Thomas U. Marron

Toll-like receptor 7 and 9 defects in common variable immunodeficiency.

BACKGROUND Common variable immunodeficiency (CVID) is characterized by hypogammaglobulinemia, reduced numbers of peripheral blood isotype-switched memory B cells, and loss of plasma cells. OBJECTIVE Because Toll-like receptor (TLR) activation of B cells can initiate and potentially sustain normal B cell functions, we examined functional outcomes of TLR7 and TLR9 signaling in CVID B cells. METHODS TLR7-mediated, TLR7/8-mediated, and TLR9-mediated cell proliferation, isotype switch, and immunoglobulin production by control and CVID B cells or isolated naive and memory B cell subsets were examined. We quantitated TNF-alpha, IL-6, and IL-12 production in response to TLR1-9 ligands and measured IFN-alpha production by TLR7-stimulated PBMCs and isolated plasmacytoid dendritic cells (pDCs). IFN-beta mRNA expression by TLR3-stimulated fibroblasts was assessed. RESULTS Unlike CD27(+) B cells of controls, TLR7-activated, TLR7/8-activated, or TLR9-activated CVID B cells or isolated CD27(+) B cells did not proliferate, upregulate CD27, or shed surface IgD. TLR-stimulated CVID B cells failed to upregulate activation-induced cytosine deaminase mRNA or produce IgG and IgA. TLR7-stimulated PBMCs and pDCs produced little or no IFN-alpha. Reconstituting IFN-alpha in TLR7-stimulated CVID B-cell cultures facilitated proliferation, CD27 upregulation, and isotype switch. These TLR defects are restricted because CVID PBMCs stimulated with TLR ligands produced normal amounts of TNF-alpha, IL-6, and IL-12; TLR3-mediated expression of IFN-beta by CVID fibroblasts was normal. CONCLUSION Defective TLR7 and TLR9 signaling in CVID B cells and pDCs, coupled with deficient IFN-alpha, impairs CVID B cell functions and prevents TLR-mediated augmentation of humoral immunity in vivo.

Toll-like receptor 4–, 7–, and 8–activated myeloid cells from patients with X-linked agammaglobulinemia produce enhanced inflammatory cytokines

BACKGROUND Bruton tyrosine kinase (BTK) is a component of signaling pathways downstream from Toll-like receptors (TLRs) 2, 4, 7, 8, and 9. Previous work in BTK-deficient mice, cell lines, and cultured cells from patients with X-linked agammaglobulinemia (XLA) suggested defective TLR-driven cytokine production. OBJECTIVE We sought to compare TLR-4-, TLR-7-, and TLR-8-induced cytokine production of primary cells from patients with XLA with that seen in control cells. METHODS PBMCs from patients with XLA, freshly isolated plasmacytoid dendritic cells, monocytes, and monocytoid dendritic cells were activated with TLR-4, TLR-7, and TLR-8 agonists. Signaling intermediates and intracellular and secreted cytokine levels were compared with those seen in control cells. RESULTS Although TLR-4, TLR-7, and TLR-8 activation of nuclear factor κB and mitogen-activated protein kinase pathways in cells from patients with XLA and control cells were comparable, TLR-activated freshly isolated monocytes and monocytoid dendritic cells from patients with XLA produced significantly more TNF-α, IL-6, and IL-10 than control cells. TLR-7/8-activated plasmacytoid dendritic cells produced normal amounts of IFN-α. In murine models BTK regulates the degradation of Toll-IL-1 receptor domain-containing adaptor protein, terminating TLR-4-induced cytokine production. Although this might explain the heightened TLR-4-driven cytokine production we observed, Toll-IL-1 receptor domain-containing adaptor protein degradation is intact in cells from patients with XLA, excluding this explanation. CONCLUSION In contrast to previous studies with BTK-deficient mice, cell lines, and cultured cells from patients with XLA suggesting impaired TLR-driven cytokine production, these data suggest that BTK inhibits TLR-induced cytokine production in primary human cells.

TLR signaling and effector functions are intact in XLA neutrophils

Toll-like receptors (TLRs) are essential components of the innate immune system, and their ligands are important activators of neutrophils. Brutons tyrosine kinase (Btk) has been reported to mediate signaling through toll-like receptors (TLRs) in many cell types, however, the role of Btk in TLR activation of neutrophils remains unclear. Impaired TLR-induced neutrophil function was found in mice with loss of Btk and in humans with TLR-signaling defects, but the integrity of TLR pathways in X-linked agammaglobulinemia (XLA) neutrophils has not been assessed. In this study LPS (TLR4) or an imidazoquinoline compound (TLR7/8) activated XLA neutrophil shedding of surface CD62L, and phosphorylated MAP kinases p38, JNK and ERK. TLR activation also induced normal respiratory burst and retarded apoptosis for XLA neutrophils, comparable to normal controls. These data demonstrate that the loss of Btk in XLA neutrophils does not impair functional responses to TLR signals.

Diagnostic Utility of Measuring Free Light Chains in the Cerebrospinal Fluid of Patients With Multiple Myeloma

Multiple myeloma involvement of the central nervous system is rare, portends a poor prognosis, and requires rapid clinical intervention. The gold standard for diagnosis is cytology, which is highly insensitive. Abnormal free light chain detection in cerebrospinal fluid was more reliably concordant with clinical diagnosis of central nervous system multiple myeloma than cytology. Free light chain levels in cerebrospinal fluid dynamically correlated with response to therapy. Assessment of free light chains in cerebrospinal fluid can be included in the diagnostic algorithm to diagnose central nervous system multiple myeloma and monitor response to therapy.

Abstract IA03: Turning a tumor into a vaccine factory: In situ vaccination for low-grade lymphoma

Background: Lymphomas are the 5th most common cancer in the U.S. and most are incurable with standard therapy. Previously, we completed three trials of ‘in situ vaccination’ - combining low-dose radiotherapy (XRT) with intratumoral administration of TLR9 agonist (CpG). We demonstrated induction of anti-tumor CD8 T cell responses and clinical remissions of patients9 non-irradiated sites of disease, lasting up to 4+ years. One limitation may have been the paucity of intratumoral dendritic cells (DC). DC are uniquely able to endocytose dying (e.g. irradiated) tumor cells for cross-presentation to anti-tumor CD8 T cells. Methods: Flt3L – the predominant DC differentiation factor– induces tumor leukocyte infiltration and regression of lymphoma tumors pre-clinically and a new formulation of this cytokine -CDX-301- was shown to mobilize BDCA-1 and BDCA-3 DC subsets in an early phase trial. These DC subsets respond to several TLR agonists and cross-present antigens more effectively than plasmacytoid DC (the CpG-responsive DC subset). We initiated a phase I/II study of a new iteration of the in situ vaccine, adding Flt3L-priming and replacing the prior TLR9 agonist with the TLR3 agonist poly-ICLC (Fig 1A). The vaccine consists of: intratumoral Flt3L administration to increase DC within the tumor low-dose XRT to induce immunogenic tumor cell death and release tumor-associated antigens, and intratumoral poly-ICLC administration to activate tumor antigen-loaded DC. Results: Six patients have been enrolled, two patients have completed therapy. Treated patients had 2-200-fold increases in BDCA1 and BDCA3 intratumoral DC after Flt3L administration and marked DC activation after XRT and poly-ICLC. Both treated patients have had partial remissions of untreated sites per Cheson criteria, persisting or improving for >6 months after vaccination. These include regressions of bulky lymph nodes (Fig 1B), as well as peripheral blood (Fig1C) and bone marrow disease. A patient with significant peripheral blood tumor burden experienced >10-fold decrease in malignant B cells with concurrent increase in non-tumor B cells, suggesting a degree of cell specificity in the tumor-killing mechanism. Conclusions: Preliminary results suggest that the Flt3L-primed in situ vaccine is feasible, safe and immunologically and clinically effective, warranting further study. Research supported by: Damon Runyon Cancer Research Foundation Clinical Investigator Award Lymphoma Research Foundation Clinical Research Mentorship Program Citation Format: Thomas Marron, Nina Bhardwaj, Elizabeth Crowley, Tibor Keler, Thomas Davis, Andres Salazar, Joshua Brody. Turning a tumor into a vaccine factory: In situ vaccination for low-grade lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA03.

Local Immunotherapies of Cancer

The past decade has seen clinical cancer research turn away from refining cytotoxic therapies to identifying immunomodulatory therapies that can harness the immune system to do the work of fighting cancer for us. The first revolution in cancer therapy brought us potent but toxic chemotherapies that were often initially good at controlling tumors; however, in the metastatic setting, recurrence was typically inevitable and more difficult to control. Immunotherapy has now emerged as among the most promising class of therapeutics for the treatment of cancer. These therapies harness the potent tumoricidal potential of cytotoxic effector cells of the patient’s own immune system. Although checkpoint-blocking antibodies approved in the past decade offer great promise, they non-specifically release the brakes on immune cells, and the majority of cancer patients are, so far, non-responders, while some patients experience toxic autoimmune adverse effects. One approach to improve on these limitations is with localized, in situ therapies, inducing a vaccinal response within the tumor microenvironment. These approaches alter the tumor microenvironment by inducing local immunogenic cell death to release tumor antigen, recruiting inflammatory leukocytes, priming of the adaptive immune response, and inhibiting tolerogenic mechanisms through which tumors dampen the immune response. Through a combined approach using some or all of these features, we are working to develop an in situ vaccine strategy that will result in systemic elimination of tumor cells as well as long-term remissions.

An Update on the Use of Immunotherapy in the Treatment of Lymphoma

Purpose of ReviewThroughout the field of oncology, immunotherapy is moving further towards the first-line setting, and there is encouraging data for the use of these novel therapies in the management of lymphomas, utilizing treatments approved for both solid and hematologic malignancies. Herein, we review promising advances in this rapidly moving field from the past year.Recent FindingsIn the last year, we have seen promising clinical data on engineered antibody therapies for the treatment of lymphomas, as well as further optimization of engineered antibody fragments fused onto linkers or chimeric T cell receptors, both of the modalities capable of transforming non-specific T cells into tumor-specific, serial killer cells.SummaryHere we will review the promising data on these advances in antibody-based therapies, as well as some of the immunomodulators and checkpoint-blocking therapies that shown to have promising results in the treatment of lymphomas within the past year.