Murine Transplantation Model Research Articles

Investigating resistance to 5-Azacytidine and Venetoclax in PDX models of MDS/AML.

Progressing myelodysplastic syndrome (MDS) into acute myeloid leukemia (AML) is an indication for hypomethylating therapy (HMA, 5-Azacytidine (AZA)) and a BCL2 inhibitor (Venetoclax, VEN) for intensive chemotherapy ineligible patients. Mouse models that engraft primary AML samples may further advance VEN + AZA resistance research. We generated a set of transplantable murine PDX models from MDS/AML patients who developed resistance to VEN + AZA and compared the differences in hematopoiesis of the PDX models with primary bone marrow samples at the genetic level. PDX were created in NSGS mice via intraosseal injection of luciferase-encoding Lentivirus-infected MDS/AML primary cells from patient bone marrow. We validated the resistance of PDX-leukemia to VEN and AZA and further tested candidate agents that inhibit the growth of VEN/AZA-resistant AML. Transplantable PDX models for MDS/AML arise with 31 % frequency. The lower frequency of transplantable PDX models is not related to peritransplant lethality of the graft, but rather to the loss of the ability of short-term proliferation of leukemic progenitors after 10 weeks of engraftment. There exist subtle genetic and cytological changes between primary and PDX-AML samples however, the PDX models retain therapy resistance observed in patients. Based on in vitro testing and in vivo validation in PDX models, Panobinostat and Dinaciclib are very promising candidate agents that overcome dual VEN + AZA resistance.

CMTM6 promotes hepatocellular carcinoma invasion and metastasis and tumor-associated neutrophil immunoinfiltration through the Wnt/β-catenin pathway

BackgroundCMTM6 has been closely associated with the onset and progression of various tumor types. However, the precise mechanism by which CMTM6 operates in hepatocellular carcinoma remains elusive, necessitating further investigation.MethodsExpression levels of CMTM6 in hepatocellular carcinoma tissues and cells were analyzed using immunohistochemistry and quantitative real-time PCR. The correlation between CMTM6 expression in hepatocellular carcinoma tissues and clinical pathological characteristics, as well as patient prognosis, was investigated. Proliferation and apoptosis of hepatocellular carcinoma cells with silenced or overexpressed CMTM6 were assessed, alongside measurements of β-catenin and Wnt1 protein expression levels. In vivo research was conducted utilizing a murine subcutaneous transplantation model. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed to elucidate the regulatory mechanism of CMTM6. Additionally, CD66b expression levels in tumor tissue were examined using immunohistochemistry, and the immune infiltration of CMTM6 and tumor-associated neutrophils (TANs) was analyzed.ResultsElevated expression levels of CMTM6 in hepatocellular carcinoma tissues and cells were found to be associated with poor patient prognosis. Overexpression of CMTM6 in hepatocellular carcinoma cells was demonstrated to promote cellular proliferation and inhibit apoptosis. Mechanistically, CMTM6 expression levels in hepatocellular carcinoma tissues were observed to positively correlate with β-catenin expression. GSEA and KEGG analysis revealed significant enrichment of CMTM6 in the Wnt/β-catenin pathway, indicating its involvement in pathway regulation. Furthermore, CMTM6 was found to be associated with immune infiltration of TANs in hepatocellular carcinoma tissues.ConclusionCMTM6 plays a pivotal role in the development and progression of hepatocellular carcinoma through regulation of the Wnt/β-catenin pathway via β-catenin. Moreover, CMTM6 demonstrates the capacity to promote immune infiltration of TANs in hepatocellular carcinoma tissues. Consequently, CMTM6 exhibits potential as both an early diagnostic marker and a novel therapeutic target for patients with hepatocellular carcinoma.

Small-molecule inhibitors of the CD40-CD40L costimulatory interaction are effective in pancreatic islet transplantation and prevention of type 1 diabetes models.

As part of our work to develop small-molecule inhibitors (SMIs) of the CD40-CD40L(CD154) costimulatory protein-protein interaction, here, we describe the ability of two of our most promising SMIs, DRI-C21041 and DRI-C21095, to prolong the survival and function of islet allografts in two murine models of islet transplantation (under the kidney capsule and in the anterior chamber of the eye) and to prevent autoimmune type 1 diabetes (T1D) onset in NOD mice. In both transplant models, a significant portion of islet allografts (50%-80%) remained intact and functional long after terminating treatment, suggesting the possibility of inducing operational immune tolerance via inhibition of the CD40-CD40L axis. SMI-treated mice maintained the structural integrity and function of their islet allografts with concomitant reduction in immune cell infiltration as evidenced by direct longitudinal imaging in situ. Furthermore, in female NODs, three-month SMI treatment reduced the incidence of diabetes from 80% to 60% (DRI-C21041) and 25% (DRI-C21095). These results (i) demonstrate the susceptibility of this TNF superfamily protein-protein interaction to small-molecule inhibition, (ii) confirm the in vivo therapeutic potential of these SMIs of a critical immune checkpoint, and (iii) reaffirm the therapeutic promise of CD40-CD40L blockade in islet transplantation and T1D prevention. Thus, CD40L-targeting SMIs could ultimately lead to alternative immunomodulatory therapeutics for transplant recipients and prevention of autoimmune diseases that are safer, less immunogenic, more controllable (shorter half-lives), and more patient-friendly (i.e., suitable for oral administration, which makes them easier to administer) than corresponding antibody-based interventions.

Abstract 4147630: Towards transplantation of hearts from donation after circulatory death - stem cell derived therapy for ameliorating donor organ damage

The use of heart transplantation (HTx), a key life-saving therapy for end-stage heart failure, is limited by the shortage of acceptable donor hearts. Although donation after circulatory death (DCD) offers a potentially promising resource for clinical transplantation, its adoption for HTx is concerned by warm ischemic damage during the death process. Mesenchymal stem cell (MSC)-derived paracrine action has been proved to mediate cardioprotection. Based on our previous study showing that MSC conditioned media (MSC CM) and MSC-exosomes (MSC Exo) confer improved donor heart preservation during cold ischemic storage, we aimed to test the effect of MSC CM and MSC Exo on ex vivo recovery of ischemia-damaged myocardium and on promoting the implanted DCD heart function using an in vivo murine heterotopic heart transplantation model. Methods: Donor hearts from adult male C57BL/6 mice with 0, 30’ or 50’ in vivo warm ischemia (WI) were divided into (n=5-7/group): 1) no WI; 2) 30’-WI; 3) 50’-WI; 4) no WI+4h cold storage at 0-4°C (no WI+CS); 5) 30’-WI+CS; 6) 50’-WI+CS; 7) 50’-WI+CS+vehicle; 8) 50’-WI+CS+MSC CM; and 9) 50’-WI+CS+MSC Exo. The CM and Exo were obtained from cultivation of human bone marrow-MSCs in serum-free media for 72h and was added into preservation solution. Donor hearts were implanted into C57BL/6 male recipients using cervical heterotopic heart transplantation. At 24h post-surgery, left ventricular (LV) function was detected in transplanted hearts. Abdominal artery pressure was measured in recipients to validate technical reproducibility of the HTx model. Mitochondrial structure was detected by electron microscopy on the fixed human LV (hDCD&hDBD). Results: WI significantly impaired LV function in transplanted DCD hearts vs. no-WI (Fig. A, B). Intriguingly, markedly improved graft function was observed in MSC CM- or Exo-treated DCD hearts vs. vehicle (Fig. C). Short and disrupted cristae in mitochondria were noticed in hDCD vs. hDBD (Fig. D). Conclusions: Our results represent the initial evidence that MSC secretome (CM&Exo) mediates ex vivo cardiac repair in DCD hearts and validate an approach on MSC derived therapy to rescue marginal DCD donor hearts and improve post-transplant graft function.

Graft-derived extracellular vesicles transport miRNAs to modulate macrophage polarization after heart transplantation

Heart transplantation, a crucial intervention for saving lives of those with end-stage cardiac failure, often faces complications from acute allograft rejection. This study focuses on the intricate dynamics of immune cell interactions and specific communication pathways between organs, which are not yet well understood. Our study investigates this interplay using a murine heterotopic transplant model, using single-cell RNA sequencing to examine CD45+ immune cells from both the heart grafts and spleens. We conduct a comprehensive analysis focused on functional enrichment, cell trajectory, and interorgan communication in heart transplants, highlighting dynamic interactions between monocyte/macrophage subtypes that is mediated by extracellular vesicles (EVs). We use unsupervised clustering and elucidate the complex cellular interactions that influence allograft outcomes. Notably, we discovered that microRNA-363 and microRNA-709, carried by EVs from CD63+ graft macrophages, can induce M1 polarization within the recipient’s spleen via the Fcho2/Notch1 signaling pathway. These insights illuminate the nuanced immune responses during acute cardiac rejection and suggest that targeting EVs from graft-resident macrophages may offer a new strategy to mitigate transplant rejection.

IL-33 Induces a Switch in Intestinal Metabolites Revealing the Tryptophan Pathway as a Target for Inducing Allograft Survival.

IL-33, a pleiotropic cytokine, has been associated with a plethora of immune-related processes, both inflammatory and anti-inflammatory. T regulatory (Treg) cells, the main leukocyte population involved in immune tolerance, can be induced by the administration of IL-33, the local microbiota, and its metabolites. Here, we demonstrate that IL-33 drastically induces the production of intestinal metabolites involved on tryptophan (Trp) metabolism. naïve mice were treated with IL-33 for 4 days and leukocyte populations were analyzed by flow cytometry, and feces were processed for microbiota and intestinal metabolites studies. Using a murine skin transplantation model, the effect of Kynurenic acid (KA) on allograft survival was tested. Under homeostatic conditions, animals treated with IL-33 showed an increment in Treg cell frequencies. Intestinal bacterial abundance analysis indicates that IL-33 provokes dysbiosis, demonstrated by a reduction in Enterobacteria and an increment in Lactobacillus genera. Furthermore, metabolomics analysis showed a dramatic IL-33 effect on the abundance of intestinal metabolites related to amino acid synthesis pathways, highlighting molecules linked to Trp metabolism, such as kynurenic acid (KA), 5-Hydroxyindoleacetic acid (5-HIAA), and 6-Hydroxynicotinic acid (6-HNA), which was supported by an enhanced expression of Ido and Kat mRNA in MLN cells, which are two enzymes involved on KA synthesis. Interestingly, animals receiving KA in drinking water and subjected to skin transplantation showed allograft acceptance, which is associated with an increment in Treg cell frequencies. Our study reveals a new property for IL-33 as a modulator of the intestinal microbiota and metabolites, especially those involved with Trp metabolism. In addition, we demonstrate that KA favors Tregs in vivo, positively affecting skin transplantation survival.

Triggering receptor expressed on myeloid cells-1 aggravates obliterative bronchiolitis via enhancing the proinflammatory phenotype of macrophages

BackgroundTriggering Receptor Expressed on Myeloid cells-1 (TREM-1) plays an important role in innate immune system. However, whether and how TREM-1 contributes to obliterative bronchiolitis (OB) progression remains unclear. MethodsA murine orthotopic tracheal transplantation model was constructed to mimic the pathogenesis of OB. qPCR and immunoblotting were used to measure TREM-1 expression. RNA sequencing was used to investigate the impact of TREM-1 on proinflammatory phenotype of macrophages. Trem-1 knockout mice and Nlrp3 knockout mice were generated to investigate the role of the TREM-1/NLRP3 pathway in the proinflammatory phenotype of macrophages. The infiltration of immune cells within the grafts was quantified using immunofluorescence staining. Flow cytometry was used to detect the proportion of different immune cells in mice spleen and the expression levels of iNOS and co-stimulatory molecules in macrophages. ResultsThe expression of TREM-1 was upregulated in the mouse OB model. Genetic ablation or pharmacological inhibition of TREM-1 ameliorated OB, whereas the stimulation of TREM-1 using anti-TREM-1 agonistic antibody exacerbated OB. Moreover, Trem-1 ablation reduced the infiltration of iNOS+ macrophages and limited the T cell responses. In vitro studies revealed that Trem-1 deletion impaired the proinflammatory function and antigen presentation ability of macrophages. Additionally, Trem-1 knockout inhibited the activation of NLRP3 signaling pathway. NLRP3 overexpression restored the proinflammatory phenotype of Trem-1 knockout macrophages. ConclusionsThese findings indicated that TREM-1 could promote the proinflammatory phenotype of macrophages through NLRP3 inflammasome activation, thereby exacerbating OB progression. These findings indicated that TREM-1 may serve as a therapeutic target for OB treatment.

Identification of Differentially Expressed Genes in Cold Storage-associated Kidney Transplantation.

Although it is acknowledged that ischemia-reperfusion injury is the primary pathology of cold storage-associated kidney transplantation, its underlying mechanism is not well elucidated. To extend the understanding of molecular events and mine hub genes posttransplantation, we performed bulk RNA sequencing at different time points (24 h, day 7, and day 14) on a murine kidney transplantation model with prolonged cold storage (10 h). In the present study, we showed that genes related to the regulation of apoptotic process, DNA damage response, cell cycle/proliferation, and inflammatory response were steadily elevated at 24 h and day 7. The upregulated gene profiling delicately transformed to extracellular matrix organization and fibrosis at day 14. It is prominent that metabolism-associated genes persistently took the first place among downregulated genes. The gene ontology terms of particular note to enrich are fatty acid oxidation and mitochondria energy metabolism. Correspondingly, the key enzymes of the above processes were the products of hub genes as recognized. Moreover, we highlighted the proximal tubular cell-specific increased genes at 24 h by combining the data with public RNA-Seq performed on proximal tubules. We also focused on ferroptosis-related genes and fatty acid oxidation genes to show profound gene dysregulation in kidney transplantation. The comprehensive characterization of transcriptomic analysis may help provide diagnostic biomarkers and therapeutic targets in kidney transplantation.

442.7: Aire-expressing cells are novel mediators of allograft tolerance in a murine heterotopic heart transplant model.

442.7: Aire-expressing cells are novel mediators of allograft tolerance in a murine heterotopic heart transplant model.

A study on the efficacy and Safety Evaluation of a novel PD-1/CTLA-4 bispecific antibody

Tumors constitute a significant health concern for humans, and PD-1 and CTLA-4 monoclonal antibodies have been proven effective in cancer treatment. Some researchers have identified that the combination of PD-1 and CTLA-4 dual blockade demonstrates superior therapeutic efficacy. However, the development of PD-1/CTLA-4 bispecific antibodies faces challenges in terms of both safety and efficacy. The present study discloses a novel PD-1/CTLA-4 bispecific antibody, designated as SH010. Experimental validation through surface plasmon resonance (SPR) confirmed that SH010 exhibits favorable binding activity with both PD-1 and CTLA-4. Flow cytometry analysis demonstrated stable binding of SH010 antibody to CHOK1 cells overexpressing human or cynomolgus monkey PD-1 protein and to 293F cells overexpressing human or cynomolgus monkey CTLA-4 protein. Moreover, it exhibited excellent blocking capabilities in protein binding between human PD-1 and PD-L1, as well as human CTLA-4 and CD80/CD86. Simultaneously, in vitro experiments indicate that SH010 exerts a significant activating effect on hPBMCs. In murine transplant models of human prostate cancer (22RV1) and small cell lung cancer (NCI-H69), administration of varying concentrations of the bispecific antibody significantly inhibits tumor growth. MSD analysis revealed that stimulation of hPBMCs from three different donors with SH010 did not induce the production of cytokine release syndrome. Furthermore, Single or repeated intravenous administrations of SH010 in cynomolgus monkeys show favorable systemic exposure without noticeable drug accumulation or apparent toxicity. In conclusion, SH010 represents a novel cancer therapeutic drug poised to enter clinical trials and obtain market approval.

Trained immunity is regulated by T cell-induced CD40-TRAF6 signaling

Trained immunity is characterized by histone modifications and metabolic changes in innate immune cells following exposure to inflammatory signals, leading to heightened responsiveness to secondary stimuli. Although our understanding of the molecular regulation of trained immunity has increased, the role of adaptive immune cells herein remains largely unknown. Here, we show that Tcells modulate trained immunity via cluster of differentiation 40-tissue necrosis factor receptor-associated factor 6 (CD40-TRAF6) signaling. CD40-TRAF6 inhibition modulates functional, transcriptomic, and metabolic reprogramming and modifies histone 3 lysine 4 trimethylation associated with trained immunity. Besides invitro studies, we reveal that single-nucleotide polymorphisms in the proximity of CD40 are linked to trained immunity responses invivo and that combining CD40-TRAF6 inhibition with cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4-Ig)-mediated co-stimulatory blockade induces long-term graft acceptance in a murine heart transplantation model. Combined, our results reveal that trained immunity is modulated by CD40-TRAF6 signaling between myeloid and adaptive immune cells and that this can be leveraged for therapeutic purposes.

Pyruvate Carboxylase Attenuates Myocardial Ischemia-Reperfusion Injury in Heart Transplantation via Wnt/β-Catenin-Mediated Glutamine Metabolism.

The ischemia-reperfusion process of a donor heart during heart transplantation leads to severe mitochondrial dysfunction, which may be the main cause of donor heart dysfunction after heart transplantation. Pyruvate carboxylase (PC), an enzyme found in mitochondria, is said to play a role in the control of oxidative stress and the function of mitochondria. This research examined the function of PC and discovered the signaling pathways controlled by PC in myocardial IRI. We induced IRI using a murine heterotopic heart transplantation model in vivo and a hypoxia-reoxygenation cell model in vitro and evaluated inflammatory responses, oxidative stress levels, mitochondrial function, and cardiomyocyte apoptosis. In both in vivo and in vitro settings, we observed a significant decrease in PC expression during myocardial IRI. PC knockdown aggravated IRI by increasing MDA content, LDH activity, TUNEL-positive cells, serum cTnI level, Bax protein expression, and the level of inflammatory cytokines and decreasing SOD activity, GPX activity, and Bcl-2 protein expression. PC overexpression yielded the opposite findings. Additional research indicated that reducing PC levels could block the Wnt/β-catenin pathway and glutamine metabolism by hindering the movement of β-catenin to the nucleus and reducing the activity of complex I and complex II, as well as ATP levels, while elevating the ratios of NADP+/NADPH and GSSG/GSH. Overall, the findings indicated that PC therapy can shield the heart from IRI during heart transplantation by regulating glutamine metabolism through the Wnt/β-catenin pathway.

Murine kidney transplant outcome is best measured by transdermal glomerular filtration rate

Mouse kidney transplantation provides a powerful preclinical model for the study of kidney transplant alloimmunity. However, accurate measurement of graft function is difficult because of the inaccuracy of traditional surrogate markers serum creatinine and urea. We report the use of transdermal glomerular filtration rate measurement under the experimental conditions of unilateral nephrectomy and allogeneic kidney transplantation. Our findings demonstrate that transdermal glomerular filtration rate measurement is easy to perform, reproducible, and has more interexperimental consistency than serum creatinine or urea measurements. Most importantly, it significantly reduces the numbers of experimental animals required to detect subtle and yet clinically relevant differences in kidney function as often is the case in experimental murine kidney transplantation models.

Graft protective effects and donor-specific antibody suppression by CD4+CD25+Foxp3+ regulatory T cell induced by HMG-CoA reductase inhibitor rosuvastatin in a murine heart transplant model

BackgroundWe previously demonstrated that the hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor (statins) play an important role in the regulation of alloimmune responses. However, little is known regarding the effects of statin on allograft protection or donor-specific antibodies (DSA). In this study, we investigated the graft-protective and immunomodulatory effects of rosuvastatin in a model of fully major histocompatibility complex-mismatched murine cardiac allograft transplantation.MethodsCBA mice underwent transplantation of C57BL/6 (B6) hearts and received 50 and 500 μg/kg/day of rosuvastatin from the day of transplantation until seven days after the completion of transplantation. To confirm the requirement for regulatory T cells (Tregs), we administered an anti-interleukin-2 receptor alpha antibody (PC-61) to rosuvastatin-treated CBA recipients. Additionally, histological and fluorescent staining, cell proliferation analysis, flow cytometry, and DSA measurements were performed.ResultsCBA recipients with no treatment rejected B6 cardiac graft acutely (median survival time [MST], 7 days). CBA mice treated with 500 μg/kg/day of rosuvastatin prolonged allograft survival (MSTs, 77 days). Fluorescent staining studies showed that rosuvastatin-treated recipients had strong aggregation of CD4+Foxp3+ cells in the myocardium and around the coronary arteries of cardiac allografts two weeks after grafting. Flow cytometry studies performed two weeks after transplantation showed an increased number of splenic CD4+CD25+Foxp3+ T cells in rosuvastatin-treated recipients. The addition of rosuvastatin to mixed leukocyte cultures suppressed cell proliferation by increasing the number of CD4+CD25+Foxp3+ Tregs. Additionally, Tregs suppressed DSA production in rosuvastatin-treated recipients.ConclusionRosuvastatin treatment may be a complementary graft-protective strategy for suppressing DSA production in the acute phase, driven by the promotion of splenic and graft-infiltrating CD4+CD25+Foxp3+ Tregs.

Investigating the effects of radiation, T cell depletion, and bone marrow transplantation on murine gut microbiota.

Microbiome research has gained much attention in recent years as the importance of gut microbiota in regulating host health becomes increasingly evident. However, the impact of radiation on the microbiota in the murine bone marrow transplantation model is still poorly understood. In this paper, we present key findings from our study on how radiation, followed by bone marrow transplantation with or without T cell depletion, impacts the microbiota in the ileum and caecum. Our findings show that radiation has different effects on the microbiota of the two intestinal regions, with the caecum showing increased interindividual variation, suggesting an impaired ability of the host to regulate microbial symbionts, consistent with the Anna Karenina principle. Additionally, we observed changes in the ileum composition, including an increase in bacterial taxa that are important modulators of host health, such as Akkermansia and Faecalibaculum. In contrast, radiation in the caecum was associated with an increased abundance of several common commensal taxa in the gut, including Lachnospiraceae and Bacteroides. Finally, we found that high doses of radiation had more substantial effects on the caecal microbiota of the T-cell-depleted group than that of the non-T-cell-depleted group. Overall, our results contribute to a better understanding of the complex relationship between radiation and the gut microbiota in the context of bone marrow transplantation and highlight the importance of considering different intestinal regions when studying microbiome responses to environmental stressors.

The DNA Methyltransferase Inhibitor 5-Aza-4'-thio-2'-Deoxycytidine Induces C>G Transversions and Acute Lymphoid Leukemia Development.

DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that decrease 5'-cytosine methylation. DNMTi are used clinically based on the hypothesis that cytosine demethylation will lead to re-expression of tumor suppressor genes. 5-Aza-4'-thio-2'-deoxycytidine (Aza-TdCyd or ATC) is a recently described thiol-substituted DNMTi that has been shown to have anti-tumor activity in solid tumor models. In this study, we investigated the therapeutic potential of ATC in a murine transplantation model of myelodysplastic syndrome. ATC treatment led to the transformation of transplanted wild-type bone marrow nucleated cells into lymphoid leukemia, and healthy mice treated with ATC also developed lymphoid leukemia. Whole-exome sequencing revealed 1,000 acquired mutations, almost all of which were C>G transversions in a specific 5'-NCG-3' context. These mutations involved dozens of genes involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Human cells treated in vitro with ATC showed 1,000 acquired C>G transversions in a similar context. Deletion of Dck, the rate-limiting enzyme for the cytidine salvage pathway, eliminated C>G transversions. Taken together, these findings demonstrate a highly penetrant mutagenic and leukemogenic phenotype associated with ATC. Significance: Treatment with a DNA methyltransferase inhibitor generates a distinct mutation signature and triggers leukemic transformation, which has important implications for the research and clinical applications of these inhibitors.

IL-6 inhibition prevents costimulation blockade-resistant allograft rejection in T cell-depleted recipients by promoting intragraft immune regulation in mice

The efficacy of costimulation blockade with CTLA4-Ig (belatacept) in transplantation is limited due to T cell-mediated rejection, which also persists after induction with anti-thymocyte globulin (ATG). Here, we investigate why ATG fails to prevent costimulation blockade-resistant rejection and how this barrier can be overcome. ATG did not prevent graft rejection in a murine heart transplant model of CTLA4-Ig therapy and induced a pro-inflammatory cytokine environment. While ATG improved the balance between regulatory T cells (Treg) and effector T cells in the spleen, it had no such effect within cardiac allografts. Neutralizing IL-6 alleviated graft inflammation, increased intragraft Treg frequencies, and enhanced intragraft IL-10 and Th2-cytokine expression. IL-6 blockade together with ATG allowed CTLA4-Ig therapy to achieve long-term, rejection-free heart allograft survival. This beneficial effect was abolished upon Treg depletion. Combining ATG with IL-6 blockade prevents costimulation blockade-resistant rejection, thereby eliminating a major impediment to clinical use of costimulation blockers in transplantation.

Blockade of CCR5 and CXCR3 attenuates murine acute graft-versus-host disease through modulating donor-derived T-cell distribution and function.

Lymphocyte trafficking via chemokine receptors such as C-C chemokine receptor 5 (CCR5) and CXCR3 plays a critical role in the pathogenesis of acute graft-versus-host disease (aGVHD). Our previous studies showed that the addition of CCR5 or CXCR3 antagonists could only slightly alleviate the development of aGVHD. Given the specificity of T lymphocytes bearing CXCR3 and CCR5, we investigated whether combined CCR5 and CXCR3 blockade could further attenuate murine aGVHD. A mouse model of aGVHD was established to assess the efficacy of CCR5 and/or CXCR3 blockade on the development of aGVHD. The distribution of lymphocytes was calculated by quantification of immunostaining cells. The immunomodulatory effect on T cells was assessed by evaluating T-cell proliferation, viability, and differentiation. Using the murine allogeneic hematopoietic stem cell transplantation model, we demonstrated that blockade of both CCR5 and CXCR3 could efficiently alleviate the development of aGVHD. Further investigation on the immune mechanisms for this prophylactic effect showed that more T cells were detained into secondary lymphoid organs (SLOs), which may lead to reduced infiltration of T cells into GVHD target organs. Our study also showed that T cells detained in SLOs dampened the activation, suppressed the polarization toward T helper type 1 (Th1) and T cytotoxic type 1 (Tc1) cells, and induced the production of Treg cells. These data suggest that concurrent blockade of CCR5 and CXCR3 attenuates murine aGVHD through modulating donor-derived T-cell distribution and function, and this might be applicable for aGVHD prophylaxis in clinical settings.

Exogenous Metabolic Modulators Improve Response to Carboplatin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) lacks targeted therapies, leaving cytotoxic chemotherapy as the current standard treatment. However, chemotherapy resistance remains a major clinical challenge. Increased insulin-like growth factor 1 signaling can potently blunt chemotherapy response, and lysosomal processes including the nutrient scavenging pathway autophagy can enable cancer cells to evade chemotherapy-mediated cell death. Thus, we tested whether inhibition of insulin receptor/insulin-like growth factor 1 receptor with the drug BMS-754807 and/or lysosomal disruption with hydroxychloroquine (HCQ) could sensitize TNBC cells to the chemotherapy drug carboplatin. Using in vitro studies in multiple TNBC cell lines, in concert with in vivo studies employing a murine syngeneic orthotopic transplant model of TNBC, we show that BMS-754807 and HCQ each sensitized TNBC cells and tumors to carboplatin and reveal that exogenous metabolic modulators may work synergistically with carboplatin as indicated by Bliss analysis. Additionally, we demonstrate the lack of overt in vivo toxicity with our combination regimens and, therefore, propose that metabolic targeting of TNBC may be a safe and effective strategy to increase sensitivity to chemotherapy. Thus, we conclude that the use of exogenous metabolic modulators, such as BMS-754807 or HCQ, in combination with chemotherapy warrants additional study as a strategy to improve therapeutic responses in women with TNBC.

Abstract ED03-01: Breaking the Obesity-Breast Cancer Link: Comparing Diet, Drug and Surgical Approaches

Abstract The prevalence of obesity, an established risk and progression factor for at least 15 types of cancer (including breast cancer), continues to rise in the US and many other countries. Lifestyle approaches to weight loss have proven challenging for most people to maintain, and bariatric surgery, while effective, is expensive and has numerous adverse effects. The recent availability of highly effective incretin therapies for weight loss raises several key questions that we will address, including: i) do these agents drive weight loss in obese female mice?; ii) do they reverse obesity-induced metabolic dysregulation?; iii) can they reverse obesity-driven cancer?; iv) how heterogeneous is the response to these agents? We will report our study of the effects of extended (13 weeks) tirzepatide (TZP, a dual GLP1R/GIPR agonist) treatment in obese female mice on weight loss, metabolic hormones, and mammary tumor growth. We found that—similar to reports in the literature using male mice—obese female mice lost >20% body weight by 4 weeks of TZP treatment. Moreover, TZP-induced weight loss was maintained through 13 weeks of treatment by progressively escalating the dose up to 40 nmol/kg. Congruent with reports in male rodents and humans, we found the majority of weight loss in female mice was attributed to loss of adipose tissue, rather than lean mass. The effects of TZP on body weight and composition were associated with reversal of obesity-induced changes in glycemic control and circulating levels of metabolic hormones, particularly leptin, insulin, and IGF-1. TZP also significantly reversed the protumor effects of obesity in our murine E0771 orthotopic transplant model of TNBC.Furthermore, daily 30% calorie restriction, which we previously reported has strong weight loss-inducing, metabolic reprogramming and anticancer effects in multiple mouse models of breast and other cancers, reversed body weight, adiposity, and TNBC tumor growth and altered metabolic hormones to a greater extent than all groups studied, including the never-obese control mice and TZP-treated mice. Citation Format: S. Hursting. Breaking the Obesity-Breast Cancer Link: Comparing Diet, Drug and Surgical Approaches [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr ED03-01.