Validation of an ICH Q2 Compliant Flow Cytometry-Based Assay for the Assessment of the Inhibitory Potential of Mesenchymal Stromal Cells on T Cell Proliferation.

Mesenchymal stromal cells (MSCs) have the potential to suppress pathological activation of immune cells and have therefore been considered for the treatment of Graft-versus-Host-Disease. The clinical application of MSCs requires a process validation to ensure consistent quality. A flow cytometry-based mixed lymphocyte reaction (MLR) was developed to analyse the inhibitory effect of MSCs on T cell proliferation. Monoclonal antibodies were used to stimulate T cell expansion and determine the effect of MSCs after four days of co-culture based on proliferation tracking with the violet proliferation dye VPD450. Following the guidelines of the International Council for Harmonisation (ICH) Q2 (R1), the performance of n = 30 peripheral blood mononuclear cell (PBMC) donor pairs was assessed. The specific inhibition of T cells by viable MSCs was determined and precision values of <10% variation for repeatability and <15% for intermediate precision were found. Compared to a non-compendial reference method, a linear correlation of r = 0.9021 was shown. Serial dilution experiments demonstrated a linear range for PBMC:MSC ratios from 1:1 to 1:0.01. The assay was unaffected by PBMC inter-donor variability. In conclusion, the presented MLR can be used as part of quality control tests for the validation of MSCs as a clinical product.

Evaluation of alloreactive T cells based on the degree of MHC incompatibility using flow cytometric mixed lymphocyte reaction assay in dogs.

It has become anticipated that regenerative medicine will extend into the field of veterinary medicine as new treatments for various disorders. Although the use of allogeneic stem cells for tissue regeneration is more attractive than that of autologous cells in emergencies, the therapeutic potential of allogeneic transplantation is often limited by allo-immune responses inducing graft rejection. Therefore, a methodology for quantifying and monitoring alloreactive T cells is necessary for evaluating allo-immune responses. The mixed lymphocyte reaction (MLR) is widely used to evaluate T cell alloreactivity. In human, flow cytometric MLR with carboxyfluorescein diacetate succinimidyl ester has been established and used as a more useful assay than conventional MLR with radioisotope labeling. However, the available information about alloreactivity based on the differences of dog major histocompatibility complex (MHC) (dog leukocyte antigen, DLA) is quite limited in dog. In this paper, we describe our established flow cytometric MLR method that can quantify the T cell alloreactivity while distinguishing cell phenotypes in dog, and T cell alloreactivity among DLA-type matched pairs was significantly lower than DLA-mismatched pairs, suggesting that our developed flow cytometric MLR method is useful for quantifying T cell alloreactivity. In addition, we demonstrated the advantage of DLA homozygous cells as a donor (stimulator) for allogeneic transplantation. We also elucidated that the frequency of alloreactive T cell precursors was almost the same as that of mouse and human (1-10%). To our knowledge, this is the first report to focus on the degree of allo-immune responses in dog based on the differences of DLA polymorphisms.

Alloanergization Method for Inducing Allospecific Hyporesponsiveness in PBMC Exposed to Allostimulation In Vitro in the Context of Costimulatory Molecule Blockade.

Alloantigen-specific hyporesponsiveness can be induced in alloreactive T cells contained within the whole peripheral blood mononuclear cell (PBMC) population by stimulating these responder cells ex vivo with HLA-mismatched stimulator PBMC as the antigen presenting cell (APC) source, in the presence of a CD28 costimulation blocking agent. As a result of this approach, specific alloreactivity is markedly decreased (by 1-2 logs), but third-party alloresponses and in vitro responses relying on the activation of pathogen- and tumor-associated antigen T-cell functional activities are not globally impinged upon (Guinan et al. N Engl J Med 340(22):1704-1714, 1999, Davies et al. Transplantation 86(6):854-864, 2008, Davies et al. Cell Transplant 21(9):2047-61, 2012). This method has been used clinically to alloanergize bone marrow and PBMC allografts, creating ex vivo cell therapies for adoptive transfer to blood cancer patients at high risk of disease relapse whose best option was to receive haploidentical hematopoietic cell transplants. These early phase trials consisting of, or containing, alloanergized T-cell infusions show promise in reducing graft-versus-host disease (GvHD), providing more rapid immune reconstitution, and decreasing severe post-transplant infectious complications and disease relapse. Herein, we describe this straightforward technique for generating alloanergized PBMC as it is performed in the research lab setting using belatacept for CD28-mediated costimulatory blockade (CSB) and PBMC isolated by Ficoll Hypaque gradient centrifugation as responders and APC. We also describe methods for evaluating subsequent alloproliferation to first and third party stimulation as well as assessment of cell division, pathogen-specific immunity, or allosuppression. The technique has successfully been transferred to collaborating labs, largely owing to the flexibility of using fresh or frozen PBMC, the lack of a requirement for specially isolated APC populations, and the ability to scale up or scale down the cell numbers that are to be anergized.

Paramunity-inducing Factors (PINDs) in dendritic cell (DC) cultures lead to impaired antileukemic functionality of DC-stimulated T-cells.

INTRODUCTION: Paramunity-inducing-Factors (PINDs) consist of attenuated/inactivated viruses of various poxvirus-genera, used in veterinary medicine as non-antigen-specific, non-immunising stimulators of the innate immune system against infectious and malignant diseases. Their danger-signaling-interactions were tested for their capacity to improve leukemic antigen-presentation on DC generated from AML-patients' blasts ('DCleu') and DC-stimulation/activation of antileukemic T-cells. METHODS: We analyzed, whether the addition of PINDs during DC cultures (15 healthy, 22 leukemic donors) and mixed lymphocyte culture (MLC, n = 15) with autologous (n = 6), allogeneic (n = 2) or T-cells after stem cell transplantation (SCT; n = 7) would alter the quality and quantity of DC, the composition of T-cell-subsets, and/or their antileukemic functionality (AF) as studied by FACS and functional Fluorolysis-cytotoxicity-assays. RESULTS: Effects on 1. DC-cultures: PINDs in DC-cultures lead to increased proportions of mature DC and DCleu, but reduced proportions of viable and overall, as well as TLR4- and TLR9-expressing DC. 2. MLC: PINDs increased early (CD8+) T-cell activation (CD69+), but reduced proportions of effector-T-cells after MLC 3. AF: Presence of PINDs in DC- and MLC-cultures reduced T-cells' as well as innate cells' antileukemic functionality. 4. Cytokine-release profile: Supernatants from PIND-treated DC- and MLC-cultures resembled an inhibitory microenvironment, correlating with impaired blast lysis. CONCLUSIONS: Our data shows that addition of PINDs to DC-cultures and MLC result in a "blast-protective-capacity" leading to impaired AF, likely due to changes in the composition of T-/innate effector cells and the induction of an inhibitory microenvironment. PINDs might be promising in treating infectious diseases, but cannot be recommended for the treatment of AML-patients due to their inhibitory influence on antileukemic functionality.

Induction of PIR-A/B+ DCs in the in vitro inflammatory condition and their immunoregulatory function.

BACKGROUND: Dendritic cells (DCs), primary antigen-presenting cells, are now well known as an immunoregulator of many aspects of immune responses including inflammatory bowel diseases (IBDs) such as Crohn's disease and ulcerative colitis. We have reported that PIR-A/Bhigh cDCs (conventional DCs) appeared in dextran sodium sulfate (DSS)-induced colitis and serve as a negative immunoregulator in an animal model of IBD. The immunoregulatory role of PIR-A/B+ cDCs was confirmed in both an in vitro culture system and an in vivo transfer experiment. Here, we have investigated the differentiation process of PIR-A/B+ cDCs in an in vitro inflammatory environment and examined their functions. METHODS: cDCs were isolated from the large intestinal lamina propria from C57BL/6 mice and cultured in an inflammatory environment (IL-1, IL-6, TNFα, and LPS). The appearance of PIR-A/B+ cDCs was determined after 24 h, and the in vitro-induced PIR-A/B+ cDCs were functionally and genetically examined. RESULTS: PIR-A/B+ cDCs were detected after a 24-h culture only in the inflammatory environment, and the cells acted as a negative immunoregulator when examined in an allogenic mixed leukocyte reaction (MLR). The message level of IL-27 was highly upregulated in PIR-A/B+ cDCs, while that of high mobility group box 1 protein (HMGB1) was downregulated in these cells. This was well in accordance with the fact that PIR-A/B+ cDCs showed a suppressive function against activated T cells. We found that PIR-A/B+ cDCs produced IL-27, as verified by an ELISA assay, and that the inhibitory effect by PIR-A/B+ cDCs was, at least partially, due to IL-27. Furthermore, CD85d+ cells, a human counterpart of mouse PIR-A/B+ cDCs, were found in the lamina propria of the colon of the patients with ulcerative colitis, but not in the similar part of the non-inflammatory area of colon specimens from patients with colon cancer. CONCLUSIONS: PIR-A/B+ cDCs induced in an in vitro inflammatory environment model showed a suppressive function against activated T cells by producing an inhibitory cytokine.

Avoiding phagocytosis-related artifact in myeloid derived suppressor cell T-lymphocyte suppression assays.

Myeloid-derived suppressor cells (MDSCs) have garnered much attention in recent years as a potential target for altering the immunosuppressive tumor microenvironment in a variety of solid tumor types. The ability to accurately assess the immunosuppressive capacity of MDSCs is fundamental to the development of therapeutic approaches aimed at disabling these immunosuppressive functions. In this article we provide evidence that the use of CD3/28 coated microbeads leads to artefactual T-lymphocyte suppression due to sequestration of beads by MDSCs isolated from the spleens of wild-type mice bearing subcutaneous syngeneic, carcinogen-induced oral cavity carcinomas. Mechanisms of this finding may include early MDSC death and acquisition of phagocytic capacity. These artefactual findings were avoided by eliminating the use of microbeads and instead using plate bound CD3/28 antibody as the T-lymphocyte stimulus. We propose model-specific validation of microbead-based MDSC assays, or use of an alternative stimulation approach such as plate bound CD3/28 antibodies.

The effect of pro-inflammatory cytokines on immunophenotype, differentiation capacity and immunomodulatory functions of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs), as cells with potential clinical utilities, have demonstrated preferential incorporation into inflammation sites. Immunophenotype and immunomodulatory functions of MSCs could alter by inflamed-microenvironments due to the local pro-inflammatory cytokine milieu. A major cellular mediator with specific function in promoting inflammation and pathogenicity of autoimmunity are IL-17-producing T helper 17 (Th17) cells that polarize in inflamed sites in the presence of pro-inflammatory cytokines such as Interleukin-1ß (IL-1ß), IL-6 and IL-23. Since MSCs are promising candidate for cell-based therapeutic strategies in inflammatory and autoimmune diseases, Th17 cell polarizing factors may alter MSCs phenotype and function. In this study, human bone-marrow-derived MSCs (BM-MSC) and adipose tissue-derived MSCs (AD-MSC) were cultured with or without IL-1ß, IL-6 and IL-23 as pro-inflammatory cytokines. The surface markers and their differentiation capacity were measured in cytokine-untreated and cytokine-treated MSCs. MSCs-mediated immunomodulation was analyzed by their regulatory effects on mixed lymphocyte reaction (MLR) and the level of IL-10, TGF-ß, IL-4, IFN-γ and TNF-α production as immunomodulatory cytokines. Pro-inflammatory cytokines showed no effect on MSCs morphology, immunophenotype and co-stimulatory molecules except up-regulation of CD45. Adipogenic and osteogenic differentiation capacity increased in CD45+ MSCs. Moreover, cytokine-treated MSCs preserved the suppressive ability of allogeneic T cell proliferation and produced higher level of TGF-ß and lower level of IL-4. We concluded pro-inflammatory cytokines up-regulate the efficacy of MSCs in cell-based therapy of degenerative, inflammatory and autoimmune disorders.

Characterization of HLA-G and Related Immunosuppressive Effects in Human Umbilical Cord Stroma-Derived Stem Cells.

Mesenchymal stem cells (MSCs) and especially those derived from fetal tissues exert a potent immunosuppressive effect that can be enhanced under inflammatory conditions. This study aimed to explore the immunosuppressive properties of human umbilical cord mesenchymal stem cells (HUCMSCs). We found that HLA-G, the nonclassical HLA allele with strong immune-inhibitory properties, was much more expressed on the HUCMSCs than on MSCs of other origins. Flow cytometry revealed that 90.8% of the HUCMSCs expressed HLA-G. RT-PCR revealed expression of HLA-G1, HLA-G5, and HLA-G7 in all of four HUCMSC lines. In a mixed lymphocyte reaction assay, the HUCMSCs inhibited the proliferation of lymphocytes by 35 ± 3% and could be reversed by treatment with an HLA-G blocking antibody. Upon coculture with the HUCMSCs, peripheral blood mononuclear cells expressed lower levels of proinflammatory mediators such as IL-6, TNF-α, and VEGF-α. This immunosuppressive effect was enhanced when the HUCMSCs were pretreated with IFN-γ, such that the expression of HLA-G was highly activated and HLA-DR diminished. The same phenomenon was not observed in MSCs derived from bone marrow or the placenta. In a xenograft rejection assay, the HUCMSCs survived in immunocompetent mice, whereas primary fibroblasts did not survive. This study confirms the HLA-G-related immunosuppressive property of HUCMSCs, which is more potent than MSCs of other origin. A good tolerance of this mesenchymal stem cell in allogeneic transplantation can thus be anticipated.

[The immunological characteristics of tonsil mesenchymal stem cells].

OBJECTIVE: To investigate the immunological characteristics of human tonsil mesenchymal stem cells (TMSCs). METHODS: Human tonsil tissues were obtained from the children patients with chronic tonsillitis. TMSCs were separated, cultured, and were detected the expression profiles of HLA-I, HLA-II, CD80, CD86 by flow cytometry. The measurement of immunogenicity, the effect on phytohemagglutinin (PHA) induced peripheral blood mononuclear cell (PBMCs) proliferation and mixed lymphocytes reaction (MLR) were performed to identify the immunological characteristics of TMSCs. The co-cultures of TMSCs + PBMCs + PHA and TMSCs + MLR were established, respectively, and the concentration of kynurenine, which is the metabolin of indoleamine 2, 3-dioxygenase, in the culture supernatant were examined. Then we added 1-methyl-L-tryptophan into the co-culture of TMSCs + PBMCs + PHA and TMSCs + MLR, respectively, and tested the proliferation of PBMCs. Each experiment was repeated three times, and there were six samples in each group. Statistical significance was assessed by analysis of variance (ANOVA), and a P value less than 0.05 was considered statistically significant. RESULTS: TMSCs expressed HLA-I, were negative for HLA-II and co-stimulatory molecules CD80 and CD86. The stimulation index in the group of TMSCs + allogeneic PBMCs was 1.38 ± 0.26, whereas the stimulation index in the group of allogeneic PBMCs was 1.22 ± 0.28, and there was no significant difference between the two groups (P > 0.05), indicating that TMSCs could not initiate the proliferation of allogeneic PBMCs. The stimulation indexes in the group of TMSCs + allogeneic PBMCs + PHA were 1.49 ± 0.29 and 1.23 ± 0.22, respectively, whereas the stimulation index in the group of allogeneic PBMCs + PHA was 4.60 ± 0.81, and the difference between the two groups had a statistical significance (P < 0.05) suggesting that TMSCs could inhibit PHA-induced PBMCs proliferation. The stimulation indexes in the group of TMSCs + MLR were 1.29 ± 0.23 and 1.26 ± 0.27, respectively, however, the stimulation index in the group of MLR was 3.04 ± 0.66, and the difference between the two groups had a statistical significance (P < 0.05), demonstrating that TMSCs could suppress MLR-induced PBMCs proliferation. The levels of kynurenine were (26.0 ± 2.3) µmol/L and (23.5 ± 4.5) µmol/L in the culture of TMSCs + PBMCs + PHA and TMSCs + MLR, respectively, thus elevating significantly. After adding of 1-methyl-L-tryptophan, TMSCs-mediated-proliferation suppression of PBMCs restored to normal levels. CONCLUSION: TMSCs possess low immunogenecity and immunosuppressive function, may be used in allogeneic transplantation.

Post-thaw non-cultured and post-thaw cultured equine cord blood mesenchymal stromal cells equally suppress lymphocyte proliferation in vitro.

Multipotent mesenchymal stromal cells (MSC) are receiving increased attention for their non-progenitor immunomodulatory potential. Cryopreservation is commonly used for long-term storage of MSC. Post-thaw MSC proliferation is associated with a lag-phase in vitro. How this lag-phase affect MSC immunomodulatory properties is unknown. We hypothesized that in vitro there is no difference in lymphocyte suppression potential between quick-thawed cryopreserved equine cord blood (CB) MSC immediately included in mixed lymphocyte reaction (MLR) and same MSC allowed post-thaw culture time prior to inclusion in MLR. Cryopreserved CB-MSC from five unrelated foals were compared using two-way MLR. For each of the five unrelated MSC cultures, paired MLR assays of MSC allowed five days of post-thaw culture and MSC included in MLR assay immediately post-thawing were evaluated. We report no difference in the suppression of lymphocyte proliferation by CB-MSC that had undergone post-thaw culture and MSC not cultured post-thaw (p<0.0001). Also, there was no inter-donor variability between the lymphocyte suppressive properties of MSC harvested from the five different donors (p = 0.13). These findings suggest that cryopreserved CB-MSC may have clinical utility immediately upon thawing. One implication hereof is the possibility of using cryopreserved CB-MSC at third party locations without the need for cell culture equipment or competencies.

Efficient generation of highly immunocompetent dendritic cells from peripheral blood of patients with hepatitis C virus-related hepatocellular carcinoma.

BACKGROUND & AIMS: Immunotherapy using dendritic cells (DCs) is a promising cancer therapy. The success of this therapy depends on the function of induced DCs. However, there has been no consensus on optimal conditions for DC preparation in vitro for immunotherapy of hepatocellular carcinoma (HCC) patients. To address relevant issues, we evaluated the procedures to induce DCs that efficiently function in hepatitis C virus (HCV)-related HCC. METHODS: We studied immunological data from 14 HCC patients. The DC preparation and the surface markers were assessed by flow cytometric analysis. Four different additional activation stimuli (Method I, medium alone; Method II, with OK-432; Method III, with IL-1ß+IL-6+TNF-α; Method IV, with IL-1ß+IL-6+TNF-α+PGE2) were tested and the functions of DCs were confirmed by examination of the ability of phagocytosis, cytokine production and allogeneic mixed lymphocyte reaction (MLR). RESULTS: The numbers of DCs induced and their cytokine production ability were not different between healthy controls and HCC patients. T-cell stimulatory activity of DCs in MLR was significantly lower in HCC patients than in healthy controls. The maturation of DCs with OK-432 boosted production of cytokines and chemokines, such as IL-2, IL-12p70, IFN-γ, TNF-α, IL-13 and MIP1α, and restored T-cell stimulatory activity of DCs in MLR. CONCLUSIONS: The clinically approved compound OK-432 is a candidate for highly immunocompetent DC preparation and may be considered as a key drug for immunotherapy of HCV-related HCC patients.

Ammonia drives dendritic cells into dysfunction.

Ammonia levels are often elevated in patients with cirrhosis or tumors. Patients with these diseases are immunocompromised. In this study, we investigated the effects of ammonia on a member of the immune cell family, the dendritic cells (DCs). Our results demonstrated that ammonia diminished cell count, phagocytosis, and lymphocyte stimulation of DCs. Ammonia also induced DC swelling, excessive reactive oxygen species production, and mitochondrial damage, which may constitute the underlying mechanism of ammonia-induced DC dysfunction. In ammonium chloride (NH4Cl)-loaded mice, DCs exhibited lowered phagocytosis and a weakened immune response to the chicken OVA vaccine. DCs from patients with cirrhosis or ammonia-treated healthy human blood both exhibited diminished phagocytosis. Moreover, tumor cell conditioned medium drove DCs into dysfunction, which could be reversed by ammonia elimination. In a murine colon carcinoma model, we found that ammonia could regulate tumor growth involving DCs and their related immune response. These findings reveal that ammonia could drive DCs into dysfunction, which contributes to the immunocompromised state of patients with cirrhosis or tumors.

Immature dendritic cells generated from cryopreserved human monocytes show impaired ability to respond to LPS and to induce allogeneic lymphocyte proliferation.

Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs), the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp)-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh)-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h) and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.

Activation-induced CD137 is a fast assay for identification and multi-parameter flow cytometric analysis of alloreactive T cells.

Detection and isolation of viable alloreactive T cells at the single-cell level requires a cell surface marker induced specifically upon T cell receptor activation. In this study, a member of the tumour necrosis factor receptor (TNFR)-family, CD137 (4-1BB) was investigated for its potential to identify the total pool of circulating alloreactive T cells. Optimal conditions for sensitive and specific detection of allogeneic-induced CD137 expression on circulating T cells were established. Thereafter, CD137(+) alloreactive T cells were phenotypically and functionally characterized by multi-parameter flow cytometry. Alloantigen-induced CD137 expression identified both alloreactive CD8(+) T cells (mean ± standard error of the mean: 0·21 ± 0·07%) and alloreactive CD4(+) T cells (0·21 ± 0·05%). CD137(+) alloreactive T cells were detected in different T cell subsets, including naive T cells, but were found preferentially in CD28(+) T cells and not in the terminally differentiated T cell subset. Upon allogeneic (re-)stimulation, the cytokine-producing as well as proliferative capacity of T cells resided mainly within the CD137-expressing fraction. About 10% of the CD137(+) alloreactive T cells produced any combination of interferon (IFN)-γ, interleukin (IL)-2 and TNF-α. Polyfunctional alloreactive T cells, defined by multiple cytokine expression, were observed infrequently. In conclusion, activation-induced CD137 expression is a fast assay allowing for detection and functional analysis of the total alloreactive T cell compartment at the single-cell level by multi-parameter flow cytometry.

Effects of tolerogenic dendritic cells generated by siRNA-mediated RelB silencing on immune defense and surveillance functions of T cells.

Dendritic cells (DCs) can initiate immune responses or induce immune tolerance, according to their level of maturation. In this study, we inhibited RelB expression in immature (im)DCs using small interfering RNA (siRNA) to maintain their immature status. RelB-silenced DCs induced donor-specific hyporesponsiveness in T cells. In contrast, T cells primed by RelB-silenced DCs maintained normal proliferation and cytokine secretion when stimulated by influenza virus antigen. Proliferation was similar between T cells stimulated with syngeneic tumor antigen or donor-specific antigen stimulation, but was significantly lower compared with T cells stimulated with influenza virus antigen. Moreover, an altered pattern of micro181a and micro155 transcriptional levels in T cells was involved in the differential regulation. This study demonstrates that RelB-silenced DCs could induce donor-specific hyporesponsiveness and slightly impair immune surveillance of T cells, while retaining their immune defense functions.

The monocytic population in chronic lymphocytic leukemia shows altered composition and deregulation of genes involved in phagocytosis and inflammation.

Macrophages reside in tissues infiltrated by chronic lymphocytic leukemia B cells and the extent of infiltration is associated with adverse prognostic factors. We studied blood monocyte population by flow cytometry and whole-genome microarrays. A mixed lymphocyte reaction was performed to evaluate proliferation of T cells in contact with monocytes from patients and normal donors. Migration and gene modulation in normal monocytes cultured with CLL cells were also evaluated. The absolute number of monocytes increased in chronic lymphocytic leukemia patients compared to the number in normal controls (792 ± 86 cells/µL versus 485 ± 46 cells/µL, P=0.003). Higher numbers of non-classical CD14(+)CD16(++) and Tie-2-expressing monocytes were also detected in patients. Furthermore, we performed a gene expression analysis of monocytes in chronic lymphocytic leukemia patients, showing up-regulation of RAP1GAP and down-regulation of tubulins and CDC42EP3, which would be expected to result in impairment of phagocytosis. We also detected gene alterations such as down-regulation of PTGR2, a reductase able to inactivate prostaglandin E2, indicating immunosuppressive activity. Accordingly, the proliferation of T cells in contact with monocytes from patients was inhibited compared to that of cells in contact with monocytes from normal controls. Finally, normal monocytes in vitro increased migration and up-regulated CD16, RAP1GAP, IL-10, IL-8, MMP9 and down-regulated PTGR2 in response to leukemic cells or conditioned media. In conclusion, altered composition and deregulation of genes involved in phagocytosis and inflammation were found in blood monocytes obtained from chronic lymphocytic leukemia patients, suggesting that leukemia-mediated "education" of immune elements may also include the establishment of a skewed phenotype in the monocyte/macrophage population.

Characterization of a novel CRAC inhibitor that potently blocks human T cell activation and effector functions.

Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC current inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4(+) T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (mixed lymphocyte reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and another calcineurin inhibitor FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC current inhibitor that potently inhibits human T cell functions.

Vascularized composite allograft rejection is delayed by intrajejunal treatment with donor splenocytes without concomitant immunosuppressants.

BACKGROUND: Mucosal or oral tolerance, an established method for inducing low-risk antigen-specific hyporesponsiveness, has not been investigated in vascularized composite allograft (VCA) research. We studied its effects on recipient immune responses and VCA rejection. METHODS: Lewis rats (n = 12; TREATED) received seven daily intrajejunal treatments of 5 × 10(7) splenocytes from semiallogeneic Lewis-Brown-Norway rats (LBN) or vehicle (n = 11; SHAM). Recipients' immune responses were assessed by mixed lymphocyte reaction (MLR) against donor antigen and controls. Other Lewis (n = 8; TREATED/VCA) received LBN hindlimb VCA and daily intrajejunal treatments of 5 × 10(7) LBN splenocytes, or LBN VCA without treatment (n = 5; SHAM/VCA), until VCAs rejected. Recipients' immune responses were characterised and VCAs biopsied for histopathology. Immunosuppressants were not used. RESULTS: LBN-specific hyporesponsiveness was induced only in treated Lewis recipients. Treatment significantly reduced MLR alloreactivity, significantly reduced VCA rejection on histopathology, and significantly delayed clinical VCA rejection (P < 0.0005; TREATED/VCA mean 9.6 versus 6.0 days for SHAM/VCA). Treatment significantly increased immunosuppressive IL-10/IL-4/TGF-ß production and significantly decreased proinflammatory IFN-γ/TNF-α. CONCLUSION: Jejunal exposure to antigen conferred donor specific hyporesponsiveness that delayed VCA rejection. This method may offer a low-risk adjunctive treatment option to help protect VCAs from rejection.

Ganglioside inhibition of CD8+ T cell cytotoxicity: interference with lytic granule trafficking and exocytosis.

Granule exocytosis-mediated cytotoxicity by CD8(+) CTL plays a crucial role in adaptive immunity to tumors and to intracellular pathogens. This T cell effector function has been shown to be defective in various murine tumor models and in human cancer. However, factors and their mechanisms that cause inhibition of CD8(+) T cell lytic function in tumor-bearing hosts remain to be fully defined. We postulate that gangliosides, highly expressed on tumor cell membranes, actively shed into the tumor microenvironment, and having well-established immunosuppressive properties, may be such a factor. We exposed primary mouse CD8(+) CTL to gangliosides derived from three sources (tumors and normal brain). This significantly inhibited cytotoxicity-mediated by granule exocytosis, that is, cytotoxicity of alloantigen-specific and polyclonal CD8(+) CTL in vitro. These molecules did not interfere with the interaction of CD8(+) T cells with their cognate targets. Rather, they inhibited lytic granule release in response both to TCR engagement and to stimuli that induce granule release in a nonpolarized manner. At the subcellular level, confocal microscopic imaging identified inhibition of polarization of lytic granules to the immunological synapse upon target cell recognition. Thus, tumor-shed gangliosides suppress lytic activity of CD8(+) T cells by a novel mechanism, that is, inhibition of trafficking of lytic granules in response to TCR engagement, as well as by interfering with the process of granule exocytosis in CD8(+) T cells.

Expression of ICOSLG on mouse hematologic neoplasm cell lines and their influence on cytotoxicity in allogeneic mixed lymphocyte reactions.

In this study, we sought to determine whether the expression of inducible co-stimulator ligand (ICOSLG) on mouse hematologic neoplasm cells influences cytotoxicity. Using flow cytometry, we examined the expression of ICOSLG in mouse hematologic neoplasm cell lines for the first time and used FBL3/A20 as targets to study. Effectors and targets were incubated at effector:target (E:T) ratios ranging from 5:1 to 50:1 for 10 h. Enzyme-linked immunosorbent assay was used to assess the expression of cytokines associated with type 2 T helper cells. We found that FBL3, A20 and P388 cells expressed ICOSLG. At E:T ratios ranging from 20:1 to 30:1, the cytotoxic activity of alloreactive T cells was significantly increased when ICOSLG was blocked (p < 0.05). We also found that ICOSLG blockade was associated with decreased expression of interleukin 4 and interleukin 10. Our results indicate that ICOSLG blockade may enhance cytotoxity in allogeneic mixed lymphocyte-hematologic neoplasm cell reactions.