Mixed Lymphocyte Culture Research Articles

Neurotoxin from Naja naja atra venom inhibits skin allograft rejection in rats.

Recent studies reported that Naja naja atra venom (NNAV) regulated immune function and had a therapeutic effect on adjunctive arthritis and nephropathy. We hypothesized that NNAV and its active component, neurotoxin (NTX), might inhibit skin allograft rejection. Skin allografts were used to induce immune rejection in rats. In addition, mixed lymphocyte culture (MLC) was used to mimic immune rejection reaction in vitro. Both NNAV and NTX were orally given starting from 5days prior to skin allograft surgery. The results showed that oral administration of NNAV or NTX prolonged the survival of skin allografts and inhibited inflammatory response. The production of Th1 cytokines (IFN-γ, IL-2) was also suppressed. NTX inhibited T-cell proliferation and CD4(+) T cell division induced by skin allografts. NTX also showed immunosuppressive activity in mixed lymphocyte culture. Atropine alone inhibited Con A-induced proliferation of T cells and potentiated NTX' s inhibitory effects on T cells, while pilocarpine only slightly enhanced Con A-induced T cell proliferation and partially reversed the inhibitory effect of NTX. On the other hand, neither nicotine nor mecamylamine had an influence on NTX's inhibitory effects on Con A-induced T cell proliferation in vitro. NTX inhibited T cell proliferation by arresting the cell cycle at the G0/G1 phase. The present study revealed that NNAV and NTX suppressed skin allograft rejection by inhibiting T cell-mediated immune responses. These findings suggest both NNAV and NTX as potential immunosuppressants for preventing the immune response to skin allografts.

NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation.

Intravesical instillation of bacillus Calmette–Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient.

Effect of SAHA on Maturation of Dendritic Cells and Its Mechanism

To investigate the effect of SAHA on the maturation of human dendritic cells (DC) and to explore its underlying mechanism. Peripheral blood mononuclear cells (PBMNC) were isolated from human peripheral blood and cultured in RPMI 1640 medium with 100 ng/ml rhGM-CSF and 500 U/ml rhIL-4. In the LPS induced maturation process, dendritic cells treated with or without SAHA were used as test group, and dendritic cells treated without LPS or SAHA were used as control group. DC was observed under inverted microscope. Flow cytometer was used to detect the surface antigen molecules expressed by DC. The mixed lymphocyte culture (MLC) was used to observe the allogeneic lymphocyte stimulation. The NF-κB signaling pathway was detected by electrophoretic mobility shift assay (EMSA). The SAHA could effectively suppress the maturation of DC induced by LPS, the DC treated with SAHA+LPS had immature morphological characteristics; the expression of CD80, CD83 and HLA-DR in SAHA+LPS group and control group were significantly down-regulated as compared with single LPS group (P<0.01); the ability of DC to stimulate the proliferation of allogeneic T lymphocytes in SAHA+LPS group and control group was significantly weaker than that in single LPS group (P<0.01); EMSA results showed that NF-κB activity decreased after SAHA and LPS treatment and was significantly lower than that of single LPS group. SAHA can effectively suppress DC maturation induced by LPS and also weaken the ability to stimulate allogeneic T lymphocyte. NF-κB signaling pathway is involved in regulating DC maturation.

Generation of tumor-infiltrating lymphocytes from pancreatic cancer lesions for cellular therapy

We investigated the influence of Panagen DNA preparations on laboratory animals and IFN-induced human dendritic cells, as well as analyzed the data from a phase II clinical trial in the therapy of breast cancer. It was shown that this treatment resulted in increased number of CD8+/perforin+ T cells in peripheral lymphoid organs of experimental animals, in mixed lymphocyte culture population and in peripheral blood of breast cancer patients. Moreover, we demonstrated that when Panagen DNA preparations are used in combination with the standard FAC-based breast cancer therapies, non-specific immune response activity remains at the same levels as observed prior to therapy, whereas in FAC-placebo patients, non-specific immunity is greatly diminished.

Dendritic cells generated with PDL-1 checkpoint blockade for treatment of advanced pancreatic cancer.

4128 Background: The efficacy of immunotherapy with monocyte derived dendritic cells (MoDC) is controlled via immune checkpoints, among them the PD-1/PDL-1 pathway. PDL-1 expressed on DC delivers an inhibitory signal to T-cells upon binding to PD-1 expressed on activated T-cells. Blocking of PDL-1 on DC may lead to improved efficacy of DC therapy for pancreatic cancer. Methods: After isolating monocytes from peripheral blood of n = 44 patients with stage IV pancreatic cancer, who failed first-line chemotherapy, antigen primed MoDC were generated using standard protocols. Patients included in the follow-up analysis received a minimum of 3 vaccines. 10 patients received MoDC modified by PDL-1 blockade after failure of previous DC therapy. PDL-1 blockade was performed by adding soluble CD80 or anti-PDL-1 to MoDC. Cytokine release and T-cell activity were measured exemplary using a mixed lymphocyte culture (MLC). Results: Median survival after onset of DC-therapy was 8 months with MoDC alone (18 months after primary diagnosis) with longest follow up of 36 months (49 months after primary diagnosis). We could induce a secondary stabilisation (4 to 8 months) in 5 from 10 patients, who failed to respond to previous DC therapy by using MoDC modified by PDL-1 blockade. Cytokine measurement using MLC, which was exemplarily performed, shows a change in the cytokine release upon PDL-1 blockade. Conclusions: An effective immune response requires both the inhibition of inhibitory signals and the activation of an antigen specific T-cell response. The efficacy of dendritic-cell based therapy may be improved by blockade of PDL-1 on dendritic cells to avoid an inhibitory signal and thus improve the T-cell specific response. Further investigations are necessary to ascertain whether the combination of systemic anti PD1-therapy with DC therapy using MoDC modified with PDL-1 blockade may further enhance therapeutic efficacy in solid tumors.

Female third party lymphocytes are effective for immunotherapy of patients with unexplained primary recurrent spontaneous abortion: A retrospective analysis of outcomes

Objectives Allogeneic lymphocytes of paternal origin or supplied by a male third party have been used for the treatment of recurrent spontaneous abortion. Few studies, however, have examined the use of female third party lymphocytes. Our purpose was to determine whether female third party lymphocytes could be used for immunotherapy of women with recurrent spontaneous abortion.Methods In this retrospective non-randomised cohort-controlled study, the medical records of patients with three or more spontaneous abortions who received immunotherapy with lymphocytes from their partner, a male third party or a female third party, as well as those who received no immunotherapy, from 1996 to 2012 were reviewed. All patients were negative for mixed lymphocyte culture reaction (MLR)-blocking antibodies. Immunotherapy was performed in 302 patients in two courses, while 53 patients received no immunotherapy.Results The pregnancy rates in patients who received lymphocytes from their partners, a male third party or a female third party, and in those not immunised, were 85.6%, 87.3%, 89.7%, and 79.3%, respectively (p = 0.523);the live birth rates were 87.3%, 75.8%, 84.6%, and 40.5%, respectively (p < 0.001).Conclusions We conclude that female third party lymphocytes can be used for immunotherapy in patients with recurrent spontaneous abortion.Chinese 摘要目的 父源同种异体淋巴细胞或第三方男性供给淋巴细胞用于复发性自然流产的治疗。少数研究调查了女性第三方淋巴细胞的应用。我们的目的是验证女性第三方淋巴细胞是否可以用于复发性自然流产的免疫治疗。方法 在这项回顾性非随机队列对照研究中回顾了自1996年至2012年,接受配偶淋巴细胞,男性第三方淋巴细胞或接受女性第三方淋巴细胞免疫治疗,或未接受免疫治疗的三次或更多次自然流产的患者病历资料。所有患者的混合淋巴细胞培养反应(MLR)-封闭抗体均为阴性。302名患者接受了两个疗程的免疫治疗,而53名患者未接受免疫治疗。结果 接受配偶淋巴细胞,男性第三方淋巴细胞,女性第三方淋巴细胞和未接受免疫治疗的患者妊娠率分别为85.6%，87.3%，89.7%，和79.3% (p = 0.523)。活产率分别为87.3%, 75.8%, 84.6%和40.5% (p < 0.001) 。结论 我们推断,女性第三方淋巴细胞可用于复发性自然流产患者的免疫治疗。关键词免疫疗法；淋巴细胞；反复自然流产

Antigen-Specific Killer Polylactic-Co-Glycolic Acid (PLGA) Microspheres Can Prolong Alloskin Graft Survival in a Murine Model

Background: The strategy of specifically depleting antigen-specific T cells can potentially be used for the treatment of allograft rejection and autoimmunity because it does not suppress the overall immune systems.Methods: In this study, we generated killer polylactic-co-glycolic acid (PLGA) microspheres by covalently coupling major histocompatibility complex (MHC) class I antigens and apoptosis-inducing anti-Fas monoclonal antibody (mAb) onto PLGA microspheres. A modified double-emulsion method was used for the preparation of cell-sized PLGA microspheres. H-2Kb/peptide monomers were generated in-house and analyzed through flow cytometry. The killer PLGA microspheres were administered intravenously into BALB/c mice (H-2Kd) that had previously been grafted with skin squares from C57BL/6 mice (H-2Kb). Tumor cell challenge and third-party mixed lymphocyte culture were used to assess the general immune functions of host.Results: The alloskin graft survival was prolonged by 4 days. The killer PLGA microspheres could specifically deplete the H-2Kb alloantigen-reactive CD8+ T cells that infiltrated into the alloskin graft but not CD4+ T cells, without impairment of host overall immune function.Conclusions: Here, we initially report that PLGA microspheres, which have been widely used as medicine-delivering carriers, were used to prepare antigen-specific killer complexes and treat allograft rejection. Our data highlight the therapeutic potential of this biocompatible and biodegradable antigen-specific killer effector for the treatment of allograft rejection and autoimmune disease.

Stronger T-Cell Alloreactivity and Diminished Suppressive Capacity of Peripheral Regulatory T Cells in Infertile Women Undergoing In Vitro Fertilization.

Increasing evidence suggests modulation of the maternal immune response to be essential for successful pregnancy. We studied the immunophenotypic profile and function of peripheral blood T lymphocytes in infertile women undergoing in vitro fertilization (IVF) and fertile control population. We collected peripheral blood mononuclear cells (PBMC) from infertile patients with recurrent implantation failure (RIF), infertile patients with successful IVF (IVFs), and normal fertile women. Cells were phenotypically analyzed, and the proliferative response and cytokine production were studied in mixed lymphocyte cultures (MLC), using lymphocytes of the own partner, or a third-party male as stimulators cells. To examine the suppressive capacity of regulatory regulatory T cells (Tregs), we performed MLC studies with a CD45(+) fraction depleted for CD4(+) CD25(bright) T cells. No significant differences in proportions of subsets of circulating T lymphocytes were observed. The proliferative allo-immune response of PBMC of IVF women (RIF and IVFs) was significantly higher, with higher production of T-helper cells (Th1) and Th2 cytokines, compared to the fertile women. This difference in proliferation and cytokine production was associated with a diminished suppressive capacity of Tregs in these women. The higher allo-immune response of the IVF women compared to fertile women might be the result of a diminished suppressive capacity of Tregs and emphasizes the important role of Tregs during conception.

Gene expression changes in HLA mismatched mixed lymphocyte cultures reveal genes associated with allorecognition.

Human leucocyte antigen (HLA) compatibility is the main factor determining the occurrence of graft‐vs‐host disease (GVHD) in patients. It has also been shown that minor histocompatibility antigen differences as well as genetic polymorphisms that are not sequenced by standard methodology for HLA typing can play a role. We used mixed lymphocyte cultures (MLCs) as a functional cellular test and investigated gene expression changes driven by HLA incompatibility in an effort to better understand the mechanisms involved in the disease. Gene expression profile of HLA matched and HLA mismatched MLC identified differentially regulated genes and pathways. We found that a great number of genes related to immune function were differentially regulated; these genes were also found to be associated with GVHD and graft rejection. The majority of differentially regulated genes were interferon‐gamma (IFNγ)‐inducible genes and IFNγ neutralisation in MLCs abrogated their induction. The microRNA‐155, a recently identified target for acute GVHD (aGVHD), was also found to be significantly induced in HLA mismatched MLC but not in the matched setting and its induction was not diminished by blocking IFNγ. In this proof‐of‐principle study we show gene expression changes in mismatched MLC that represent alloreactive responses, correlate with markers involved in GVHD and can potentially be useful in the study of the biological processes involved in this disease.

Recipient Myd88 Deficiency Promotes Spontaneous Resolution of Kidney Allograft Rejection.

The myeloid differentiation protein 88 (MyD88) adapter protein is an important mediator of kidney allograft rejection, yet the precise role of MyD88 signaling in directing the host immune response toward the development of kidney allograft rejection remains unclear. Using a stringent mouse model of allogeneic kidney transplantation, we demonstrated that acute allograft rejection occurred equally in MyD88-sufficient (wild-type [WT]) and MyD88(-/-) recipients. However, MyD88 deficiency resulted in spontaneous diminution of graft infiltrating effector cells, including CD11b(-)Gr-1(+) cells and activated CD8 T cells, as well as subsequent restoration of near-normal renal graft function, leading to long-term kidney allograft acceptance. Compared with T cells from WT recipients, T cells from MyD88(-/-) recipients failed to mount a robust recall response upon donor antigen restimulation in mixed lymphocyte cultures ex vivo. Notably, exogenous IL-6 restored the proliferation rate of T cells, particularly CD8 T cells, from MyD88(-/-) recipients to the proliferation rate of cells from WT recipients. Furthermore, MyD88(-/-) T cells exhibited diminished expression of chemokine receptors, specifically CCR4 and CXCR3, and the impaired ability to accumulate in the kidney allografts despite an otherwise MyD88-sufficient environment. These results provide a mechanism linking the lack of intrinsic MyD88 signaling in T cells to the effective control of the rejection response that results in spontaneous resolution of acute rejection and long-term graft protection.

The Src tyrosine kinase Lck binds to CD2, CD4-1, and CD4-2 T cell co-receptors in channel catfish, Ictalurus punctatus

The binding of the lymphocyte specific protein tyrosine kinase (Lck) to T cell co-receptors is required for T cell development and activation. In mammals, Lck initiates signal transduction by binding to CD4 and CD8 co-receptors and phosphorylating ITAMs in the cytoplasmic tail of the CD3 molecules and the ζ chains. In addition, Lck can also bind to the adhesion molecule CD2 and trigger T cell activation. In this study, Lck and CD2 homologs were identified and characterized in channel catfish, Ictalurus punctatus. Lck and CD2 mRNAs were specifically expressed by clonal T cell lines, including both CD4+ and CD4−CD8− CTL lines, and in mixed lymphocyte cultures (MLC). Western blot analyses using anti-trout Lck and anti-human p-Lck antibodies demonstrated that Lck protein is expressed in catfish clonal CTL and is phosphorylated at a conserved tyrosine residue. Because of the lack of CD8+ CTL lines as well as the absence of CD8 message in MLC, we performed magnetic bead binding assays to correlate CD2, CD4, and CD8 co-receptor expression with Lck binding ability. Recombinant Lck reproducibly bound to CD2, CD4-1, and CD4-2, but not to CD8α or CD8β. These data provide one possible explanation for the apparent low numbers of CD8+ CTL and the presence of CD4+ and CD4−CD8−CD2+ CTL in catfish.

Preclinical characterization of DUOC-01, a cell therapy product derived from banked umbilical cord blood for use as an adjuvant to umbilical cord blood transplantation for treatment of inherited metabolic diseases

Background aimsCord blood (CB) transplantation slows neurodegeneration during certain inherited metabolic diseases. However, the number of donor cells in the brain of patients does not appear to be sufficient to provide benefit until several months after transplant. We developed the cell product DUOC-01 to provide therapeutic effects in the early post-transplant period. MethodsDUOC-01 cultures initiated from banked CB units were characterized by use of time-lapse photomicroscopy during the 21-day manufacturing process. Antigen expression was measured by means of flow cytometry and immunocytochemistry; transcripts for cytokines and enzymes by quantitative real-time polymerase chain reaction; activities of lysosomal enzymes by direct biochemical analysis; alloreactivity of DUOC-01 and of peripheral blood (PB) mononuclear cells (MNC) to DUOC-01 by mixed lymphocyte culture methods; and cytokine secretion by Bioplex assays. ResultsDUOC-01 cultures contained highly active, attached, motile, slowly proliferating cells that expressed common (cluster of differentiation [CD]11b, CD14 and Iba1), M1 type (CD16, inducible nitric oxide synthase), and M2-type (CD163, CD206) macrophage or microglia markers. Activities of 11 disease-relevant lysosomal enzymes in DUOC-01 products were similar to those of normal PB cells. All DUOC-01 products secreted interleukin (IL)-6 and IL-10. Accumulation of transforming growth factor-β, IL-1β, interferon-γ and TNF-α in supernatants was variable. IL-12, IL-2, IL-4, IL-5 and IL-13 were not detected at significant concentrations. Galactocerebrosidase, transforming growth factor-β and IL-10 transcripts were specifically enriched in DUOC-01 relative to CB cells. PB MNCs proliferated and released cytokines in response to DUOC-01. DUOC-01 did not proliferate in response to mismatched MNC. ConclusionsDUOC-01 has potential as an adjunctive cell therapy to myeloablative CB transplant for treatment of inherited metabolic diseases.

B-Cells induce regulatory T cells through TGF-β/IDO production in A CTLA-4 dependent manner

A number of studies have suggested that B cell mediated-regulation contributes to the establishment of immunological tolerance. However, the precise mechanisms by which regulatory B cells establish and maintain tolerance in humans remain to be determined. The objective of the current study is to understand the cellular and molecular bases of B-cell regulatory functions in humans. To describe the mechanisms regulating the functional plasticity of regulatory B cells, we used an in vitro co-culture model based on autologous mixed lymphocyte cultures involving freshly isolated B and T cells. The results show that activated B cells regulate T cell proliferation through producing transforming growth factor (TGF)-β and indoleamine 2,3-dioxygenase (IDO). The production of TGF-β and IDO leads to the induction of not only “natural” regulatory T cells but also of TGF-β-producing CD4+ T cells and IL-10-producing regulatory T cells. Furthermore, we evidenced for the first time that CTLA-4 induces B-cells to produce IDO and to become effective induced regulatory B cells (iBregs). This study emphasizes a novel regulatory axis and open news insights in how to manage regulatory B cell functions in autoimmunity.

ITOC2 – 014. Immunomodulation of blasts in AML-patients (AML-pts) with clinically approved response modifiers to improve antileukemic T-cell reactivity: An ex vivo simulation of the clinical

Antileukemic T-cell-reactivity has to be improved in AML-pts. Leukaemia-derived DC (DCleu) are most effective antileukemic T-cell-stimulators. We tested combinations of clinically approved immune modifiers to convert blasts into a ‘DCleu-vaccine', that induces antileukemic T-cell (memory) without induction of blast proliferation in 52 AML-samples. 11 KITS with combinations of seven clinically approved compounds (GMCSF, INTRON, CALCIMYCIN, PICIBANIL, PGE1,2, TNF<i>α</i>) tested in Whole Blood (‘WB') culture system to generate DCleu, to induce (unwanted) blast proliferation and to stimulate antileukemic activity of pts' T-cells. 1. Generation of DC, DCleu from WB: seven of 11 Kits produced DCleu comparable to standard methods. 2. Some KITS induced ex vivo blast proliferation in individual pts' WB. 3. Best five of 11 blast modulating KITs evaluated by ranking. 4. Antileukemic reactivity of T-cells stimulated with KIT-treated WB was improved compared to controls. 5. Blast lysis correlated with proportions of DC-subtypes (DC, DCleu), CD8+ Tcells after mixed lymphocyte culture and concentrations of IL-12, IFNg, IL-6 and −8 in culture supernatants. <i>Conclusion:</i> KITs can create DCleu from blasts in WB cultures. Blast-stimulating potential and capacity to generate antileukemic T-cells can be evaluated. A ranking based on DC, DCleu subtypes, antileukemic reactivity and cytokine profiles should be created to allow an individualised selection of, best KIT' for <i>in vivo</i> applications. (Patent pending).

Liver Transplantation for Hepatic Graft-Versus-Host-Disease: A United Network for Organ Sharing (UNOS) Database Study

rabbit anti-human CD83 antibodies prevented GVHD in mouse and human preclinical alloHCT models. A second generation human mAb 3C12C has been evaluated as a novel therapeutic (Seldon et al, in prep) and its ability to deplete activated DC and preserve protective T cell responses was investigated. Methods: Control human IgG1 (trastazumab) and 3C12C mAb were tested in human peripheral blood mononuclear cell (PBMC) cultures and in allogeneic mixed lymphocyte cultures (alloMLC) for their ability to deplete DC. The antibody was tested in a human xenogeneic TBI and anti-NK conditioned SCID mouse model of GVHD. Survival, clinical scoring and histology were used to document the effect of 3C12C compared to the control mAb. Flow cytometry and immunohistology were used to investigate CD83 induction and the effect of 3C12C on human DC and T cell biology. Results: 3C12C but not the control mAb reduced the number of CD83+ DC in PBMC cultures. The 3C12C mAb reduced T cell proliferation in the alloMLC but did not affect Cytomegalovirus specific CD8+ T cell numbers. Human cells were identified in the liver, lung, spleen and gut of the control mAb treated PBMC transplanted mice, which developed histological GVHD d+8-13. Human DC were activated by d+2 and expressed the CMRF-44 activation marker plus CD83, CD80 and CD86. Treatment with 3C12CmAb eliminated CD83+ CMRF44+ DC early post-transplant and reduced T cell activation in terms of CD25, CD69 and CD137 and cytokine expression but maintained Treg cells. Conclusion: A potential therapeutic human anti-CD83 mAb appears to induce significant immunosuppression, associated with the depletion of CMRF-44+ activated DC, whilst preserving T cell numbers.

Research on Preparation of Human Immune Cell &lt;em&gt;in vitro&lt;/em&gt; with Response to Shrimp Allergen

Shrimp is one of the most important food allergens. Tropomyosin is its major allergen. Wherein Pen a 1, contains five antibody binding regions, has been identified as the only major shrimp allergen. However, the study on IgE with response to shrimp allergen is still a serious lack, compared with the allergenic proteins. Particularly in the aspects of the preparation of IgE in vitro, it is restricted and can only obtain the complete IgE molecules by polyclonal or monoclonal technology. As for the preparation of small molecule IgE to the shrimp allergen has not yet been reported. This study attempts to carry out research on obtaining of cell materials that are used to clone. It sets up a convenient and efficient immune system in vitro which combines dendritic cell differentiation, allergens immune, mixed lymphocyte culture and so on. Finally the system successfully activates the proliferation of specific B cells and the secretion of a large number of specific IgE antibodies to shrimp allergen.

Impairments of Antigen-Presenting Cells in Pulmonary Tuberculosis.

The phenotype and functional properties of antigen-presenting cells (APC), that is, circulating monocytes and generated in vitro macrophages and dendritic cells, were investigated in the patients with pulmonary tuberculosis (TB) differing in lymphocyte reactivity to M. tuberculosis antigens (PPD-reactive versus PPD-anergic patients). We revealed the distinct impairments in patient APC functions. For example, the monocyte dysfunctions were displayed by low CD86 and HLA-DR expression, 2-fold increase in CD14+CD16+ expression, the high numbers of IL-10-producing cells, and enhanced IL-10 and IL-6 production upon LPS-stimulation. The macrophages which were in vitro generated from peripheral blood monocytes under GM-CSF were characterized by Th1/Th2-balance shifting (downproduction of IFN-γ coupled with upproduction of IL-10) and by reducing of allostimulatory activity in mixed lymphocyte culture. The dendritic cells (generated in vitro from peripheral blood monocytes upon GM-CSF + IFN-α) were characterized by impaired maturation/activation, a lower level of IFN-γ production in conjunction with an enhanced capacity to produce IL-10 and IL-6, and a profound reduction of allostimulatory activity. The APC dysfunctions were found to be most prominent in PPD-anergic patients. The possible role of APC impairments in reducing the antigen-specific T-cell response to M. tuberculosis was discussed.

Description of New More Effective Photochemotherapeutic Compounds for Improving Effectiveness of Extracorporeal Photopheresis

Introduction: Extracorporeal photopheresis (ECP) is a clinically used cell-based immunomodulatory therapy that involves exposure of peripheral blood autologous mononuclear cells to the photosensitizer 8-methoxypsoralen (8-MOP) and UVA radiation before reinfusion. ECP has demostrated efficacy in the treatment of multiple diseases caused by unregulated T cell populations such as graft-versus-host disease (GvHD). During UVA irradiation, 8-MOP covalently binds to DNA, inducing apoptosis. It has been previously reported that 8-MOP-mediated apoptosis triggers some immumodulatory effects that finally lead to the acquisition of peripheral immunotolerance, including the generation of tolerogenic dendritic cells and T regulatory cells (Treg), production of anti-inflammatory cytokines such as IL-10 and TGF-β and the deletion of effector CD4+ and CD8+ T cells in lymph nodes, mechanisms that account for the clinical benefit of this therapy. In the present work the in vitro activity and immunomodulatory effects mediated by 8-MOP in comparison with other two new photosensitizer (BB01 and BB02) has been analyzed, aiming to find novel more effective photochemotherapeutic compounds to use in ECP. Moreover, the therapeutic effectiveness of BB01 and BB02 by ECP was also evaluated in an experimental murine model of GvHD.Methods: Human mononuclear cells (MNC) were incubated with increasing concentrations of 8-MOP, BB01 and BB02 and irradiated with UVA light. The MNC apoptosis percentage was measured by flow cytometry after 48h of culture at 37ºC. Also, mixed lymphocyte cultures (MLC) with either myeloid immature (iDC) or mature dendritic cells (mDC) and MNC treated with the different compounds and UVA radiation were performed. After 48h of MLC, CCL21-stimulated migration of iDC and mDC was studied. In other assays, after 6 days of MLC the proliferation of treated MNC, production of pro-inflammatory and anti-inflammatory cytokines, generation of tolerogenic DC and differentiation of Treg were analyzed. On the other hand, murine GvHD was induced after transplanting bone marrow cells and splenocytes from donor Balb/c mice into C57Bl/6J recipients previously conditioned with a lethal dose of 10 Gy. For doing the ECP procedure, splenocytes from separate cohorts of C57Bl/6J with ongoing GvHD were isolated 12 days after transplantation, incubated with the different compounds and UVA light and injected intravenously once a week for four weeks. Survival after transplantation was monitored daily and clinical GvHD was graded using a previously described score analyzing weight loss, posture, activity, skin integrity and fur texture.Results: There was a significative increase of MNC apoptosis when usingBB02 (from 50 ng/ml, p<0,001) and BB01 (from 200 ng/ml, p<0,05) over that achieved using 8-MOP, as well as a significant upregulation of CCL21-promoted migration of iDC and lower migration of mDC, mainly with BB02 (p<0,05 and p<0,001, respectively). Moreover, MNC proliferation after MLC with allogeneic iDC or mDC was partially inhibited after treatment with these compounds (p<0,01), together with a significative reduction of levels of pro-inflammatory cytokines (p<0,01, p<0,001) and increase of anti-inflammatory IL-10 and TGF-β (p<0,01, p<0,001). In addition, analysis of the expression profile of DC’s maturation and co-stimulatory/MHC-II molecules, as well as modulation in CCR7 expression after MLC demonstrated a more tolerogenic phenotype. Also, iDC co-cultured with MNC pretreated with BB02 were more efficient than 8-MOP in the induction of autologous Treg (1,88-fold increase). Finally, mice treated weekly with BB02 showed a significant higher survival than those treated with 8-MOP (p<0,05), while BB01 had a similar effect to that of 8-MOP. Mice treated with either compound improved their clinical GvHD score compared to untreated mice group, being significantly lower with BB02 than with BB01 and 8-MOP (p<0,05).Conclusions: BB01 and specially BB02 showed higher in vitro activity and in vivo effectiveness and could be considered as a possible better therapeutic alternative than 8-MOP.Acknowledgments: Work financed by the Spanish Ministry of Economy and Competitiveness (Grant BFU2010-19599), together with FEDER Funds, and Spanish Net of Cell Therapy (TerCel) from Institute of Health Carlos III (RD07/0010/2012 and RD12/0019/0001). DisclosuresNo relevant conflicts of interest to declare.

Glycolytic Inhibitor 3-Brpa Inhibits Proliferation and Induces Apoptosis of Mouse Splenic Lymphocytes in Mixed Lymphocytes Culture

Background: T-cell activation plays a critical role in the pathogenesis of acute graft-versus-host disease (GVHD). Quiescent T cells utilize oxidative phosphorylation to generate ATP, whereas activated T cells utilize glycolysis, so use glycolysis inhibitor may be a metabolically regulator needed to control T cells induced GVHD. The mixed lymphocytes culture (MLC) was used as a model to evaluate the effect of treatment for GVHD in vitro. Glucolysis inhibitor 3-Bromopyruvic acid (3-BrPA), a glucolysis inhibitor, can effectively induce multidrug resistance leukemia cell lines apoptosis and enhanced chemotherapy-induced cytotoxity to leukemia cells.Objective : This study aimed to study the effects of glycolytic inhibitor 3-Bromopyruvate (3-BrPA) on the proliferation, the apoptosis, the T lymphocyte subsets and the contents of cytokine IL-4 and IFN-γ in mouse spleen cells harvested from mixed lymphocyte culture.Methods An one-way mixed lymphocyte culture system characterized by labeled responder cells with BALB/c mouse spleen cells (H-2kd) and stimulator cells with C57BL/6 mouse spleen cells (H-2kb) was established. With treatment of 3-BrPA at different concentrations (0-200 μmol/L), the CCK-8 method was applied for lymphoproliferation activity, flow cytometry for cell surface markers of CD3, CD4 and CD8, and ELISA method for the levels of cytokine IL-4 and IFN-γ in the supernatant.Results: The CCK-8 test revealed that 3-BrPA in middle or high concentrations (over IC 30, 20 μmol/L) displayed a dose-dependent inhibitory effect on T-cell proliferation of MLC system. The IC50 were 48.6、41.2 and 41.9 μmol/L after 24 h, 36 h and 48 h of culture, respectively. FCM test discovered that the inhibitory effect mainly occurred in the CD4+ cells. After 48 h of culture, the apoptosis rate of 0, 10, 20, 50 and 100 μmol/L group were 4.86±0.88%, 5.2±1.13%, 12.63±2.97%, 18.55±4.06% and 22.47±3.61%, respectively. With treatment of 20 or 50μmol/L 3-BrPA, the levels of IFN-γ decreased obviously to 243.37±15.64 ng/L and 164.25±20.14 ng/L, compared with the control group (277.61±18.46 ng/L). The levels of IL-4 increased mildly to 33.18±5.69 ng/L and 31.06±6.06 ng/L, compared with the control group (28.64±3.97ng/L). Thus, the IFN-γ/IL-4 ratio decreased significantly.Conclusions :The results indicated that 3-BrPA could inhibit T cells proliferation, induce apoptosis and contribute to the Th2 cytokine environment in murine mixed lymphocyte culture system. DisclosuresLiu:National Natural Science Foundation of China (81270647, 81300445, 81200388): Research Funding; National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; National Public Health Grand Research Foundation (201202017): Research Funding; Natural Science Foundation of Guangdong Province (S2012010009299): Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1): Research Funding; the Technology Plan of Guangdong Province of China (2012B031800403): Research Funding; the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding.

Specificity of JAK-Kinase Inhibition Determines Impact on T-Cell Function

Inhibitors of JAK2-kinase (Ruxolitinib, Momelotinib) are already approved or currently investigated in advanced clinical trials for treatment of myeloproliferative neoplasia (MPN). Besides their effect on mutated JAK2-kinase these compounds inhibit wildtype JAK and thereby impair JAK-STAT-signaling, which is an important pathway for proliferation and activation of other cell types such as human T-cells. Accumulating evidence suggests that they may also exert substantial immunosuppressive activity. Very recent reports highlighting hepatitis B reactivation complemented the series of severe infections in ruxolitinib-treated patients among which cryptococcus neoformans pneumonia, toxoplasmosis retinitis, disseminated tuberculosis, and progressive multifocal leukencephalopathy are the most alarming.We hypothesized that JAK-kinase inhibitors may act as immunosuppressant drugs by impairment of T-cell responses through inhibition of T-cell signaling (JAK-STAT pathway) and that specificity of JAK-kinase inhibition may be of major importance for the degree of T-cell inhibition. Therefore we investigated the effects of pharmacological JAK-kinase inhibition on healthy donor (HD-) and MPN patient T-cells. Selective inhibitors of JAK2-kinase (BSK805) and JAK3-kinase (BQM245) as well as clinically relevant inhibitors of JAK1/2-kinases (Ruxolitinib and Momelotinib) were used for pharmacologic inhibition. The SRC-kinase inhibitor Dasatinib served as a positive control for T-cell inhibition. Knockdown of specific JAK-kinases by RNAi was used to control for target specificity.In regard to T-cell receptor (TCR)-mediated signaling we investigated bona fide signaling molecules downstream of the TCR by Western Blotting. Besides SRC-kinases like LCK also ZAP70, PLCG1 and the MAPK/ERK pathway have been described to play a pivotal role in T-cell activation. In our data set, selectivity of JAK-kinase inhibition (JAK2, JAK3 or JAK1/2) influenced TCR-signaling in regard to overall tyrosine phosphorylation but also in regard to downstream effectors such as ERK.As activation and proliferation of primary T-cells is a critical step in immune responses against viral and tumor antigens we aimed to investigate the influence of JAK-kinase inhibition on activation and proliferation of human T-cells. T-cells from healthy donors were stimulated using either PHA 0.5% or CD3/CD28 beads to ensure a more T-cell receptor specific stimulation. CD69 expression was used as a marker for T-cell activation and CFSE staining was applied to assess for T-cell proliferation. Using CD3/CD28 stimulation, CD69 expression was almost abrogated following Dasatinib treatment and proliferation was significantly reduced. Applying relevant doses of specific JAK2 and JAK3 inhibitors to isolated T-cells did neither influence CD69 expression nor T-cell proliferation. These findings are confirmed by RNAi. In contrast, clinically relevant doses of JAK1/2 inhibitors Ruxolitinib and Momelotinib, respectively reduced CD69 expression and T-cell proliferation. Likewise, T-cells derived from MPN patients treated with Ruxolitinib revealed decreased CD69 expression and decreased proliferative capacity upon stimulation, compared to untreated patients or HD-controls.In order to investigate T-cell function, we assessed for allo-reactivity in a mixed lymphocyte culture. Human pan-T-cells were co-cultured with allogeneic antigen presenting cells. T-cell reactivity – as measured by 3H-thymidine incorporation – was significantly impaired by Ruxolitinib and Momelotinib. Specific inhibition of JAK2 or JAK3 kinase, however, did not affect T-cell reactivity. These effects could be confirmed using T-cells derived from Ruxolitinib-treated MPN patients. Investigation of leukemia- and virus-antigen-specific T-cell responses are currently under way to gain deeper insight regarding this clinically relevant scenario.Taken together, specificity of JAK-kinase inhibition influences the inhibitory potential on T-cell function. JAK1 kinase seems to play an important role in T-cell activation, as unspecific inhibitors of JAK1 & JAK2 Kinase inhibit T-cell function while selective inactivation of JAK2 kinase leaves T-cell function almost unaffected. Heterogeneity in T-cell function of Ruxolitinib-treated patients is an important finding that deserves detailed investigation. DisclosuresHeidel:Novartis: Consultancy.