High expression alleles of the innate cytokine, macrophage migration inhibitory factor (MIF), are associated with the development or the severity of autoimmune inflammatory diseases, including rheumatoid arthritis. Numerous studies support MIF's role in activating inflammatory pathways and MIF inhibition reduces joint pathology in different experimental models of arthritis. We examined the impact of gene deletion of MIF or its cognate receptor CD74 in the T cell-dependent model of collagen-induced arthritis (CIA) and observed the complete absence of arthritis development, suggesting an unforeseen role for MIF/CD74 signaling in the development of arthritogenic T cells. While MIF has been shown in model systems to contribute to T cell activation by augmenting innate responses, fewer than 1% of T lineage cells express CD74 in naive spleens and lymph nodes, and its functional consequences in pathogenic T cell subpopulations have not been studied. We found CD74+ T cells to expand during CIA and to increase in number within joint synovium, where they express an effector memory phenotype and recapitulate CIA development upon transfer into naive mice. We further found evidence for the presence of CD74+ T cells in the circulation and joint synovium of patients with rheumatoid arthritis. MIF-dependent, CD74+ T cells may contribute to the chronicity of rheumatoid synovitis and to disease relapse in previously inflamed joints.

RNA-based vaccines allow for rapid development and deployment. We evaluated immune responses over 1 yr in mice administered with two equivalent doses of a first generation self-amplifying RNA (saRNA) or mRNA vaccine encoding the SARS-CoV-2 spike protein. IgG1 concentrations were higher at 42 d post-vaccination in the mRNA group (mRNA: 2839.6 mg/mL, saRNA: 39.0 mg/mL; p < 0.001) without discernable differences in IgG2a, IgG3, or neutralizing antibody levels. At 180 d post-vaccination, the saRNA vaccinated group showed better retention of a neutralizing antibody response (saRNA: 87.6 mg/mL, mRNA: 32.8 mg/mL; p = 0.011), with elevated IgG3 titers (saRNA: 4.4 mg/mL, mRNA: 2.2 mg/mL; p = 0.031). One-year post-vaccination, saRNA vaccinated mice maintained elevated neutralizing antibody response (saRNA: 2.8 mg/mL, mRNA: 1.3 mg/mL; p = 0.015). Analysis of the B and T cell compartments at 42 d showed increased B cell antibody class switching (saRNA: 5.1%, mRNA: 4.3%; p = 0.033) and an increase in CD4+ cmT cells (saRNA: 1.7%, mRNA: 1.3%; p = 0.027) in the saRNA vaccinated cohort. scRNAseq analysis showed an increased inflammatory character of antigen-presenting cells at the injection site and a greater differentiation of cytotoxic CD8 T cells in the saRNA vs mRNA groups. These data provide evidence for an enhancement in humoral and cellular immunity by saRNA that may be beneficial in circumstances where antibody differentiation and antigen-experienced memory responses are desirable. Ludwig and Mathers Foundations Vaccines and Immunotherapy (VAC)

Macrophage migration inhibitory factor (MIF) is a key modulator of innate and adaptive immunity that has been extensively reported to promote tumor cell survival, proliferation, and metastasis. A recent study focusing on the microenvironment of acute myeloid leukemia (AML) showed that pharmacological inhibition of MIF signaling, in vitro as well as in vivo, reduces AML cell survival. Such data highlights the crucial role of MIF in AML pathogenesis and support the efforts for developing selective MIF modulators. Here, we report the identification and crystallographic characterization of a MIF inhibitor (compound 1) with an allosteric binding motif. Single point screening of 1 against a panel of National Cancer Institute (NCI) 60 human tumor cell lines revealed a selective antitumor activity for the AML cell line HL-60. After confirming the protein's expression in multiple AML cell lines, we utilized 1 to extract mechanistic insights into MIF action. Our findings demonstrate that AML cells utilize an MIF-dependent proliferation mechanism, which upon inhibition triggers a G0/G1 cell cycle arrest of the malignant cells. Complementary analysis of the MIF receptors utilizing neutralizing antibodies and selective small molecule antagonists associates this effect with inhibition of CD74 activation. The collection of data presented herein highlights the important role of MIF in proliferation of AML cells and points to the need of developing small molecule anticancer therapeutics that target MIF signaling.

Generating effective neutralizing antibodies is the goal of RNA-based vaccine platforms. We evaluated the comparative efficacy of antibody responses in C57BL/6 mice vaccinated at days 0 and 28 with a first-generation self-amplifying RNA (saRNA) versus a conventional mRNA directed against the SARS-CoV-2 spike protein. Analysis of circulating antibody titers at day 42 showed a significantly higher level of total spike-specific IgG in the mRNA-vaccinated mice (mRNA: 574.4 mg/mL vs saRNA: 169 mg/mL, p = 0.0003). Our measurement of the individual IgG subclass titers revealed mRNA IgG to be primarily composed of IgG1 (IgG1: 99%, IgG2a: 0.02%, IgG2b: 0.8%, IgG3: 0.2%), while the saRNA-vaccinated mice had a greater diversity of IgG subclasses (IgG1: 33%, IgG2a: 0.7%, IgG2b: 56%, IgG3: 10%). We found the greatest neutralization activity in the IgG2b subclass of the saRNA-vaccinated mice (saRNA: 17.5%, mRNA: 0.8%, expressed per microgram of IgG2b; p > 0.0001). By contrast, IgG1 (1 µg) from either vaccinated group showed similar neutralizing activity (saRNA: 7.2%, mRNA: 6.5%; p = NS). B cell repertoire analysis by scRNAseq showed increased BCR somatic hypermutation and clonal diversity in saRNA-vaccinated mice. These results suggest that single vaccination and boost by an saRNA vaccine, while generating less total antibody than a comparable mRNA vaccine targeting the SARS-CoV-2 spike protein, generates a greater diversity of IgG subclasses with higher neutralization activity. Ludwig Foundation Mathers Foundation Vaccines and Immunotherapy (VAC)

Macrophage migration inhibitory factor (MIF) is an upstream regulatory cytokine that is associated with advanced disease and poor outcomes in multiple cancer types, including melanoma. We investigated whether anti-MIF therapy could enhance the antitumor effects of the immune checkpoint inhibitor anti-programmed cell death 1 (anti-PD-1) in 2 murine tumor models. The therapeutic efficacy of anti-MIF, alone or combined with anti-PD-1, was tested in the YUMMER1.7 melanoma and MC38 colorectal cancer models. Tumor growth and survival were assessed in untreated Mif-knockout (KO) and low-expression human MIF allele (CATT5) mice and compared with wild-type (WT) or high-expression MIF allele (CATT7) mice. Tumor-bearing animals underwent cytokine profiling, tumor immunohistochemistry, flow cytometry, and scRNA-Seq. We also correlated functional variant MIF alleles with melanoma incidence and progression in patients. Our results showed that combined anti-MIF and anti-PD-1 significantly reduced tumor growth, improved survival, and promoted tumor regression, accompanied by enhanced TH1 cytokine levels, increased macrophage activation-related cytokines, and increased type 1 conventional dendritic cells. scRNA-Seq analysis revealed an expansion of intratumor Cd74/C1q/Aif1-expressing macrophages, which exhibited an antitumor phenotype, in response to anti-MIF therapy. MIF-KO and CATT5 mice exhibited reduced tumor burdens compared with WT or CATT7 mice alone and in the presence of anti-PD-1. In patients with melanoma, the high-MIF expression genotype (-173C/C) occurred at higher frequencies compared with healthy controls. These findings highlight that the addition of anti-MIF to anti-PD-1 reduces tumor growth, enhances antitumor responses, prolongs survival, and augments key intratumor immune cell populations involved in immune activation against tumors. This approach merits further consideration for clinical trial development.

Lipoprotein lipase (LPL) hydrolyzes circulating triglycerides (TGs), releasing fatty acids (FA) and promoting lipid storage in white adipose tissue (WAT). However, the mechanisms regulating adipose LPL and its relationship with the development of hypertriglyceridemia are largely unknown. WAT from obese humans exhibited high PAR2 expression, which was inversely correlated with the LPL gene. Decreased LPL expression was also inversely correlated with elevated plasma TG levels, suggesting that adipose PAR2 might regulate hypertriglyceridemia by downregulating LPL. In mice, aging and high palmitic acid diet (PD) increased PAR2 expression in WAT, which was associated with a high level of macrophage migration inhibitory factor (MIF). MIF downregulated LPL expression and activity in adipocytes by binding with CXCR2/4 receptors and inhibiting Akt phosphorylation. In a MIF overexpression model, high-circulating MIF levels suppressed adipose LPL, and this suppression was associated with increased plasma TGs but not FA. Following PD feeding, adipose LPL expression and activity were significantly reduced, and this reduction was reversed in Par2-/- mice. Recombinant MIF infusion restored high plasma MIF levels in Par2-/- mice, and the levels decreased LPL and attenuated adipocyte lipid storage, leading to hypertriglyceridemia. These data collectively suggest that downregulation of adipose LPL by PAR2/MIF may contribute to the development of hypertriglyceridemia.

Bladder pain is a prominent symptom in Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). We studied spinal mechanisms of bladder pain in mice using a model where repeated activation of intravesical Protease Activated Receptor-4 (PAR4) results in persistent bladder hyperalgesia (BHA) with little or no bladder inflammation. Persistent BHA is mediated by spinal macrophage migration inhibitory factor (MIF), and is associated with changes in lumbosacral proteomics. We investigated the contribution of individual spinal MIF receptors to persistent bladder pain as well as the spinal proteomics changes associated with relief of persistent BHA by spinal MIF antagonism. Female mice with persistent BHA received either intrathecal (i.t.) MIF monoclonal antibodies (mAb) or mouse IgG1 (isotype control antibody). MIF antagonism temporarily reversed persistent BHA (peak effect: 2 h), while control IgG1 had no effect. Moreover, i.t. antagonism of the MIF receptors CD74 and C-X-C chemokine receptor type 4 (CXCR4) partially reversed persistent BHA. For proteomics experiments, four separate groups of mice received either repeated intravesical scrambled peptide and sham i.t. injection (control, no pain group) or repeated intravesical PAR4 and: sham i.t.; isotype IgG1 i.t. (15 μg); or MIF mAb (15 μg). L6-S1 spinal segments were excised 2 h post-injection and examined for proteomics changes using LC-MS/MS. Unbiased proteomics analysis identified and relatively quantified 6739 proteins. We selected proteins that showed significant changes compared to control (no pain group) after intravesical PAR4 (sham or IgG i.t. treatment) and showed no significant change after i.t. MIF antagonism. Six proteins decreased during persistent BHA (V-set transmembrane domain-containing protein 2-like confirmed by immunohistochemistry), while two proteins increased. Spinal MIF antagonism reversed protein changes. Therefore, spinal MIF and MIF receptors mediate persistent BHA and changes in specific spinal proteins. These novel MIF-modulated spinal proteins represent possible new targets to disrupt spinal mechanisms that mediate persistent bladder pain.

CD74 is a cell-surface receptor for the cytokine macrophage migration inhibitory factor (MIF). MIF binding to CD74 induces a signaling cascade resulting in the release of its cytosolic intracellular domain (CD74-ICD), which regulates transcription in naïve B and chronic lymphocytic leukemia (CLL) cells. In the current study, we investigated the role of CD74 in the regulation of the immunosuppressive tumor microenvironment (TME) in triple-negative breast cancer (TNBC). TNBC is the most aggressive breast cancer subtype and is characterized by massive infiltration of immune cells to the tumor microenvironment, making this tumor a good candidate for immunotherapy. The tumor and immune cells in TNBC express high levels of CD74; however, the function of this receptor in the tumor environment has not been extensively characterized. Regulatory B cells (Bregs) and tolerogenic dendritic cells (tol-DCs) were previously shown to attenuate the antitumor immune response in TNBC. Here, we demonstrate that CD74 enhances tumor growth by inducing the expansion of tumor-infiltrating tol-DCs and Bregs. Utilizing CD74-KO mice, Cre-flox mice lacking CD74 in CD23+ mature B cells, mice lacking CD74 in the CD11c+ population, and a CD74 inhibitor (DRQ), we elucidate the mechanism by which CD74 inhibits antitumor immunity. MIF secreted from the tumor cells activates CD74 expressed on DCs. This activation induces the binding of CD74-ICD to the SP1 promotor, resulting in the up-regulation of SP1 expression. SP1 binds the IL-1β promotor, leading to the down-regulation of its transcription. The reduced levels of IL-1β lead to decreased antitumor activity by allowing expansion of the tol-DC, which induces the expansion of the Breg population, supporting the cross-talk between these 2 populations. Taken together, these results suggest that CD74+ CD11c+ DCs are the dominant cell type involved in the regulation of TNBC progression. These findings indicate that CD74 might serve as a novel therapeutic target in TNBC.

Macrophage migration inhibitory factor (MIF) is an innate cytokine that regulates both inflammatory and homeostatic responses. MIF is expressed by cardiomyocytes, where it exerts a protective action against ischemia-reperfusion (I/R) injury by activating AMP-activated protein kinase (AMPK). This effect is attenuated in the senescent heart due to an intrinsic, age-related reduction in MIF expression. We hypothesized that treating the aged heart with the small molecule MIF agonist (MIF20) can reinforce protective MIF signaling in cardiomyocytes, leading to a beneficial effect against I/R stress. The administration of MIF20 at the onset of reperfusion was found to not only decrease myocardial infarct size but also preserves systolic function in the aged heart. Protection from I/R injury was reduced in mice with cardiomyocyte-specific Mif deletion, consistent with the mechanism of action of MIF20 to allosterically increase MIF affinity for its cognate receptor CD74. We further found MIF20 to contribute to the maintenance of mitochondrial fitness and to preserve the contractile properties of aged cardiomyocytes under hypoxia/reoxygenation. MIF20 augments protective metabolic responses by reducing the NADH/NAD ratio, leading to a decrease in the accumulation of reactive oxygen species (ROS) in the aged myocardium under I/R stress. We also identify alterations in the expression levels of the downstream effectors PDK4 and LCAD, which participate in the remodeling of the cardiac metabolic profile. Data from this study demonstrates that pharmacologic augmentation of MIF signaling provides beneficial homeostatic actions on senescent myocardium under I/R stress.

OBJECTIVE:Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by abnormal activation of the type I interferon (IFN) pathway, which results in tissue inflammation and organ damage. We explored the role of the RhoA GTPase in the type I IFN activation pathway to provide a potential basis for targeting GTPase signaling for the treatment of SLE.METHODS:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy controls, and the mRNA expression levels of RhoA and IFN-stimulated genes were measured by SYBR Green quantitative reverse transcriptase-polymerase chain reaction. IFN-a-stimulated response element (ISRE)-luciferase reporter gene assays and Western blotting were conducted to assess the biologic function of RhoA. An enzyme-linked immunoassay (ELISA) measured C-X-C motif chemokine ligand 10 (CXCL10) protein expression.RESULTS:Our studies demonstrate that the expression of RhoA in the PBMCs of SLE subjects was significantly higher than in healthy controls and positively correlated with type I IFN scores and type I IFN-stimulated gene (ISGs) expression levels. SiRNA-mediated knockdown of RhoA and the RhoA/ROCK inhibitor Y27632 reduced the activity of the type I IFN-induced ISRE, the signal transducer and activator of transcription 1 (STAT-1) phosphorylation, and the expression of CXCL10 and 2'-5'-oligoadenylate synthetase 1 (OAS1). Finally, we verified that Y27632 could significantly down-regulate the OAS1 and CXCL10 expression levels in the PBMCs of SLE patients.CONCLUSION:Our study shows that RhoA positively regulates the activation of the type I IFN response pathway. Reducing the expression level of RhoA inhibits the abnormal activation of the type I IFN system, and the RhoA/ROCK inhibitor Y27632 decreases aberrant type I IFN signaling in SLE PBMCs, suggesting the possibility of targeting the RhoA GTPase for the treatment of SLE.

Functional variants of the gene for the cytokine macrophage migration inhibitory factor (MIF) are defined by a 4-nucleotide promoter microsatellite (-794 CATT5-8, rs5844572) and confer risk for autoimmune, infectious, and oncologic diseases. We describe herein the discovery of a prototypic, small molecule inhibitor of MIF transcription with selectivity for high microsatellite repeat number and correspondingly high gene expression. Utilizing a high-throughput luminescent proximity screen, we identify 1-carbomethoxy-5-formyl-4,6,8trihydroxyphenazine (CMFT) to inhibit the functional interaction between the transcription factor ICBP90 (namely, UHRF1) and the MIF -794 CATT5-8 promoter microsatellite. CMFT inhibits MIF mRNA expression in a -794 CATT5-8 length-dependent manner with an IC50 of 470 nM, and preferentially reduces ICBP90-dependent MIF mRNA and protein expression in high-genotypic versus low-genotypic MIF- expressing macrophages. RNA expression analysis also showed CMFT to downregulate MIF-dependent, inflammatory gene expression with little evidence of off-target metabolic toxicity. These findings provide proof-of-concept for advancing the pharmacogenomic development of precision-based MIF inhibitors for diverse autoimmune and inflammatory conditions.

Metabolic abnormalities affect clinical recovery and long-term survival in patients with acute myocardial infarction, but the underlying mechanisms remain unclear. D-dopachrome tautomerase (DDT) in cardiomyocytes protects the heart from injury during hypoxia-ischemia in mice. Our present study found that metabolic dysfunction induced by high fat diet (HFD) was associated with reduced cardiac DDT expression and aggravated cardiac injury following ischemia-reperfusion. Supplementation of DDT prior to ischemia decreased post-ischemic injury in these hearts, suggesting that DDT reduction is an important mechanism regulating post-ischemic cardiac injury associated with metabolic dysfunction. Among all the major fatty acid species in HFD, high palmitic acid (PA) triggered the expression and activation of protease activated receptor 2 (PAR2), which upregulated the transcriptional factors, CREB1 and FOXO1 leading to downregulation of DDT expression in the heart. PAR2 stimulated ERK phosphorylation, thereby upregulating CREB1 phosphorylation. Attenuation of CREB by siRNA significantly decreased FOXO1 expression and accumulation in the nucleus, thereby reducing cardiac DDT expression. Accordingly, PAR2 deficient mice exhibited normal ERK and CREB phosphorylation and DDT levels in the heart following HFD and reversed cardiac function recovery following reperfusion. Overall, our data reveal for the first time a novel role for DDT in mediating myocardial ischemia-reperfusion injury associated with metabolic dysfunction. Disclosure L. Li: None. Y. Qi: None. N. Cui: None. L. Leng: None. H. Wu: None. R. Bucala: None. D. Qi: None. Funding This study was supported by National Sciences and Engineering Research Council of Canada (NSERC: RGPIN-2017-04542) and Canadian Institutes of Health Research (CIHR Project Grant: PJT-156116).

Attenuation of adipose hormone sensitive lipase (HSL) may impair lipolysis and exacerbate obesity. We investigate the role of cytokine, macrophage migration inhibitory factor (MIF) in regulating adipose HSL and adipocyte hypertrophy. Extracellular MIF downregulates HSL in an autocrine fashion, by activating the AMPK/JNK signaling pathway upon binding to its membrane receptor, CD74. WT mice fed high fat diet (HFD), as well as mice overexpressing MIF, both had high circulating MIF levels and showed suppression of HSL during the development of obesity. Blocking the extracellular action of MIF by a neutralizing MIF antibody significantly reduced obesity in HFD mice. Interestingly, intracellular MIF binds with COP9 signalosome subunit 5 (Csn5) and JNK, which leads to an opposing effect to inhibit JNK phosphorylation. With global MIF deletion, adipocyte JNK phosphorylation increased, resulting in decreased HSL expression, suggesting that the loss of MIF's intracellular inhibitory action on JNK was dominant in Mif−/− mice. Adipose tissue from Mif−/− mice also exhibited higher Akt and lower PKA phosphorylation following HFD feeding compared with WT, which may contribute to the downregulation of HSL activation during more severe obesity. Both intracellular and extracellular MIF have opposing effects to regulate HSL, but extracellular actions predominate to downregulate HSL and exacerbate the development of obesity during HFD.

While insulin resistance (IR) is associated with inflammation in white adipose tissue, we report a non-inflammatory adipose mechanism of high fat-induced IR mediated by loss of Pref-1. Pref-1, released from adipose Pref-1+ cells with characteristics of M2 macrophages, endothelial cells or progenitors, inhibits MIF release from both Pref-1+ cells and adipocytes by binding with integrin β1 and inhibiting the mobilization of p115. High palmitic acid induces PAR2 expression in Pref-1+ cells, downregulating Pref-1 expression and release in an AMPK-dependent manner. The loss of Pref-1 increases adipose MIF secretion contributing to non-inflammatory IR in obesity. Treatment with Pref-1 blunts the increase in circulating plasma MIF levels and subsequent IR induced by a high palmitic acid diet. Thus, high levels of fatty acids suppress Pref-1 expression and secretion, through increased activation of PAR2, resulting in an increase in MIF secretion and a non-inflammatory adipose mechanism of IR.

Activation of intravesical protease activated receptors-4 (PAR4) results in bladder pain through the release of urothelial macrophage migration inhibitory factor (MIF) and high mobility group box-1 (HMGB1). We aimed to identify HMGB1 downstream signaling events at the bladder that mediate HMGB1-induced bladder pain in MIF-deficient mice to exclude any MIF-related effects. We studied whether oxidative stress and ERK activation are involved by examining bladder tissue in mice treated with intravesical disulfide HMGB1 for 1 h and analyzed with Western blot and immunohistochemistry. HMGB1 intravesical treatment increased urothelium 4HNE and phospho-ERK1/2 staining, suggesting that HMGB1 increased urothelial oxidative stress and ERK activation. Furthermore, we examined the functional roles of these events. We evaluated lower abdominal mechanical thresholds (an index of bladder pain) before and 24 h after intravesical PAR4 or disulfide HMGB1. Intravesical pre-treatments (10 min prior) included: N-acetylcysteine amide (NACA, reactive oxygen species scavenger) and FR180204 (FR, selective ERK1/2 inhibitor). Awake micturition parameters (voided volume; frequency) were assessed at 24 h after treatment. Bladders were collected for histology at the end of the experiment. Pre-treatment with NACA or FR significantly prevented HMGB1-induced bladder pain. No significant effects were noted on micturition volume, frequency, inflammation, or edema. Thus, HMGB1 activates downstream urothelial oxidative stress production and ERK1/2 activation to mediate bladder pain. Further dissection of HMGB1 downstream signaling pathway may lead to novel potential therapeutic strategies to treat bladder pain.

Background Activation of stimulator of interferon genes (STING) has shown great potential to enhance antitumor immunity. Several synthetic STING agonists have been tested preclinically and in the clinic. However, these molecules are susceptible to enzymatic degradation leading to low bioavailability in target tissues, unwanted toxicities, and narrow therapeutic windows. We have previously reported a novel STING agonist, ISAC-8803, with marked anti-cancer activity in mouse tumor models and in canine companion animals with glioblastoma. Here, we tested ISAC-8803 in combination with an antibody against both PD-L1 and PD-L2 with effector function (IMGS-27907), developed to diminish the immune suppression in immune excluded tumors. Methods ISAC-8803 was analyzed in vitro for potency in activating the human (THP-1 reporter cells) and mouse (293 reporter cells) STING pathways. ISAC-8803 was tested in vivo, alone and in combination with IMGS-27907, against mouse models of melanoma (B16F10 expressing mouse PD-L2) and mammary adenocarcinoma (TS/A). Mice with established tumors were treated with ISAC-8803 intratumorally at 10 ug/dose twice (days 11 and 14 for B16F10-PDL2 and days 23 and 26 for TS/A) and with IMGS-27907 at 10 mg/kg twice a week for 3 weeks, starting with the first ISAC-8803 treatment. TS/A tumors were removed 32 days post-tumor challenge and analyzed via H&E, IHC and FACS to assess overall necrosis, T cell infiltration, and macrophage content. Results ISAC-8803 more potently activated human and mouse STING relative to clinical benchmarks. It's EC50 of 0.1ug/ml and 0.28ug/ml in mouse and human cell lines, respectively, was 12–175X lower than the other compounds. In vivo, the combination of ISAC-8803 with IMGS-27907 resulted in 70% overall survival in B16F10-PDL2, compared to ≤10% in monotherapy and control groups, and significant extension of survival in the TS/A model that shows <10% PD-L1 and no PD-L2 expression. In the TS/A model, the combination therapy resulted in a large necrotic area compared to the respective control and individual treatments. CD3+ and CD8+ cells were more numerous in the tumors treated with the combination, while F4–80+ cells showed a decrease in comparison with the controls. Conclusions We demonstrated that ISAC-8803 is a potent STING agonist that when used in conjunction with a novel anti-PD-L1/PD-L2 monoclonal antibody induces curative responses in checkpoint-refractory tumor models. The increased survival is associated with an increase of T cells and potential decrease of M2 macrophages at the tumor site suggesting that delivery of STING intratumorally can potentiate the systemic activity of a novel checkpoint inhibitor. Acknowledgements This work was conducted with support from the Cancer Prevention and Research Institute of Texas (CPRIT).

CD74 is receptor for the cytokine macrophage migration inhibitory factor (MIF). MIF binding to CD74 induces a signaling cascade resulting in the release of its cytosolic intracellular domain (CD74-ICD) that serves as a transcriptional regulator in chronic lymphocytic leukemia (CLL) cells. In the current study, we investigated the transcriptional and regulatory function of CD74-ICD in normal B cells. We show that following activation, CD74-ICD forms a complex in the cytosol with transcription factors, like PAX5, and binds the chromatin at a significantly higher number of sites compared with its binding in CLL cells. The expression of a major portion of these bound genes is shut down in the malignant cells. The CD74-ICD:PAX5 complex binds the promoter areas of a tumor-suppressor gene, DMTF1, and downregulates its expression through inhibition of transcription. These findings can help identify novel therapeutic pathways that are regulated during onco-genic transformation and are targets for future treatments.