Mixed Lymphocyte Reaction Research Articles

Cyclosporine and Voclosporin Resistant Immune Effector Cells to Improve Outcomes after Stem Cell Transplantation

Introduction: Relapse of a patient's underlying malignancy, and complications related to viral reactivation are prominent causes of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Immune effector cells (IEC) are increasingly deployed to prevent and treat these complications. These include anti-CD19 CAR-T-cells used for post-HCT B-ALL relapse, as well as viral specific T cells (VSTs) targeting EBV, CMV, and others. However, the efficacy of these IECs early after HCT may be compromised by the inhibitory effects of immunosuppressive agents used to prevent graft-versus host-disease (GVHD). A commonly employed immunosuppressant used for GVHD prophylaxis is the calcineurin inhibitor (CNI) cyclosporine (CsA). A structural analog, voclosporin (VCS), is now FDA approved for lupus nephritis and in trials to prevent rejection after solid organ transplantation. Given its favorable pharmacodynamic and toxicity profile versus CsA and tacrolimus, VCS is poised to become an important option for HCT patients. Both CsA and VCS interact with calcineurin only in the presence of their immunophilin binding partner - cyclophilin A ( PPIA). Tacrolimus and sirolimus interact with a distinct immunophilin, FKBP12. This creates the opportunity to delete a single immunophilin (eg. PPIA) to create IECs resistant to one class of CNI (CsA/VCS) but sensitive to other immunosuppressive agents, an important safety feature. Here we developed CsA/VCS-resistant IECs by CRISPR/Cas9 knockout (KO) of PPIA. These cells exhibited superior proliferation and preserved effector function in the presence of CsA and VCS. Methods: We developed IECs that are simultaneously resistant to both CsA and VCS via CRISPR-mediated KO of PPIA. Proliferation, cytotoxicity, and cytokine production of PPIA KO conventional T cells (Tcon), CMV (pp65)-specific VSTs, and CD19 CAR-T cells were tested against mock-edited (MOCK) cells in functional in vitro assays with escalating doses of CsA and VCS. Results: PPIA KO Tcon demonstrated high editing efficiency (88-92%), high viability, and robust expansion in culture. Indeed, PPIA KO efficiency was maintained over several weeks in culture, suggestive of no survival disadvantage of cells bearing this deletion. Importantly, when PPIA KO cells were used as Responders in a mixed lymphocyte reaction in the presence of CsA, the edited cells demonstrated significant resistance to drug-mediated suppression of CD4+ and CD8+ T cell proliferation across the dose-response curve (p < 0.01; Fig. 1). Similar results were observed with VCS, with PPIA KO cells significantly resistant to VCS-mediated suppression across otherwise highly suppressive doses (p<0.0001; Fig. 1). Having demonstrated that edited Tcon were resistant to CsA and VCS, we next sought to make PPIA KO IECs. CRISPR-edited VSTs and CD19 CAR-T cells showed similar editing efficiencies to Tcon. Consistent with prior results, CMV-specific PPIA KO responder VSTs were significantly resistant to suppression of proliferation by CsA vs MOCK cells at all doses tested (p=0.0004, not shown). Proliferation in the presence of drug was then assessed in KO CAR-T cells using the CD19+ NALM6 line as stimulators. Division of PPIA KO responder CD19 CAR-T cells was almost completely unaffected by CsA and VCS across escalating doses ( Fig. 2). For example, at an effector-to-target (E:T) ratio of 1:0.1 and a CsA dose of 50 ng/mL, there was 40.6 ± 2.3 % suppression of division in MOCK CD8+ CAR-T cells vs. -0.2 ± 0.4 % in PPIA KO CD8+ CAR-T cells (p<0.0001). Similar findings were observed with CD4+ responder CAR-T cells (p<0.0001, not shown). PPIA KO CAR-T cells also demonstrated intact cytotoxic function with no significant differences in % killing between the MOCK and PPIA KO groups at 3 different E:T ratios. For example, at a 1:1 E:T ratio, there was 59 ± 15% killing vs. 55 ± 12% killing in MOCK vs PPIA KO groups, respectively (not shown). Conclusion: KO of PPIA is an effective strategy for engineering resistance to CsA and VCS incellular therapies. Our results demonstrate that PPIA KO conventional T cells, VSTs, and CAR-T cells were markedly resistant to the otherwise suppressive effects on T cell proliferation that are hallmarks of both CsA and VCS. Engineering CNI-resistance into IECs will enhance the ability to deploy these cells early after transplant to prevent or treat both malignant relapse and viral reactivation.

Characteristics of Alloreactive T-Cell Clones That Predict Early Development of Acute GvHD Following Allogeneic HCT

Acute Graft-versus-Host Disease (GvHD) is mediated by T-cells recognizing alloantigens. Despite advances in prophylactic regimens that broadly deplete T-cells, incidence of acute GvHD remains high, suggesting that there is a need to further characterize alloreactive T-cells specifically. Using mixed-lymphocytes reactions (MLR) and high-throughput T cell receptor (TCR) sequencing of third complementarity-determining region (CDR3) of the TCRβ chain, we identified and characterized alloreactive T-cell clones in donor-recipient pairs of allogeneic hematopoietic cell transplantation. We sequenced and identified a total of 34,903 unique donor-derived CDR3β functional sequences (FS) from 20 donor-recipient in vitro MLRs using peripheral blood mononuclear cells (PBMC) collected from donors and patients pre-transplant. We also sequenced the respective unstimulated donor PBMC samples (Donor Type: 13 HLA-Matched/7 Mismatched, GvHD prophylaxis: 11 Tacrolimus (Tac)-Methotrexate/8 Tac-MMF/1 Sirolimus, PTCy: 12 Yes/8 No). On average, our identified alloreactive FS make up a small fraction of the sampled donor repertoire in comparison to nonalloreactive FS. (Mean fraction: 0.004 vs. 0.996, p < 0.0001) Surprisingly, we found that alloreactive FS have significantly shorter CDR3β length compared to nonalloreactive FS (mean nt 43.50 vs. 44.40, p < 10E-15). We also found that alloreactive FS which can be located within the unstimulated donor sample (n= 4522) are shorter in comparison to all derived alloreactive FS (mean nt 42.43 vs. 43.50, p < 10E-15) and to nonalloreactive FS (mean nt 42.43 vs. 44.40, p < 10E-15) (Figure 1A). Publicly shared FS have, on average, shorter CDR3β length and are overrepresented among FS associated with autoreactivity and cross-reactivity (Khosravi-Maharlooei et al., J Clin Invest, 2019). Thus, we hypothesized that alloreactive FS may be also publicly shared. We derived a list of shared FS from our donor samples, where if a FS is found in more than one donor, it is considered shared. Alloreactive clones were significantly more likely to be shared compared to nonalloreactive (OR 1.94; CI 95%: 1.88 to 2.01). In addition, using a publicly available set of cross-reactive FS (https://vdjdb.cdr3.net/search), we found that alloreactive sequences are also highly cross-reactive compared to nonalloreactive sequences (OR 6.6; CI 95%: 5.18 to 8.42). FS with greater levels of sharedness appear more frequently within a repertoire (Madi et al., Genome Res, 2014). As alloreactive sequences are highly shared, we hypothesized that donor repertoires with greater proportion of shared and alloreactive FS will increase the odds of developing clinically significant GvHD (Grade 2-4) in the recipient. Indeed, within donor repertoires, we found a higher fraction of alloreactive sequences amongst the highly expressed FS (The top 50% of FS in repertoire frequency) which correlated with development of GvHD in recipients post-transplant (mean fraction: GvHD+ group 0.013 vs GvHD- group 0.004, p < 10E-7). Due to the higher portion of shared and alloreactive FS in the top half of the donor T-cell repertoire, we used R50, an index that estimates the diversity of the top 50% of a donor repertoire, to predict the development of GvHD. Utilizing an initial validation cohort of 12 donors, we found that donors whose corresponding recipients developed GvHD within the first 3 months post-transplant had significantly lower R50 compared to those that did not develop GvHD (R50 of 0.096 vs 0.345 p < 0.0001). We also conducted a time-to-event analysis regarding GvHD development in the first 90 days post-transplant. Using the average R50 of the validation cohort as the cutoff for low and high R50 (Average R50: 0.20), we found, in an expanded bi-institutional cohort (n=57), that those with lower R50 values had a significantly greater risk of GvHD compared to those with higher R50 (Figure 1B). In conclusion, our data suggests that functional alloreactive T lymphocytes are characterized by shorter TCRβ CDR3 length. These FS are highly shared and cross-reactive, suggesting a common mechanism underlying the selection of alloreactive and autoreactive clones. From there, we were able to develop and validate a diversity metric that predicts GvHD development pre-transplant based on the donor T-cell repertoire. These findings have implications for donor selection and need to be examined prospectively.

Developing “Off-the-Shelf” CLL1 CAR-DNT Therapeutics for the R/R Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is one of the most common and highly heterogeneous hematological malignancy. The prognosis is poor especially for the elderly patients. For Relapsed/Refractory(R/R) AML patients, the one-year overall survival rate is merely 20%. CAR-T cells have shown tremendous therapeutic benefits for ALL, DLBCL and MM. One of the limitations of current CAR-T therapies is the use of autologous T cells. This can be limiting due to the cost, complicated manufacturing process, rapid disease progression, and manufacturing failures etc. Therefore, there is a need to develop the ready-to-use allogeneic cell product. Double negative T (DNT) cells are the ideal candidate for AML cell therapy as they naturally kill AML cells, and have been successfully administered to AML patients with therapeutic benefit without causing GvHD. Relying on their own natural immune activated receptors, DNT cells could lyse a wide range of tumors independent of TAA expression, which have more advantages compared to the conventional CAR-T cells. In our study, we developed the allogeneic product of CAR-DNT cells targeting C-type lectin-like molecule-1 (CLL1), which is a promising target due to its over-expression in primary AML cells and leukemia stem cells, while absence on hematopoietic stem/progenitor cells. Methods: We developed a humanized single chain variable fragment (scFv) with specific recognition of human CLL1 antigen by screening from 62 murine-derived CLL1 antibodies. Then, we designed a second-generation CLL1 CAR structure containing 4-1BB-CD3ζ and transduced it into the DNT cells by lentivirus. To clarify the GvH and HvG reactions of DNT cells, we performed the mixed lymphocyte reaction experiments in vitro and compared DNT cells with TCR and B2M double knockout T(DKO) cells and TCR knockout T(TKO) cells. We evaluated the anti-tumor function of CLL1 CAR-DNT cells through killing experiments by different effector-to-target ratios and multiple stimulations with AML cell lines (THP1 and HL60). Furthermore, we speculated that the CAR structure plays a critical role in both tumoricidal function and persistence of CAR-DNT cells. We designed nearly 20 enhanced CAR structures based on the signaling pathway of DNT cells. These newly designed structures have been tested in vitro and will be confirmed in vivo. Results: The humanized anti-CLL1 scFv candidate H1-3Q2 demonstrated high specificity toward human CLL1 antigen at the Membrane Protein Array. H1-3Q2 based CAR-T cells showed good tumor suppression in vivo. However, due to the heterogenous expression of CLL1 in AML, CLL1 CAR-T cells may not be able to eliminate AML cancer cells completely. While DNT cells could naturally lyse tumor cells through the receptors like NKG2D and DNAM-1, providing them with lots of advantages. Moreover, DNT cells have the potential to be used as allogenic cell therapy product. Co-culturing allogenic PBMCs or umbilical cord blood with DNT cells for up to 72 hours resulted in little or no killing reaction, indicated a low risk of GvHD. Conversely, the PBMCs from three different donors were used to attack allogenic DNT cells or DKO/TKO T cells in vitro. The killing effect on DKO/TKO T cells gradually increased over time, while no effect on DNT cells was observed even after 192 hours. This suggested that DNT cells are not easily cleared by host immune cells. DNT cells could efficiently lyse various hematological and solid tumor cell lines, particularly showing high sensitivity towards AML cells. The introduction of the CAR moiety further enhanced the specificity of DNT cells. The in vitro killing results demonstrated that CLL1 CAR-DNT cells exhibited stronger and faster AML resistance compared with CAR-T cells, and even eliminated tumor cells that have lost TAA. However, CLL1 CAR-DNT cells showed limited proliferation in the multiple-stimulation assays. To address this, we designed enhanced CAR structures. Screening by multiple-challenge assays, the fifth-generation CAR exhibited outstanding proliferation and superior tumor lysis ability in vitro. Conclusions: Our findings demonstrate that CAR-DNT product is an ideal candidate for allogeneic adoptive immunotherapy and exhibits superior tumoricidal function. This study underscores the potential of the “off-the-shelf” CLL1 CAR-DNT cell product for treating R/R AML.

Coordinated Immune Cell Networks in the Bone Marrow Microenvironment Define the Graft Versus Leukemia Response with Adoptive Cellular Therapy

Donor lymphocyte infusion (DLI) is an established therapy for relapsed acute myeloid leukemia (AML) after hematopoietic stem cell transplant (HSCT), but response rates are poor (~20%). Interactions between leukemia and immune cells within the leukemia microenvironment may determine responsiveness to adoptive cellular immunotherapies. We hypothesized that systematic characterization of the leukemic marrow microenvironment over treatment course with DLI would define leukemia-immune cell interactions critical to response and hence provide mechanistic understanding of the graft-versus-leukemia (GvL) effect. In particular, we anticipated that use of multi-modal single-cell sequencing across patients and time points before and after therapy accounting for temporal dependencies would facilitate disentangling of the complex microenvironment of leukemic marrow. To infer cell-cell interactions in a temporally resolved fashion from single cell RNA-sequencing (scRNA-seq) data, we developed DIISCO, a Bayesian model using a Gaussian process regression network. In total, we profiled 30 bone marrow aspirates (14 pre- and 16 post-DLI) from 9 patients (5 responder [R], 4 nonresponder [NR]) with post-HSCT relapsed AML treated with DLI by scRNA-seq, single cell TCR sequencing (scTCR-seq) and surface protein characterization (scCITE-seq). We obtained 51,371 high-quality transcriptomes from the marrow samples along with 43,918 cells from the DLI products of these patients (4 R, 3 NR) for a total of 95,289 cells. Cells were clustered with Phenograph resulting in 57 clusters, including 2 CD4+ T cell, 6 CD8+ T cell (C0, C5, C21, C25, C26, C40), 3 NK, 10 B cell, 3 AML leukemia, and several myelo-erythroid clusters. DIISCO revealed a cascading response post-DLI that centered around CD8 C0, CD8 C5, AML, and a B cell cluster in R, not observed in NR. Furthermore, C0 appeared to expand after DLI only in R. We observed a strong negative interaction from CD8 C0 to AML after DLI, suggesting an anti-leukemia immune response, supporting the notion that immune cell populations formed a coordinated interactome associated with effective GvL. Evaluation of inferred interacting pairs using DIISCO and with the tool CellPhoneDB pointed toward an activation circuit in R between CD226 on CD8 C0 and PVR/NECTIN2 on AML. Conversely, inhibitory interactions between the exhaustion marker TIGIT on CD8 C0 and AML were observed in NR. Because DIISCO pinpointed CD8 C0 as a central hub for response, we characterized the identity of this cluster and its post-DLI dynamics. scTCR-seq revealed that this cluster, but not other T cell clusters, underwent marked clonal expansion after DLI ( Figure 1A). scRNA-seq revealed high expression of activation/effector genes ( GZMB, IFNG, CX3CR1, PRF1) compared to other T cells. The clonally expanded cells of C0 also highly expressed ZNF683, a transcription factor associated with T cell cytotoxicity. scCITE-seq demonstrated expression of CD45RA and CD57 but not CD28, consistent with an effector memory re-expressing RA (TEMRA) phenotype. We validated post-DLI expansion of CD8 TEMRA cells (CD3+, CD8+, CD62L-, CD45RA+) in AML R by multi-parameter flow cytometry in an independent cohort of 53 AML patients (29 R, 24 NR) with post-HSCT relapse treated with DLI at our institution (fold change 2.56 pre to post for R, p<0.001; 1.3 pre to post for NR, p=0.474). Moreover, to examine whether CD8+ TEMRA cells were specifically activated after encounter with leukemia, we devised an in vitromodified mixed lymphocyte reaction. For 6 AML R, post-DLI donor-derived CD8+ TEMRA cells that were cultured with pre-DLI recipient leukemia cells consistently demonstrated greater CD137 (p=0.004) and IFN-γ secretion compared to co-culture of the leukemia cells with other T cell subsets (p=0.015) ( Figure 1B). CD8+ TEMRA cells cultured in the absence of leukemia cells did not show elevated expression of either CD137 or IFN-γ, indicating antigen-specific functional activation of CD8+ TEMRAs but not other cell types after encounter with autologous leukemia. Our results reveal clonal expansion and tumor-specific activation of CD8+ TEMRA cells, supporting an antigen specific functional role of these cells in mediating GvL in AML DLI responders. Further evaluation of this CD8 TEMRA population with the ability to recognize leukemia cells may translate to strategies to optimize adoptive cellular therapy or predict responsiveness to DLI.

XFab-α4-1BB/CD40L fusion protein activates dendritic cells, improves expansion of antigen-specific T cells, and exhibits antitumour efficacy in multiple solid tumour models.

Additional immunotherapies are still warranted for non-responders to checkpoint inhibitors with refractory or relapsing cancers, especially for patients with "cold" tumours lacking significant immune infiltration at treatment onset. We developed XFab-α4-1BB/CD40L, a bispecific antibody targeting 4-1BB and CD40 for dendritic cell activation and priming of tumour-reactive T cells to inhibit tumours. XFab-α4-1BB/CD40L was developed by engineering an anti-4-1BB Fab arm into a CD40L trimer based on XFab® platform. Characterisation of the bispecific antibody was performed by cell-based reporter assays, maturation of dendritic cell assays, and mixed lymphocyte reactions. The abilities of antigen-specific T-cell expansion and antitumour efficacy were assessed in syngeneic mouse tumour models. Toxicological and pharmacodynamic profiles were investigated in non-human primates. XFab-α4-1BB/CD40L demonstrated independent CD40 agonistic activity and conditional 4-1BB activity mediated by CD40 crosslinking, leading to dendritic cell maturation and T-cell proliferation in vitro. We confirmed the expansion of antigen-specific T cells in the vaccination model and potent tumour regression induced by the bispecific antibody alone or in combination with gemcitabine in vivo, concomitant with improved tumour-reactive T-cell infiltration. XFab-α4-1BB/CD40L showed no signs of liver toxicity at doses up to 51mg/kg in a repeated-dose regimen in non-human primates. XFab-α4-1BB/CD40L is capable of enhancing antitumour immunity by modulating dendritic cell and T-cell functions via targeting 4-1BB agonism to areas of CD40 expression. The focused, potent, and safe immune response induced by the bispecific antibody supports further clinical investigations for the treatment of solid tumours.

Epstein-Barr virus–encoded EBNA2 downregulates ICOSL by inducing miR-24 in B-cell lymphoma

AbstractHematological malignancies such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B-cell lymphoma (DLBCL) cause significant morbidity in humans. A substantial number of these lymphomas, particularly HL and DLBCLs have poorer prognosis because of their association with Epstein-Barr virus (EBV). Our earlier studies have shown that EBV-encoded nuclear antigen (EBNA2) upregulates programmed cell death ligand 1 in DLBCL and BLs by downregulating microRNA-34a. Here, we investigated whether EBNA2 affects the inducible costimulator (ICOS) ligand (ICOSL), a molecule required for efficient recognition of tumor cells by T cells through the engagement of ICOS on the latter. In virus-infected and EBNA2-transfected B-lymphoma cells, ICOSL expression was reduced. Our investigation of the molecular mechanisms revealed that this was due to an increase in microRNA-24 (miR-24) by EBNA2. By using ICOSL 3′ untranslated region–luciferase reporter system, we validated that ICOSL is an authentic miR-24 target. Transfection of anti–miR-24 molecules in EBNA2-expressing lymphoma cells reconstituted ICOSL expression and increased tumor immunogenicity in mixed lymphocyte reactions. Because miR-24 is known to target c-MYC, an oncoprotein positively regulated by EBNA2, we analyzed its expression in anti–miR-24 transfected lymphoma cells. Indeed, the reduction of miR-24 in EBNA2-expressing DLBCL further elevated c-MYC and increased apoptosis. Consistent with the in vitro data, EBNA2-positive DLBCL biopsies expressed low ICOSL and high miR-24. We suggest that EBV evades host immune responses through EBNA2 by inducing miR-24 to reduce ICOSL expression, and for simultaneous rheostatic maintenance of proproliferative c-MYC levels. Overall, these data identify miR-24 as a potential therapeutically relevant target in EBV-associated lymphomas.

Advances of the novel immunosuppressant brasilicardin A

Brasilicardin A (BraA) is a natural diterpene glycoside isolated from the pathogenic actinomycete Nocardia brasiliensis IFM 0406 with highly potent immunosuppressive activity (IC50=0.057 μg/mL). BraA potently inhibits the uptake of amino acids that are substrates for amino acid transport system L of T cells, which is different from the existing clinical immunosuppressants. BraA is more potent in a mouse mixed lymphocyte reaction and less toxic against various human cell lines compared with the known clinical immunosuppressants, such as cyclosporin A, ascomycin and tacrolimus. Therefore, BraA attracted more attention as a new promising immunosuppressant. However, the development of this promising immunosuppressant as drug for medical use is so far hindered because BraA has the unusual and synthetically challenging skeleton and shows the low-yield production in the natural pathogenic producer. This review introduces the molecular structure of BraA, its activity, mechanism of action, chemical synthesis of BraA analogs, heterologous expression of gene cluster, and an application of combining microbial and chemical synthesis for production of BraA, with the aim to facilitate the efficient production of BraA and its analogs.

Modeling human anti-pig xenoimmune responses in a pig artery tissue grafted humanized mouse model.

Blood vessels that contain endothelial cells (ECs) on the surface are in direct contact with host blood and are the first target of xenograft rejection. Currently, our understanding of human anti-pig vessel immune responses is primarily based on in vitro assays using pig ECs. Therefore, it is necessary to develop an animal model that permits in vivo study of human immunological rejection of pig vessels. Pig artery tissues (PAT) were transplanted into human immune system (HIS) mice or immunodeficient NSG mice (as controls). Intragraft human immune cell infiltration and antibody deposition were quantified using histology and immunohistochemistry. Donor antigen-specific immune responses were quantified using a mixed lymphocyte reaction and a complement-dependent killing assay. Pig CD31+ ECs were detected and increased 2-fold from weeks 3 to 5 in PAT xenografts from immunodeficient NSG mice. However, compared with NSG mice, PAT xenografts in HIS mice had significantly lower numbers of porcine CD31+ ECs and showed a marked reduction from week 3 to week 5. PAT xenograft rejection in HIS mice is associated with intensive infiltration of human immune cells, deposition of human IgM and IgG antibodies, and the formation of a tertiary lymphoid structure. Robust donor pig antigen-specific human T cells and antibody responses were detected in PAT-transplanted HIS mice. We have developed a humanized mouse model to evaluate human anti-pig xenoimmune responses by PAT transplantation in vivo. This model is expected to facilitate the refinement of pig gene-editing strategies (the expression on EC surface) and the testing of local immunosuppressive strategies for clinical pig organ xenotransplantation.

Activated protein C modulates T-cell metabolism and epigenetic FOXP3 induction via α-ketoglutarate.

A direct regulation of adaptive immunity by the coagulation protease activated protein C (aPC) has recently been established. Preincubation of T cells with aPC for 1 hour before transplantation increases FOXP3+ regulatory T cells (Tregs) and reduces acute graft-versus-host disease (aGVHD) in mice, but the underlying mechanism remains unknown. Because cellular metabolism modulates epigenetic gene regulation and plasticity in T cells, we hypothesized that aPC promotes FOXP3+ expression by altering T-cell metabolism. To this end, T-cell differentiation was assessed invitro using mixed lymphocyte reaction or plate-bound α-CD3/CD28 stimulation, and exvivo using T cells isolated from mice with aGVHD without and with aPC preincubation, or analyses of mice with high plasma aPC levels. In stimulated CD4+CD25- cells, aPC induces FOXP3 expression while reducing expression of T helper type 1 cell markers. Increased FOXP3 expression is associated with altered epigenetic markers (reduced 5-methylcytosine and H3K27me3) and reduced Foxp3 promoter methylation and activity. These changes are linked to metabolic quiescence, decreased glucose and glutamine uptake, decreased mitochondrial metabolism (reduced tricarboxylic acid metabolites and mitochondrial membrane potential), and decreased intracellular glutamine and α-ketoglutarate levels. In mice with high aPC plasma levels, T-cell subpopulations in the thymus are not altered, reflecting normal T-cell development, whereas FOXP3 expression in splenic T cells is reduced. Glutamine and α-ketoglutarate substitution reverse aPC-mediated FOXP3+ induction and abolish aPC-mediated suppression of allogeneic T-cell stimulation. These findings show that aPC modulates cellular metabolism in T cells, reducing glutamine and α-ketoglutarate levels, which results in altered epigenetic markers, Foxp3 promoter demethylation and induction of FOXP3 expression, thus favoring a Treg-like phenotype.

The Potential Use of THP-1, a Monocytic Leukemia Cell Line, to Predict Immune-Suppressive Potency of Human Bone-Marrow Stromal Cells (BMSCs) In Vitro: A Pilot Study.

Adoptive transfer of cultured BMSCs was shown to be immune-suppressive in various inflammatory settings. Many factors play a role in the process, but no master regulator of BMSC-driven immunomodulation was identified. Consequently, an assay that might predict BMSC product efficacy is still unavailable. Below, we show that BMSC donor variability can be monitored by IL-10 production of monocytes/macrophages using THP-1 cells (immortalized monocytic leukemia cells) co-cultured with BMSCs. Using a mixed lymphocyte reaction (MLR) assay, we also compared the ability of the different donor BMSCs to suppress T-cell proliferation, another measure of their immune-suppressive ability. We found that the BMSCs from a donor that induced the most IL-10 production were also the most efficient in suppressing T-cell proliferation. Transcriptome studies showed that the most potent BMSC batch also had higher expression of several known key immunomodulatory molecules such as hepatocyte growth factor (HGF), PDL1, and numerous members of the PGE2 pathway, including PTGS1 and TLR4. Multiplex ELISA experiments revealed higher expression of HGF and IL6 by the most potent BMSC donor. Based on these findings, we propose that THP-1 cells may be used to assess BMSC immunosuppressive activity as a product characterization assay.

Novel Human Umbilical Di-Chimeric (HUDC) cell therapy for transplantation without life-long immunosuppression.

Cell-based therapies are promising for tolerance induction in bone marrow (BM), solid organs, and vascularized composite allotransplantation (VCA). The toxicity of bone marrow transplantation (BMT) protocols precludes this approach from routine clinical applications. To address this problem, we developed a new therapy of Human Umbilical Di-Chimeric (HUDC) cells for tolerance induction in transplantation. This study established in vitro characterization of the created HUDC cells. We performed sixteen ex vivo polyethylene glycol (PEG)-mediated fusions of human umbilical cord blood (UCB) cells from two unrelated donors. Fusion feasibility was confirmed in vitro by flow cytometry (FC) and confocal microscopy (CM). The HUDC cells' genotype was assessed by lymphocytotoxicity test and short tandem repeat-polymerase chain reaction (STR-PCR) analysis, phenotype by FC, viability by LIVE/DEAD® assay, and apoptosis level by Annexin V staining. We used COMET assay to assess HUDC cells' genotoxicity after the fusion procedure. Clonogenic properties of HUDC cells were evaluated by colony forming unit (CFU) assay. Mixed lymphocyte reaction (MLR) assay assessed immunogenic and tolerogenic properties of HUDC cells. We confirmed the creation of HUDC cells from two unrelated human donors of UCB cells by FC and CM. Human leukocyte antigen (HLA) class I and II typing, and STR-PCR analysis of HUDC cells confirmed the presence of alleles and loci from both unrelated UCB donors (donor chimerism: 49%±8.3%, n=4). FC confirmed the hematopoietic phenotype of HUDC cells. We confirmed high HUDC cells' viability (0.47% of dead cells) and a low apoptosis level of fused HUDC cells (15.9%) compared to positive control of PKH-stained UCB cells (20.4%) before fusion. COMET assay of HUDC cells revealed a lack of DNA damage. CFU assay confirmed clonogenic properties of HUDC cells, and MLR assay revealed a low immunogenicity of HUDC cells. This study confirmed creation of a novel HUDC cell line by ex vivo PEG-mediated fusion of UCB cells from two unrelated donors. The unique concept of creating a HUDC cell line, representing the genotype and phenotype of both, transplant donor and the recipient, introduces a promising approach for tolerance induction in BM, solid organs, and VCA transplantation.

Human Dendritic Cell Enrichment and Their Activation of T Cells.

Dendritic cells (DCs) enable the immune system to mount and modulate precisely targeted responses to various threats across the organism by bridging innate and adaptive immunity. Historically, DCs have been classified as conventional (cDC) and plasmacytoid (pDC). More recently, cDCs were acknowledged as a heterogenous population composed of several subsets. Examining the functional diversity of cDCs in healthy homeostasis and pathology requires a robust experimental pipeline, beginning with an efficient enrichment protocol in preparation for cell sorting. Unfortunately, several commercial DC enrichment kits were developed before the discovery of the more recently described DC populations. Here, we detail two approaches to enrich human blood DCs or certain DC subsets and an in vitro protocol to examine DC stimulation of naïve T cells. The methods employed here overcome many hurdles encountered while enriching human DC subsets. Basic Protocol 1 describes a method that will enrich pDC, Axl Siglec6-DC (AS-DC), cDC1, DC2, DC3, monocytes, and human HLA+ cells by crosslinking unwanted cells to erythrocytes. Basic Protocol 2 describes the enrichment of pDC, AS-DC, cDC1, and DC2 but not DC3 via a highly efficient negative magnetic selection that is valuable in circumstances where DC3 is not required. Finally, Basic Protocol 3 describes a conventional protocol to perform a Mixed leucocyte Reaction (MLR) following the isolation of these DC subsets. These methods detail the advantages and pitfalls when isolating a heterogeneous population of cells. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Human peripheral mononuclear phagocyte enrichment Basic Protocol 2: DC enrichment of pDC, cDC1, AS-DC, and DC2 but not DC3 Basic Protocol 3: Basic mixed lymphocyte reaction protocol with sorted human DC subsets.

Single-cell transcriptomic analysis of renal allograft rejection reveals insights into intragraft TCR clonality.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/β sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8+ T cell clonotypes (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCR-α/β cDNAs from CD8EXP into Jurkat 76 cells (TCR-/-) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection.

CDX-585, a Bispecific Antibody with Dual Targeting of ILT4 and PD-1 Checkpoint Pathways

Immunoglobulin-like transcript 4 (ILT4) is an immunosuppressive molecule predominantly expressed on myeloid cells. Recent studies combining ILT4 suppression with programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) blockade have shown promising signs of activity in immune checkpoint inhibitor refractory patients. We theorized that coupling ILT4 and PD-1/PD-L1 blockade in a bispecific antibody (bsAb) may provide greater immune activating properties than combining the individual mAbs due to enhanced bridging of APCs to T cells. To test this approach, we developed CDX-585, a tetravalent ILT4xPD-1 IgG1-scFv bsAb from novel PD-1 and ILT-4 mAbs. CDX-585 is a potent antagonist of both PD-1 and ILT4. CDX-585 promotes M1 macrophage polarization and enhances pro-inflammatory cytokine secretion in response to lipopolysaccharide or CD40 agonist mAb treatment. In mixed lymphocyte reaction (MLR) assays, CDX-585 is more potent than the combination of parental antibodies. In a humanized NCG mouse SK-MEL-5 tumor model, CDX-585 exhibits greater antitumor activity than the combination of parental mAbs. A pilot study of CDX-585 in cynomolgus macaques confirmed a mAb-like pharmacokinetic profile without noted toxicities. These studies demonstrate that CDX-585 effectively combines ILT4 and the PD-1 blockade into one molecule that is more potent than the combination of the parental antibodies, providing the rationale to advance this bsAb into clinical studies.

Scale-Up of Academic Mesenchymal Stromal Cell Production

Background: Many clinical trials have reported the use of mesenchymal stromal cells (MSCs) following the indication of severe SARS-CoV-2 infection. However, in the COVID19 pandemic context, academic laboratories had to adapt a production process to obtain MSCs in a very short time. Production processes, especially freezing/thawing cycles, or culture medium have impacts on MSC properties. We evaluated the impact of an intermediate cryopreservation state during MSC culture to increase production yields. Methods: Seven Wharton’s jelly (WJ)-MSC batches generated from seven different umbilical cords with only one cryopreservation step and 13 WJ-MSC batches produced with intermediate freezing were formed according to good manufacturing practices. The identity (phenotype and clonogenic capacities), safety (karyotype, telomerase activity, sterility, and donor qualification), and functionality (viability, mixed lymphocyte reaction) were analyzed. Results: No significant differences between MSC production processes were observed, except for the clonogenic capacity, which was decreased, although it always remained above our specifications. Conclusions: Intermediate cryopreservation allows an increase in the production yield and has little impact on the basic characteristics of MSCs.

#3211 INHIBITION OF TRAINED INNATE IMMUNITY BY MYELOID CD40-TRAF6 BLOCKADE PROMOTES ALLOGRAFT TOLERANCE

Abstract Background and Aims Allograft rejection is largely mediated by adaptive immune cells. Although innate immune cells are also involved, their role is less clear. We hypothesize that suppression of innate immune responses, particularly trained innate immunity, can contribute to graft tolerance in transplantation. Trained immunity refers to the long-term epigenetic and metabolic reprogramming of innate immune cells, which potentiates responses to secondary stimuli. Here, we studied the effect of blocking CD40-TRAF6 signaling in myeloid cells on trained immunity induction using in vitro stimulations, ChIP-sequencing and metabolic analyses. We evaluated the effect of myeloid CD40-TRAF6 inhibition on T cell responses in mixed lymphocyte reactions (MLR) and the effect of CD40-TRAF6 inhibitor (TRAF6i) nanobiologic (NB) treatment on allograft rejection in a murine heterotopic heart transplantation model. Method Human peripheral blood mononuclear cells (PBMCs) were stimulated for 24 h with heat-killed Candida albicans (HKCA), a well-described inducer of trained immunity, in presence or absence of TRAF6i, or RPMI medium (control) followed by 5 days of rest. Interleukin-6 (IL-6) and tumor necrosis factor (TNF) production upon restimulation with lipopolysaccharide (LPS) was assessed using ELISA. We performed ChIP-sequencing and metabolic analyses with Seahorse assays at day 6. To investigate the effect of TRAF6i on T cell responses to allogeneic stimuli, we performed 7-day MLR with HKCA- or HKCA+TRAF6i trained monocytes and CellTrace Violet-labeled naïve T cells. Proliferation and FOXP3 expression of naïve T cells was measured by flow cytometry. The effect of TRAF6i-NB on graft survival was assessed by injection of myeloid-directed TRAF6i-NB 0, 2 and 5 days post-transplantation in C57BL/6J mice heterotopically transplanted with BALB/c hearts, that did or did not receive pre-operative CTLA4-Ig treatment. Results TRAF6i treatment inhibited IL-6 and TNF production upon LPS restimulation of HKCA-treated PBMCs in vitro (Fig. 1A). ChIP-sequencing analysis and Seahorse technology respectively revealed that the epigenetic changes (Fig. 1B) and metabolic alterations (Fig. 1C) underlying trained immunity were prevented by TRAF6i. Induction of trained immunity in monocytes reduced differentiation of naïve T cells to FOXP3+CD4+ T cells upon allogeneic stimulation, which was partially reversed by TRAF6i treatment of monocytes (Fig. 1D). Treatment with TRAF6i-NB, combined with CTLA4-Ig, induced >100 days graft survival in 5 out of 6 C57BL/6J mice heterotopically transplanted with BALB/c hearts, while one graft was rejected at 99 days post-transplantation (Fig. 1E). Conclusion Inhibition of TRAF6 prevents trained immunity in monocytes at the functional, epigenetic and metabolic level. We show in vitro that inhibiting trained immunity has the effect of modulating T cell responses to allogeneic stimuli towards FOXP3+CD4+ T cell differentiation. Using a mouse heart transplant model, we show that the combination of TRAF6i and CTLA4-Ig treatment prevents allograft rejection in vivo. This study identifies CD40-TRAF6 signaling as a novel target to inhibit trained immunity and to promote allograft tolerance.

Siplizumab combination therapy with belatacept or abatacept broadly inhibits human T cell alloreactivity in vitro

Combined antigen-specific T cell receptor stimulation and costimulation are needed for complete T cell activation. Belatacept and abatacept are nondepleting fusion proteins blocking CD28/B7 costimulation, whereas siplizumab is a depleting antiCD2 immunoglobulin G1 monoclonal antibody targeting CD2/CD58 costimulation. Herein, the effect of siplizumab combination therapy with abatacept or belatacept on T cell alloreactivity in mixed lymphocyte reactions was investigated. In contrast to monotherapy, the combination of siplizumab with belatacept or abatacept induced near-complete suppression of T cell proliferation and increased the potency of siplizumab-mediated T cell inhibition. Furthermore, dual targeting of CD2 and CD28 costimulation enhanced the selective depletion of memory T cells compared with monotherapy. Although siplizumab monotherapy leads to significant regulatory T cell enrichment, high doses of cytotoxic T-lymphocyte-associated antigen 4 and a human IgG1 Fc fragment in the combination therapy reduced this effect. These results support the clinical evaluation of dual costimulation blockade, combining siplizumab with abatacept or belatacept, for the prophylaxis of organ transplant rejection and improvement of long-term outcomes following transplantation. Ongoing investigative research will elucidate when other forms of siplizumab-based dual costimulatory blockade may be able to induce similarly strong inhibition of T cell activation although still allowing for enrichment of regulatory T cells.

Long-Term Effects of Low-Dose Naltrexone on Immunomodulatory Properties of Human Adipose-Derived Mesenchymal Stem Cells.

Low-dose naltrexone (LDN) is involved in the treatment of inflammatory and immune system diseases and can affect immune cells. Mesenchymal stem cells (MSCs) are known for their immunomodulatory effects and the potential for the treatment of certain types of autoimmune diseases. To investigate the long-term effects of LDN on human adipose-derived mesenchymal stem cells (ASCs) to see how their immunomodulatory properties are affected and also how LDN-treated ASCs interact with other immune cells present in peripheral blood mononuclear cells (PBMCs). After 14 days of treatment, the ability of LDN-treated ASCs to modulate PBMC proliferation in a two-way mixed lymphocyte reaction (MLR) model was assessed using XTT. The relative expression of IDO, PD-L1, COX-2, HGF genes, and the level of IL-6 and TGF-β cytokines were measured in IFN-γ stimulated and unstimulated ASCs (treated and not treated cells) using real-time PCR and ELISA respectively. Unstimulated ASCs treated with 10-8 M Naltrexone (10-8 M NTX) showed higher levels of TGF-β, compared with the controls (P<0.05). Stimulated ASCs treated with 10-6 M NTX showed elevated expression of IDO, PD-L1 genes, and IL-6 level (P<0.05). Our results demonstrated that various LDN concentrations have dissimilar effects on ASCs' immunomodulatory properties. A higher LDN concentration induced an alteration in the immunomodulatory features of ASCs.

A bispecific antibody AP203 targeting PD-L1 and CD137 exerts potent antitumor activity without toxicity

BackgroundBispecific antibody has garnered considerable attention in the recent years due to its impressive preliminary efficacy in hematological malignancies. For solid tumors, however, the main hindrance is the suppressive tumor microenvironment, which effectively impedes the activation of infiltrating T cells. Herein, we designed a bispecific antibody AP203 with high binding affinity to PD-L1 and CD137 and assessed its safety and anti-tumor efficacy, as well as explored the mechanism of action.MethodsThe optimal antibody binders against PD-L1 and CD137 were screened from the OmniMab phagemid library. The binding affinity of the constructed AP203 were evaluated using enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). T-cell stimulatory capacity was assessed using the allogeneic mixed lymphocyte reaction (MLR), antigen-specific recall response, and coculture with PD-L1-expressing cells. In vivo antitumor efficacy was evaluated using two models of tumor-xenografted humanized mice with profiling of tumor infiltrating lymphocytes (TILs). The possible toxicity of AP203 was examined using in vitro cytokine release assay by human PBMCs.ResultsAP203, which simultaneously targeted PD-L1 and costimulatory CD137, elicit superior agonistic effects over parental antibodies alone or in combination in terms of T cell activation, enhanced memory recall responses, and overcoming Treg-mediated immunosuppression (P < 0.05). The agonistic activity of AP203 was further demonstrated PD-L1-dependent by coculturing T cells with PD-L1-expressing cells. In vivo animal studies using immunodeficient or immunocompetent mice both showed a dose-related antitumor efficacy superior to parental antibodies in combination (P < 0.05). Correspondingly, AP203 significantly increased tumor infiltrating CD8 + T cells, while decreased CD4 + T cells, as well as Treg cells (P < 0.05), resulting in a dose-dependent increase in the CD8 + /CD4 + ratio. Moreover, either soluble or immobilized AP203 did not induce the production of inflammatory cytokines by human PBMCs.ConclusionsAP203 exerts potent antitumor activity not only by blocking PD-1/PD-L1 inhibitory signaling, but also by activating CD137 costimulatory signaling in effector T cells that consequently counteracts Treg-mediated immunosuppression. Based on promising preclinical results, AP203 should be a suitable candidate for clinical treatment of solid tumors.

Maternal dendritic cells influence fetal allograft response following murine in-utero hematopoietic stem cell transplantation

BackgroundIntrauterine hematopoietic stem cell transplantation (IUT), potentially curative in congenital haematological disease, is often inhibited by deleterious immune responses to donor cells resulting in subtherapeutic donor cell chimerism (DCC). Microchimerism of maternal immune cells (MMc) trafficked into transplanted recipients across the placenta may directly influence donor-specific alloresponsiveness, limiting DCC. We hypothesized that dendritic cells (DC) among trafficked MMc influence the development of tolerogenic or immunogenic responses towards donor cells, and investigated if maternal DC-depletion reduced recipient alloresponsiveness and enhanced DCC.MethodsUsing transgenic CD11c.DTR (C57BL/6) female mice enabled transient maternal DC-depletion with a single dose of diphtheria toxin (DT). CD11c.DTR females and BALB/c males were cross-mated, producing hybrid pups. IUT was performed at E14 following maternal DT administration 24 h prior. Bone marrow-derived mononuclear cells were transplanted, obtained from semi-allogenic BALB/c (paternal-derived; pIUT), C57BL/6 (maternal-derived; mIUT), or fully allogenic (aIUT) C3H donor mice. Recipient F1 pups were analyzed for DCC, while maternal and IUT-recipient immune cell profile and reactivity were examined via mixed lymphocyte reactivity functional assays. T- and B-cell receptor repertoire diversity in maternal and recipient cells were examined following donor cell exposure.ResultsDCC was highest and MMc was lowest following pIUT. In contrast, aIUT recipients had the lowest DCC and the highest MMc. In groups that were not DC-depleted, maternal cells trafficked post-IUT displayed reduced TCR & BCR clonotype diversity, while clonotype diversity was restored when dams were DC-depleted. Additionally, recipients displayed increased expression of regulatory T-cells and immune-inhibitory proteins, with reduced proinflammatory cytokine and donor-specific antibody production. DC-depletion did not impact initial donor chimerism. Postnatal transplantation without immunosuppression of paternal donor cells did not increase DCC in pIUT recipients; however there were no donor-specific antibody production or immune cell changes.ConclusionsThough maternal DC depletion did not improve DCC, we show for the first time that MMc influences donor-specific alloresponsiveness, possibly by expanding alloreactive clonotypes, and depleting maternal DC promotes and maintains acquired tolerance to donor cells independent of DCC, presenting a novel approach to enhancing donor cell tolerance following IUT. This may have value when planning repeat HSC transplantations to treat haemoglobinopathies.