Chandra Sekhar Boddupalli

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.

Type II NKT-TFH cells against Gaucher lipids regulate B-cell immunity and inflammation

Chronic inflammation including B-cell activation is commonly observed in both inherited (Gaucher disease [GD]) and acquired disorders of lipid metabolism. However, the cellular mechanisms underlying B-cell activation in these settings remain to be elucidated. Here, we report that β-glucosylceramide 22:0 (βGL1-22) and glucosylsphingosine (LGL1), 2 major sphingolipids accumulated in GD, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT) cells. Human βGL1-22- and LGL1-reactive CD1d tetramer-positive T cells have a distinct T-cell receptor usage and genomic and cytokine profiles compared with the classical type I NKT cells. In contrast to type I NKT cells, βGL1-22- and LGL1-specific NKT cells constitutively express T-follicular helper (TFH) phenotype. Injection of these lipids leads to an increase in respective lipid-specific type II NKT cells in vivo and downstream induction of germinal center B cells, hypergammaglobulinemia, and production of antilipid antibodies. Human βGL1-22- and LGL1-specific NKT cells can provide efficient cognate help to B cells in vitro. Frequency of LGL1-specific T cells in GD mouse models and patients correlates with disease activity and therapeutic response. Our studies identify a novel type II NKT-mediated pathway for glucosphingolipid-mediated dysregulation of humoral immunity and increased risk of B-cell malignancy observed in metabolic lipid disorders.

Clonal Immunoglobulin against Lysolipids in the Origin of Myeloma

Antigen-driven selection has been implicated in the pathogenesis of monoclonal gammopathies. Patients with Gauchers disease have an increased risk of monoclonal gammopathies. Here we show that the clonal immunoglobulin in patients with Gauchers disease and in mouse models of Gauchers disease-associated gammopathy is reactive against lyso-glucosylceramide (LGL1), which is markedly elevated in these patients and mice. Clonal immunoglobulin in 33% of sporadic human monoclonal gammopathies is also specific for the lysolipids LGL1 and lysophosphatidylcholine (LPC). Substrate reduction ameliorates Gauchers disease-associated gammopathy in mice. Thus, long-term immune activation by lysolipids may underlie both Gauchers disease-associated gammopathies and some sporadic monoclonal gammopathies.

Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW 264.7 cells also involves c-rel transcription factor

Interleukin‐10 (IL‐10) is known to inhibit IL‐12 production in macrophages primarily at the transcriptional level with the involvement of p50 and p65 nuclear factor‐κB (NF‐κB). We demonstrate that the c‐rel transcription factor also plays a major role in IL‐10‐mediated IL‐12 suppression. Treatment of macrophages with recombinant IL‐10 inhibited nuclear c‐rel levels, whereas addition of neutralizing anti‐IL‐10 antibody up‐regulated both nuclear c‐rel levels and IL‐12 production by macrophages. Decreased nuclear c‐rel was associated with a reduction in phosphorylation of inhibitory kappa B alpha (IκBα) in the cytoplasm, indicating that IL‐10 prevents degradation of IκBα and the subsequent translocation of c‐rel into the nucleus. Treatment with leptomycin B, a known inhibitor of c‐rel at a concentration of 10 nm, when used with anti‐IL‐10 antibody, resulted in reduced expression of IL‐12. In a complementary experiment, in vitro transient expression of p65 NF‐κB could not rescue the inhibitory effect of IL‐10 on IL‐12 production, suggesting that NF‐κB alone was not sufficient to restore IL‐12 production during IL‐10 treatment. However, over‐expression of c‐rel resulted in IL‐12 restoration upon stimulation with lipopolysaccharide plus interferon‐γ during IL‐10 treatment. Our studies highlight the involvement of c‐rel in IL‐10‐mediated IL‐12 regulation.

Interlesional diversity of T cell receptors in melanoma with immune checkpoints enriched in tissue-resident memory T cells

Heterogeneity of tumor cells and their microenvironment can affect outcome in cancer. Blockade of immune checkpoints (ICPs) expressed only on a subset of immune cells leads to durable responses in advanced melanoma. Tissue-resident memory T (TRM) cells have recently emerged as a distinct subset of memory T cells in nonlymphoid tissues. Here, we show that functional properties and expression of ICPs within tumor-infiltrating lymphocytes (TILs) differ from those of blood T cells. TILs secrete less IL-2, IFN-γ, and TNF-α compared with circulating counterparts, and expression of VEGF correlated with reduced TIL infiltration. Within tumors, ICPs are particularly enriched within T cells with phenotype and genomic features of TRM cells and the CD16+ subset of myeloid cells. Concurrent T cell receptor (TCR) and tumor exome sequencing of individual metastases in the same patient revealed that interlesional diversity of TCRs exceeded differences in mutation/neoantigen load in tumor cells. These findings suggest that the TRM subset of TILs may be the major target of ICP blockade and illustrate interlesional diversity of tissue-resident TCRs within individual metastases, which did not equilibrate between metastases and may differentially affect the outcome of immune therapy at each site.

Conditional overexpression of TGFβ1 promotes pulmonary inflammation, apoptosis and mortality via TGFβR2 in the developing mouse lung.

BackgroundEarlier studies have reported that transforming growth factor beta 1(TGFβ1) is a critical mediator of hyperoxia-induced acute lung injury (HALI) in developing lungs, leading to impaired alveolarization and a pulmonary phenotype of bronchopulmonary dysplasia (BPD). However, the mechanisms responsible for the TGFβ1-induced inflammatory signals that lead to cell death and abnormal alveolarization are poorly understood. We hypothesized that TGFβ1 signaling via TGFβR2 is necessary for the pathogenesis of the BPD pulmonary phenotype resulting from HALI.MethodsWe utilized lung epithelial cell-specific TGFβ1 overexpressing transgenic and TGFβR2 null mutant mice to evaluate the effects on neonatal mortality as well as pulmonary inflammation and apoptosis in developing lungs. Lung morphometry was performed to determine the impaired alveolarization and multicolor flow cytometry studies were performed to detect inflammatory macrophages and monocytes in lungs. Apoptotic cell death was measured with TUNEL assay, immunohistochemistry and western blotting and protein expression of angiogenic mediators were also analyzed.ResultsOur data reveals that increased TGFβ1 expression in newborn mice lungs leads to increased mortality, macrophage and immature monocyte infiltration, apoptotic cell death specifically in Type II alveolar epithelial cells (AECs), impaired alveolarization, and dysregulated angiogenic molecular markers.ConclusionsOur study has demonstrated the potential role of inhibition of TGFβ1 signaling via TGFβR2 for improved survival, reduced inflammation and apoptosis that may provide insights for the development of potential therapeutic strategies targeted against HALI and BPD.

ABC transporters and NR4A1 identify a quiescent subset of tissue-resident memory T cells

Immune surveillance in tissues is mediated by a long-lived subset of tissue-resident memory T cells (Trm cells). A putative subset of tissue-resident long-lived stem cells is characterized by the ability to efflux Hoechst dyes and is referred to as side population (SP) cells. Here, we have characterized a subset of SP T cells (Tsp cells) that exhibit a quiescent (G0) phenotype in humans and mice. Human Trm cells in the gut and BM were enriched in Tsp cells that were predominantly in the G0 stage of the cell cycle. Moreover, in histone 2B-GFP mice, the 2B-GFP label was retained in Tsp cells, indicative of a slow-cycling phenotype. Human Tsp cells displayed a distinct gene-expression profile that was enriched for genes overexpressed in Trm cells. In mice, proteins encoded by Tsp signature genes, including nuclear receptor subfamily 4 group A member 1 (NR4A1) and ATP-binding cassette (ABC) transporters, influenced the function and differentiation of Trm cells. Responses to adoptive transfer of human Tsp cells into immune-deficient mice and plerixafor therapy suggested that human Tsp cell mobilization could be manipulated as a potential cellular therapy. These data identify a distinct subset of human T cells with a quiescent/slow-cycling phenotype, propensity for tissue enrichment, and potential to mobilize into circulation, which may be harnessed for adoptive cellular therapy.

Antigen-mediated regulation in monoclonal gammopathies and myeloma

A role for antigen-driven stimulation has been proposed in the pathogenesis of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) based largely on the binding properties of monoclonal Ig. However, insights into antigen binding to clonal B cell receptors and in vivo responsiveness of the malignant clone to antigen-mediated stimulation are needed to understand the role of antigenic stimulation in tumor growth. Lysolipid-reactive clonal Ig were detected in Gaucher disease (GD) and some sporadic gammopathies. Here, we show that recombinant Ig (rIg) cloned from sort-purified single tumor cells from lipid-reactive sporadic and GD-associated gammopathy specifically bound lysolipids. Liposome sedimentation and binding assays confirmed specific interaction of lipid-reactive monoclonal Ig with lysolipids. The clonal nature of lysolipid-binding Ig was validated by protein sequencing. Gene expression profiling and cytogenetic analyses from 2 patient cohorts showed enrichment of nonhyperdiploid tumors in lipid-reactive patients. In vivo antigen-mediated stimulation led to an increase in clonal Ig and plasma cells (PCs) in GD gammopathy and also reactivated previously suppressed antigenically related nonclonal PCs. These data support a model wherein antigenic stimulation mediates an initial polyclonal phase, followed by evolution of monoclonal tumors enriched in nonhyperdiploid genomes, responsive to underlying antigen. Targeting underlying antigens may therefore prevent clinical MM.

Clinical and Serologic Responses After a Two-dose Series of High-dose Influenza Vaccine in Plasma Cell Disorders: A Prospective, Single-arm Trial

Micro‐Abstract The goal of the present study was to evaluate a novel prospective influenza vaccination strategy for patients with plasma cell disorders. Fifty‐one patients were treated with a 2‐dose series of high‐dose inactivated trivalent influenza vaccine. This vaccination strategy was well tolerated and led to very high rates of seroprotection against influenza. Background: Patients with multiple myeloma (MM) and other plasma cell disorders are highly susceptible to influenza infections, which are major causes of morbidity in this population, despite the routine administration of a seasonal influenza vaccination. Existing data are limited by small and retrospective studies, which suggest poor seroprotection rates of < 20% after standard influenza vaccination in patients with MM. Patients and Methods: Patients with plasma cell dyscrasia (n = 51) were treated with a 2‐dose series of high‐dose inactivated trivalent influenza vaccine during the 2014 to 2015 influenza season. Laboratory‐confirmed influenza infections were identified through seasonal surveillance, sera were collected for influenza hemagglutination antibody inhibition (HAI) titer assays, and logistic regression models were used to identify the clinical correlates to the HAI serologic responses. Results: Influenza vaccine was well tolerated, without any vaccine‐related grade ≥ 2 adverse events. Only 3 patients (6%) experienced laboratory‐confirmed influenza. The rates of HAI seroprotection against all 3 vaccine strains (A/California/7/2009 [H1N1] pdm09‐like virus; A/Texas/50/2012 [H3N2]‐like virus; and a B/Massachusetts/2/2012‐like virus) increased from 4% at baseline to 49% and 65% after 1 and 2 doses, respectively. The risk factors associated with a lower likelihood of HAI serologic response included plasma cell disorder requiring therapy, less than a partial response found on disease response assessment, and active conventional chemotherapy. Alternatively, active therapy with an immunomodulatory drug alone or with a proteasome inhibitor was associated with a greater likelihood of an HAI serologic response. Conclusion: These data have demonstrated that, in contrast to the historically poor results with standard influenza vaccination, this novel high‐dose booster vaccination strategy leads to high rates of seroprotection. Randomized controlled studies are needed to compare this novel strategy to the standard vaccination strategy.

Tissue resident memory T cells create genetically distinct immune microenvironments within melanoma metastasis

Blockade of inhibitory immune checkpoints (ICPs) improves survival in melanoma patients. Interestingly, the expression of these ICPs in most tumor tissues including melanoma is restricted to only a subset of tumor-infiltrating immune cells (TIICs). Here, we utilize single cell mass cytometry (CyTOF), functional profiling and TCR sequencing of freshly isolated cells to evaluate the phenotypic, functional and genetic heterogeneity of TIICs in patients (n=43) with advanced melanoma to define the subsets of immune cells involved in ICP-mediated regulation. Using paired blood and tumor samples from melanoma patients and CyTOF analysis with 36 different markers, we show that T and myeloid cells within tumor metastases are distinct from their circulating counterparts. T cells within metastases are highly enriched for CD45RO+, CD69+, CD103+ tissue resident CD8+ memory T cells (TRM). Expression of some ICPs (such as PD-1, Tim3 and PD-L1) in TIICs is primarily restricted to TRM cells and to CD16+ subset of inflammatory/patrolling myeloid cells. Functional analysis of freshly isolated tumor T cells using multiplex luminex ELISA revealed that despite expression of multiple ICPs, tumor-associated T cells are functional, but have an altered cytokine profile compared to circulating T cells. In contrast to blood T cells, tumor infiltrating T cells secrete lower levels of IL-2, IFN-γ and TNF-α, but comparable levels of IL4, IL5 and IL17. Tissue restriction of murine TRM cells without recirculation has been extensively demonstrated in the form of lack of equilibrium in parabiotic mice that share a systemic circulation. The finding that melanoma metastatic lesions are enriched for TRM cells suggested the possibility that each of the metastatic lesions may contain a distinct microcosm of T cells without equilibration, in spite of shared systemic circulation within the same host, analogous to the biology in parabiotic mice. In order to test this directly, we compared the TCR sequences of memory CD4 and CD8 T cells isolated from patients with biopsies of multiple metastatic sites. Using high throughput deep sequencing of rearranged TCRβ loci, we find that each biopsy site has a distinct TCR usage with only partial overlap with the other metastatic site from the same patient. Together these data suggest that immunity to melanoma is highly regional and enriched for tumor-resident memory T cells creating genetically unique immune-microenvironments within each metastatic lesion. Regional nature of immunity within metastases has several implications for current strategies to harness and measure tumor immunity.