Mixed Lymphocyte Reaction Research Articles

Association of PIRCHE-II score with anti-donor T-cell response and risk of de novo donor-specific antibody production in kidney transplant recipients

BackgroundDe novo donor-specific antibodies (dnDSAs) affect long-term outcomes of kidney transplantation (KT). A higher Predicted Indirectly ReCognizable Human Leukocyte Antigen (HLA) Epitopes (PIRCHE-II) score correlates with various clinical outcomes, including dnDSA formation. However, a detailed analysis of the relationship between the PIRCHE-II score and anti-donor T-cell response is lacking. Therefore, this study investigated the relationship between PIRCHE-II scores associated with dnDSA formation and mixed lymphocyte reaction results of anti-donor T-cell response. MethodsData of 105 adult living-donor KT recipients were retrospectively assessed. ResultsOf the 105 patients, 13.3 % developed dnDSAs during the observation period. The PIRCHE-II score at the HLA-DQ locus (PIRCHE-DQ) was significantly higher in patients with dnDSA formation than in those without. The incidence of dnDSA formation was significantly higher in the PIRCHE-DQ ≥ 77 group than in the PIRCHE-DQ < 77 group. The proportion of patients with increased anti-donor T-cell response was significantly higher in the PIRCHE-DQ ≥ 77 group than in the PIRCHE-DQ < 77 group before KT and at 4 and 5 years after KT. ConclusionsPIRCHE-DQ may predict dnDSA formation and anti-donor T-cell response. Reducing the immunosuppressive drug dose in cases of high PIRCHE-DQ might not be prudent.

Fresh Human Umbilical Cord Arteries as a Potential Source for Small-Diameter Vascular Grafts.

The demand for small-diameter vascular grafts has been globally increased but still lacks optimal solutions in this category. This study evaluated the feasibility of utilizing human pretreated fresh and nondecellularized umbilical cord arteries (hUCAs) as vascular grafts without needing any immunosuppression process. A mixed lymphocyte reaction assay revealed that hUCAs did not induce lymphocyte proliferation or cytokine production. To assess the in vivo inflammatory response, hUCAs were buried in fatty tissue under the skin of the abdominal wall in the left and right iliac fossas of rats. The average sizes of the implanted hUCAs remained consistent at 30 days post implantation. To evaluate xenogeneic transplantation, hUCAs were grafted to the abdominal aorta below the kidney of Wister rats. Remarkably, all rats exhibited positive revascularization and perfusion, maintaining blood pressure values of around 110/70 mmHg. Doppler ultrasound consistently indicated good circulation, with the three separate echogenic layers corresponding to the three arterial wall layers throughout the assessment period. Grafted rats exhibited normal motor behavior, accompanied by positive responses to thermal and pain stimulation. Blood biochemical values and whole blood cell counts showed no significant differences between pre and post-transplantation. Histological analysis of the grafts revealed no calcification or thrombosis, and a mild chronic inflammatory response was presented. In conclusion, hUCAs maintained their structural and functional properties after transplantation in rats without immunosuppression. This highlights their potential as a source for allogeneic, readily accessible, small-diameter vascular grafts.

Durable Mixed Chimerism May Permit Subsequent Immunosuppression-Free Intestinal Transplantation – a Proof-of-Principle Study

Intestinal transplantation (ITx) is the definitive treatment for intestinal failure but has the highest rejection rate among solid organ transplants, requiring high doses of immunosuppression with high rates of infection, graft-versus-host disease, and malignancy. Transplant tolerance would overcome the need for long-term immunosuppression. Using non-myeloablative conditioning, our laboratory has developed a novel swine model of hematopoietic stem cell transplantation (HSCT) that produces durable mixed chimerism (MC) and immune tolerance without toxicity. We investigated whether durable MC would promote tolerance of subsequently transplanted donor-matched intestinal allografts without immunosuppression. Using miniature swine with defined MHC, we performed HSCT across an MHC-Class-I haplotype mismatch. Immunosuppression was stopped by day 45. MC was evaluated by flow cytometry, and mixed lymphocyte reaction (MLR) assays were used to evaluate cellular responses. Subsequently, orthotopic ITx was performed without immunosuppression using a donor that was MHC-matched to the HSCT donor. Recipients were observed for four weeks and euthanized for tissue collection and mechanistic assays. After HSCT, the recipients developed durable multilineage MC and apparent deletional tolerance. After ITx, recipients showed no clinical or histological signs of rejection, and chimerism was unchanged. These results demonstrate the potential value of generating durable MC to achieve transplant tolerance.

Targeting CD36-Mediated Lipid Metabolism by Selective Inhibitor-Augmented Antitumor Immune Responses in Oral Cancer.

The fatty acid receptor CD36 is expressed on various malignant cells and is suggested to contribute to tumor progression. CD36 is also expressed by several immune cells and involved in immune responses and may be a potential target in cancer immunotherapy. In this study, we investigated whether the selective inhibition of CD36 can inhibit tumor progression and facilitate an antitumor immune response in oral squamous carcinoma cells (OSCCs). We assessed the effects of sulfosuccinimidyl oleate sodium (SSO), a CD36 inhibitor, on the proliferation apoptosis and alteration in tumor cell surface expression levels of immune accessory molecules in vitro. We also assessed whether SSO-treated OSCCs could promote a T cell response via a Mixed Lymphocyte Reaction (MLR) assay. We also investigated the direct antitumor effects and immunomodulatory effects of SSO using a mouse oral cancer OSCC model. SSO treatment significantly inhibited OSCC proliferation, increased apoptotic cell death, and upregulated the cell surface expression of several immune accessory molecules, including CD83, MHC-Class II, and PD-L1. SSO-treated OSCCs augmented T cell proliferation following MLR. In vivo SSO administration significantly attenuated mouse tumor growth with an increased proportion of immune cells, including CD4+ T, CD8+ T, and dendritic cells; it also decreased the proportion of immune suppressive cells, such as myeloid-derived suppressor and regulatory T cells. These results suggest that the selective inhibition of CD36 can induce direct and indirect antitumor effects by facilitating host antitumor immune responses in OSCCs.

Indirubin induces tolerogenic dendritic cells via aryl hydrocarbon receptor activation and ameliorates allergic asthma in a murine model by expanding Foxp3-expressing regulatory T cells

BackgroundAllergic asthma is a chronic bronchial inflammatory disease closely associated with abnormal immune responses of dendritic cells (DCs) and allergen-specific type 2 T helper (Th2) cells. Indirubin (IR), a natural aryl hydrocarbon receptor (AhR) ligand, exerts anti-inflammatory and immunomodulatory properties. PurposeIn this study, we aimed to clarify whether IR exhibits immunomodulatory action on DCs via AhR activation and investigated the antiallergic effects of IR in a mouse model of allergic asthma. MethodsLipopolysaccharide (LPS)-activated bone marrow-derived DCs were treated with IR. Their mRNA expressions, cytokine production, and phenotype patterns were determined by a quantitative real-time PCR, ELISA, flow cytometry, and RNA sequencing. The mixed lymphocyte reaction was utilized to evaluate the regulatory function of IR-treated DCs on T-cell differentiation. Moreover, mice with ovalbumin (OVA)-induced allergic asthma were treated with IR. Thereafter, the airway hyperresponsiveness (AHR), allergen-specific IgE production, cytokine levels, airway inflammation, and T-cell responses were evaluated. ResultsTreatment of LPS-stimulated DCs with 20 μM IR significantly reduced IL-12 and TNF-α production while increasing IL-10 secretion. Meanwhile, these DCs expressed decreased levels of CD80 but increased levels of Jagged 1 surface molecules. However, the effects of IR on DCs were reversed by pretreatment with the AhR antagonist, CH223191. Additionally, the coculture of these tolerogenic-like DCs with allogeneic CD4+T cells promoted the generation of Foxp3+ regulatory T (Treg) cells. A transcriptomic analysis identified several downregulated genes that are involved in regulating cell migration, cytokine secretion, and inflammatory responses in DCs after IR treatment. In an asthmatic murine model, oral administration of 25 mg kg-1 body weight of IR efficiently alleviated the development of AHR, OVA-specific IgE production, and levels of Th2-type cytokines (IL-4, IL-5, and IL-13) and the CCL11 chemokine. IR treatment also attenuated inflammatory cell recruitment and mucus production in the lungs. Notably, an enhanced frequency of Foxp3+ Treg cells and reduced effector T-cell proliferation associated with increased levels of IL-10 and TGF-β were observed in IR-treated mice. ConclusionIR can induce tolerogenic-like BMDCs which promote the differentiation of Treg cells. Importantly, the expansion of Foxp3+ Treg cells contributed to the therapeutic efficacy of IR against allergic asthma.

Dual A2A/A2B Adenosine Receptor Antagonist M1069 Counteracts Immunosuppressive Mechanisms of Adenosine and Reduces Tumor Growth In Vivo.

While A2A adenosine receptor (AR) was considered as a major contributor to adenosine-mediated immunosuppression, A2B, having the lowest affinity to adenosine, has also emerged as a potential contributor to tumor promotion. Therefore, in adenosine-rich tumor microenvironment (TME), where A2B could be complementary and/or compensatory to A2A, simultaneous targeting of A2A and A2B ARs can provide higher potential for cancer immunotherapy. We developed M1069-a highly selective dual antagonist of the A2A and A2B AR. In assays with primary human and murine immune cells, M1069 rescued IL2 production from T cells (A2A dependent) and inhibited VEGF production by myeloid cells (A2B dependent) in adenosine-high settings. M1069 also demonstrated superior suppression of the secretion of protumorigenic cytokines CXCL1, CXCL5, and rescue of IL12 secretion from adenosine-differentiated dendritic cells compared to an A2A-selective antagonist (A2Ai). In a one-way mixed lymphocyte reaction (MLR) assay, adenosine-differentiated human and murine dendritic cells treated with M1069 demonstrated superior T-cell stimulatory activity compared to dendritic cells differentiated in presence of A2Ai. In vivo, M1069 decreased tumor growth as a monotherapy and enhanced antitumor activity of bintrafusp alfa (BA) or cisplatin in syngeneic adenosinehi/CD73hi 4T1 breast tumor model, but not in the CD73 knockout 4T1 tumor model or in adenosinelow/CD73low MC38 murine colon carcinoma model. In summary, our dual A2A/A2B AR antagonist M1069 may counteract immune-suppressive mechanisms of high concentrations of adenosine in vitro and in vivo and enhance the antitumor activity of other agents, including BA and cisplatin.

CD19-chimeric antigen receptor-invariant natural killer T cells transactivate NK cells and reduce alloreactivity

Invariant natural killer T (iNKT) cells are a small fraction of T lymphocytes with strong cytotoxic and immunoregulatory properties. We previously showed that human culture-expanded iNKT cells prevent alloreactivity and lyse primary leukemia blasts. Here, iNKT cells have several advantages over T cells based on their immunoregulatory capabilities. Since chimeric antigen receptors (CARs) increase the benefit of immune effector cells, they play a crucial role in improvement of cytotoxic abilities of novel cellular therapeutics such as iNKT cells. In the present study, we investigated transactivation of NK cells and prevention of alloreactivity through iNKT cells transduced with a CD19-directed CAR. iNKT cells were isolated by magnetic cell separation from peripheral blood mononuclear cells and transduced with a CD19-CAR retrovirus. Transduction efficiency, purity and cell subsets were measured by flow cytometry. Transactivation and cytotoxicity assays have been established to investigate the ability of CD19-CAR-iNKT cells to transactivate primary NK cells. A mixed lymphocyte reaction (MLR) was performed to explore the inhibition of alloreactive CD3+ T cells by CD19-CAR-iNKT cells. CD19-CAR-iNKT cells are able to transactivate NK cells independent of cell contact: The expression of activation marker CD69 was significantly increased and also production of the proinflammatory cytokine interferon-gamma was higher in NK cells pretreated with CD19-CAR-iNKT cells. Consequently, the cytotoxic activity of such NK cells was significantly increased being able to lyse leukemia cells more effectively than without prior transactivation. Adding CD19-CAR-iNKT cells to an MLR resulted in a decreased expression of the T cell activation marker CD25 on alloreactive CD3+ T lymphocytes stimulated with HLA mismatched dendritic cells. Also, the proliferation of alloreactive CD3+ T lymphocytes was significantly reduced in this setting. We demonstrate that CD19-CAR-iNKT cells keep their immunoregulatory properties despite transduction with a CAR making them an attractive effector cell population for application after allogeneic hematopoietic cell transplantation. By transactivating NK cells, increasing their cytotoxic activity and suppressing alloreactive T cells, they might further improve outcomes through prevention of both relapse and graft-versus-host disease.

Clara cell 10 (CC10) protein attenuates allergic airway inflammation by modulating lung dendritic cell functions

Allergic asthma is a complex inflammatory disorder predominantly orchestrated by T helper 2 (Th2) lymphocytes. The anti-inflammatory protein Clara Cell 10-kDa (CC10), also known as secretoglobin family 1A member 1 (SCGB1A1), shows promise in modulating respiratory diseases. However, its precise role in asthma remains unclear. This study examines the potential of CC10 to suppress allergic asthma inflammation, specifically assessing its regulatory effects on Th2 cell responses and dendritic cells (DCs). Lower CC10 levels in asthma were observed and correlated with increased IgE and lymphocytes. Cc10−/− mice exhibited exacerbated allergic airway inflammation marked by increased inflammatory cell infiltration, Th2 cytokines, serum antigen-specific IgE levels, and airway hyperresponsiveness (AHR) in house dust mite (HDM)-induced models. Conversely, recombinant CC10 significantly attenuated these inflammatory responses. Intriguingly, CC10 did not directly inhibit Th cell activation but significantly downregulated the population of CD11b+CD103− DCs subsets in lungs of asthmatic mice and modulated the immune activation functions of DCs through NF-κB signaling pathway. The mixed lymphocyte response assay revealed that DCs mediated the suppressive effect of CC10 on Th2 cell responses. Collectively, CC10 profoundly mitigates Th2-type allergic inflammation in asthma by modulating lung DC phenotype and functions, highlighting its therapeutic potential for inflammatory airway conditions and other related immunological disorders.

Normothermic liver perfusion derived extracellular vesicles have concentration-dependent immunoregulatory properties.

Extracellular vesicles (EVs) are major contributors to immunological responses following solid organ transplantation. Donor derived EVs are best known for their role in transplant rejection through transferring donor major histocompatibility complex proteins to recipient antigen presenting cells, a phenomenon known as ‛cross-decoration'. In contrast, donor liver-derived EVs are associated with organ tolerance in small animal models. Therefore, the cellular source of EVs and their cargo could influence their downstream immunological effects. To investigate the immunological effects of EVs released by the liver in a physiological and transplant-relevant model, we isolated EVs being produced during normothermic ex vivo liver perfusion (NEVLP), a novel method of liver storage prior to transplantation. We found EVs were produced by the liver during NEVLP, and these EVs contained multiple anti-inflammatory miRNA species. In terms of function, liver-derived EVs were able to cross-decorate allogeneic cells and suppress the immune response in allogeneic mixed lymphocyte reactions in a concentration-dependent fashion. In terms of cytokine response, the addition of 1×109 EVs to the mixed lymphocyte reactions significantly decreased the production of the inflammatory cytokines TNF-α, IL-10 and IFN-γ. In conclusion, we determined physiologically produced liver-derived EVs are immunologically regulatory, which has implications for their role and potential modification in solid organ transplantation.

Extracellular vesicles from seminal plasma interact with T cells in vitro and drive their differentiation into regulatory T-cells.

Seminal plasma induces immune tolerance towards paternal allogenic antigens within the female reproductive tract and during foetal development. Recent evidence suggests a role for extracellular vesicles in seminal plasma (spEVs). We isolated spEVs from seminal plasma that was donated by vasectomized men, thereby excluding any contributions from the testis or epididymis. Previous analysis demonstrated that such isolated spEVs originate mainly from the prostate. Here we observed that when isolated fluorescently labelled spEVs were mixed with peripheral blood mononuclear cells, they were endocytosed predominantly by monocytes, and to a lesser extent also by T-cells. In a mixed lymphocyte reaction, T-cell proliferation was inhibited by spEVs. A direct effect of spEVs on T-cells was demonstrated when isolated T cells were activated by anti-CD3/CD28 coated beads. Again, spEVs interfered with T cell proliferation, as well as with the expression of CD25 and the release of IFN-γ, TNF, and IL-2. Moreover, spEVs stimulated the expression of Foxp3 and IL-10 by CD4+CD25+CD127- T cells, indicating differentiation into regulatory T-cells (Tregs). Prior treatment of spEVs with proteinase K revoked their effects on T-cells, indicating a requirement for surface-exposed spEV proteins. The adenosine A2A receptor-specific antagonist CPI-444 also reduced effects of spEVs on T-cells, consistent with the notion that the development of Tregs and their immune suppressive functions are under the influence of adenosine-A2A receptor signalling. We found that adenosine is highly enriched in spEVs and propose that spEVs are targeted to and endocytosed by T-cells, after which they may release their adenosine content into the lumen of endosomes, thus allowing endosome-localized A2A receptor signalling in spEVs targeted T-cells. Collectively, these data support the idea that spEVs can prime T cells directly for differentiation into Tregs.

Allostimulation leads to emergence of a human B cell population with increased expression of HLA class I antigen presentation–associated molecules and the immunoglobulin receptor FcRL5

In the extensive literature characterizing lymphocyte contributions to transplant-related pathologies including allograft rejection and graft-versus-host disease, T cell–focused investigation has outpaced investigation of B cells. Most B cell–related reports describe regulatory and antibody-producing functions, with less focus on the potential role of antigen-presenting capacity. Using in vitro human mixed lymphocyte reactions (MLRs) to model allostimulation, we analyzed responder B cells using transcriptional analysis, flow cytometry, and microscopy. We observed emergence of an activated responder B cell subpopulation phenotypically similar to that described in individuals with graft-versus-host disease or allograft rejection. This population had markedly increased expression of FcRL5 (Fc receptor like 5) and molecules associated with human leukocyte antigen class I antigen presentation. Consistent with this phenotype, these cells demonstrated increased internalization of irradiated cell debris and dextran macromolecules. The proportion of this subpopulation within MLR responders also correlated with emergence of activated, cytotoxic CD8+ T cells. B cells of similar profile were quite infrequent in unstimulated blood from healthy individuals but readily identifiable in disaggregated human splenocytes and increased in both cases upon allostimulation. Further characterization of the emergence and function of this subpopulation could potentially contribute to identification of novel biomarkers and targeted therapeutics relevant to curbing transplant-related pathology.

Correlation analysis of patients with diabetic foot ulcers treated with tibial cortex transverse transport surgery and platelet-to-lymphocyte ratio and monocyte-to-neutrophil ratio.

Diabetic foot ulcers (DFUs) represent one of the most severe late-stage complications of diabetes. Tibial cortex transverse transport (TTT) surgery stands as the prevailing method for addressing DFUs. This surgical intervention holds the promise of expediting DFU wound healing and diminishing the rate of amputations, with the mitigation of inflammatory responses playing a pivotal role. In this study, we aim to explore the correlation between inflammation and TTT surgery, with the overarching goal of facilitating swift prognostic assessments in clinical practice. The correlation between the severity of DFUs and clinical test results remains ambiguous. A clinical prediction model was devised to explore the connection between DFU severity and the efficacy of TTT surgery, utilizing straightforward and efficient clinical indicators. Clinical data and examination results were gathered by tracking hospitalized DFU patients who underwent TTT surgery at the First Affiliated Hospital of Guangxi Medical University (Nanning, China). Indicators associated with DFU severity and wound healing time post-surgery were identified through logistic regression and least absolute shrinkage and selection operator (LASSO) regression analyses. Subsequently, a clinical prediction model was constructed. Finally, the intersection of these 2 sets of indicators revealed factors correlated with wound severity and post-operative healing duration. Our study was comprised of 202 patients who were categorized into 2 groups based on Wagner's grading classifications. Utilizing Student's t-tests, LASSO regression and logistic regression analyses, we identified 3 factors indicative of DFU severity: platelet-to-lymphocyte ratio (PLR), mixed lymphocyte reaction (MLR) and hemoglobin (HGB). Univariate COX regression analysis revealed 12 factors such as: white blood cells (WBC), neutrophils (NEUT), monocytes (MO), PLR, MLR, neutrophil-to-lymphocyte ratio (NLR), erythrocyte sedimentation rate (ESR), age, lymphocytes (LY), monocyte-to-neutrophil ratio (MNR), uric acid (UA), and albumin (ALB) associated with the postoperative healing duration. Ultimately, we identified 2 factors, PLR and MNR, at the intersection of these 2 datasets. Platelet-to-lymphocyte ratio and MNR were identified as factors associated with both the severity of DFUs and the prognosis following TTT surgery.

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.

CS12192: A novel selective and potent JAK3 inhibitor mitigates acute graft-versus-host disease in bone marrow transplantation

BackgroundDespite the significant role of JAK3 in various autoimmune diseases, including graft-versus-host disease (GVHD), there has been a lack of potent and selective JAK3 inhibitors specifically studied for GVHD. In our preclinical investigations, we evaluated a novel JAK3 inhibitor called CS12192, which is already undergoing clinical investigation in autoimmune diseases. MethodsWe evaluated the efficacy of CS12192 in GVHD through mixed lymphocyte reaction (MLR) in both mouse and human cells, as well as allogeneic bone marrow transplantation (BMT) in a murine model. ResultsCS12192, starting at a concentration of 0.5 μM, dose-dependently reduced the intracellular positivity for cytokines TNF-α and IFN-γ in CD4+ T cells (p < 0.05 to p < 0.0001) and CD8+ T cells (p < 0.01 to p < 0.0001) during mouse allogeneic MLR assays. This effect was observed for both single and double positivity of the cytokines. Moreover, In MLR assays with three different human donors, CS12192 also demonstrated a dose-dependent reduction in the proportion of IFN-γ positive CD4+ T cells (p < 0.0001) and CD8+ T cells (p < 0.01 to p < 0.0001). Additionally, it suppressed T cell proliferation in the mouse MLR (p < 0.05 to p < 0.0001), but this effect was observed in only one human donor (p < 0.001 to p < 0.0001). Furthermore, the administration of CS12192 at 40 and 80 mg/kg BID significantly improved the survival rate in the BMT model, resulting in cumulative 62-day survival rates of 88.89% (p < 0.01) and 100% (p < 0.001), respectively, compared with prednisolone (p < 0.05). ConclusionsCS12192 is a novel, potent and selective JAK3 inhibitor demonstrating great potential to mitigate acute GVHD.

Effect of TT-816, a novel immune response modifier targeting cannabinoid CB2 receptor, on antitumor immunity and cancer growth.

e14595 Background: The endocannabinoid system is widely expressed in the human body, including the innate and adaptive immune system, where endocannabinoids, Δ9-tetrahydrocannabinol and synthetic ligands regulate immune suppression. The effects of endocannabinoids on immune regulation are primarily mediated by G-protein coupled cannabinoid CB2 receptors (CB2R) via several mechanisms, including immune cell development, migration, proliferation and T and NK cell effector functions. The upregulated expression of CB2R and elevated levels of endocannabinoids have been observed in a variety of tumor microenvironments and are associated with the aggressiveness of cancer. Methods: NK cell function was determined by co-culturing TT-816 pretreated NK cells with K562 cancer cells for 24 hours. The mixed lymphocyte reaction assay was conducted by co-culturing human CD4+ T cells with monocyte-derived dendritic cells. Cell viability was measured by FACS and IFN-g by MSD. The in vivo efficacy of TT-816 was determined in the murine syngeneic B16F10 melanoma and MC38 colorectal cancer models. Tumor-bearing C57BL/6 mice were randomized 6 days after inoculation and treated with vehicle (0.5% CMC, QD) or with TT-816 at 1, 4 or 10 mg/kg (QD) orally, or anti-PD1 (RMP1 14) at 5 mg/kg (Q3D), intraperitoneally. Tumor size and body weight were determined every 2 to 3 days. Data represent mean±SEM. Results: TT-816 concentration-dependently enhanced the functions of NK cells, dendritic cells and T cells. It increased NK cell killing of the human cancer cells and IFN-g production, significantly stimulated the expression of CD86, HLA-DR, IL-12 and TNF-a in monocyte-derived dendritic cells, and enhanced CD4+ T cell proliferation and IFN-g production in a mixed lymphocyte reaction assay. In B16F10 melanoma and MC38 colorectal cancer models, TT-816 dose-dependently inhibited the tumor growth. Furthermore, combination of TT-816 with anti-PD1 synergistically inhibited both tumor growth. In B16F10 model, treatment with TT 816 (4 mg/kg, QD) alone significantly inhibited the tumor growth by 45%. By contrast, anti-PD1 had little effect (TGI 5%). However, the combination of TT-816 with anti-PD1 resulted in a TGI of 72%. In the MC38 model, treatment with TT-816 (4 mg/kg, QD) and anti-PD1 alone, achieved TGI of 50% and 36%, respectively. When combined, TGI was increased to 88%. Conclusions: TT-816 is a novel, oral small molecule immune response modifier that selectively blocks CB2R on immune cells and cancer cells. Preclinical data have demonstrated that it stimulates antitumor immune response and inhibits cancer growth in both hot and cold tumor models. TT-816 is currently undergoing phase 1 clinical trials for the treatment of a broad range of solid tumors.

Lung Transplant Immunomodulation with Genetically Engineered Mesenchymal Stromal Cells-Therapeutic Window for Interleukin-10.

Lung transplantation results are compromised by ischemia-reperfusion injury and alloimmune responses. Ex vivo lung perfusion (EVLP) is used to assess marginal donor lungs before transplantation but is also an excellent platform to apply novel therapeutics. We investigated donor lung immunomodulation using genetically engineered mesenchymal stromal cells with augmented production of human anti-inflammatory hIL-10 (MSCsIL-10). Pig lungs were placed on EVLP for 6 h and randomized to control (n = 7), intravascular delivery of 20 × 106 (n = 5, low dose) or 40 × 106 human MSCs IL-10 (n = 6, high dose). Subsequently, single-lung transplantation was performed, and recipient pigs were monitored for 3 days. hIL-10 secretion was measured during EVLP and after transplantation, and immunological effects were assessed by cytokine profile, T and myeloid cell characterization and mixed lymphocyte reaction. MSCIL-10 therapy rapidly increased hIL-10 during EVLP and resulted in transient hIL-10 elevation after lung transplantation. MSCIL-10 delivery did not affect lung function but was associated with dose-related immunomodulatory effects, with the low dose resulting in a beneficial decrease in apoptosis and lower macrophage activation, but the high MSCIL-10 dose resulting in inflammation and cytotoxic CD8+ T cell activation. MSCIL-10 therapy during EVLP results in a rapid and transient perioperative hIL-10 increase and has a therapeutic window for its immunomodulatory effects.

Extracellular release of damaged mitochondria induced by prehematopoietic stem cell transplant conditioning exacerbates GVHD

AbstractDespite therapeutic advancements, graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HSCT). In current models of GVHD, tissue injury induced by cytotoxic conditioning regimens, along with translocation of microbes expressing pathogen-associated molecular patterns, result in activation of host antigen-presenting cells (APCs) to stimulate alloreactive donor T lymphocytes. Recent studies have demonstrated that in many pathologic states, tissue injury results in the release of mitochondria from the cytoplasm to the extracellular space. We hypothesized that extracellular mitochondria, which are related to archaebacteria, could also trigger GVHD by stimulation of host APCs. We found that clinically relevant doses of radiation or busulfan induced extracellular release of mitochondria by various cell types, including cultured intestinal epithelial cells. Conditioning-mediated mitochondrial release was associated with mitochondrial damage and impaired quality control but did not affect the viability of the cells. Extracellular mitochondria directly stimulated host APCs to express higher levels of major histocompatibility complex II (MHC-II), costimulatory CD86, and proinflammatory cytokines, resulting in increased donor T-cell activation, and proliferation in mixed lymphocyte reactions. Analyses of plasma from both experimental mice and a cohort of children undergoing HSCT demonstrated that conditioning induced extracellular mitochondrial release in vivo. In mice undergoing MHC-mismatched HSCT, administration of purified syngeneic extracellular mitochondria increased host APC activation and exacerbated GVHD. Our data suggest that pre-HSCT conditioning results in extracellular release of damaged mitochondria, which increase alloreactivity and exacerbate GVHD. Therefore, decreasing the extracellular release of damaged mitochondria after conditioning could serve as a novel strategy for GVHD prevention.

Stealth transgenes enable CAR-T cells to evade host immune responses

BackgroundAdoptive cell therapy, such as chimeric antigen receptor (CAR)-T cell therapy, has improved patient outcomes for hematological malignancies. Currently, four of the six FDA-approved CAR-T cell products use the FMC63-based...

Calcium chloride declotted human platelet lysate promotes the expansion of mesenchymal stromal cells and allows manufacturing of immunomodulatory active extracellular vesicle products

BackgroundMesenchymal stromal cells (MSCs) exert immunomodulatory effects, primarily through released extracellular vesicles (EVs). For the clinical-grade manufacturing of MSC-EV products culture conditions need to support MSC expansion and allow the manufacturing of potent MSC-EV products. Traditionally, MSCs are expanded in fetal bovine serum-supplemented media. However, according to good manufacturing practice (GMP) guidelines the use of animal sera should be avoided. To this end, human platelet lysate (hPL) has been qualified as an animal serum replacement. Although hPL outcompetes animal sera in promoting MSC expansion, hPL typically contains components of the coagulation system that need to be inhibited or removed to avoid coagulation reactions in the cell culture. Commonly, heparin is utilized as an anticoagulant; however, higher concentrations of heparin can negatively impact MSC viability, and conventional concentrations alone do not sufficiently prevent clot formation in prepared media. MethodsTo circumvent unwanted coagulation processes, this study compared various clotting prevention strategies, including different anticoagulants and calcium chloride (CaCl2)-mediated declotting methods, which in combination with heparin addition was found effective. We evaluated the influence of the differently treated hPLs on the proliferation and phenotype of primary bone marrow-derived MSCs and identified the CaCl2-mediated declotting method as the most effective option. To determine whether CaCl2 declotted hPL allows the manufacturing of immunomodulatory MSC-EV products, EVs were prepared from conditioned media of MSCs expanded with either conventional or CaCl2 declotted hPL. In addition to metric analyses, the immunomodulatory potential of resulting MSC-EV products was assessed in a recently established multi-donor mixed lymphocyte reaction assay. Results and ConclusionsOur findings conclusively show that CaCl2-declotted hPLs support the production of immunomodulatory-active MSC-EV products.

Restore Veto Property in Low Dose Aspirin/ASA Treated Preeclampsia Placenta Mesenchymal Stem Cells: Insights Into ASA-mediated Clinical Response*

Preeclampsia (PE) is a pregnancy complication characterized by elevated blood pressure, proteinuria, and other laboratory abnormalities. PE affects 2–8% of pregnancies globally and can lead to preterm birth and other complications for the mother and fetus. Successful pregnancy depends on the ability of the mother’s immune system to tolerate the allogeneic fetus. However, in PE, this immune tolerance is exacerbated by inflammation. Transforming growth factor β (TGF-β) is important to retain an immune balance in healthy pregnancy. In PE, TGF-β level is reduced, with an imbalance of the T-cell subset pool to favor inflammation. Omics studies by our group reported an increase in TGF-β signaling when PE-derived placenta mesenchymal stem cells (P-MSCs) were treated with low dose aspirin (ASA). This correlated with increased cycling quiescence and epigenetic changes, resembling healthy P-MSCs. This study tested the hypothesis that ASA could restore the veto property of P-MSCs to mitigate inflammation. ASA (10 mM) treated P-MSCs from PE and healthy placentas increased TGFβ1 and its receptor. The ASA treated MSCs, when added as third-party cells to a one-way mixed lymphocyte reaction, suppressed T-cell proliferation. Prediction studies with omics data indicated that ASA-mediated TGFβ signaling could explain ASA-induced blunting of cell apoptosis. Together, the findings support ASA-mediated expression of TGFβ1 and its receptor on P-MSCs from PE to restore the ability to be licensed as immune suppressor to mitigate PE inflammation. The findings provide new insight into the benefit of ASA treatment for PE.