Phillip D. Fromm

Long-term survival in multiple myeloma is associated with a distinct immunological profile, which includes proliferative cytotoxic T-cell clones and a favourable Treg/Th17 balance

Despite improved outcomes in multiple myeloma (MM), a cure remains elusive. However, even before the current therapeutic era, 5% of patients survived >10 years and we propose that immune factors contribute to this longer survival. We identified patients attending our clinic, who had survived >10 years (n=20) and analysed their blood for the presence of T-cell clones, T-regulatory cells (Tregs) and T helper 17 (Th17) cells. These results were compared with MM patients with shorter follow-up and age-matched healthy control donors. The frequency of cytotoxic T-cell clonal expansions in patients with <10 years follow-up (MM patients) was 54% (n=144), whereas it was 100% (n=19/19) in the long-survivors (LTS-MM). T-cell clones from MM patients proliferated poorly in vitro, whereas those from LTS-MM patients proliferated readily (median proliferations 6.1% and 61.5%, respectively (P<0.0001)). In addition, we found significantly higher Th17 cells and lower Tregs in the LTS-MM group when compared with the MM group. These results indicate that long-term survival in MM is associated with a distinct immunological profile, which is consistent with decreased immune suppression.

Mechanism of Cytotoxicity and Cellular Uptake of Lipophilic Inert Dinuclear Polypyridylruthenium(II) Complexes

The accumulation, uptake mechanism, cytotoxicity, cellular localisation of—and mode of cell death induced by—dinuclear ruthenium(II) complexes ΔΔ/ΛΛ‐[{Ru(phen)2}2{μ‐bbn}]4+ (Rubbn), where phen is 1,10‐phenanthroline, bbn is bis[4(4′‐methyl‐2,2′‐bipyridyl)]‐1,n‐alkane (n=2, 5, 7, 10, 12 or 16), and the corresponding mononuclear complexes containing the bbn ligands, were studied in L1210 murine leukaemia cells. Cytotoxicity increased with linker chain length, and the ΔΔ‐Rubb16 complex displayed the highest cytotoxicity of the series, with an IC50 value of 5 μM, similar to that of carboplatin in the L1210 murine leukaemia cell line. Confocal microscopy and flow cytometry studies indicated that the complexes accumulate in the mitochondria of L1210 cells, with the magnitude of cellular uptake and accumulation increasing with linking chain length in the bbn bridge of the metal complex. ΔΔ‐Rubb16 entered the L1210 cells by passive diffusion (with a minor contribution from protein‐mediated active transport), inducing cell death via apoptosis. Additionally, metal‐complex uptake in leukaemia cells was approximately 16‐times that observed in healthy B cells highlighting that the bbn series of complexes may have potential as selective anticancer drugs.

CD86+ or HLA-G+ can be transferred via trogocytosis from myeloma cells to T cells and are associated with poor prognosis

The transfer of membrane proteins between cells during contact, known as trogocytosis, can create novel cells with a unique phenotype and altered function. We demonstrate that trogocytosis is more common in multiple myeloma (MM) than chronic lymphocytic leukemia and Waldenstrom macroglobulinaemia; that T cells are more probable to be recipients than B or natural killer cells; that trogocytosis occurs independently of either the T-cell receptor or HLA compatibility; and that after trogocytosis, T cells with acquired antigens can become novel regulators of T-cell proliferation. We screened 168 patients with MM and found that CD86 and human leukocyte antigen G (HLA-G) were antigens commonly acquired by T cells from malignant plasma cells. CD3+ CD86acq+ and CD3+ HLA-Gacq+ cells were more prevalent in bone marrow than peripheral blood samples. The presence of either CD86 or HLA-G on malignant plasma cells was associated with a poor prognosis. CD38++ side population cells expressed HLA-G, suggesting that these putative myeloma stem cells could generate immune tolerance. HLA-G+ T cells had a regulatory potency similar to natural Tregs, thus providing another novel mechanism for MM to avoid effective immune surveillance.

Dendritic Cells as Pharmacological Tools for Cancer Immunotherapy

Although the earliest—rudimentary—attempts at exploiting the immune system for cancer therapy can be traced back to the late 18th Century, it was not until the past decade that cancer immunotherapeutics have truly entered mainstream clinical practice. Given their potential to stimulate both adaptive and innate antitumor immune responses, dendritic cells (DCs) have come under intense scrutiny in recent years as pharmacological tools for cancer immunotherapy. Conceptually, the clinical effectiveness of this form of active immunotherapy relies on the completion of three critical steps: 1) the DCs used as immunotherapeutic vehicles must properly activate the antitumor immune effector cells of the host, 2) these immune effector cells must be receptive to stimulation by the DCs and be competent to mediate their antitumor effects, which 3) requires overcoming the various immune-inhibitory mechanisms used by the tumor cells. In this review, following a brief overview of the pivotal milestones in the history of cancer immunotherapy, we will introduce the reader to the basic immunobiological and pharmacological principles of active cancer immunotherapy using DCs. We will then discuss how current research is trying to define the optimal parameters for each of the above steps to realize the full clinical potential of DC therapeutics. Given its high suitability for immune interventions, acute myeloid leukemia was chosen here to showcase the latest research trends driving the field of DC-based cancer immunotherapy.

Myeloid derived suppressor cells are numerically, functionally and phenotypically different in patients with multiple myeloma

Abstract Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells that have been implicated as inhibitors of lymphopoiesis in patients with malignancies. They have a consensus phenotype of CD33+/CD11b+/HLA-DRlo/− and can be further divided into CD15 + granulocytic (G-MDSC) and CD14 + monocytic (M-MDSC) subsets. We characterized MDSCs in patients with multiple myeloma (MM) and found a significant increase in G-MDSCs in the blood of patients with progressive MM. Flow-sorted MDSCs from patients with MM induced the generation of regulatory T cells (Treg). MDSCs from both patients with MM and aged-matched controls demonstrated a dose-dependent inhibition of lymphocyte proliferation in carboxyfluorescein succinimidyl ester (CFSE)-tracking experiments. Granulocyte colony stimulating factor (G-CSF) administered to induce stem cell mobilization caused an increase in the number of MDSCs in the peripheral blood of patients with MM and a concentration of these immune-suppressive cells in peripheral blood stem cell collections. MDSCs are likely to cause immune dysfunction in patients with MM.

The role of TNF in parasitic diseases: Still more questions than answers

The inhibition of TNF with therapeutic monoclonal antibodies or antibody/receptor fusion proteins in rheumatoid arthritis still constitutes the benchmark for a successful intervention in an ongoing auto-immune-inflammatory disease and underlines the importance of this cytokine. TNF plays a central role in the defence against intracellular infections and is responsible for the promotion of different aspects of the innate immune response such as inflammatory cell recruitment and cell differentiation. While this cytokine generally displays pro-inflammatory activities supporting the early stages of the inflammatory response, it has been demonstrated to be especially important during infection with intracellular pathogens and, consequently, leishmaniasis of TNF(-/-) mice ends fatally. However, the specific activities of TNF that confer protection are not yet fully understood. This review will summarize the current understanding of TNF function and signalling, and will discuss recent work in the models of malaria, toxoplasmosis, trypanosomiasis and leishmaniasis with particular emphasis on work with gene-deficient mouse models.

Interleukin-15-Induced CD56(+) Myeloid Dendritic Cells Combine Potent Tumor Antigen Presentation with Direct Tumoricidal Potential

Dendritic cells (DCs) are the quintessential antigen-presenting cells of the human immune system and play a prime role in coordinating innate and adaptive immune responses, explaining the strong and still growing interest in their application for cancer immunotherapy. Much current research in the field of DC-based immunotherapy focuses on optimizing the culture conditions for in vitro DC generation in order to assure that DCs with the best possible immunogenic qualities are being used for immunotherapy. In this context, monocyte-derived DCs that are alternatively induced by interleukin-15 (IL-15 DCs) have attracted recent attention due to their superior immunostimulatory characteristics. In this study, we show that IL-15 DCs, in addition to potent tumor antigen-presenting function, possess tumoricidal potential and thus qualify for the designation of killer DCs. Notwithstanding marked expression of the natural killer (NK) cell marker CD56 on a subset of IL-15 DCs, we found no evidence of a further phenotypic overlap between IL-15 DCs and NK cells. Allostimulation and antigen presentation assays confirmed that IL-15 DCs should be regarded as bona fide myeloid DCs not only from the phenotypic but also from the functional point of view. Concerning their cytotoxic activity, we demonstrate that IL-15 DCs are able to induce apoptotic cell death of the human K562 tumor cell line, while sparing tumor antigen-specific T cells. The cytotoxicity of IL-15 DCs is predominantly mediated by granzyme B and, to a small extent, by tumor necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL) but is independent of perforin, Fas ligand and TNF-α. In conclusion, our data provide evidence of a previously unappreciated role for IL-15 in the differentiation of human monocytes towards killer DCs. The observation that IL-15 DCs have killer DC capacity lends further support to their implementation in DC-based immunotherapy protocols.

Myeloma skews regulatory T and pro-inflammatory T helper 17 cell balance in favor of a suppressive state

Abstract Discrepancies in the literature between regulatory T cell (Treg) and pro-inflammatory T helper 17 (Th17) numbers in multiple myeloma (MM) can be largely explained by technical differences in methodology and patient selection. In this study, Treg cells were defined as CD3+CD4+CD25++CD127lo cells. Patients with MM (n = 20) had a significant imbalance in Treg/Th17 ratio when compared with either aged-matched controls (n = 28) or other monoclonal gammopathies, and this was associated with a significantly worse survival. The percent Treg in bone marrow of patients with MM was higher than that in matched peripheral blood samples (p = 0.02), although FOXP3 expression within bone marrow T cells was lower (p = 0.02). We observed increased Treg function, both in vivo and in vitro, due at least partially to an increase in CTLA-4 expression by concurrent treatment with dexamethasone and immune modulatory compounds (iMiDs). We suggest that immunoregulatory balance is important during active chemotherapy and that conclusions related to the immunostimulatory effect of iMiDs based on in vitro testing must be considered with caution.

CCR6 is transiently upregulated on B cells after activation and modulates the germinal center reaction in the mouse

The CC‐chemokine receptor 6 (CCR6) is expressed constitutively at an intermediate level on naïve B cells and is upregulated after activation on pregerminal center (GC) B cells. We hypothesized that it could be involved in the events leading to GC reaction and high‐affinity antibody production, and therefore investigated the potential role of CCR6 in B‐cell differentiation in vivo. After antigenic challenge of CCR6−/− mice with the T‐cell‐dependent antigen nitrophenyl‐keyhole limpet hemocyanin (NP‐KLH), GC B‐cell development was found to be accelerated and the number of GC had increased significantly compared with control mice, but the antibodies produced by CCR6−/− B cells were on average of lower affinity. We conclude from these data that the CCR6/CCL20 axis has an important role in regulating the kinetics and efficiency of the GC reaction.

Loss of TNF Signaling Facilitates the Development of a Novel Ly-6C low Macrophage Population Permissive for Leishmania major Infection

In the absence of TNF, the normally resistant C57BL/6 (B6.WT) strain develops a fatal, progressive form of leishmaniasis after infection with Leishmania major. It is not yet understood which TNF activity or the lack thereof is responsible for the dramatic progression of leishmaniasis in TNF-negative (B6.TNF−/−) mice. To elucidate the underlying mechanisms resulting in the fatal outcome of L. major infection in this gene-deficient mouse strain, we analyzed the monocytic component of the inflammatory infiltrate in the draining popliteal lymph node and the site of the infection using multicolor flow cytometry. The leukocytic infiltrate within the draining lymph node and footpad of B6.TNF−/− mice resembled that of B6.WT mice over the first 2 wk of cutaneous L. major infection. Thereafter, the B6.TNF−/− mice showed an increase of CD11c+Ly-6C+CCR2+ monocytic dendritic cells within the popliteal lymph node in comparison with B6.WT mice. This increase of inflammatory dendritic cells was paired with the accumulation of a novel CD11b+Ly-6ClowCCR2low population that was not present in B6.WT mice. This B6.TNF−/−- and B6.TNFR1−/−-specific cell population was CD115+Ly-6G−iNOS−, not apoptotic, and harbored large numbers of parasites.