Combined immunotherapy with whole tumor lysate-pulsed interleukin-15-activated dendritic cells and cucurbitacin I promotes strong CD8(+) T-cell responses and cures highly aggressive lymphoma.

BACKGROUND AIMS: Dendritic cell (DC)-based therapies could be important strategies for lymphoma treatment. METHODS: AKR/J mice with Dalton's lymphoma were treated with recombinant interleukin-15 (rIL-15)-activated autologous DCs and pulsed with whole tumor cell lysates in the presence or absence of suboptimal doses of the STAT3 inhibitor cucurbitacin I. One group of treated mice received an additional dose of rIL-15 to boost the DC-based adoptive cell therapy (ACT). Kaplan-Meier survival analysis and multiple immunologic and enzymatic parameters were assessed to demonstrate the efficacy of the vaccination protocol. RESULTS: Therapy with tumor lysate-pulsed, rIL-15-activated DCs plus cucurbitacin I significantly prolongs the survival of tumor-bearing mice but fails to provide a complete cure. Additional treatment of vaccinated mice with rIL-15 dramatically improves the therapeutic efficacy and provides a lifelong cure with no relapse. DCs derived from the surviving vaccinated mice regained their anti-tumor potential against the lymphoma cells with respect to growth inhibition and cytotoxicity. Both cluster of differentiation (CD)4(+) and CD8(+) T cells were mobilized in metastatic organs of successfully vaccinated mice in large numbers and demonstrated antigen-specific proliferation and tumor cell cytotoxicity. ACT also augments DC function by upregulating tumor necrosis factor-related apoptosis-inducing ligand and tumor necrosis factor-α expression. In addition, combinatorial immunotherapy restores the levels of antioxidant enzymes and liver function enzyme activities that are severely repressed in untreated tumor-bearing mice. CONCLUSIONS: Effective vaccination for a complete cure against aggressive lymphoma requires DC-based ACT in combination with chemotherapy and cytokine therapy.

Downregulation of endogenous STAT3 augments tumoricidal activity of interleukin 15 activated dendritic cell against lymphoma and leukemia via TRAIL.

Effector functions in tumor resistance by dendritic cells (DCs) are less well characterized. In this study, we describe that the murine DCs upon stimulation with recombinant IL-15 in vitro or in vivo, expresses TNF superfamily member TRAIL which mediates cytotoxicity and growth inhibition against a murine lymphoma called Dalton lymphoma (DL) via apoptosis. Presence of tumor lysate or intact tumor cells significantly reduces the DC mediated tumoricidal effect, possibly via masking and down-regulating TRAIL in DCs. The antitumor effect of DC derived TRAIL was further augmented by deactivation of STAT3 in tumor cells by cucurbitacin I, which makes it more susceptible to DC derived TRAIL Treatment of tumor cells with cucurbitacin I upregulates TRAIL receptor expression in addition to activation of caspases. Compared to naïve DCs, DCs from tumor bearing mice are significantly impaired in TRAIL expression and consequent antitumor functions against DL which was partially restored by activation with IL-15 or LPS. Priming with recombinant IL-15 prolongs the survival of tumor bearing mice treated with cucurbitacin I. Naïve peripheral blood DCs derived from chronic myeloid leukemia (CML) patients have significant impairment in expression of TRAIL and consequent tumoricidal properties against TRAIL sensitive lymphoma cell lines and primary tumor cells compared to normal control.

Galunisertib synergistically potentiates the doxorubicin-mediated antitumor effect and kickstarts the immune system against aggressive lymphoma.

In clinical practice, major efforts are underway to identify appropriate drug combinations to boost anticancer activity while suppressing unwanted adverse effects. In this regard, we evaluated the efficacy of combination treatment with the widely used chemotherapeutic drug doxorubicin along with the TGFßRI inhibitor galunisertib (LY2157299) in aggressive B-cell non-Hodgkin lymphoma (B-NHL). The antiproliferative effects of these drugs as single agents or in combination against several B-NHL cell lines and the synergism of the drug combination were evaluated by calculating the combination index. To understand the putative molecular mechanism of drug synergism, the TGF-ß and stress signaling pathways were analyzed after combination treatment. An aggressive lymphoma model was used to evaluate the anticancer activity and post-therapeutic immune response of the drug combination in vivo. Galunisertib sensitized various B-NHL cells to doxorubicin and in combination synergistically increased apoptosis. The antitumor activity of the drug combinations involved upregulation of p-P38 MAPK and inhibition of the TGF-ß/Smad2/3 and PI3K/AKT signaling pathways. Combined drug treatment significantly reduced tumor growth and enhanced survival, indicating that the synergism between galunisertib and Dox observed in vitro was most likely retained in vivo. Based on the tumor-draining lymph node analysis, combination therapy results in better prognosis, including disappearance of disease-exacerbating regulatory T cells and prevention of CD8+ T-cell exhaustion by downregulating MDSCs. Galunisertib synergistically potentiates the doxorubicin-mediated antitumor effect without aggravating the toxic effects and the ability to kickstart the immune system, supporting the clinical relevance of targeting TGF-ßRI in combination with doxorubicin against lymphoma.

Targeting thymidine phosphorylase alleviates resistance to dendritic cell immunotherapy in colorectal cancer and promotes antitumor immunity.

T-cell exhaustion plays a pivotal role in the resistance of microsatellite-stable colorectal cancer (CRC) to immunotherapy. Identifying and targeting T-cell exhaustion-activating mechanisms is a promising strategy to augment the effects of immunotherapy. Here, we found that thymidine phosphorylase (TYMP) plays a decisive role in inducing systemic T-cell exhaustion and abrogating the efficacy of dendritic cell (DC) therapy in a CRC model. Targeting TYMP with tipiracil hydrochloride (TPI) induces immunological cell death (ICD). The combined effects of TPI and imiquimod-activated DCs turn CT26 tumors into immunologically 'hot' tumors by inducing ICD in vivo. High-dimensional cytometry analysis revealed T-cell and IFN-γ dependency on the therapeutic outcome. In addition, chemoimmunotherapy converts intratumoral Treg cells into Th1 effector cells and eliminates tumor-associated macrophages, resulting in higher cytotoxic T lymphocyte infiltration and activation. This effect is also associated with the downregulation of PD-L1 expression in tumors, leading to the prevention of T-cell exhaustion. Thus, cooperative and cognitive interactions between dendritic cells and immunogenic cell death induced by therapy with TPI promote the immune response and tumoricidal activities against microsatellite stable colorectal cancer. Our results support TYMP targeting to improve the effects of DC immunotherapy and outcomes in CRC.

Pattern of autologous stem cell transplants at a tertiary care government hospital, with emphasis on transplant outcomes with pre-harvest CD34+ level.

Purpose: Autologous stem cell transplantation (ASCT) is an established therapy for many hematological diseases. This study assessed the pattern of ASCTs at a tertiary care center and associated factors, including pre-harvest CD34+ stem cell levels, leading to improved engraftment outcomes. Methodology: A retrospective study was conducted in India, between February 2009-August 2020. Patients who underwent ASCT for different hematological malignancies (n=65) were included, and the patients' age, sex, type and stage of disease, pre- and post-harvest CD34+ counts, and time to attain platelet/neutrophil engraftment or febrile neutropenia were analyzed. The post-harvest CD34+ dose was calculated. Pre-conditioning was performed using Granulocyte Colony Stimulating Factor (GCSF)±Plerixafor. Progression-free survival (PFS) was calculated using relapse/death as the endpoint. Results: The median age of the cohort (n=65) was 49 years, with a male preponderance. Multiple myeloma was the most common malignancy (70.8% [46/65]), requiring ASCT. The median time to ASCT was 13 months. All patients had received GCSF, while Plerixafor was used in 17 patients with a pre-harvest CD34+ count of <10 cells/µL. The median pre-harvest CD34+ concentration and post-harvest CD34+ cell dose was 27.54 cells/µL (n=26) and 5.23×106 cells/kg body weight (n=65), respectively. The median time to engraftment was 11 and 12 days, for neutrophils and platelets, respectively. One patient did not engraft and was excluded from the analysis. The time required to attain neutrophil engraftment was significantly lower (p=0.02) among freshly harvested stem cells (n=48) than that of cryopreserved products (n=17). Platelet engraftment associated with CD34+ pre- and post-harvest levels was not significant (p=0.06). The time to attain neutropenia and subsequent febrile neutropenia was significantly lower with an adequate post-harvest CD34+ dose (p=0.009). Febrile neutropenia was seen in 83.1% (54/65) patients. The median time for febrile neutropenia was 4 days post-ASCT. Pre- and post-harvest CD34+ concentrations were directly proportional to each other (p<0.001). The median PFS was 112 months (n=65). Survival was better in males (median PFS: 112 months) vs. females (median PFS: 59 months) (p=0.27). Eight patients relapsed, and eight patients had died. Conclusion: Although unrelated to age or sex, the post-harvest CD34+ dose was inversely related to febrile neutropenia. As pre- and post-harvest CD34+ levels were directly proportional, pre-harvest CD34+ concentrations may be reliably used to assess engraftment outcomes. Rapid neutrophil engraftment was noted in fresh stem cells with PFS of 112 months, and was better among males, the exact reason being unknown. Thus, a larger number of patients should be followed up to obtain an accurate picture.

Galunisertib Drives Treg Fragility and Promotes Dendritic Cell-Mediated Immunity against Experimental Lymphoma.

Galunisertib (LY2157299) is a selective ATP-mimetic inhibitor of TGF-ß receptor-I activation, currently under clinical trial in a variety of cancers. We have tested the combined effects of galunisertib- and interleukin-15-activated dendritic cells in an aggressive and highly metastatic murine lymphoma. Based on the tumor-draining lymph node architecture, and its histology, the combination therapy results in better prognosis, including disappearance of the disease-exacerbating regulatory T cells. Our data suggest that galunisertib significantly enhances the success of immunotherapy with IL-15-activated dendritic cells by limiting the regulatory T cells generation with consequent downregulation of regulatory T cells in the tumor-draining lymph nodes and vascularized organ like spleen. This is also associated with consistent loss p-SMAD2 and downregulation of Neuropilin-1, leading to better prognosis and positive outcome. These results connect the role of combined therapy with the consequent elimination of disease-exacerbating T regulatory cells in a metastatic murine lymphoma.

Downregulation of STAT3 phosphorylation enhances tumoricidal effect of IL-15-activated dendritic cell against doxorubicin-resistant lymphoma and leukemia via TNF-α.

Although disputed by some, increasing evidence suggests that TNF-α synergies with traditional chemotherapeutic drugs to exert a heightened antitumor effect. The present study investigated the antitumor efficacy of recombinant IL-15 in combination with the STAT3 inhibitor cucurbitacin-I in a doxorubicin-resistant murine lymphoma model. The significance of the work is to understand and design effective strategies in doxorubicin resistant lymphomas. TNF-α is downregulated in dendritic cells from mice with Dalton's lymphoma and shows an inverse relationship with disease progression. Doxorubicin-resistant DL cells have elevated levels of Bcl-2 and Mcl-1 and increased phosphorylation of STAT3. These cells are refractory to dendritic cell derived TNF-α. Doxorubicin resistant Dalton's lymphoma is susceptible to dendritic cell derived TNF-α upon stimulation with the STAT3 inhibitor cucurbitacin-I, which downregulates STAT3 and other survival molecules. The combined treatment of low dose of cucurbitacin-I and rIL-15 is ineffective in mice with doxorubicin resistant Dalton's lymphoma, but a similar therapy prolongs the survival of mice transplanted with parental Dalton's lymphoma. Doxorubicin resistant Dalton's lymphoma responds to therapy with high doses of cucurbitacin-I and rIL-15. Dendritic cell derived from mice responded positively to the therapy and regained their tumoricidal properties with respect to growth inhibition and killing of DL tumor cells. Similar to DL, DC derived from CML patients are impaired in TNF-α expression and are unable to restrict the growth of drug-resistant lymphoma and leukemia cells. This combination approach could be used as a new therapeutic strategy for aggressive and highly metastatic doxorubicin resistant lymphoma.