PD-L1 expression predicts efficacy in the phase II SPiReL trial with MVP-S, pembrolizumab, and low-dose CPA in R/R DLBCL.

BACKGROUND: Patients with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) have limited treatment options. METHODS: R/R DLBCL patients, who were mostly ineligible for ASCT due to age or comorbidities, were treated with maveropepimut-S (MVP-S, previously DPX-Survivac) a survivin directed T cell educating therapy, pembrolizumab, and intermittent low-dose cyclophosphamide. FINDINGS: We identified, using univariate analysis, a subset of patients with enhanced ORR, PFS and DOR. Patients with baseline CD20+/PD-L1 expression had an ORR of 46% (6/13) and the disease control rate was 10/13 (77%). The PFS and OS of the positive CD20+/PD-L1 patients were 7.1 months and 17.4 months, whereas in the intent-to-treat (ITT) population of 25 enrolled patients, the ORR was 28% (7/25), median PFS and OS were 4.2 months and 10.1 months respectively. A total of 6/7 clinical responders occurred in CD20+/PD-L1 patients. The regimen was well-tolerated, requiring only minor dose modifications and one discontinuation. Grade 1 or 2 injection site reactions occurred in 14/25, (56%). Statistically significant associations were also seen between PFS and; injection site reactions; and ELISpot response to survivin peptides, both identifying the mechanistic importance of specific immune responses to survivin. INTERPRETATION: This immunotherapy combination was found to be active and safe in this clinically challenging patient population.

Low-depth sequencing for copy number abnormalities in multiple myeloma supersedes fluorescent in situ hybridization in scope and resolution.

Multiple myeloma (MM) is an incurable hematological malignancy that relies on cytogenetic determination of copy number abnormalities (CNAs) for prognosis and management. Low-depth whole genome sequencing (LD-WGS) is a cost-effective alternative to targeted genomics for CNA detection, but its value has yet to be explored in MM. DNA from CD138+ cells from MM patients were sequenced using an Illumina NextSeq at <1x depth (ultralow-depth). Subsampling analysis and window size adjustment were performed for determining sensitivity limits and results compared to fluorescent in-Situ hybridization (FISH). CNA calls made down to 5 million (M) reads were comparable to those at 20 M reads at a window size of 100 kb had a sensitivity and specificity of 93%, 92% and an area under the curve of 0.94. All CNAs detected by FISH on the MM samples were also detected by LD-WGS; the latter detected a further 36 focal CNAs not detected by FISH. Cost per sample of LD-WGS was significantly lower for our organization than FISH testing. LD-WGS for MM is significantly more sensitive than targeted technologies such as FISH in CNA detection and resolution, provides a more cost-effective option for clinical purposes and potential for exploring prognostically relevant and drug discovery targets.

Induction of CD4(+)CD25(+)Foxp3(-) regulatory T cells by Thy-1 stimulation of CD4(+) T cells.

Thy-1 (CD90) on mouse T cells has been reported to have both T-cell activating and regulatory roles. In this study, we show that monoclonal antibody (mAb)-mediated crosslinking of Thy-1 on CD4(+) mouse T-cells-induced regulatory T (T(reg)) cells that expressed CD25, CD39 and glucocorticoid-induced tumor necrosis factor receptor family-related gene, but not CD73, CD122 or Foxp3. The proliferation of CD4(+) T(responder) cells in response to anti-CD3/anti-CD28mAb-coated T-cell expander beads or syngeneic dendritic cells and soluble anti-CD3mAb was inhibited by Thy-1-induced T(reg) cells, in spite of elevated IL-2 levels in the co-cultures. Interestingly, stimulation with T-cell expander beads caused Thy-1-induced T(reg) cells to synthesize large amounts of interleukin-2 (IL-2). IL-10 was also elevated in co-cultures of activated T(responder) cells and Thy-1-induced T(reg) cells. However, mAb-mediated neutralization of IL-10 did not restore T(responder)-cell proliferation to control levels, which excluded IL-10 as a potential mediator of Thy-1-induced T(reg)-cell suppressor function. In addition, Thy-1-induced T(reg) cells did not inhibit IL-2-dependent proliferation of CTLL-2 cells, suggesting that IL-2 receptor signaling remained intact in the presence of Thy-1-induced T(reg) cells. We suggest that T(reg) cells induced by Thy-1 ligation in vivo may contribute to the maintenance of T-cell homeostasis.

Regulator of calcineurin 1 (Rcan1) is required for the development of pulmonary eosinophilia in allergic inflammation in mice.

The presence of eosinophils in the lung is often regarded as a defining feature of asthma. On allergen stimulation, numbers of eosinophils and their progenitors are increased in both the bone marrow and lungs. Eosinophil progenitors provide an ongoing supply of mature eosinophils. Here, we report that deficiency in the regulator of calcineurin 1 gene (Rcan1) leads to a near-complete absence of eosinophilia in ovalbumin-induced allergic asthma in mice. In the absence of Rcan1, bone marrow cells produce significantly fewer eosinophils in vivo and in vitro on interleukin-5 stimulation. Importantly, eosinophil progenitor populations are significantly reduced in both naïve and ovalbumin-challenged Rcan1(-/-) mice. Bone marrow cells from Rcan1(-/-) mice are capable of developing into fully mature eosinophils, suggesting that Rcan1 is required for eosinophil progenitor production but may not be necessary for eosinophil maturation. Thus, Rcan1 represents a novel contributor in the development of eosinophilia in allergic asthma through regulation of eosinophil progenitor production.

A Canadian Perspective on the Treatment of Waldenström Macroglobulinemia.

Waldenström macroglobulinemia (WM) is a slowly progressing B-cell non-Hodgkin lymphoma characterized by monoclonal IgM gammopathy in the blood and infiltration of the bone marrow by clonal lymphoplasmacytic cells. As an incurable disease, the goals for therapy for WM are to relieve symptoms, slow disease progression, prevent organ damage, and maintain quality of life. However, given the rarity of WM, clinical trials comparing treatments for WM are limited and there is no definitive standard of care. The selection of first-line WM therapy is thus based on patient factors, disease characteristics, and drug access, with bendamustine-rituximab and Bruton's tyrosine kinase (BTK) inhibitor therapy considered preferred treatments. Other treatments such as proteasome inhibitor- or purine analogue-based therapy, alternative chemoimmunotherapy, and autologous stem cell transplantation are generally reserved for the relapsed setting but may be used in rare circumstances in earlier lines of therapy. This paper summarizes the efficacy and safety of these WM therapies and discusses considerations for treatment from a Canadian perspective.

Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling.

Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4(+) T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca(2+) release to inhibit IκB phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4(+)CD25(+) regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

Mast cells down-regulate CD4+CD25+ T regulatory cell suppressor function via histamine H1 receptor interaction.

Mast cells promote both innate and acquired immune responses, but little is known about the effect of mast cells on T regulatory (T(reg)) cell function. In this study, we show for the first time that the capacity of murine CD4(+)CD25(+) T(reg) cells to suppress in vitro proliferation by CD4(+)CD25(-) T responder (T(resp)) cells in response to anti-CD3/anti-CD28 mAb-coated beads was reduced in the presence of syngeneic bone marrow-derived mast cells (BMMC) activated by FcepsilonR cross-linking. Activated BMMC culture supernatants or exogenous histamine also inhibited T(reg) cell suppressor function while the histamine H1 receptor-specific antagonist loratadine, but not the H2 receptor-specific antagonist famotidine, restored T(reg) cell suppressor function in the presence of activated BMMC or activated BMMC culture supernatants. Moreover, treatment of T(reg) cells with loratadine, but not famotidine, rescued T(reg) cell suppressor function in the presence of exogenous histamine. In addition, the H1 receptor-specific agonist 2-pyridylethylamine dihydrochloride inhibited T(reg) cell suppressor function to an extent that was comparable to histamine, whereas the H2 receptor-specific agonist amthamine dihydrobromide was without effect. Both T(reg) cells and T(resp) cells expressed H1 receptors. Exposure to histamine caused T(reg) cells to express lower levels of CD25 and the T(reg) cell-specific transcription factor Foxp3. Taken together, these data indicate that BMMC-elaborated histamine inhibited T(reg) cell suppressor function by signaling through the H1 receptor. We suggest that histamine released as a result of mast cell activation by microbial products might cause a transient decrease in T(reg) cell suppressor function, thereby enhancing the development of protective immunity.

DMG26: A Targeted Sequencing Panel for Mutation Profiling to Address Gaps in the Prognostication of Multiple Myeloma.

Multiple myeloma presents with numerous primary genomic lesions that broadly dichotomize cases into hyperdiploidy or IgH translocated. Clinically, these large alterations are assessed by fluorescence in situ hybridization (FISH) for risk stratification at diagnosis. Secondary focal events, including indels and single-nucleotide variants, are also reported; however, their clinical correlates are poorly described, and FISH has insufficient resolution to assess many of them. This study examined the exonic sequences of 26 genes reported to be mutated in >1% of patients with myeloma using a custom panel. These exons were sequenced to approximately 1000 times in a cohort of 76 patients from Atlantic Canada with detailed clinical correlates and in four multiple myeloma cell lines. Across the 76 patients, 255 mutations and 33 focal copy number variations were identified. High-severity mutations and mutations predicted by FATHMM-XF to be pathogenic identified patients with significantly reduced progression-free survival. These mutations were mutually exclusive from the Revised International Staging System high-risk FISH markers and were independent of all biochemical parameters of the Revised International Staging System. Applying our panel to patients classified by FISH to be standard risk successfully reclassified patients into high- and standard-risk groups. Furthermore, three patients in our cohort each had two high-risk markers; two of these patients developed plasma cell leukemia, a rare and severe clinical sequela of multiple myeloma.

Signaling through TLR7 enhances the immunosuppressive activity of murine CD4+CD25+ T regulatory cells.

Although signaling through certain TLRs is known to modulate the function of T lymphocytes, the effect of TLR7 stimulation on CD4(+)CD25(+) T(reg) cell activity has not yet been elucidated. In this study, we show that mouse CD4(+)CD25(+) T(reg) cells express TLR7 mRNA and protein. We therefore used the TLR7 agonists imiquimod, gardiquimod, and single-stranded poly(U) to show that TLR7 stimulation enhanced the ability of murine T(reg) cells to suppress anti-CD3/anti-CD28 mAb-coated bead-stimulated proliferation of syngeneic CD4(+)CD25(-) T(resp) cells. In contrast, imiquimod failed to enhance the suppressor function of T(reg) cells from mice deficient in the MyD88 adaptor protein involved in TLR7 and other TLR signal transduction. Imiquimod increased murine T(reg) cell-mediated suppression of T(resp) cell proliferation induced by anti-TCRbeta mAb in the presence of syngeneic BMDCs, and T(reg) cells from gardiquimod-treated mice exhibited enhanced in vitro suppressor function. Moreover, levels of T(resp) cell-secreted IL-2 and IFN-gamma were reduced further in the presence of T(reg) cells plus imiquimod in comparison with T(reg) cells alone. In addition, imiquimod treatment increased CD25 expression by T(reg) cells and caused exogenous IL-2 to enhance T(reg) cell suppressor function. Furthermore, combined treatment with imiquimod and IL-2 increased Foxp3 expression by T(reg) cells. Collectively, these findings suggest that TLR7 signaling enhanced the suppressor function of T(reg) cells by sensitizing T(reg) cells to IL-2-induced activation. We speculate that TLR7-stimulated enhancement of T(reg) cell suppressor function may modulate host T cell responses against ssRNA viruses.

Depletion of natural CD4+CD25+ T regulatory cells with anti-CD25 antibody does not change the course of Pseudomonas aeruginosa-induced acute lung infection in mice.

Pseudomonas aeruginosa is a common cause of lung infection in immune compromised individuals. Studies in humans and mice have demonstrated that P. aeruginosa lung infection is associated with a predominant Th2 immune response, whereas Th1 responses are accompanied by a better pulmonary outcome. Regulatory T cells (Tregs) are a subpopulation of T cells with unique immunologic characteristics that suppress effector T cell functions. Whether Tregs contribute to P. aeruginosa-induced host responses has not been studied previously. We found that P. aeruginosa lung infection induced an increase in natural Treg cells (CD4+CD25+FOXP3+ T cells) in the spleen of mice. To investigate a role of natural CD4+CD25+ Tregs in the host response to P. aeruginosa lung infection in vivo, anti-CD25 Ab was used to deplete endogenous CD4+CD25+ Tregs. Anti-CD25 treatment depleted 90% of CD4+CD25+FOXP3+ cells. Surprisingly, no differences of P. aeruginosa-induced NF-kappaB activation and cytokine/chemokine production (IL-1beta, TNF, IL-6, IL-10, RANTES or MIP-2) were observed between anti-CD25-treated and isotype control Ab-treated animals. Similarly, no differences in lung histology and airway neutrophil infiltration were observed between anti-CD25 and control Ab-treated animals. Furthermore, no difference in survival outcome was found between anti-CD25 and control Ab-treated animals. These data demonstrate that although P. aeruginosa lung infection causes an increase of Tregs, the endogenous natural CD4+CD25+ Treg cells do not contribute significantly to the host response to this bacterium.