CD40 signaling augments IL-10 expression and the tolerogenicity of IL-10-induced regulatory dendritic cells.

CD40 expressed on stimulatory dendritic cells (DC) provides an important accessory signal for induction of effector T cell responses. It is also expressed at lower levels on regulatory DC (DCreg), but there is little evidence that CD40 signaling contributes to the tolerogenic activity of these cells. Indeed, CD40 silencing within DCreg has been reported to induce T cell tolerance in multiple disease models, suggesting that CD40 is superfluous to DC-induced tolerance. We critically assessed whether CD40 does have a role in tolerance induced by IL-10-differentiated DC (DC10) by using DC10 generating from the bone marrow of wild-type (w.t.) or CD40-/- donor mice, or IL-10-complemented CD40-/- DC10 to treat asthmatic mice. Wild-type DC10 ablated the OVA-asthma phenotype via induction of Foxp3+ Treg responses, but CD40-/- DC10 had no discernible effects on primary facets of the phenotype (e.g., IL-5, IL-9, IL-13 levels, IgE & IgG1 antibodies; p>0.05) and were ≤40% effective in reversal of others. Foxp3+ T cells from the lungs of CD40-/- DC10-treated mice expressed reduced levels of a panel of six Treg-specific activation markers relative to Treg from w.t. DC10-treated mice. Coculture with effector T cells from asthmatic mice induced a marked upregulation of cell surface CD40 on w.t. DC10. While untreated CD40-/- and w.t. DC10 secreted equally low levels of IL-10, stimulation of w.t. DC10 with anti-CD40 for 72 h increased their expression of IL-10 by ≈250%, with no parallel induction of IL-12. Complementing IL-10 expression in CD40-/- DC10 by IL-10 mRNA transfection fully restored the cells' abilities to suppress the asthma phenotype. In summary, CD40 signaling in DC10 contributes importantly to their expression of IL-10 and to a robust induction of tolerance, including activation of induced Treg.

225Ac-labeled CD33-targeting antibody reverses resistance to Bcl-2 inhibitor venetoclax in acute myeloid leukemia models.

PURPOSE: Despite the availability of new drugs, many patients with acute myeloid leukemia (AML) do not achieve remission and outcomes remain poor. Venetoclax is a promising new therapy approved for use in combination with a hypomethylating agent or with low-dose cytarabine for the treatment of newly diagnosed older AML patients or those ineligible for intensive chemotherapy. 225 Actinium-lintuzumab (225 Ac-lintuzumab) is a clinical stage radioimmunotherapy targeting CD33 that has shown evidence of single-agent activity in relapsed/refractory AML. Increased expression of MCL-1 is a mediator of resistance to venetoclax in cancer. EXPERIMENTAL DESIGN: Here we investigated the potential for 225 Ac-lintuzumab-directed DNA damage to suppress MCL-1 levels as a possible mechanism of reversing resistance to venetoclax in two preclinical in vivo models of AML. RESULTS: We demonstrated that 225 Ac-lintuzumab in combination with venetoclax induced a synergistic increase in tumor cell killing compared to treatment with either drug alone in venetoclax-resistant AML cell lines through both an induction of double-stranded DNA breaks (DSBs) and depletion of MCL-1 protein levels. Further, this combination led to significant tumor growth control and prolonged survival benefit in venetoclax-resistant in vivo AML models. CONCLUSIONS: There results suggest that the combination of 225 Ac-lintuzumab with venetoclax is a promising therapeutic strategy for the treatment of patients with venetoclax-resistant AML. Clinical trial of this combination therapy (NCT03867682) is currently ongoing.

Targeted lymphodepletion with a CD45-directed antibody radioconjugate as a novel conditioning regimen prior to adoptive cell therapy.

Chimeric antigen receptor (CAR) T cell therapies, and adoptive cell therapy (ACT) in general, represent one of the most promising anti-cancer strategies. Conditioning has been shown to improve the immune homeostatic environment to enable successful ACT or CAR-T engraftment and expansion in vivo following infusion, and represents potential point of intervention to decrease serious toxicities following CAR-T treatment. In contrast to relatively non-specific chemotherapy-derived lymphodepletion, targeted lymphodepletion with radioimmunotherapy (RIT) directed to CD45 may be a safer and more effective alternative to target and deplete immune cells. Here we describe the results of preclinical studies with an anti-mouse CD45 antibody 30F11, labeled with two different beta-emitters 131Iodine (131I) and 177Lutetium (177Lu), to investigate the effect of anti-CD45 RIT lymphodepletion on immune cell types and on tumor control in a model of adoptive cell therapy. Treatment of mice with 3.7 MBq 131I-30F11 or 1.48 MBq 177Lu-30F11 safely depleted immune cells such as spleen CD4+ and CD8+ T Cells, B and NK cells as well as Tregs in OT I tumor model while sparing RBC and platelets and enabled E. G7 tumor control. Our results support the application of CD45-targeted RIT lymphodepletion with a non-myeloablative dose of 131I-30F11 or 177Lu-30F11 antibody prior to adoptive cell therapy.

Daratumumab-225Actinium conjugate demonstrates greatly enhanced antitumor activity against experimental multiple myeloma tumors.

Daratumumab is an anti-CD38 directed monoclonal antibody approved for the treatment of multiple myeloma (MM) and functions primarily via Fc-mediated effector mechanisms such as complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and T-cell activation. However, not all patients respond to daratumumab therapy and management of MM remains challenging. Radioimmunotherapy with alpha particle-emitting radionuclides represents a promising approach to significantly enhance the potency of therapeutic antibodies in cancer treatment. Here we report the results of mechanistic and feasibility studies using daratumumab radiolabeled with an alpha-emitter 225Actinium for therapy of MM. CD38-positivelymphoma Daudi cell line and MM cell lines KMS-28BM and KMS-28PE were treated in vitro with 225Ac-daratumumab. 225Ac-daratumumab Fc-functional properties were assessed with C1q binding and ADCC assays. The pharmacokinetics and tumor uptake of 111In-daratumumab in Daudi tumor-bearing severe combined immunodeficiency (SCID) mice were measured with microSPECT/CT. The therapeutic effects of 225Ac-daratumumab on Daudi and KSM28BM tumors in mice and treatment side effects were evaluated for 50 days posttreatment. The safety of 225Ac-labeled antimurine CD38 mAb in immunocompetent mice was also evaluated. 225Ac-daratumumab efficiently and specifically killed CD38-positive tumor cells in vitro, while its complement binding and ADCC functions remained unaltered. MicroSPECT/CT imaging demonstrated fast clearance of the radiolabeled daratumumab from the circulation and tissues, but prolonged retention in the tumor up to 10 days. Therapy and safety experiments with 225Ac-daratumumab showed a significant increase in the antitumor potency in comparison to naked antibody without any significant side effects. Our results highlight the potential of targeting alpha-emitters to tumors as a therapeutic approach and suggest that 225Ac-daratumumab may be a promising therapeutic strategy for the treatment of hematologic malignancies.

Detection and targeting insulin growth factor receptor type 2 (IGF2R) in osteosarcoma PDX in mouse models and in canine osteosarcoma tumors.

Osteosarcoma (OS) represents 3.4% of all childhood cancers with overall survival of 70% not improving in 30 years. The consistent surface overexpression of insulin-like growth factor-2 receptor (IGF2R) has been reported in commercial and patient-derived xenograft (PDX) OS cell lines. We aimed to assess efficacy and safety of treating PDX and commercial OS tumors in mice with radiolabeled antibody to IGF2R and to investigate IGF2R expression on canine OS tumors. IGF2R expression on human commercial lines 143B and SaOS2 and PDX lines OS-17, OS-33 and OS-31 was evaluated by FACS. The biodistribution and microSPECT/CT imaging with 111Indium-2G11 mAb was performed in 143B and OS-17 tumor-bearing SCID mice and followed by radioimmunotherapy (RIT) with 177Lutetium-2G11 and safety evaluation. IGF2R expression in randomly selected canine OS tumors was measured by immunohistochemistry. All OS cell lines expressed IGF2R. Biodistribution and microSPECT/CT revealed selective uptake of 2G11 mAb in 143B and OS-17 xenografts. RIT significantly slowed down the growth of OS-17 and 143B tumors without local and systemic toxicity. Canine OS tumors expressed IGF2R. This study demonstrates the feasibility of targeting IGF2R on OS in PDX and spontaneous canine tumors and sets the stage for further development of RIT of OS using comparative oncology.

Prospective Analysis of the Effects of Maternal Immune Activation on Rat Cytokines during Pregnancy and Behavior of the Male Offspring Relevant to Schizophrenia.

Influenza during pregnancy is associated with the development of psychopathology in the offspring. We sought to determine whether maternal cytokines produced following administration of viral mimetic polyinosinic-polycytidylic acid (polyI:C) to pregnant rats were predictive of behavioral abnormalities in the adult offspring. Timed-pregnant Sprague Dawley rats received a single intravenous injection of 4-mg/kg polyI:C or saline on gestational day (GD)15. Blood was collected 3 h later for serum analysis of cytokine levels with ELISA. Male offspring were tested in a battery of behavioral tests during adulthood and behavior was correlated with maternal cytokine levels. Maternal serum levels of CXCL1 and interleukin (IL)-6, but not tumor necrosis factor (TNF)-α or CXCL2, were elevated in polyI:C-treated dams. PolyI:C-treated dams experienced post-treatment weight loss and polyI:C pups were smaller than controls at postnatal day (PND)1. Various behavior alterations were seen in the polyI:C-treated offspring. Male polyI:C offspring had enhanced MK-801-induced locomotion, and reduced sociability. PolyI:C offspring failed to display crossmodal and visual memory, and oddity preference was also impaired. Set-shifting, assessed with a lever-based operant conditioning task, was facilitated while touchscreen-based reversal learning was impaired. Correlations were found between maternal serum concentrations of CXCL1, acute maternal temperature and body weight changes, neonatal pup mass, and odd object discrimination and social behavior. Overall, while the offspring of polyI:C-treated rats displayed behavior abnormalities, maternal serum cytokines were not related to the long-term behavior changes in the offspring. Maternal sickness effects and neonatal pup size may be better indicators of later effects of maternal inflammation in the offspring.

Metformin inhibits the development, and promotes the resensitization, of treatment-resistant breast cancer.

Multiple drug resistant (MDR) malignancy remains a predictable and often terminal event in cancer therapy, and affects individuals with many cancer types, regardless of the stage at which they were originally diagnosed or the interval from last treatment. Protein biomarkers of MDR are not globally used for clinical decision-making, but include the overexpression of drug-efflux pumps (ABC transporter family) such as MDR-1 and BCRP, as well as HIF1α, a stress responsive transcription factor found elevated within many MDR tumors. Here, we present the important in vitro discovery that the development of MDR (in breast cancer cells) can be prevented, and that established MDR could be resensitized to therapy, by adjunct treatment with metformin. Metformin is prescribed globally to improve insulin sensitivity, including in those individuals with Type 2 Diabetes Mellitus (DM2). We demonstrate the effectiveness of metformin in resensitizing MDR breast cancer cell lines to their original treatment, and provide evidence that metformin may function through a mechanism involving post-translational histone modifications via an indirect histone deacetylase inhibitor (HDACi) activity. We find that metformin, at low physiological concentrations, reduces the expression of multiple classic protein markers of MDR in vitro and in preliminary in vivo models. Our demonstration that metformin can prevent MDR development and resensitize MDR cells to chemotherapy in vitro, provides important medical relevance towards metformin's potential clinical use against MDR cancers.

Rapamycin reduces fibroblast proliferation without causing quiescence and induces STAT5A/B-mediated cytokine production.

Rapamycin is a well-known inhibitor of the Target of Rapamycin (TOR) signaling cascade; however, the impact of this drug on global genome function and organization in normal primary cells is poorly understood. To explore this impact, we treated primary human foreskin fibroblasts with rapamycin and observed a decrease in cell proliferation without causing cell death. Upon rapamycin treatment chromosomes 18 and 10 were repositioned to a location similar to that of fibroblasts induced into quiescence by serum reduction. Although similar changes in positioning occurred, comparative transcriptome analyses demonstrated significant divergence in gene expression patterns between rapamycin-treated and quiescence-induced fibroblasts. Rapamycin treatment induced the upregulation of cytokine genes, including those from the Interleukin (IL)-6 signaling network, such as IL-8 and the Leukemia Inhibitory Factor (LIF), while quiescent fibroblasts demonstrated up-regulation of genes involved in the complement and coagulation cascade. In addition, genes significantly up-regulated by rapamycin treatment demonstrated increased promoter occupancy of the transcription factor Signal Transducer and Activator of Transcription 5A/B (STAT5A/B). In summary, we demonstrated that the treatment of fibroblasts with rapamycin decreased proliferation, caused chromosome territory repositioning and induced STAT5A/B-mediated changes in gene expression enriched for cytokines.

Behavioral alterations in rat offspring following maternal immune activation and ELR-CXC chemokine receptor antagonism during pregnancy: implications for neurodevelopmental psychiatric disorders.

Research suggests that maternal immune activation (MIA) during pregnancy increases the risk of neurodevelopmental disorders including schizophrenia and autism in the offspring. Current theories suggest that inflammatory mediators including cytokines and chemokines may underlie the increased risk of these disorders in humans. For example, elevated maternal interleukin-8 (IL-8) during pregnancy is associated with increased risk of schizophrenia in the offspring. Given this association, the present experiments examined ELR-CXC chemokines CXCL1 and CXCL2, rodent homologues of human IL-8, and activation of their receptors (CXCR1 and CXCR2) in an established rodent model of MIA. Pregnant Long Evans rats were treated with the viral mimetic polyinosinic-polycytidylic acid (polyI:C; 4 mg/kg, i.v.) on gestational day 15. Protein analysis using multiplex assays and ELISA showed that polyI:C significantly increased maternal serum concentrations of interleukin-1ß, tumor necrosis factor, and CXCL1 3h after administration. Subsequent experiments tested the role of elevated maternal CXCL1 on behavior of the offspring by administering a CXCR1/CXCR2 antagonist (G31P; 500 µg/kg, i.p.; 1h before, 48 and 96 h after polyI:C treatment). The male offspring of dams treated with polyI:C demonstrated subtle impairments in prepulse inhibition (PPI), impaired associative and crossmodal recognition memory, and altered behavioral flexibility in an operant test battery. While G31P did not completely reverse the behavioral impairments caused by polyI:C, it enhanced PPI during adolescence and strategy set-shifting and reversal learning during young adulthood. These results suggest that while polyI:C treatment significantly increases maternal CXCL1, elevations of this chemokine are not solely responsible for the effects of polyI:C on the behavior of the offspring.

Tissue eosinophilia in a mouse model of colitis is highly dependent on TLR2 and independent of mast cells.

The mechanisms initiating eosinophil influx into sites of inflammation have been well studied in allergic disease but are poorly understood in other settings. This study examined the roles of TLR2 and mast cells in eosinophil accumulation during a nonallergic model of eosinophilia-associated colitis. TLR2-deficient mice (TLR2(-/-)) developed a more severe colitis than wild-type mice in the dextran sodium sulfate (DSS) model. However, they had significantly fewer eosinophils in the submucosa of the cecum (P < 0.01) and mid-colon (P < 0.01) than did wild-type mice after DSS treatment. Decreased eosinophilia in TLR2(-/-) mice was associated with lower levels of cecal CCL11 (P < 0.01). Peritoneal eosinophils did not express TLR2 protein, but TLR2 ligand injection into the peritoneal cavity induced local eosinophil recruitment, indicating that TLR2 activation of other cell types can mediate eosinophil recruitment. After DSS treatment, mast cell-deficient (Kit(W-sh/W-sh)) mice had similar levels of intestinal tissue eosinophilia were observed as those in wild-type mice. However, mast cell-deficient mice were partially protected from DSS-induced weight loss, an effect that was reversed by mast cell reconstitution. Overall, this study indicates a critical role for indirect TLR2-dependent pathways, but not mast cells, in the generation of eosinophilia in the large intestine during experimental colitis and has important implications for the regulation of eosinophils at mucosal inflammatory sites.

Mast cells, histamine, and IL-6 regulate the selective influx of dendritic cell subsets into an inflamed lymph node.

In response to bacterial stimuli, multiple dendritic cell (DC) populations accumulate within the draining lymph node, thus enhancing opportunities for effective T cell-DC interaction. DC subpopulations, such as plasmacytoid, CD8(+), and CD11b(+) subsets, have distinct roles in determining the nature of the immune response. The mechanisms whereby individual DC subpopulations are mobilized and the extent to which these processes are linked to increases in overall lymph node cellularity have not been determined. In the current study, the mechanisms of DC subset mobilization to the draining auricular lymph node were examined after intradermal injection of Staphylococcus aureus-derived peptidoglycan. Using mast cell-deficient mice and local mast cell reconstitution, plasmacytoid and CD8(+) DC responses were shown to be mast cell dependent, whereas the CD11b(+) DC response was not. A histamine H2 receptor-dependent, CXCL9-independent pathway controlled the selective influx of both plasmacytoid and CD11b(+) DC into the lymph node, but not lymph node cellularity. In contrast, IL-6 was important for the mobilization of CD8(+) and CD11b(+) DC. TNF and IL-1 receptor were dispensable for plasmacytoid, CD11b(+), and CD8(+) DC responses. These findings provide novel opportunities for the selective mobilization of specific DC subsets to lymph nodes and demonstrate critical roles for both histamine and IL-6 in this process.

4-1BB and OX40 act independently to facilitate robust CD8 and CD4 recall responses.

Mice deficient in OX40 or 4-1BB costimulatory pathways show defects in T cell recall responses, with predominant effects on CD4 vs CD8 T cells, respectively. However, OX40L can also stimulate CD8 T cells and 4-1BBL can influence CD4 T cells, raising the possibility of redundancy between the two TNFR family costimulators. To test this possibility, we generated mice deficient in both 4-1BBL and OX40L. In an adoptive transfer model, CD4 T cells expressed 4-1BB and OX40 sequentially in response to immunization, with little or no overlap in the timing of their expression. Under the same conditions, CD8 T cells expressed 4-1BB, but no detectable OX40. Thus, in vivo expression of 4-1BB and OX40 can be temporally and spatially segregated. In the absence of OX40L, there were decreased CD4 T cells late in the primary response and no detectable secondary expansion of adoptively transferred CD4 T cells under conditions in which primary expansion was unaffected. The 4-1BBL had a minor effect on the primary response of CD4 T cells in this model, but showed larger effects on the secondary response, although 4-1BBL(-/-) mice show less impairment in CD4 secondary responses than OX40L(-/-) mice. The 4-1BBL(-/-) and double knockout mice were similarly impaired in the CD8 T cell response, whereas OX40L(-/-) and double knockout mice were similarly impaired in the CD4 T cell response to both protein Ag and influenza virus. Thus, 4-1BB and OX40 act independently and nonredundantly to facilitate robust CD4 and CD8 recall responses.

Role of T cell costimulation in anti-viral immunity.

Members of both the CD28 and TNFR families can have costimulatory roles in T cell activation. Gene targeted mice as well as in vivo blocking experiments have established distinct roles for CD28/B7; ICOS/ICOSL; CD27/CD70; 4-1BB/4-1BBL and OX40/OX40L during viral infection. Many issues remain to be addressed, including the timing and location of the interactions, the possibility of partial redundancy between related family members and the molecular basis for the specific phenotypes observed in the different gene targeted mice.

Expression and function of 4-1BB during CD4 versus CD8 T cell responses in vivo.

4-1BBL(-/-) mice have a defect in recall CD8+ T cell responses to viruses, whereas CD4+ T cell responses to virus are unimpaired in these mice. In contrast, both CD4+ and CD8+ T cells respond to 4-1BB ligand (4-1BBL) in vitro. To clarify the role of 4-1BB/4-1BBL in CD4+ versus CD8+ T cell responses in vivo, we compared CD4 (OT-II) and CD8 (OT-I) TCR transgenic T cells responding to the same antigen in an in vivo adoptive transfer model in 4-1BBL(+/+) versus 4-1BBL(-/-) mice. During primary and secondary responses, expression of 4-1BB on in vivo-activated TCR transgenic T cells was earlier and more transient than previously observed in vitro, correlating with expression of the early activation antigen CD69 and preceding the transition to the CD44hi state. Although 4-1BB is expressed early in the primary response, there was no effect of 4-1BBL deficiency on initial CD8 T cell expansion and only a minor effect on initial CD4 T cell expansion. The major effect of 4-1BB/4-1BBL interaction is on the T cell recall response. This is due to effects of 4-1BBL on maintenance of T cell numbers at the end of the primary response with additional effects of 4-1BBL on secondary expansion of T cells.

A switch in costimulation from CD28 to 4-1BB during primary versus secondary CD8 T cell response to influenza in vivo.

4-1BBL(-/-) mice exhibit normal primary CD8 T cell responses to influenza virus, but show decreased CD8 T cell numbers late in the primary response as well as decreased secondary responses. In contrast, CD28(-/-) mice are defective in initial CD8 T cell expansion. Using agonistic anti-4-1BB Ab to replace the CD28 or 4-1BB signal, we examined the timing of the required signals for CD28 vs 4-1BB costimulation. A single dose of agonistic anti-4-1BB Ab added only during priming restores the secondary CD8 T cell response in CD28(-/-) mice. Once the T cell numbers in the primary response reach a minimum threshold, a full secondary response is achieved even in the absence of CD28. In contrast, anti-4-1BB added during priming fails to correct the defective secondary response in 4-1BBL(-/-) mice, whereas addition of anti-4-1BB during challenge fully restores this response. Thus, there is a switch in costimulatory requirement from CD28 to 4-1BB during primary vs recall responses. Adoptive transfer studies show that T cells primed in 4-1BBL(-/-) or wild-type mice are equally capable of re-expansion when rechallenged in wild-type mice. These studies rule out a model in which signals delivered through 4-1BB during priming program the T cells to give a full recall response and suggest that 4-1BB-4-1BBL interactions take place at later stages in the immune response. The results indicate that anti-4-1BB or 4-1BBL therapy will be most effective during the boost phase of a prime-boost vaccination strategy.

4-1BB (CD137) differentially regulates murine in vivo protein- and polysaccharide-specific immunoglobulin isotype responses to Streptococcus pneumoniae.

4-1BB (CD137) is induced on activated CD4(+) and CD8(+) T cells and delivers a costimulatory signal upon binding the 4-1BB ligand (4-1BBL) expressed on antigen-presenting cells. Induction of 4-1BB is dependent on activation via the T-cell receptor (TCR) and possibly CD28. It was previously demonstrated that both an in vivo protein (pneumococcal surface protein A [PspA])- and polysaccharide (phosphorylcholine [PC] determinant of teichoic acid)-specific immunoglobulin (Ig) isotype response to Streptococcus pneumoniae was dependent on CD4(+) TCRalphabeta(+) T cells and B7-dependent costimulation through CD28. We thus postulated that 4-1BB costimulation would also play a role in regulating the in vivo anti-PspA and anti-PC response to S. pneumoniae. We demonstrate that mice genetically deficient in 4-1BBL elicit a markedly reduced IgM and IgG anti-PC but normal primary and secondary IgG anti-PspA responses to S. pneumoniae relative to those for wild-type mice. However, injection of an agonistic anti-4-1BB monoclonal antibody (MAb), while having no significant effect on the anti-PC response, strongly inhibits the primary anti-PspA response, the generation of PspA-specific memory, and germinal center formation but does not induce a lasting state of tolerance. In contrast, anti-4-1BB MAb has no effect on the anti-PspA response when injected only at the time of secondary immunization. Delay of the addition of anti-4-1BB leads to progressively less inhibition of the primary response up to day 8. This inhibition is independent of CD8(+) T cells and is associated with the expansion of CD4(+) T cells with an activated phenotype, which is partly dependent on B7-dependent costimulation. These data are the first to suggest a stimulatory role for endogenous 4-1BB-4-1BBL interactions during a humoral immune response to a pathogen and further underscore significant differences in costimulation requirements for an in vivo protein- versus polysaccharide-specific Ig isotype response to an extracellular bacterium.