade instead leads to changes in genes implicated in cytolysis and NK cell function. Combination blockade leads to nonoverlapping changes in gene expression, including proliferation-associated and chemokine genes. These therapies also have differential effects on plasma levels of CXCL10, soluble IL-2R, and IL-1α. Importantly, PD-1 receptor occupancy following anti–PD-1 therapy may be incomplete in the tumor T cells even in the setting of complete receptor occupancy in circulating T cells. These data demonstrate that, despite shared property of checkpoint blockade, Abs against PD-1, CTLA-4 alone, or in combination have distinct immunologic effects in vivo. Improved understanding of pharmacodynamic effects of these agents in patients will support rational development of immune-based combinations against cancer.

Dendritic cells mediate the induction of polyfunctional human IL17-producing cells (Th17-1 cells) enriched in the bone marrow of patients with myeloma.

IL17-producing (Th17) cells are a distinct lineage of T helper cells that regulate immunity and inflammation. The role of antigen-presenting cells in the induction of Th17 cells in humans remains to be fully defined. Here, we show that human dendritic cells (DCs) are efficient inducers of Th17 cells in culture, including antigen-specific Th17 cells. Although most freshly isolated circulating human Th17 cells secrete IL17 alone or with IL2, those induced by DCs are polyfunctional and coexpress IL17 and IFNgamma (Th17-1 cells). The capacity of DCs to expand Th17-1 cells is enhanced upon DC maturation, and mature DCs are superior to monocytes for the expansion of autologous Th17 cells. In myeloma, where tumors are infiltrated by DCs, Th17 cells are enriched in the bone marrow relative to circulation. Bone marrow from patients with myeloma contains a higher proportion of Th17-1 cells compared with the marrow in preneoplastic gammopathy (monoclonal gammopathy of undetermined significance [MGUS]). Uptake of apoptotic but not necrotic myeloma tumor cells by DCs leads to enhanced induction of Th17-1 cells. These data demonstrate the capacity of DCs to induce expansion of polyfunctional IL17-producing T cells in humans, and suggest a role for DCs in the enrichment of Th17-1 cells in the tumor bed.

Enhancement of clonogenicity of human multiple myeloma by dendritic cells

Infiltration by dendritic cells (DCs) is a common feature of most human tumors. Prior studies evaluating the interaction of DCs with tumors have focused largely on their immunologic properties (for review see Banchereau, J., and R.M. Steinman. 1998. Nature. 392:245–252). In this study, we show that the clonogenicity of several human tumor cell lines and primary tumor cells from myeloma patients is enhanced by their interactions with DCs. Myeloma cells cultured in the presence of DCs have an altered phenotype with an increased proportion of cells lacking terminal plasma cell differentiation marker CD138. DC–tumor interaction also leads to the up-regulation of B cell lymphoma 6 expression in myeloma cells. Effects of DCs on myeloma cells are inhibited by blockade of the receptor activator of NF-kB (RANK)–RANK ligand and B cell–activating factor–APRIL (a proliferation-inducing ligand)-mediated interactions. Together, these data suggest that tumor–DC interactions may directly impact the biology of human tumors, particularly multiple myeloma, and may be a target for therapeutic intervention.

Selective blockade of the inhibitory Fcγ receptor (FcγRIIB) in human dendritic cells and monocytes induces a type I interferon response program

The ability of dendritic cells (DCs) to activate immunity is linked to their maturation status. In prior studies, we have shown that selective antibody-mediated blockade of inhibitory FcγRIIB receptor on human DCs in the presence of activating immunoglobulin (Ig) ligands leads to DC maturation and enhanced immunity to antibody-coated tumor cells. We show that Fcγ receptor (FcγR)–mediated activation of human monocytes and monocyte-derived DCs is associated with a distinct gene expression pattern, including several inflammation-associated chemokines, as well as type 1 interferon (IFN) response genes, including the activation of signal transducer and activator of transcription 1 (STAT1). FcγR-mediated STAT1 activation is rapid and requires activating FcγRs. However, this IFN response is observed without a detectable increase in the expression of type I IFNs themselves or the need to add exogenous IFNs. Induction of IFN response genes plays an important role in FcγR-mediated effects on DCs, as suppression of STAT1 by RNA interference inhibited FcγR-mediated DC maturation. These data suggest that the balance of activating/inhibitory FcγRs may regulate IFN signaling in myeloid cells. Manipulation of FcγR balance on DCs and monocytes may provide a novel approach to regulating IFN-mediated pathways in autoimmunity and human cancer.

Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC

ABSTRACT Dendritic cell-derived exosomes (Dex) are small extracellular vesicles secreted by viable dendritic cells. In the two phase-I trials that we conducted using the first generation of Dex (IFN-γ-free) in end-stage cancer, we reported that Dex exerted natural killer (NK) cell effector functions in patients. A second generation of Dex (IFN-γ-Dex) was manufactured with the aim of boosting NK and T cell immune responses. We carried out a phase II clinical trial testing the clinical benefit of IFN-γ-Dex loaded with MHC class I- and class II-restricted cancer antigens as maintenance immunotherapy after induction chemotherapy in patients bearing inoperable non-small cell lung cancer (NSCLC) without tumor progression. The primary endpoint was to observe at least 50% of patients with progression-free survival (PFS) at 4 mo after chemotherapy cessation. Twenty-two patients received IFN-γ-Dex. One patient exhibited a grade three hepatotoxicity. The median time to progression was 2.2 mo and median overall survival (OS) was 15 mo. Seven patients (32%) experienced stabilization of >4 mo. The primary endpoint was not reached. An increase in NKp30-dependent NK cell functions were evidenced in a fraction of these NSCLC patients presenting with defective NKp30 expression. Importantly, MHC class II expression levels of the final IFN-γ-Dex product correlated with expression levels of the NKp30 ligand BAG6 on Dex, and with NKp30-dependent NK functions, the latter being associated with longer progression-free survival. This phase II trial confirmed the capacity of Dex to boost the NK cell arm of antitumor immunity in patients with advanced NSCLC.

Clinical regressions and broad immune activation following combination therapy targeting human NKT cells in myeloma

Natural killer T (iNKT) cells can help mediate immune surveillance against tumors in mice. Prior studies targeting human iNKT cells were limited to therapy of advanced cancer and led to only modest activation of innate immunity. Clinical myeloma is preceded by an asymptomatic precursor phase. Lenalidomide was shown to mediate antigen-specific costimulation of human iNKT cells. We treated 6 patients with asymptomatic myeloma with 3 cycles of combination of α-galactosylceramide-loaded monocyte-derived dendritic cells and low-dose lenalidomide. Therapy was well tolerated and led to reduction in tumor-associated monoclonal immunoglobulin in 3 of 4 patients with measurable disease. Combination therapy led to activation-induced decline in measurable iNKT cells and activation of NK cells with an increase in NKG2D and CD56 expression. Treatment also led to activation of monocytes with an increase in CD16 expression. Each cycle of therapy was associated with induction of eosinophilia as well as an increase in serum soluble IL2 receptor. Clinical responses correlated with pre-existing or treatment-induced antitumor T-cell immunity. These data demonstrate synergistic activation of several innate immune cells by this combination and the capacity to mediate tumor regression. Combination therapies targeting iNKT cells may be of benefit toward prevention of cancer in humans.

Natural immunity to pluripotency antigen OCT4 in humans.

OCT4 is a transcription factor critical for the pluripotency of human embryonal stem (ES) and induced pluipotency stem (IPS) cells. OCT4 is commonly expressed in germ-cell tumors as well as putative cancer stem cells in several tumors, and is a key determinant of oncogenic fate in germ-cell tumors. The capacity of the human immune system to recognize this critical stem-cell gene is not known, but has implications for preventing tumors with ES/IPS-based therapies and targeting stem-cell pathways in cancer. Here we show that OCT4-specific T cells can be readily detected in freshly isolated T cells from most (>80%) healthy donors. The reactivity to OCT4-derived peptides resides primarily in the CD45RO+ memory T-cell compartment and consists predominantly of CD4+ T cells. T cells reactive against OCT4-derived peptides can be readily expanded in culture using peptide-loaded dendritic cells. In contrast to healthy donors, immunity to OCT4 was detected in only 35% of patients with newly diagnosed germ-cell tumors. However, chemotherapy of germ-cell tumors led to the induction of anti-OCT4 immunity in vivo in patients lacking such responses at baseline. These data demonstrate the surprising lack of immune tolerance to this critical pluripotency antigen in humans. Harnessing natural immunity to this antigen may allow immune-based targeting of pluripotency-related pathways for prevention of cancers, including those in the setting of ES/IPS-based therapies.

Targeting human dendritic cells in situ to improve vaccines.

Dendritic cells (DCs) provide a critical link between innate and adaptive immunity. The potent antigen presenting properties of DCs makes them a valuable target for the delivery of immunogenic cargo. Recent clinical studies describing in situ DC targeting with antibody-mediated targeting of DC receptor through DEC-205 provide new opportunities for the clinical application of DC-targeted vaccines. Further advances with nanoparticle vectors which can encapsulate antigens and adjuvants within the same compartment and be targeted against diverse DC subsets also represent an attractive strategy for targeting DCs. This review provides a brief summary of the rationale behind targeting dendritic cells in situ, the existing pre-clinical and clinical data on these vaccines and challenges faced by the next generation DC-targeted vaccines.