Primary Mixed Lymphocyte Reaction Research Articles

Differentiation of regulatory myeloid and T-cells from adult human hematopoietic stem cells after allogeneic stimulation.

Donor hematopoietic stem cell (DHSC) infusions are increasingly being studied in transplant patients for tolerance induction. To analyze the fate of infused DHSCs in patients, we developed an in vitro culture system utilizing CD34+DHSCs stimulated with irradiated allogeneic cells in cytokine supplemented medium long-term. Flow cytometric analyses revealed loss of the CD34 marker and an increase in CD33+ myeloid and CD3+ T-cell proportion by 10.4% and 72.7%, respectively, after 21 days in culture. T-cells primarily expressed TcR-αβ and were of both CD4+ and CD8+ subsets. Approximately 80% of CD3+ T cells lacked expression of the co-stimulatory receptor CD28. The CD4+ compartment was predominated by CD4+CD25+CD127-FOXP3+ Tregs (>50% CD4+CD127- compartment) with <1% of all leukocytes exhibiting a CD4+CD127+ phenotype. Molecular analyses for T-cell receptor excision circles showed recent and increased numbers of TcR rearrangements in generated T cells over time suggesting de novo differentiation from DHSCs. CD33+ myeloid cells mostly expressed HLA-DR, but lacked expression of co-stimulatory receptors CD80 and CD83. When studied as modulators in primary mixed lymphocyte reactions where the cells used to stimulate the DHSC were used as responders, the DHSC-lines and their purified CD8+, CD4+, CD33+ and linage negative subsets inhibited the responses in a dose-dependent and non-specific fashion. The CD8+ cell-mediated inhibition was due to direct lysis of responder cells. Extrapolation of these results into the clinical situation would suggest that DHSC infusions into transplant recipients may generate multiple subsets of donor "chimeric" cells and promote recipient Treg development that could regulate the anti-donor immune response in the periphery. These studies have also indicated that T cell maturation can occur in vitro in response to allogeneic stimulation without the pre-requisite of a thymic-like environment or NOTCH signaling stimulatory cell line.

IL-10-Engineered Dendritic Cells Modulate Allogeneic CD8+ T Cell Responses.

Tolerogenic dendritic cells (tolDC) play a central role in regulating immune homeostasis and in promoting peripheral tolerance. These features render tolDC a promising tool for cell-based approaches aimed at inducing tolerance in T-cell mediated diseases and in allogeneic transplantation. We developed a protocol to generate genetically engineered human tolDC overexpressing IL-10 (DCIL-10) by means of a bidirectional lentiviral vector (LV) encoding for IL-10. DCIL-10 promote allo-specific T regulatory type 1 (Tr1) cells, modulate allogeneic CD4+ T cell responses in vitro and in vivo, and are stable in a pro-inflammatory milieu. In the present study, we investigated the ability of DCIL-10 to modulate cytotoxic CD8+ T cell responses. We demonstrate that DCIL-10 reduces allogeneic CD8+ T cell proliferation and activation in primary mixed lymphocyte reactions (MLR). Moreover, long-term stimulation with DCIL-10 induces allo-specific anergic CD8+ T cells without signs of exhaustion. DCIL-10-primed CD8+ T cells display limited cytotoxic activity. These findings indicate that stable over-expression of IL-10 in human DC leads to a population of cells able to modulate cytotoxic allogeneic CD8+ T cell responses, overall indicating that DCIL-10 represent a promising cellular product for clinical applications aimed at inducing tolerance after transplantation.

IL-10 and TGF-β induce alloreactive CD4+CD25– T cells to acquire regulatory cell function

AbstractWe previously reported that interleukin-10 (IL-10) and transforming growth factor (TGF)–β treatment of primary mixed lymphocyte reaction (MLR) cultures resulted in secondary alloantigen-specific hyporesponsiveness and protection from graft-versus-host disease (GVHD) lethality. Here, we report that CD4+ T cells recovered from the IL-10– and TGF-β–treated primary MLR cultures have immunoregulatory function. Tolerized cells significantly inhibited proliferation of naive alloreactive CD4+ T cells in a primary MLR. Inhibition of the naive alloresponse was observed with as few as 1 tolerized cell to 10 naive responder cells. Tolerized cells were able to significantly reduce GVHD lethality when injected with naive alloreactive CD4+ T cells into major histocombatibility class (MHC) II disparate recipients. Rigorous CD25 depletion of the primary MLR had no effect on generation of a regulatory capacity, suggesting that the regulatory cells likely originated from CD4+CD25– T cells. Immune suppression was mediated independently of IL-10 and TGF-β production, as neutralizing antibodies for IL-10, IL-10R, and TGF-β were unable to revert suppression, and IL-10– deficient CD4+ T cells were able to mediate in vitro and in vivo suppression. The generation of immunoregulatory cells from a CD4+CD25– population during tolerization with IL-10 and TGF-β provides an additional mechanism to prevent GVHD lethality by T cells that may escape full tolerance induction.

FS222, a CD137/PD-L1 Tetravalent Bispecific Antibody, Exhibits Low Toxicity and Antitumor Activity in Colorectal Cancer Models.

With the increased prevalence in checkpoint therapy resistance, there remains a significant unmet need for additional therapies for patients with relapsing or refractory cancer. We have developed FS222, a bispecific tetravalent antibody targeting CD137 and PD-L1, to induce T-cell activation to eradicate tumors without the current toxicity and efficacy limitations seen in the clinic. A bispecific antibody (FS222) was developed by engineering CD137 antigen-binding sites into the Fc region of a PD-L1 IgG1 mAb. T-cell activation by FS222 was investigated using multiple in vitro assays. The antitumor efficacy, survival benefit, pharmacodynamics, and liver pharmacology of a murine surrogate molecule were assessed in syngeneic mouse tumor models. Toxicology and the pharmacokinetic/pharmacodynamic profile of FS222 were investigated in a non-human primate dose-range finding study. We demonstrated simultaneous binding of CD137 and PD-L1 and showed potent T-cell activation across CD8+ T-cell activation assays in a PD-L1-dependent manner with a CD137/PD-L1 bispecific antibody, FS222. FS222 also activated T cells in a human primary mixed lymphocyte reaction assay, with greater potency than the monospecific mAb combination. FS222 showed no signs of liver toxicity up to 30 mg/kg in a non-human primate dose-range finding study. A surrogate molecule caused significant tumor growth inhibition and survival benefit, concomitant with CD8+ T-cell activation, in CT26 and MC38 syngeneic mouse tumor models. By targeting CD137 agonism to areas of PD-L1 expression, predominantly found in the tumor microenvironment, FS222 has the potential to leverage a focused, potent, and safe immune response augmenting the PD-(L)1 axis blockade.

Safety and pharmacodynamics of anti-CD2 monoclonal antibody treatment in cynomolgus macaques - an experimental study.

Anti-CD2 treatment provides targeted immunomodulatory properties that have demonstrated clinical usefulness to condition the immune system and to treat transplant rejection. The treatment is species-specific due to structural CD2 antigen differences between nonhuman primates and humans. Herein, we report the safety profile and efficacy of two modifications of the same anti-CD2 monoclonal antibody in cynomolgus macaques. Twelve subjects received one i.v. anti-CD2 (of rat or rhesus type) dose each, range 1-4mg/kg, and were followed for 1-7days. Treatment effects were evaluated with flow cytometry on peripheral blood and histopathological evaluation of secondary lymphoid organs. In vitro inhibitory activity on primary MHC disparate mixed lymphocyte reactions (MLRs) was determined. Upon anti-CD2 treatment, CD4+ , CD8+ memory subsets were substantially depleted. Naïve T cells and Tregs were relatively spared and exhibited lower CD2 expression than memory T cells. Early immune reconstitution was noted for naïve cells, while memory counts had not recovered after one week. Both antibodies displayed a concentration-dependent MLR inhibition. Lymph node examination revealed no significant lymphocyte depletion. None of the animals experienced any significant study drug-related adverse events. This study outlines the safety and pharmacodynamic profile of primate-specific anti-CD2 treatment, relevant for translation of anti-CD2-based animal models into clinical trials.

PD-L2 generates a signal through the PD-1 co-receptor distinct from that of PD-L1

Abstract Immune checkpoint blockade of the PD-1/PD-L1/PD-L2 signalling axis yields potent anti-tumor responses by disinhibiting T cell effector function. Mouse and human data demonstrating an inhibitory role for PD-L1 has resulted in FDA approval of anti-PD-L1 antibodies; however, PD-L2 remains sparsely studied despite being the higher affinity of the two ligands for PD-1. Recent findings demonstrate that PD-L2 is widely expressed by human malignancies. PD-L1 and PD-L2 have also been shown to induce distinct conformational changes on engaging human PD-1. We utilized a Jurkat T cell based bioluminescent assay of T cell activation and PD-1 inhibition to determine the unique mechanism of action of human PD-L2. In this system, human PD-L2 generates a solely co-inhibitory signal, but at reduced inhibitory potential relative to PD-L1. We find that unlike murine PD-L2, human PD-L2 appears to have a dedicated co-inhibitory function as we find no significant engagement of the human homolog of its murine co-stimulatory partner RGMb. Reverse phase protein array analysis of human PD-L1 and PD-L2 engagement of PD-1 on Jurkat T cells revealed significant differences in downstream T cell signalling generated by each ligand. We also find that human PD-L1 and PD-L2 have disparate impacts on proliferation wherein PD-L2 supports a preferential arrest of T cells in the S-phase of the cell cycle. We observed that combination blockade of PD-L1 and PD-L2 improves on the blockade of PD-L1 alone resulting in increased production of IL-2 and IFNγ in primary human mixed lymphocyte reactions. Our findings address critical gaps in our knowledge about the basic function of PD-L2 and will inform future attempts to manipulate the PD-1 co-inhibitory circuit.

Early expansion of donor-specific Tregs in tolerant kidney transplant recipients

Allograft tolerance, in which a graft is accepted without long-term immunosuppression, could overcome numerous obstacles in transplantation. Human allograft tolerance has been intentionally induced across HLA barriers via combined kidney and bone marrow transplantation (CKBMT) with a regimen that induces only transient chimerism. Tregs are enriched early after CKBMT. While deletional tolerance contributes to long-term tolerance, the role of Tregs remains unclear. We have optimized a method for identifying the donor-specific Treg repertoire and used it to interrogate the fate of donor-specific Tregs after CKBMT. We expanded Tregs with several different protocols. Using functional analyses and T cell receptor sequencing, we found that expanding sorted Tregs with activated donor B cells identified the broadest Treg repertoire with the greatest potency and donor specificity of suppression. This method outperformed both alloantigen stimulation with CTLA4Ig and sequencing of CFSElo cells from the primary mixed lymphocyte reaction. In 3 tolerant and 1 nontolerant CKBMT recipients, we sequenced donor-specific Tregs before transplant and tracked them after transplant. Preexisting donor-specific Tregs were expanded at 6 months after CKBMT in tolerant patients and were reduced in the nontolerant patient. These results suggest that early expansion of donor-specific Tregs is involved in tolerance induction following CKBMT.

Enhancement of the Immunostimulatory Functions of Ex Vivo–Generated Dendritic Cells from Early-Stage Colon Cancer Patients by Consecutive Exposure to Low Doses of Sequential-Kinetic-Activated IL-4 and IL-12. A Preliminary Study

Dendritic cells (DCs), specialized antigen-presenting cells bridging innate and adaptive immunity, play a crucial role in determining specific immune response to tumors. Because of their potent immunoregulatory capacities, DCs have been exploited in anticancer vaccination, with limited success thus far. This pilot study compared low-dose interleukin (IL)-4 and IL-12 prepared by sequential kinetic activation (SKA) with standard doses of the same recombinant human cytokines on functional activity of ex vivo–generated monocyte-derived (Mo) DCs from colon carcinoma patients and normal subjects. MoDCs were exposed to medium alone, SKA-IL-4 (0.5 fg/ml), or SKA-IL-12 (2 fg/ml), alone or consecutively combined, in parallel with rhIL-4 (50 ng/ml) and rhIL-12 (1 ng/ml). Primary allogeneic one-way mixed lymphocyte reaction (MLR) was the end point to assess in vitro T-lymphocyte proliferation in response to MoDCs, and secreted IL-12p70 and interferon-γ in MLR supernatants measured by ELISA to assay for T-helper 1–promoting MoDC phenotype. No single agent enhanced the compromised allostimulatory activity of MoDCs from colon cancer patients, unlike healthy donors. However, MoDCs from nonmetastatic colon cancer patients, after sequential exposure to SKA-IL-4 (48 hours) and SKA-IL-12 (24 hours), displayed increased T-cell stimulatory capacity by MLR and acquired driving T-helper 1 polarization activity, although less markedly than the effects induced by recombinant human cytokines or found in normal subjects. These results point to an immunomodulatory capacity of low-dose SKA-IL-4 and SKA-IL-12 and encourage further investigation to provide clues for the rational development of new and more effective immunotherapeutic strategies against cancer.

Repeated Injections of IL-2 Break Renal Allograft Tolerance Induced via Mixed Hematopoietic Chimerism in Monkeys.

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1-10 years after withdrawal of immunosuppression received subcutaneous injections of IL-2 cytokine (0.6-3 × 10(6) IU/m(2) ). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro-inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti-CD8 antibody treatment. Finally, this process was reversible in that cessation of IL-2 administration aborted the rejection process and restored normal kidney graft function.

Generation of mouse pluripotent stem cell-derived proliferating myeloid cells as an unlimited source of functional antigen-presenting cells.

The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow-derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2K(b)-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs.

The study of prolongation of rat renal allograft survival by CD4+ CD25- T cells induced by recipient dendritic cells transfected with dominant negative form of I.KAPPA.B kinases 2

Objective To observe whether CD4 + CD25-T cells induced by recipient-derived immature dendritic cells (imDC) transfected with dominant negative form of IκB kinases 2 (IKK2dn) gene and loaded with donor antigen can prolong the survival time of kidney transplantation in rats and investigate its probably.Methods DC were cultured from recipient rats (Lewis) bone marrow,transfected with AdvIKK2dn and loaded with donor antigen,then cultured with Lewis rats lymphocytes in primary mixed lymphocyte reaction (MLR).The CD4 + CD25-T cells were separated by immune magnetic beads method after 72 hours.Male Brown Norway rats and Lewis rats were used as donors and recipients respectively,Four groups were set up(naive T cells group,Adv0-CD4 + T cells group,CD4 + CD25-T cells group and rejection group),receiving 1 × 107 naive CD4 + T-cell,Adv-0-DC-generated CD4 + CD25-T-cell,Adv-IKK2dnDC-generated CD4 + CD25 T-cell and equal volume of normal saline,respectively 7 days before transplantation.In the third party donor group,wistar rats as donors were treated the same as CD4 + CD25 T cells group before transplantation.After transplantation,the survival time of recipients were observed; the T lymphocyte proliferation in recipients was measured; the levels of serum interleukin (IL)-2,IL-10,interferon-γ(IFN-γ),transforming growth factor-β (TGF-β) were detected; serum creatinine levels were monitored ; pathological changes were examined to identify the grade of rejection,Results Compared with other groups,CD4 + CD25-T cells group markedly prolonged the survival time of renal allografts [(28.50 ± 2.36) d,P ＜0.01],and had higher expression of IL-10 and TGF-β (P ＜0.05).Compared with Adv0CD4 + T cells group,rejection group,the third party donor group,expression levels of IL-2 and IFN-γwere lower(P ＜0.05).In contrast to other groups,CD4 + CD25-T cells group elicited markedly lower proliferative responses (P ＜ 0.05) and lower grade as estimated by pathological examination.Conclusion These findings suggested that CD4 + CD25 T cells induced by recipient-derived immature DC transfected by IKK2dn and loaded with donor antigen could prolong renal allograft survival in a donor-specific manner,and it maybe by expressing high levels of IL-10 and TGF-β. Key words: Dendritic cells ; Dominant negative form of IκB kinases 2 ; CD4+ CD25-T cells; Kidney allotransplantation ; Rat

Cathepsin H Is an Additional Convertase of Pro-granzyme B

The serine protease granzyme B (GrB) is the most potent proapoptotic cytotoxin of the granule exocytosis pathway of cytotoxic lymphocytes. GrB is synthesized as a zymogen (proGrB) and activated in cytotoxic granules by the lysosomal cysteine protease cathepsin C (CatC) which removes the N-terminal dipeptide Gly-Glu. It has been shown recently that mice lacking CatC nonetheless express significant residual GrB activity, indicating the presence of additional proGrB convertases. Here, we describe an assay to assess activation of proGrB and show that the amino-peptidase cathepsin H (CatH) has proGrB convertase activity in vitro, whereas dipeptidylpeptidase II does not. We generated mice lacking both CatC and CatH expression (CatCH(-/-)) and found that their lymphocytes have reduced convertase activity compared with those from CatC-deficient mice. Despite this, cytotoxic lymphocytes from CatCH(-/-) mice retain cytotoxic activity and some residual GrB activity. We conclude that CatH can act as an additional proGrB convertase and that other protease/s (apart from dipeptidylpeptidase II) must also possess convertase activity. This indicates a great deal of functional redundancy in GrB maturation, which would prevent pathogen-mediated immune suppression by via convertase inhibition.

The Triterpenoid CDDO-Me Delays Murine Acute Graft-versus-Host Disease with the Preservation of Graft-versus-Tumor Effects after Allogeneic Bone Marrow Transplantation

The occurrence of acute graft-versus-host disease (aGVHD) and tumor relapse represent the two major obstacles impeding the efficacy of allogeneic bone marrow transplantation (BMT) in cancer. We have previously shown that the synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO) can inhibit murine early aGVHD, but antitumor effects were not assessed. In the current study, we found that a new derivative of CDDO, CDDO-Me, had an increased ability to inhibit allogeneic T cell responses and induce cell death of alloreactive T cells in vitro. Administration of CDDO-Me to mice following allogeneic BMT resulted in significant and increased protection from lethal aGVHD compared to CDDO. This correlated with reduced TNF-alpha production, reduced donor T cell proliferation, and decreased adhesion molecule (alpha(4)beta(7) integrin) expression on the donor T cells. CDDO-Me was also superior to CDDO in inhibiting leukemia growth in vitro. When CDDO-Me was administered following an allogeneic BMT to leukemia-bearing mice, significant increases in survival were observed. These findings suggest that CDDO-Me is superior to CDDO in delaying aGVHD, while preserving or possibly even augmenting GVT effects.

Transformation of breast milk macrophages by HTLV-I: implications for HTLV-I transmission via breastfeeding

Human T cell leukemia virus type I (HTLV-I), a causative agent of adult T-cell leukemia (ATL), is transmitted from mother to child predominantly by breastfeeding. The source of HTLV-I-infected cells in breast milk has been thought to be T cells, however, the majority of cells in breast milk are CD14(+) macrophages but not CD3(+) T lymphocytes, and no data are available regarding HTLV-I transmission through breast milk macrophages (BrMMpsi). To explore the potential of BrMMpsi as a possible source of infection in mother to child transmission (MTCT) of HTLV-I, an immortalized cell line (HTLV-BrMMpsi) has been established from BrMMpsi by infection with HTLV-I. HTLV-BrMMpsi retained macrophage characteristics and did not express a complete dendritic cell (DC) phenotype; nevertheless, HTLV-BrMMpsi efficiently promoted T cell proliferation in primary allogeneic mixed lymphocyte reaction (MLR) like DC. Moreover, HTLV-I infection could be transmitted from HTLV-BrMMpsi to activated T cells in the peripheral blood. These findings suggested that BrMMpsi might be an appropriate HTLV-I reservoir involved in MTCT transmission via breastfeeding.

Differential cytokine expression and regulation of human anti‐pig xenogeneic responses by modified porcine dendritic cells

Porcine dendritic cells (DC) are likely to be pivotal cells in the initiation of stimulatory and potential tolerogenic responses to xenoantigens, however, there are limited studies characterizing these antigen presenting cells. Porcine PBMC (CD172a(+)) were cultured with GM-CSF and IL-4 and phenotype and functional capabilities assessed. Lipopolysaccharide (LPS), IL-10, and IL-3 were added to the GM-CSF/IL-4 DC cultures to determine phenotypic and functional changes. Quantitative real-time polymerase chain reaction (PCR) for key cytokines was performed and the modified porcine DC were further assessed by primary mixed lymphocyte reaction to determine the effect of LPS, IL-10, and IL-3 on stimulatory capability. Porcine PBMC (CD172(+)) cultured with GM-CSF and IL-4 produced cells with DC morphology, which were major histocompatability complex (MHC) class II(+), CD14(-/lo), and CD1a(lo). Addition of IL-10 or IL-3 to GM-CSF/IL-4 DC cultures produced cells with lower levels of MHC class II and higher levels of antigen uptake consistent with less mature DC. Quantitative real-time PCR of DC showed the addition of IL-10 induced an increase in IL-10 mRNA, no detectable IL-12, and reduced IL-6 mRNA. The addition of IL-3 to DC cultures decreased IL-12, IL-6 and tumor necrosis factor (TNF), with no change in IL-10 mRNA. GM-CSF/IL-4 DC induced strong human lymphocyte proliferation, compared with significantly reduced stimulatory capacity induced by IL-10 and IL-3 treated DC cultures. The profound effect on differential DC cytokine profile and reduced human anti-pig responses has important therapeutic implications in xenotransplantation. The mechanism of altered regulation warrants further investigation.

Costimulatory Blockade (CSB) during Mixed Lymphocyte Reaction (MLR) Prevents IL27 Upregulation and Signalling.

Numerous methods for selectively depleting or manipulating alloantigen (alloAg) specific CD4 T cells (CD4 T) are being studied for their potential in improving transplant outcomes by limiting GVHD or graft rejection. Although some methods target specific molecules (e.g. costimulatory receptors and ligands, activation antigens), the effects of these methods on “off-target” CD4 T cellular pathways and functions as well as effects on other PBMC, direct or indirect, have not been well characterized. Such effects may impact significantly on clinical efficacy and/or may shed light on the mechanism of action of manipulated PBMC in a complex in vivo milieu. To understand the molecular impact on bystander PBMC of inducing alloAg specific CD4 T anergy by CSB via disruption of the positive costimulatory signal between B7.1 and B7.2 on antigen presenting cells (APC) and CD28 on CD4 T by humanized anti B7.1/B7.2 monoclonal antibodies (MoAbs), we have used global gene expression profiling (GEP). Mimicking our ex vivo clinical anergization protocol, we used PBMC obtained from fully-HLA mismatched healthy volunteers (n=12 pairs) to perform an ex vivo primary MLR +/− anti B7.1/B7.2 MoAbs. CD28:B7 CSB inhibited mean proliferation by 73% after 72 hours of MLR. GEP was performed using Affymetrix hu133 plus2 chips on monocytes (Mo), CD4 and CD8 T cells, and B and NK cells purified from the MLR. Differentially expressed genes between cells from MLR +/− CSB were determined by SAM and EBAM analysis. Despite the low published frequency (1–10%) of alloAg specific CD4 T, we could detect global gene expression variance between CD4 T isolated from MLR +/− CSB (P≤0.05) suggesting effects on non-alloAg specific CD4 T. Use of the Ingenuity pathway analyzer to classify these differentially expressed genes by specific cellular pathways showed most were involved in cell proliferation and differentiation. Differences in IL27 downstream signaling molecules (DSM) in Mo and CD4 T were pronounced. IL27 is a member of IL12 cytokine family produced by APC and is a heterodimer of p28 and EBV-Induced gene 3 (EBI3). The IL27 receptor (IL27R) WSX1 is expressed on CD4 T. IL27 regulates adaptive immunity by controlling T cell proliferation, Th1 cell differentiation and IFNγ synthesis. Both p28 and EBI3 transcriptional and translational levels were decreased in Mo from MLR + CSB. Expression of STAT3, an IL27 pathway DSM was also decreased. After CD28:B7 CSB, CD4 T exhibited decreased expression of the IL27R and also inactivation of IL27 DSM including pSTAT1 and NFκB at both the transcriptional and translational levels. Consistent with a negative effect on Th1 differentiation, intracytoplasmic cytokine (ICC) analysis showed decreased expression of type 1 cytokines IL2 and IFNγ in CD4 T from MLR + CSB. ICC also showed decreased expression of the type 1 cytokine IL15 and increased expression of the type 2 cytokine IL10 in Mo from MLR + CSB. These results suggest that CD28:B7 signaling during MLR is required for Mo production of IL27. Decreased expression of IL27 and its DSMs pSTAT1 and NFκB1 after CSB with antiB7 MoAbs may contribute to CD4 T alloAg anergy induction by suppressing effector cytokines and Th1 cell differentiation. The observation that CD28:B7 modulates IL27 and IL27R emphasizes that targeted therapies used in the complex environment of human PBMC may have effects not predicted by in vitro clonal systems. Such effects may be important in the functional outcome of the intervention.

Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses. To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC. Semi-allogeneic DC were generated from the F(1) progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8(+) and CD4(+) T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo. In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8(+) OT-I transgenic T-cells, primed naïve CD4(+) OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4(+) response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival. Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.

Elevation of Intracellular Cyclic AMP in Alloreactive CD4 + T Cells Induces Alloantigen-Specific Tolerance That Can Prevent GVHD Lethality In Vivo

Cyclic AMP (cAMP) is an important negative regulator of T cell activation, and an increased level of cAMP is associated with T cell hyporesponsiveness in vitro. We sought to determine whether elevating intracellular cAMP levels ex vivo in alloreactive T cells during primary mixed lymphocyte reactions (MLR) is sufficient to induce alloantigen-specific tolerance and prevent graft-versus-host disease (GVHD). Primary MLRs were treated with exogenous 8Br-cAMP and IBMX, a compound that increases intracellular cAMP levels by inhibition of phosphodiesterases. T cell proliferation and IL-2 responsiveness in the treated primary MLR cultures were greatly reduced, and viable T cells recovered on day 8 also had impaired responses to restimulation with alloantigen compared to control-treated cells, but without an impairment to nonspecific mitogens. Labeling experiments showed that cAMP/IBMX inhibited alloreactive T cell proliferation by limiting the number of cell divisions, increasing susceptibility to apoptosis, and rendering nondeleted alloreactive T cells hyporesponsive to alloantigen restimulation. cAMP/IBMX-treated CD4 + T cells had a markedly reduced capacity for GVHD lethality in major histocompatibility complex class II disparate recipients, but maintained the capacity to mediate other CD4 + T cell responses in vivo. Thus, our results provide the first preclinical evidence of using cAMP-elevating pharmaceutical reagents to achieve long-term alloantigen-specific T cell tolerance that is sufficient to prevent GVHD.

Granulocyte–macrophage colony‐stimulating factor drives monocytes to CD14low CD83+ DCSIGN– interleukin‐10‐producing myeloid cells with differential effects on T‐cell subsets

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has long been found to have growth-promoting effects on multipotent haematopoietic lineages, specifically granulocytes and macrophages. GM-CSF combined with interleukin-4 (IL-4) drives monocytes to become myeloid dendritic cells (mDCs) in vitro. We report that culturing human monocytes with GM-CSF alone generates myeloid cells (GM-Mono) that have lower expression of CD14 than monocytes and that fail to express DC-SIGN. GM-Monos, however, express CD83 and the transcription factor PU.1, although at a lower level than the conventional mDCs generated in the presence of GM-CSF and IL-4. On stimulation with tumour necrosis factor-alpha, interferon-gamma and anti-CD40 monoclonal antibody, the GM-Monos predominantly produced IL-10 but were less efficient in IL-12 production. In a primary allogeneic mixed lymphocyte reaction, GM-Monos induced hyporesponsiveness and IL-10-biased cytokine production in CD4(+) T cells. In fresh mixed lymphocyte reaction, GM-Monos inhibited conventional mDC-induced allogeneic CD4(+) T-cell proliferation. GM-Mono-induced inhibition of allogeneic CD4(+) T-cell proliferation was partially attributed to IL-10. Interestingly, GM-Monos neither induced hyporesponsiveness in allogeneic CD8(+) T cells nor inhibited conventional mDC-induced allogeneic CD8(+) T-cell proliferation. Taken together, we characterize monocyte-derived CD14(low) CD83(+) cells generated by GM-CSF that can induce tolerance or stimulation of T cells depending on T-cell subsets.

DnIKK2-Transfected Dendritic Cells Induce a Novel Population of Inducible Nitric Oxide Synthase???Expressing CD4+CD25??? Cells with Tolerogenic Properties

We previously documented that rat bone marrow-derived dendritic cells (DCs), transfected with an adenovirus encoding a dominant negative form of IKK2 (dnIKK2), have impaired allostimulatory capacity and generate CD4 T cells with regulatory function. Here we investigate the potency, the phenotype, and the mechanism of action of dnIKK2-DC-induced regulatory cells and we evaluated their tolerogenic properties in vivo. Brown Norway (BN) transfected dnIKK2-DCs were cultured with Lewis (LW) lymphocytes in primary mixed lymphocyte reaction (MLR). CD4 T cells were purified from primary MLR and incubated in secondary coculture MLR with LW lymphocytes. Phenotypic characterization was performed by fluorescence-activated cell sorting and real-time polymerase chain reaction. The tolerogenic potential of CD4 T cells pre-exposed to dnIKK2-DCs was evaluated in vivo in a model of kidney allotransplantation. CD4 T cells pre-exposed to dnIKK2-DCs were CD4CD25 and expressed interleukin (IL)-10, transforming growth factor-beta, interferon-gamma, IL-2, and inducible nitric oxide synthase (iNOS). These cells (dnIKK2-Treg), cocultured (at up to 1:10 ratio) with a primary MLR, suppressed T-cell proliferation to alloantigens. The regulatory effect was cell-to-cell contact-independent since it was also observed in a transwell system. A nitric oxide synthase inhibitor significantly reverted dnIKK2-Treg-mediated suppression, whereas neutralizing antibodies to IL-10 and TGF-beta had no significant effect. DnIKK2-Treg given in vivo to LW rats prolonged the survival of a kidney allograft from BN rats (the donor rat strain used for generating DCs). DnIKK2-Treg is a unique population of CD4CD25 T cells expressing high levels of iNOS. These cells potently inhibit T-cell response in vitro and induce prolongation of kidney allograft survival in vivo.