Mixed Lymphocyte Reaction Response Research Articles

Early allogeneic immune modulation after establishment of donor hematopoietic cell-induced mixed chimerism in a nonhuman primate kidney transplant model.

Mixed lymphohematopoietic chimerism is a proven strategy for achieving operational transplant tolerance, though the underlying immunologic mechanisms are incompletely understood. A post-transplant, non-myeloablative, tomotherapy-based total lymphoid (TLI) irradiation protocol combined with anti-thymocyte globulin and T cell co-stimulatory blockade (belatacept) induction was applied to a 3-5 MHC antigen mismatched rhesus macaque kidney and hematopoietic cell transplant model. Mechanistic investigations of early (60 days post-transplant) allogeneic immune modulation induced by mixed chimerism were conducted. Chimeric animals demonstrated expansion of circulating and graft-infiltrating CD4+CD25+Foxp3+ regulatory T cells (Tregs), as well as increased differentiation of allo-protective CD8+ T cell phenotypes compared to naïve and non-chimeric animals. In vitro mixed lymphocyte reaction (MLR) responses and donor-specific antibody production were suppressed in animals with mixed chimerism. PD-1 upregulation was observed among CD8+ T effector memory (CD28-CD95+) subsets in chimeric hosts only. PD-1 blockade in donor-specific functional assays augmented MLR and cytotoxic responses and was associated with increased intracellular granzyme B and extracellular IFN-γ production. These studies demonstrated that donor immune cell engraftment was associated with early immunomodulation via mechanisms of homeostatic expansion of Tregs and early PD-1 upregulation among CD8+ T effector memory cells. These responses may contribute to TLI-based mixed chimerism-induced allogenic tolerance.

Characterization of CD4+ and CD8+ T cells responses in the mixed lymphocyte reaction by flow cytometry and single cell RNA sequencing.

The Mixed Lymphocyte Reaction (MLR) consists in the allogeneic co-culture of monocytes derived dendritic cells (MoDCs) with T cells from another donor. This in vitro assay is largely used for the assessment of immunotherapy compounds. Nevertheless, the phenotypic changes associated with lymphocyte responsiveness under MLR have never been thoroughly evaluated. Here, we used multiplex cytokine and chemokine assays, multiparametric flow cytometry and single cell RNA sequencing to deeply characterize T cells activation and function in the context of CD4+- and CD8+-specific MLR kinetics. We showed that CD4+ and CD8+ T cells in MLR share common classical markers of response such as polyfunctionality, increased proliferation and CD25 expression but differ in their kinetics and amplitude of activation as well as their patterns of cytokines secretion and immune checkpoints expression. The analysis of immunoreactive Ki-67+CD25+ T cells identified PBK, LRR1 and MYO1G as new potential markers of MLR response. Using cell-cell communication network inference and pathway analysis on single cell RNA sequencing data, we also highlighted key components of the immunological synapse occurring between T cells and the stimulatory MoDCs together with downstream signaling pathways involved in CD4+ and CD8+ T cells activation. These results provide a deep understanding of the kinetics of the MLR assay for CD4+ or CD8+ T cells and may allow to better characterize compounds impacting MLR and eventually identify new strategies for immunotherapy in cancer.

#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.

Gene Expression Profiling and Signaling Pathway Analysis of Tolerant T cells Circulating in Adult Patients after PTCy Haplotransplantation

Abstract The mechanisms for the signaling pathways of T cell-dependent tolerance after haploidentical hematopoietic stem cell transplantation (haplo-HSCT), are incompletely understood. We tested 8 patients (Pt) with leukemia or myelodysplastic syndromes (median age:51-yrs old) following IRB approved consenting. All received Flu+TBI (+/− Cy) pre-conditioning and haplo-HSCT with post-transplant Cytoxan (PTCy) and FK506 as GvHD prophylaxis. The circulating T cells were hyporeactive in mixed lymphocyte reaction (MLR) assay to recipient DC in Pts at 1yr (or 2yrs in 1 Pt) post-haplo-HSCT (~6m post-ISD withdrawal), while these T cells responded vigorously to 3rd party APC. Neither depletion of Tregs, nor blockade of IL10R or PD1 could reverse T-cell hyporeactivity. Low-dose IL2 triggered mild anti-host responses. Cytokine profiling further confirmed the MLR responses. ImmunoSEQ™ revealed the disappearance of alloreactive T-cell clones from peripheral pool with time, even though a few alloreactive T clones remained. Low dose IL2 had minimal impact to reverse the diminishment. Unlike graft T-cell responses against host DC, tolerant T cells in circulation against hDC showed relative downregulation of allograft rejection, T-helper response, co-stimulation, and cell proliferation pathway, that was paired with upregulation of co-inhibition, PD1-PDL1, Ferroptosis and CREB pathways. In summary, hyporeactivity of donor-derived circulating T cells to recipient DC were evident. We found Treg, Tr1 activity to be insignificant in maintaining long-term tolerance. T-cell clonal deletion appears to be the dominant mechanism, along with systemically altered signaling pathways in tolerant T-cells.

Gene Expression Profiling and Signaling Pathway Analysis of Tolerant T Cells Circulating in Adult Patients after Ptcy Haplotransplantation

The mechanisms of T cell-dependent tolerance, especially its signaling pathways after haploidentical hematopoietic stem cell transplantation (haplo-HSCT), are incompletely understood. Both central and peripheral regulation mechanisms are suspected. Subjects and Methods: We tested 8 patients with leukemia (5) or myelodysplastic syndromes (MDS, 3) (age 35-73-years old, median: 51-years old) following IRB approved consenting. All received Flu+TBI (+/- Cy) pre-conditioning and haplo-HSCT with post-transplant Cytoxan (PTCy) and FK506 as GvHD prophylaxis. Chimerism at tolerance studies was donor dominant (97-100%, mean: 99% for both CD33+ myeloid cells and CD3+T cells). Immunosuppression drug (ISD) was withdrawn ~6 months before the time of the in vitro tolerance study described below. We described previously Treg depletion from purified T cells with IL2-immunotoxin (Denileukin Diftitox, Seragen Inc™) to test the involvement of these cells in long-term tolerance. The contribution of Tr1 or anergy was examined by either blocking IL10R or adding low dose IL2, respectively. T-cell exhaustion was tested by blocking the PD1 pathway with anti-PD1 MoAb (OPDIVO, Bristol Myers Squibb™). Purified T cell responses to recipient dendritic cells (DC) were measured in mixed lymphocyte reaction (MLR) assay, and further tested by quantifying cytokine secretion in MLR culture supernatants using Bio-Plex immunoassays (Bio-Rad Lab™). To test for clonal deletion, alloreactive T-cell clones with frequency >0.1% identified in vitro from the graft pre-infusion were tracked by monitoring their T-cell receptor CDR3 sequence serially, in an ImmunoSEQ™ assay (Adaptive Biotech™). Gene expression profiling and signaling pathway analysis on alloreactive or tolerant T-cells were performed by collecting T cells from in vitro MLR reactions, purifying total RNAs from these cells, running RNA-sequencing and analyzing with software of CLC Genomic Workbench®v21, GSEA®v4.1.0 and IPA®. Results: We have previously shown that circulating T cells were hyporeactive to recipient DC in patients at 1yr (or 2yrs in 1 patient) post-haplo-HSCT [6 months, (or 2-5 months in 3 patients) post-ISD withdrawal], while these T cells responded vigorously to 3rd party APC, As positive control, host DC triggered robust 3rd party T-cell response. Neither depletion of Tregs, nor blockade of IL10R or PD1 could reverse T-cell hypo-reactivity. Low-dose IL2 triggered mild anti-host proliferation responses. Similar scenarios were detected in graft T cells against self-DC, (negative control in autologous set up), while vigorous responses were shown in the graft T cells against host-DC pre-haplo-HSCT. Cytokine profiling further confirmed the MLR responses. ImmunoSEQ™ revealed disappearance of alloreactive T-cell clones from peripheral blood with time. Low dose IL2 had minimal impact to reverse hyporeactivity. While most alloreactive T clones were undetectable, a few remained in the peripheral pool in tolerant patients tested (Nee, T et al, Transplantation and Cellular Therapy, 2022;28(3):S44-S45). Unlike graft T-cell responses against host DC, tolerant T-cells in circulation (including remaining alloreactive T cells) against hDC showed distinct patterns of gene expressions, with relative downregulation of allograft rejection (GSEA-Hallmark) (Fig 1A, B, C) and T-helper response, co-stimulation, cell proliferation pathway, that was paired with upregulation of co-inhibition, PD1-PDL1, Ferroptosis, PTEN and CREB pathways (Fig1D). The profiles suggested systemic alterations in the signaling pathways on tolerant T cells post-haplo-HSCT. Conclusion: Hypo-reactivity of donor-derived circulating T cells to recipient DC and graft T cells to graft DC (negative control) were evident. We found Treg, Tr1 activity or T-cell exhaustion to be insignificant as their blockade or removal did not lead to 'flare’ in circulating tolerant T-cell reactivity against patient DC. T-cell clonal deletion appears to be the dominant mechanism explaining the in vitro hyporeactive proliferation, cytokine secretion, and alloreactive TCR clonotype attrition, along with systemically altered signaling pathways in tolerant T-cells including the alloreactive T cells remained. Anergy may play a minor supportive role. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DNA Aptamer Against Anti- Programmed Cell Death-1 (Anti-PD1-Apt) Induces Robust Anti-Leukemic Activity In Vitro and In Vivo Humanized NSG Mice with Myeloid Leukemia Xenografts

Background: Cancer cells have an extraordinary ability to escape immune response by modulating proteins that regulate immune checkpoints and their cognate ligands. Programmed cell death protein (PD-1), a cell surface receptor expressed on T cells is one such immune checkpoint receptor when bound to its ligands- PDL1 or PDL2 transmits an inhibitory signal. Such modulation often leads to inhibition of T-cell activation allowing tumors to escape immune surveillance. Recently, several antibodies that inhibit the signaling from PD-1/PD-L1 axis were developed that showed enhanced anti-cancer immune reponse leading to their FDA approval for therapeutic use. Aptamers are synthetic small molecule ligands composed of short, single-stranded RNA or DNA oligonucleotides with specific 3D conformation. Aptamers mimic antibodies to recognize and bind to their targets with high affinity. The nucleic acid component has several advantages over the protein counterparts- such as ease of production under less stringent conditions, long shelf life and low cost. FDA has approved several aptamers for therapeutic use that include Macugen, a RNA aptamer that inhibits VEGF signaling to treat age-related macular degeneration, Vitravene to treat cytomegalovirus (CMV) retinitis and Kynamro to treat homozygous familial hypercholesterolemia. There are several other potentially promising aptamers in ongoing preclinical and clinical tests.Methods: Here, we report the development of several PD-1 specific aptamers by systematic evolution of ligands by exponential enrichment (SELEX) technology against endogenous immunoprecipitated PD-1 protein using DNA library with a complexity of 1014. Following several rounds of SELEX, the selected aptamers sequenced by high throughput Next-Generation Sequencing (NGS) were found to have highly conserved regions. Six PD-1 specific aptamers (Anti-PD1-Apt) were then assessed for target validation using leukemic cell lysates (cell lines and primary patient samples) and were found to bind to the PD-1 in its native state.Results: The selected Anti-PD1-Apt were able to specifically pull down the PD-1 protein from the lysates mimicking PD-1 antibody. The specific interaction of the Anti-PD1-Apt was also demonstrated by flow cytometry and fluorescent microscopy. As expected, Anti-PD1-Apt was able to bind to PD-1 with Kd of ~ 500 pM as assessed by Bio-Layer Interferometry. Furthermore, we also characterized and confirmed Anti-PD1-Apt biological activity using an PD-1/PD-L1 cell-based assay using PD-1/NFAT reporter-Jurkat cells. We have observed several fold induction of NFAT luciferase reporter activity (Relative Luciferase Units) in PD-1/NFAT reporter-Jurkat cells co-cultured with HEK293 cells overexpressing PD-L1 and TCR activator in the presence of Anti-PD1-Apt compared to control. Our preliminary data also demonstrate robust Anti-PD1 blockade in Mixed Lymphocyte Reaction (MLR) along with induction of Th1 cytokines Interferon-gamma and IL-2 from different donor sets of PBMCs. In vitro cytotoxicity T- cell assays demonstrated the ability of PD-1 aptamer to potently enhance T-cell response and cytokine production in MLR. Our findings from pharmacokinetics in vivo studies demostrated that PEG-conjugation of PD-1 aptamer has improved the stability of DNA in mouse plasma. A PEGylated form of PD-1 DNA aptamer has demonstrated the ability to block PD-1:PD-L1 interaction and significantly reduced the growth of HEL92.1.7 leukemic tumor cells in vivo in humanized NSG mice with a potency equivalent to an antagonistic anti-PD-1 therapeutic agents. Tumor growth inhibition in humanized NSG mice treated with PD-1 DNA aptamer was 22.59% and comparable to PD-1 antibody over the control group.Conclusions: Overall, our findings demonstrate a comprehensive preclinical characterization of PD-1 aptamer for which antitumor activity was demonstrated in mouse studies. DisclosuresIyer:Genentech: Research Funding; Takeda: Research Funding.

The effect of a novel immunosuppressive drug, a PAK-2 inhibitor, on macrophage differentiation/polarization in a rat small intestinal transplantation model

ObjectivePQA-18 (Prenylated quinolinecarboxylic acid-18) has been reported to be a novel immunosuppressant that attenuates the production of various cytokines, and the differentiation of macrophages by inhibiting PAK2. In this study, we investigated the function of this drug mainly on macrophages using a rat small intestinal transplant model. MethodsMale Dark Agouti (DA) and Lewis rats (LEW), 7–9 weeks of age, were used as donor and recipient, respectively. Approximately 15 cm intestinal grafts were heterotopically transplanted to the recipient rats. The recipient rat was treated with PQA-18 (4 mg/kg/day) by intraperitoneal injection (ip) from postoperative day 1 for 2 weeks. The in vivo effects of this drug were evaluated based on changes in body weight, and the population of each type of blood cell. Mixed lymphocyte reaction (MLR) was also assessed, using the T cells from intestinal mesenteric lymph nodes (MLN) of the grafts on POD6. Total cells from MLN and graft Payer's patch (PP) were next collected on POD6, and the number of infiltrated macrophages was determined. ResultsWhile the survival time was 7.0 ± 0.77 days for the control group (n = 9), that for the PQA-18 group was 10.7 ± 1.26 days (n = 10) (p < .001). Histological examinations showed a relatively clear difference in the grafts for both groups. In addition, the MLR response was significantly lower in recipients treated with PQA-18, suggesting PQA-18 well suppressed the T cells. Moreover, while a significant increase of both MHC class II and CD11b/c positive cells, estimated as differentiated/polarized macrophages, in MLN & PP was observed in the control group, PQA-18-administration significantly suppressed the differentiation of macrophages in the MLN & PP. ConclusionPQA-18 significantly prolonged the survival of the rats with intestinal grafts, and also suppressed the infiltration of lymphocytes, and macrophages to the grafts.

Immunomodulatory Effects of Fentanyl or Dexmedetomidine Hydrochloride Infusion After Allogeneic Heart Transplantation in Mice.

Postoperatively, transplant recipients receive immunosuppressants, as well as sedatives and analgesics. The immunomodulatory effects of these other agents during the induction period following transplantation remain unclear. We aimed to determine whether the agents dexmedetomidine hydrochloride (Dex) and fentanyl (Fen) have immunomodulatory effects during the induction period following heart transplantation (HTx). Fifty mice were used for antinociception tests after administration of Dex and Fen, and T cells from 3 naive animals were used for in vitro lymphocyte transformation test (study 1). Fifty-four B6 mice received HTx from BALB/c mice and were treated with Dex, Fen, or neither (study 2). Thirty-six recipients were used for graft survival data and were humanely killed at the time of cessation of heart graft contraction. The remaining 18 were humanely killed at either postoperative day (POD) 4 or 6 for histologic examination of graft survival, as well as in vitro analysis. Based on the results of study 1, daily intraperitoneal administration of Dex at 30 μg/kg or Fen at 0.25 mg/kg was determined to be the optimal dose to induce analgesia without oversedation following HTx. Graft survivals in both Dex- or Fen-treated animals were statistically prolonged compared with control (P < 0.01). Graft survival of Fen-treated recipients was increased up to 15 days, and graft survival of Dex-treated animals was also increased up to 10 days, whereas control mice rejected heart grafts by POD 7. Mixed lymphocyte reaction responses on POD 4 showed statistically lower responses in Dex-treated recipients and Fen-treated recipients when compared with controls (P < 0.01). Cytokine profiles of splenocytes showed markedly fewer interferon γ-positive splenocytes in Fen-treated recipients on POD 4. These data suggest that both Dex and Fen have immunomodulatory properties in the induction period following transplantation.

Abstract B194: Identification and characterization of monoclonal antibodies targeting the Tyro3, Axl and MerTK (TAM) family of receptor tyrosine kinases

Abstract Members of the TAM family of receptor tyrosine kinases (RTKs) play important roles in tumorigenesis and homeostatic regulation of the immune system. Two closely related ligands, Protein S (PROS1) and Growth Arrest Specific 6 (GAS6), bind and activate the TAMs, with GAS6 activating all TAMs and PROS1 activating MerTK and Tyro3. TAM RTKs are aberrantly expressed in many types of cancer where they may promote survival, metastasis, and chemoresistance. These receptors are also expressed on innate immune cells, particularly macrophages and dendritic cells, where they play important roles in clearance of apoptotic cells and negative regulation of inflammation. In tumors, activation of TAM signaling cascades appears to promote an antiinflammatory and immunosuppressive microenvironment, in turn driving tumor growth. Thus, inhibition of TAM signaling pathways through selective targeting with monoclonal antibodies could result in direct antitumor effects and promote antitumor immunity, whereas activation of TAMs could have antiinflammatory and immunosuppressive effects. We have used a variety of antibody-discovery strategies to identify potent and selective antagonists and agonists of human and rodent TAMs. Robust preclinical model systems for characterizing the mechanistic and functional effects of these antibodies have been established using engineered and nonengineered cell lines and primary cells. These include models to assess TAM activation and downstream signaling in cells and effects on cytokine secretion in LPS-stimulated U937 monocyte-derived and primary macrophages and primary dendritic cells. Effects of TAM inhibition on T-cell responses in mixed lymphocyte reactions are also being evaluated, as are in vivo mouse models. Utilizing these models, we have identified anti-TAM antibodies with antagonist or agonist activity, suggesting they may have potential in modulating the activity of these pathways. Citation Format: Diego Alvarado, Laura Vitale, Michael Murphy, Thomas O'Neill, Andrew Proffitt, Jay Lillquist, Gwenda Ligon, Craig Polson, James R. Storey, Jenifer Widger, Laura Mills-Chen, Karuna Sundarapandiyan, Andrea Crocker, Colleen Patterson, Biwei Zhao, Russell A. Hammond, Li-Zhen He, Venky Ramakrishna, Joel Goldstein, Lawrence Thomas, Henry C. Marsh, Tibor Keler, Richard Gedrich. Identification and characterization of monoclonal antibodies targeting the Tyro3, Axl and MerTK (TAM) family of receptor tyrosine kinases [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B194.

The protein kinase C inhibitor sotrastaurin allows regulatory T cell function

The novel immunosuppressant sotrastaurin is a selective inhibitor of protein kinase C isoforms that are critical in signalling pathways downstream of the T cell receptor. Sotrastaurin inhibits nuclear factor (NF)-κB, which directly promotes the transcription of forkhead box protein 3 (FoxP3), the key regulator for the development and function of regulatory T cells (Tregs). Our center participated in a randomized trial comparing sotrastaurin (n = 14) and the calcineurin inhibitor Neoral (n = 7) in renal transplant recipients. We conducted ex vivo mixed lymphocyte reaction (MLR) and flow cytometry studies on these patient samples, as well as in vitro studies on samples of blood bank volunteers (n = 38). Treg numbers remained stable after transplantation and correlated with higher trough levels of sotrastaurin (r = 0·68, P = 0·03). A dose-dependent effect of sotrastaurin on alloresponsiveness was observed: the half maximal inhibitory concentration (IC50 ) to inhibit alloactivated T cell proliferation was 45 ng/ml (90 nM). In contrast, Treg function was not affected by sotrastaurin: in the presence of in vitro-added sotrastaurin (50 ng/ml) Tregs suppressed the proliferation of alloactivated T effector cells at a 1:5 ratio by 35 versus 47% in the absence of the drug (P = 0·33). Signal transducer and activator of transcription 5 (STAT)-5 phosphorylation in Tregs remained intact after incubation with sotrastaurin. This potent Treg function was also found in cells of patients treated with sotrastaurin: Tregs inhibited the anti-donor response in MLR by 67% at month 6, which was comparable to pretransplantation (82%). Sotrastaurin is a potent inhibitor of alloreactivity in vitro, while it did not affect Treg function in patients after kidney transplantation.

TL1A-Ig induces transplantation tolerance (P1003)

Abstract Tumor necrosis factor (TNF) superfamily member 15 (TL1A) is the natural ligand for TNFRSF25. We previously reported that TNFRSF25 stimulation with an agonist antibody 4C12 selectively expands pre-existing Tregs in vivo. To compare TL1A to 4C12, we generated a soluble mouse hexameric TL1A-Ig fusion protein. TL1A-Ig mediates proliferation of Foxp3+ Treg in vitro. TNFRSF25 stimulation by TL1A-Ig in vivo induces expansion of Tregs to 30-35% of all CD4+ T cells in the peripheral blood within 5 days of treatment. With daily injections of TL1A-Ig elevated levels of Tregs can be maintained for at least 20 days. TL1A-Ig expanded Tregs express high levels of the activation/memory markers KLRG1 and CD103 and are highly suppressive ex vivo. TL1A-Ig mediated Treg expansion in vivo is protective against allergic lung inflammation by reversing the ratio of Tconvs:Tregs in the lung and blocking eosinophil exudation into the bronchoaveolar fluid. We tested the potential of TNFRSF25 agonists in transplant tolerance in several models. (1) TL1A-Ig injections 12 days after injection of allogeneic splenocytes caused diminished alloantigen-specific T cell responses in mixed lymphocyte reactions as compared to mice treated with allogeneic splenocytes only. (2) TNFRSF25 mediated Treg expansion in vivo significantly prolonged allogeneic skin graft survival. Thus, TL1A-Ig fusion proteins are potent and tightly controllable agents that may provide therapeutic benefit in asthma and transplant tolerance.

Sunset yellow FCF, a permitted food dye, alters functional responses of splenocytes at non-cytotoxic dose

Sunset yellow FCF (SY), a permitted food color, is extensively used in various food preparations and quite often exceeds the permissible levels (100–200mg/kg). Several toxicity studies on SY are reported, however immunomodulatory properties have not been explored yet. To investigate the immunotoxic properties of SY, splenocytes were isolated, cultured and subjected to mitogen stimulated proliferation assay (lipopolysaccharide, LPS or concanavalin A, Con A), mixed lymphocyte reaction (MLR) assay, immunophenotypic analysis of cell surface receptor expression and assay for cytokines release in the culture supernatants were performed in the presence of SY. Since SY did not exhibit any cytotoxicity up to 250μg/ml, this dose was used for further studies. It was observed that SY (250μg/ml) significantly (p<0.05) suppressed the mitogen induced proliferation of splenocytes and MLR response. Further, immunophenotypic analysis revealed that SY alters the relative expression of CD3e/CD4/CD8 in T cells and CD19 in B-cells. Consistent with the suppression of T-cell and B-cell responses and altered surface receptor expression, SY also lowered the expression of IL2, IL4, IL6, IL-17, IFN-γ and TNF-α cytokines. These results suggest that non-cytotoxic dose of SY may have immunomodulatory effects.

Functional Characteristics of C-terminal Lysine to Cysteine Mutant Form of CTLA-4Ig

CTLA-4Ig is regarded as an inhibitory agent of the T cell proliferation via blocking the costimulatory signal which is essential for full T cell activation. To improve applicability, we developed the CTLA-4Ig-CTKC in which the c-terminal lysine had been replaced by cysteine through single amino acid change. The single amino acid mutation of c-terminus of CTLA-4Ig was performed by PCR and was checked by in vitro transcription and translation. DNA construct of mutant form was transfected to Chinese hamster ovary (CHO) cells by electroporation. The purified proteins were confirmed by Western blot and B7-1 binding assay for their binding ability. The suppressive capacity of CTLA-4Ig-CTKC was evaluated by the mixed lymphocyte reaction (MLR) and in the allogeneic pancreatic islet transplantation model. CTLA-4Ig-CTKC maintained binding ability to B7-1 molecule and effectively inhibits T cell proliferation in MLR. In the murine allogeneic pancreatic islet transplantation, short-term treatment of CTLA-4Ig-CTKC prolonged the graft survival over 100 days. CTLA-4Ig-CTKC effectively inhibits immune response both in MLR and in allogeneic islet transplantation model, indicating that single amino acid mutation does not affect the inhibitory function of CTLA-4Ig. CTLA-4Ig-CTKC can be used in vehicle-mediated drug delivery system such as liposome conjugation.

Mizoribine as Sole Immunosuppressive Agent in Islet Xenotransplantation Models: A Candidate Immunosuppressant Causing no Adverse Effects on Islets

Mizoribine (MZ) inhibits the differentiation and proliferation of helper T and B cells after antigen recognition by suppressing the purine biosynthesis pathway and nucleic acid synthesis. MZ has been used in kidney transplantation, but distinct data are unavailable for islet transplantation. The present study investigated the efficacy of MZ for islet xenotransplantation. Immunosuppressive effects of MZ were determined by mixed lymphocyte reaction (MLR) assay in vitro. Toxicities for Wistar rat islets were determined by adenosine triphosphate (ATP) contents of islets during 3-day culture and stimulation index in response to glucose after culture. Immunosuppressive effects in vivo were tested in a Wistar-to-B6 islet xenotransplantation model. MZ was administered continuously for 28 days subcutaneously or intramuscularly. MZ inhibited MLR response by approximately 50% at 0.1 μg/ml. ATP contents decreased with MZ >100 μg/ml, while stimulation index was maintained. Continuous infusion of MZ at 10 mg/kg maintained blood concentrations at 0.13-0.19 μg/ml, while intramuscular injection of MZ at 100 mg/kg/day (peak 520 μg/ml at 1 h postinjection) resulted in below measurable levels (<0.03 μg/ml) within 24 h. Graft survival was significantly prolonged following continuous infusion of 10 mg/kg/day compared to controls (31.0 ± 9.5 vs. 13.2 ± 5.2 days; p = 0.002). Furthermore, animals with intramuscular injection at doses of 3.2, 10, or 100 mg/kg/day showed significantly longer graft survival (20.0 ± 7.5, 22.0 ± 7.31, and 24.5 ± 8.1 days, respectively; p < 0.05 each). Histological examination showed significant suppression of lymphocyte infiltration by MZ administration. MZ showed immunosuppressive effects in an experimental islet xenotransplantation model without adverse effects on endocrine function of islet grafts.

In vitro studies on immunotoxic potential of Orange II in splenocytes

Orange II, an azo dye, is not permitted in food preparations, but high levels of the dye have been detected in different food commodities. Though there are reports on the toxicity of Orange II but knowledge based on the immunomodulatory properties of Orange II is scanty. The present investigation was undertaken to study the in vitro immunotoxic potential of Orange II in splenocytes. Splenocytes were isolated, cultured and subjected to immunophenotypic analysis, mixed lymphocyte reaction (MLR) assay or stimulated with lipopolysaccharide (LPS) or concanavalin A (Con A) for 72 h. The supernatant was collected for cytokine assays. Orange II showed cytotoxic effects at 100–1000 μg/ml concentrations and 50 μg/ml was determined as the highest non-cytotoxic dose. Orange II at the non-cytotoxic dose (50 μg/ml) significantly altered the relative distribution of T and B-cells, MLR response and the mitogen induced proliferative response of T-cells and B-cells. Consistent with the hypo-responsiveness of the T and B-lymphocytes, Orange II induced a concomitant decline in the secretion of cytokines IL-2, IL-4, IL-6, IFN-γ, TNF-α and IL-17. On the contrary, there was an increase in the production of IL-10, an anti-inflammatory regulatory cytokine, which may be one of the causative factor for immunosuppressive property of Orange II. These results suggest that non-cytotoxic dose of Orange II may have immunomodulatory effects.

Human Mesenchymal Stem Cells and Immunosuppressive Drug Interactions in Allogeneic Responses: An In Vitro Study Using Human Cells

Objectives The use of mesenchymal stem cells (MSC), which display immunosuppressive activity, seems to be a promising therapeutic approach in solid organ transplantation. However, little is known about their interactions with immunosuppressive drugs. The objective of this study was to assess these interactions in allogeneic responses. Methods We studied the effects on alloimmune responses in mixed lymphocyte reactions of MSC plus five agents—cyclosporine, tacrolimus, rapamycin, mycophenolate acid (MPA), and dexamethasone (DEX). Results Human MSC isolated from bone marrow were characterized by their phenotype and their ability to differentiate into adipocytes or osteoblastes. MSC plus the agents inhibited allogeneic lymphocyte proliferation in a dose-dependent manner. Calcineurin inhibitors and rapamycin antagonized the inhibitory effect of MSC, whereas MPA promoted it and DXM did not modify it. Conclusion MPA seems to be the best immunosuppressant to associated with MSC for transplanted patients.

Increased Interleukin-10 Production Without Expansion of CD4+CD25+ T-Regulatory Cells in Early Stable Renal Transplant Patients on Calcineurin Inhibitors

Increasing long-term allograft survival is the main challenge in organ transplantation, and allograft loss due to chronic rejection has been found to correlate with episodes of early acute rejection. It is important to understand the mechanisms that maintain the donor-specific hyporesponsive state. This study prospectively evaluates immune events related to donor-specific hyporesponsiveness in the stable transplant patients on calcineurin inhibitors (CNIs). Peripheral blood mononuclear cells of transplant recipients on CNI (n=19) were tested in mixed lymphocyte reaction (MLR) against donor and third-party peripheral blood mononuclear cells pretransplant and at 6 to 8 weeks, 14 to 18 weeks, and 6 to 8 months posttransplant. Interleukin (IL)-10 and transforming growth factor-beta were quantitated in cultures supernatants by enzyme-linked immunosorbent assay. CD4CD25 T-regulatory cells (Tregs) were enumerated using flow cytometry. All patients showed sharp decline in anti-donor and third-party MLR response at 6 to 8 weeks posttransplant with progressive decline up to 6 to 8 months. This was accompanied by increased IL-10 levels in the supernatant at all the follow-ups. Transforming growth factor-beta level in the supernatant was significantly lower at 14 to 18 weeks. Frequency of CD4CD25 Tregs showed a significant decrease at 6 to 8 weeks posttransplant, which was sustained up to 6 to 8 months. The study shows that the maintenance of good graft function in early posttransplant period in recipients on CNI is associated with a decrease in donor-specific and third-party MLRs. There is a decline in Treg numbers along with increased IL-10 levels. High IL-10, probably from a non-Tregs source, may have an important role in maintaining hyporesponsiveness and good graft function.

Th1/Th17 Immune Response Is Induced by Mesenteric Lymph Node Dendritic Cells in Crohn's Disease

Dendritic cells (DCs) possess the most potent ability to induce acquired immunity. However, their involvement in the pathogenesis of Crohn's disease (CD) has not yet been determined. We aimed to establish the immune status of mesenteric lymph nodes, the major gut-associated lymphoid tissue, and isolated DCs and determine their involvement in the pathogenesis of CD. CD4(+) T cells and DCs were isolated from mesenteric lymph nodes of CD, ulcerative colitis, and normal control. The immune status of CD4(+) T cells was analyzed by cytokine production and transcriptional profile. Surface phenotype of DCs was analyzed by flow cytometry. Cytokine production by myeloid DCs was analyzed by real-time polymerase chain reaction and exogenous bacterial stimulation. Immune stimulating activity of DCs was determined by mixed lymphocyte reaction. In CD, mesenteric lymph node CD4(+) T cells produced higher amounts of interferon-gamma and interleukin (IL)-17 compared with ulcerative colitis and normal control, and this was dictated by increased T-bet and retinoic acid-related orphan receptor-gamma expression. Three subtypes of DCs, myeloid DC, plasmacytoid DC, and mature DC, were identified in all groups. When stimulated with exogenous bacterial derivative, myeloid DCs from CD produced a higher amount of IL-23 and a lower amount of IL-10. Myeloid DCs from CD induced stronger T helper cell (Th)1 immune response in mixed lymphocyte reaction compared with those from ulcerative colitis and normal control. Our findings revealed that mesenteric lymph node is the key pathogenic location of CD elicited by the unique cytokine milieu produced by DCs leading to a dysregulated Th1/Th17 immune response.

Cell contact, prostaglandin E(2) and transforming growth factor beta 1 play non-redundant roles in human mesenchymal stem cell induction of CD4+CD25(High) forkhead box P3+ regulatory T cells.

Adult human mesenchymal stromal or stem cells (MSC) can differentiate into a variety of cell types and are candidate cellular therapeutics in regenerative medicine. Surprisingly, these cells also display multiple potent immunomodulatory capabilities, including allosuppression, making allogeneic cell therapy a possibility. The exact mechanisms involved in regulatory T cell induction by allogeneic human MSC was examined, using purified CD4+ populations and well-characterized bone marrow-derived adult human MSC. Allogeneic MSC were shown to induce forkhead box P3 (FoxP3)+ and CD25+ mRNA and protein expression in CD4+ T cells. This phenomenon required direct contact between MSC and purified T cells, although cell contact was not required for MSC induction of FoxP3 expression in an unseparated mononuclear cell population. In addition, through use of antagonists and neutralizing antibodies, MSC-derived prostaglandins and transforming growth factor (TGF)-beta1 were shown to have a non-redundant role in the induction of CD4+CD25+FoxP3+ T cells. Purified CD4+CD25+ T cells induced by MSC co-culture expressed TGF-beta1 and were able to suppress alloantigen-driven proliferative responses in mixed lymphocyte reaction. These data clarify the mechanisms of human MSC-mediated allosuppression, supporting a sequential process of regulatory T cell induction involving direct MSC contact with CD4+ cells followed by both prostaglandin E(2) and TGF-beta1 expression. Overall, this study provides a rational basis for ongoing clinical studies involving allogeneic MSC.

The immunomodulatory activity of human umbilical cord blood‐derived mesenchymal stem cells in vitro

Bone marrow-derived mesenchymal stem cells (BM-MSC) are currently being investigated in preclinical and clinical settings because of their self-renewal and multipotent differentiative capacity or their immunosuppressive function. However, BM may be detrimental because of the highly invasive donation procedure and BM-MSC decline with age. Therefore, MSC derived from other sources have been considered as an alternative. However, there is only limited knowledge on their immunomodulatory properties. Human umbilical cord blood (UCB) cells are good substitutes for BM-MSC because of the immaturity of newborn cells. In this study, we successfully isolated MSC from UCB. The morphological phenotypes, cell cycle status, surface markers and differentiation potential of these clonally expanded cells are consistent with BM-MSC. Furthermore, UCB-MSC expanded in vitro retain low immunogenicity and an immunomodulatory effect. Flow cytometry analysis showed that UCB-MSC did not express CD40, CD40 ligand, CD80, CD86 and major histocompatibility complex class II molecules. We have demonstrated that UCB-MSC are incapable of inducing allogeneic peripheral blood mononuclear cell (PBMC) proliferation and have a dose-dependent inhibition of PBMC immune responses in mixed lymphocyte reactions (MLR) and phytohaemagglutinin activation assays, even after interferon-gamma treatment. Additionally, we have found that UCB-MSC can suppress the function of mature dendritic cells. Using transwell systems, we have demonstrated an inhibition mechanism that depends on both cell contact and soluble factors. Based on the findings we conclude that banked UCB could serve as a potential alternative source of MSC for allogeneic application in the future.