Stimulators In Mixed Lymphocyte Reaction Research Articles

Prospective Tracking of Donor-Reactive T-Cell Clones in the Circulation and Rejecting Human Kidney Allografts.

BackgroundAntigen recognition of allo-peptides and HLA molecules leads to the activation of donor-reactive T-cells following transplantation, potentially causing T-cell-mediated rejection (TCMR). Sequencing of the T-cell receptor (TCR) repertoire can be used to track the donor-reactive repertoire in blood and tissue of patients after kidney transplantation.Methods/DesignIn this prospective cohort study, 117 non-sensitized kidney transplant recipients with anti-CD25 induction were included. Peripheral mononuclear cells (PBMCs) were sampled pre-transplant and at the time of protocol or indication biopsies together with graft tissue. Next-generation sequencing (NGS) of the CDR3 region of the TCRbeta chain was performed after donor stimulation in mixed lymphocyte reactions to define the donor-reactive TCR repertoire. Blood and tissue of six patients experiencing a TCMR and six patients without rejection on protocol biopsies were interrogated for these TCRs. To elucidate common features of T-cell clonotypes, a network analysis of the TCR repertoires was performed.ResultsAfter transplantation, the frequency of circulating donor-reactive CD4 T-cells increased significantly from 0.86 ± 0.40% to 2.06 ± 0.40% of all CD4 cells (p < 0.001, mean dif.: -1.197, CI: -1.802, -0.593). The number of circulating donor-reactive CD4 clonotypes increased from 0.72 ± 0.33% to 1.89 ± 0.33% (p < 0.001, mean dif.: -1.168, CI: -1.724, -0.612). No difference in the percentage of donor-reactive T-cells in the circulation at transplant biopsy was found between subjects experiencing a TCMR and the control group [p = 0.64 (CD4+), p = 0.52 (CD8+)]. Graft-infiltrating T-cells showed an up to six-fold increase of donor-reactive T-cell clonotypes compared to the blood at the same time (3.7 vs. 0.6% and 2.4 vs. 1.5%), but the infiltrating TCR repertoire was not reflected by the composition of the circulating TCR repertoire despite some overlap. Network analysis showed a distinct segregation of the donor-reactive repertoire with higher modularity than the overall TCR repertoire in the blood. These findings indicate an unchoreographed process of diverse T-cell clones directed against numerous non-self antigens found in the allograft.ConclusionDonor-reactive T-cells are enriched in the kidney allograft during a TCMR episode, and dominant tissue clones are also found in the blood.Trial RegistrationClinicaltrials.gov: NCT: 03422224 (https://clinicaltrials.gov/ct2/show/NCT03422224).

Pathogen Inactivated Platelet Components Used without Leukofiltration, or Gamma Irradiation Show Comparable Therapeutic Efficacy and Safety to Gamma Irradiated and Non-Leukofiltered Conventional Products for the Support of HSCT Patients in Hong Kong

▪Introduction: Depending on the conditioning intensity and graft source, mosthematopoietic stem cell transplant(HSCT) patients invariably experience a cytopenic phase of at least 2-3 weeks and require transfusion support until engraftment. All transfused blood components post-transplant need to be gamma irradiated to prevent transfusion associated graft versus host disease (TA-GVHD). Platelet component (PC) manufacturing methodologies differ around the globe. Currently, Hong Kong HSCT patients are supported with individual platelet rich plasma (PRP) PCs without leukocyte filtration (NLF), tested by short-term aerobic cultures for bacterial contamination (STABC), and gamma irradiated. When new PC manufacturing methodologies are considered for adoption, it is important to assess the immediate and longer term effects on transfused patients. Amotosalen+UVA light (A-UVA) pathogen reduction technology (INTERCEPT™ Blood System, Cerus Corporation, Concord, CA) inactivates bacteria, viruses, parasites and leukocytes by efficient covalent adduct modification of DNA and RNA. Treatment with A-UVA prevents TA- GVHD in an animal model, inhibits clonal T cell proliferation, prevents allogeneic antigen stimulation in mixed lymphocyte reactions, and inhibits transcription mediated cytokine production and early activation antigen expression (Corash et al, BMT 2004). The common strategy of prescribing gamma irradiated PC only for suspected "high risk" patients is sub-optimal, as failure to identify recipients at risk for TA-GVHD was responsible for 50% of reported cases (Kopolovic et al. Blood 2016). Treatment of PC with A-UVA addresses two key issues for HSCT patients: bacterial contamination, and universal prevention of TA-GVHD. Here, we compared the clinical support of HSCT patients with conventional PCs (C-PC) vs. an equivalent HSCT group supported with A-UVA PCs (I-PC) using a sequential patient cohort design within a single HSCT clinical center.Methods: I-PCs were prepared from 5 pooled ABO-matched, NLF buffy coat PCs in platelet additive solution and treated with A-UVA replacing gamma irradiation and bacterial detection. C-PCs were prepared from 5 ABO-matched, STABC-negative, NLF PRP PCs, and treated with gamma irradiation. Each patient could receive up to 5 PC transfusions. The primary efficacy endpoint was the 1-hour corrected count increment (CCI) and the primary safety endpoint was the proportion of patients (P) with acute transfusion reactions (ATR). Follow-up data on mortality, hematopoiesis engraftment, and immune status were collected up to 100 days post HSCT.Results: Patient demographics and type of HSCT were similar between patient cohorts. 33 patients received 76 A-UVA PCs and 31 patients received 89 C-PCs. The mean days of platelet support (p=0.618) and mean 1-hour CCIs per patient averaged for all transfusions were comparable (p=0.296) between the I- PC and conventional C-PC cohorts (Table). The proportion of patients with AEs was lower (p=0.021) for the I-PC group (Table), and no related SAEs were observed during the entirety of trial. Survival at 100 days post HSCT and rates of remission were similar between the cohorts (Table). The ATR rate trended lower, although not significantly different (p=0.296), in the I- PC group (Table). Follow-up data showed that the patients had comparable neutrophil and platelet engraftment (Table) with comparable immune system reconstitution by 100 days post HSCT. No cases of TA-GVHD were observed in either cohort.Conclusions: A-UVA-treated PCs prepared without LF, gamma irradiation, and bacterial detection can replace C-PCs for support of HSCT patients resulting in comparable post transfusion CCI responses and short and intermediate term clinical outcomes, while offering additional protection against transfusion transmitted bacteria and emerging or untested pathogens. [Display omitted] DisclosuresSim Pui Yin:Cerus Corporation: Other: Investigator sponsored trial. Leung Yuk Yan:Cerus Corporation: Other: Investigator sponsored trial. Lam Chun Kit:Cerus Corporation: Other: Investigator sponsored trial. Tsoi Wai:Cerus Corporation: Other: Investigator sponsored trial. Lee Cheuk:Cerus Corporation: Other: Investigator sponsored trial. Lie Kwok Wai:Cerus Corporation: Other: Investigator sponsored trial. Huang:Cerus Corporation: Employment. Rico:Cerus Corporation: Employment. Lin:Cerus corp: Employment. Corash:Cerus Corporation: Employment. Stassinopoulos:Cerus Corporation: Employment.

Treatment Use Study of Platelet Components Treated with Amotosalen and Ultraviolet a Light in Response to Emerging Arboviruses in Puerto Rico - True Study Final Results

▪Background: In 2014, epidemic chikungunya virus (CHIKV) emerged in Puerto Rico, concurrent with endemic dengue (DENV). The Department of Health mandated blood donation quarantine for 72h pending follow-up donor contact for infectious symptoms. The American Red Cross (ARC) halted local platelet collections and began importing blood components. Imported platelet components (PCs) from the continental U.S. were used to supply hospitals in the Caribbean. An alternative strategy is local PC production with amotosalen +UVA (A-UVA) pathogen reduction (PR) technology (INTERCEPT™ Blood System, Cerus Corporation, Concord, CA) as utilized by the French Transfusion Service in the Overseas Territories, including the Caribbean since 2007. The major logistical advantage of such a strategy is earlier PC availability to hospitals since there is no need to wait for the results of bacterial culture, or transport of PCs from the continental U.S. A-UVA technology inactivates bacteria, viruses, parasites and leukocytes by efficient covalent adduct modification of DNA and RNA, thus addressing risks of bacterial contamination and reducing the risk of transfusion-transmitted infection (TTI). Treatment with A-UVA also prevents transfusion associated graft vs. host disease (TA-GVHD) in an animal model, inhibits clonal T cell proliferation, prevents allogeneic antigen stimulation in mixed lymphocyte reactions, and inhibits transcription mediated cytokine production and early activation antigen expression (Corash et al, BMT 2004) and is recognized by the AABB as a replacement for gamma irradiation in the prevention of TA-GVHD.Aims: A treatment use clinical study (TRUE) was conducted in Puerto Rico under the Investigational Device Exemption (IDE) early/expanded access regulations while the INTERCEPT Premarket approval (PMA) application was under review by the Food and Drug Administration (FDA). The objective of the TRUE study was to provide early access to INTERCEPT to reduce the risk of TTIs during the ongoing CHIKV/DENV epidemic.Methods: A prospective, open label, single arm, treatment use, multi-center study was conducted involving 7 hospitals and the ARC. Apheresis (Trima® or Amicus® collectors) leukocyte-reduced PR PCs, suspended in 100% plasma without gamma irradiation, bacterial contamination testing, or the need for CMV serology were produced. Safety outcome measures included all adverse events (AEs), serious adverse events (SAEs) and acute transfusion reactions. Patients were transfused with study PCs according to local practices for as long as clinically indicated.Results: 90 patients (55.6% male, mean age 67.2 y) were transfused with 256 PR PCs (2.8 ± 3.5/patient). The vast majority of the patients were hematology oncology patients (76.7%). Most patients (81.1%) received 1 transfusion cycle (transfusion support without > 5 days between transfusions) with 2 PR PCs (40.0%), while 32.2% received 1 PR PC, and 25.6% received between 3-10 PR PCs. Mean duration of platelet support was 14.7 days. Thirty-three unrelated AEs were reported in 26 patients. Nineteen patients (21.1%) had unrelated SAEs (including 17 deaths [18.9%]). Two febrile non-hemolytic reactions and one allergic transfusion reaction were reported and deemed unrelated to INTERCEPT by the investigators. No suspected cases of CHIKV or DENV infections or other TTIs were reported. Reported AEs were within the expected spectrum of co-morbidity and mortality for patients of similar age with advanced hematology-oncology diseases.Conclusions: The INTERCEPT Blood System was rapidly deployed in response to the CHIKV and DENV epidemic with replacement of bacterial detection, gamma irradiation and the need for CMV serology. No PR PC related AEs were reported. INTERCEPT was approved in late 2014 by the FDA to treat Amicus apheresis PCs suspended in platelet additive solution and in 2016 for Trima PCs suspended in 100% plasma. The importance of this proactive technology is highlighted by the recent emergence of Zika virus (ZIKV). FDA has recently requested that all blood establishments in Puerto Rico and in certain counties in Florida cease collecting blood immediately until investigational donor screening test for ZIKV RNA is implemented or until they implement the use of an approved or investigational pathogen reduction technology. DisclosuresRico:Cerus Corporation: Employment. Stramer:Cerus Corporation: Other: fee for service study with Cerus Corporation. Benjamin:Cerus corp.: Employment. Cartagena:Cerus Corporation: Other: fee for service study with Cerus Corporation. Arroyo:Cerus Corporation: Other: fee for service study with Cerus Corporation. Robles:Cerus Corporation: Other: fee for service study with Cerus Corporation. Carlo:Cerus Corporation: Other: fee for service study with Cerus Corporation. Vera:Cerus Corporation: Other: fee for service study with Cerus Corporation. Solivan-Ortiz:Cerus Corporation: Other: fee for service study with Cerus Corporation. Huang:Cerus Corporation: Employment. Koontz:Cerus Corporation: Employment. Berry:Cerus Corporation: Employment. Corash:Cerus Corporation: Employment.

Ketamine affects in vitro differentiation of monocyte into immature dendritic cells.

Monocytes (MOs) have the unique ability to differentiate into immature dendritic cells (iDCs) (MO→iDC) under the influence of interleukin-4 and granulocyte-monocyte colony-stimulating factor (IL-4&GM-CSF). In this study, the authors investigated the influence of ketamine on the process of MO→iDC. iDCs were cultured from MO obtained from 36 subjects in the presence of IL-4 and GM-CSF and ketamine at 100, 10, and 1 μg/ml for 5 days. In some of the experiments, the authors used nonspecific N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, NMDA, or a neutralizing antibody for transforming growth factor β (TGFβ). The expression of surface markers and functional assays were used to assess the effect of ketamine on IL-4&GM-CSF-stimulated MO. IL-4&GM-CSF-stimulated MO's supernatants were assessed for cytokine levels. Ketamine at 10 μg/ml, and higher concentrations, diminished the expression of CD1a on IL-4&GM-CSF-stimulated MO and retarded both their ability to process DQ ovalbumin and mixed lymphocyte reaction stimulation. The addition of ketamine to IL-4&GM-CSF-differentiated MO resulted in the persistent expression of CD14 and unchanged expression of CD86 and CD206. The phagocytic abilities of IL-4&GM-CSF-differentiated MO were not changed by ketamine. MK-801, a nonselective NMDA agonist, mimicked ketamine's effect on MO→iDC differentiation. Adding exogenous NMDA to IL-4&GM-CSF-stimulated MO in the presence of ketamine partially restored the level of CD1a. TGFβ was elevated in supernatants of IL-4&GM-CSF-stimulated MO in the presence of ketamine. Adding neutralizing TGFβ antibody or TGFβR1 blocker (SB431542) resulted in the full recovery of MO→iDC, despite the presence of ketamine. Ketamine diminishes the process of MO→iDC in vitro. This is mediated via NMDA-dependent mechanisms and TGFβ.

TNFRSF14 aberrations in Follicular Lymphoma B Cells Result in Increased Alloresponses in Vitro and in Vivo

IntroductionGenetic aberrations of Tumor Necrosis Factor Receptor Superfamily 14 (TNFRSF14, also known as HVEM) have been shown to occur at high frequencies in patients (pts) with follicular lymphoma (FL). HVEM is a ligand for B and T lymphocyte attenuator (BTLA) which negatively regulates T cell responses and BTLA stimulation reduces acute graft-versus-host disease (aGvHD) in murine allogeneic hematopoietic cell transplantation (AHCT) models. As activated FL B cells are potent alloantigen presenting cells, we hypothesized that TNFRSF14 aberrations in FL B cells would reduce expression of HVEM and potentiate capacity of FL B cells to stimulate allogeneic T cell responses. We therefore sought to determine the functional effect of TNFRSF14 aberrations on FL B cell-stimulated donor T cell alloresponses in vitro. We also examined the impact of TNFRSF14 aberrations on the outcome of FL pts after HLA-matched reduced intensity conditioning (RIC) AHCT.ResultsFL B cells from lymph nodes were FACS-sorted (>90% purity and > 95% light chain restriction), activated and used as stimulators in mixed lymphocyte reactions with purified allogeneic responder CD3+ T cells. HVEM expression on FL B cells from pts with biallelic TNFRSF14 aberrations (Mut/Del cases) was undetectable whereas 40% of FL B cells from TNFRSF14 WT cases expressed HVEM (Fig 1 A). In contrast, FL B cells from Mut/Del and WT cases expressed similar levels of MHC class I/II, CD80, CD86 and CD58 before and after activation. Allostimulation with Mut/Del FL B cells resulted in significantly greater expression of activation markers on responder CD4+ T cells, increased secretion of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-2) measured by ELISA and increased frequencies of cytokine-secreting CD4+ and CD8+ T cells enumerated by intracellular cytokine staining. Responder T cell proliferation by thymidine incorporation was significantly greater after stimulation with Mut/Del FL B cells compared to WT FL B cells. CFSE labeling studies demonstrated that this effect resulted from increased proliferation of CD4+ and CD8+ responder T cells after both primary (Fig 1B) and secondary allostimulation. To determine if the increased alloresponses we observed using FL B cells from TNFRSF14 Mut/Del cases was due to reduced HVEM-BTLA signaling, we performed allogeneic co-cultures in the presence of antagonist or agonist BTLA antibodies (ab). Antagonist anti-BTLA ab increased proliferation of responder T cells after stimulation with WT FL B cells confirming that BTLA limits alloresponses in our in vitro model. Importantly, agonist BTLA ab reduced alloresponses stimulated by Mut/Del FL B cells.We next sought to determine if the increased alloresponses we detected in vitro in FL pts with TNFRSF14 aberrations resulted in an increase in clinical alloreactivity after AHCT. DNA from lymph nodes from FL pts undergoing T-cell replete RIC AHSCT was screened for TNFRSF14 mutations and deletions by Sanger sequencing and multiplex ligation-probe amplification respectively. Cumulative incidences (CI) of aGvHD and GvHD-related death were calculated with FL progression as a competing risk. TNFRSF14 aberrations were identified in 10/21 pts prior to RIC AHCT (4 Mut/Del, 1 Del/Del, 1 Mut/WT, 4 Del/WT). Most (18/21) pts had evidence of ongoing FL pre-transplant. Disease and donor characteristics were similar in pts with and without aberrations. There was no significant difference in CI of aGvHD in pts with or without TNFRSF14 aberrations. However there was a significantly higher CI of fatal aGvHD in patients with TNFRSF14 aberrations (45%) compared to those without aberrations (0%, p<0.01). Interestingly, relapse was less frequent in patients with TNFRSF14 aberrations consistent with increased graft-versus-tumor effects, although this did not reach statistical significance.ConclusionThis study is the first to describe the impact of TNFRSF14 aberrations on the allostimulatory capacity of FL B cells. TNFRSF14 aberrations were associated with enhanced T-cell alloresponses in vitro and increased death from aGvHD. Importantly, our results suggest FL patients with TNFRSF14 aberrations may benefit from more aggressive immunosuppression to prevent fatal aGvHD after AHCT. The increased antigen-presenting capacity of FL B cells with TNFRSF14 aberrations could also influence autologous anti-tumor responses and impact outcome after other treatment modalities. [Display omitted] DisclosuresGribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

Genetically engineered fixed K562 cells: potent “off-the-shelf” antigen-presenting cells for generating virus-specific T cells

Background aimsThe human leukemia cell line K562 represents an attractive platform for creating artificial antigen-presenting cells (aAPC). It is readily expandable, does not express human leukocyte antigen (HLA) class I and II and can be stably transduced with various genes. MethodsIn order to generate cytomegalovirus (CMV) antigen-specific T cells for adoptive immunotherapy, we transduced K562 with HLA-A∗0201 in combination with co-stimulatory molecules. ResultsIn preliminary experiments, irradiated K562 expressing HLA-A∗0201 and 4-1BBL pulsed with CMV pp65 and IE-1 peptide libraries failed to elicit antigen-specific CD8+ T cells in HLA-A∗0201+ peripheral blood mononuclear cells (PBMC) or isolated T cells. Both wild-type K562 and aAPC strongly inhibited T cell proliferation to the bacterial superantigen staphylococcal enterotoxin B (SEB) and OKT3 and in mixed lymphocyte reaction (MLR). Transwell experiments suggested that suppression was mediated by a soluble factor; however, MLR inhibition was not reversed using transforming growth factor-β blocking antibody or prostaglandin E2 inhibitors. Full abrogation of the suppressive activity of K562 on MLR, SEB and OKT3 stimulation was only achieved by brief fixation with 0.1% formaldehyde. Fixed, pp65 and IE-1 peptide-loaded aAPC induced robust expansion of CMV-specific T cells. ConclusionsFixed gene-modified K562 can serve as effective aAPC to expand CMV-specific cytotoxic T lymphocytes for therapeutic use in patients after stem cell transplantation. Our findings have implications for broader understanding of the immune evasion mechanisms used by leukemia and other tumors.

Regulatory T Cells Are Resistant to Cyclophosphamide (Cy) Through Expression of Aldehyde Dehydrogenase (ALDH) Upon Allogeneic Stimulation

High-dose, post-transplantation Cy (PT/Cy) is a novel and effective strategy for preventing graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (alloBMT). We previously reported that regulatory T cells (Tregs) are resistant to Cy in allogeneic reactions; activated Tregs (CD4+CD45RA-Foxp3+hi) are relatively increased 30 and 60 days after PT/Cy in vivo and both naïve (CD4+CD45RA+Foxp3+) and activated Tregs are resistant to cytotoxicity induced by activated Cy (mafosfamide) in mixed lymphocyte reactions (MLR) in vitro. However, the mechanisms of Treg resistance to Cy have not been understood. As resistance of hematopoietic stem cells to Cy is dependent on expression of the ALDH-1 family (retinaldehyde dehydrogenases), we hypothesized that Tregs also might express ALDH as a potential mechanism of resistance to Cy. To examine ALDH expression, we used Aldefluor as an established and reproducible flow cytometric-based method. There was minimal to no Aldefluor-positivity in unstimulated Tregs. However, after allogeneic stimulation in MLR, significant subsets of both naïve and activated Tregs were Aldefluor-positive. These findings were confirmed by PCR which showed undetectable mRNA transcript levels in unstimulated cells but significant upregulation of ALDH1A1 in naïve and activated Tregs at Days 3 and 7 of MLR. These ALDH+ cells persisted after mafosfamide treatment but were not present in significant amounts in the presence of cyclosporine or rapamycin (activated Tregs, P = .027). ALDH expression correlated with expression of the activation markers CD69 and CD25 and with proliferation. The clinical relevance of these findings was confirmed by showing Aldefluor+CD4+ T cells in peripheral blood retrieved from patients on Day 3 post-alloBMT at the time of receiving PT/Cy. To dissect functional implications of these findings, we explored pharmacological inhibition of ALDH using diethylaminobenzaldehyde (DEAB). DEAB treatment drastically reduced activation and proliferation of CD4+CD25+ T cells in MLR (P = .031 for both comparisons) and abolished Treg resistance to mafosfamide-induced cytotoxicity (activated Tregs, P < .001). Therefore, resistance of Tregs to Cy is dependent at least in part on ALDH expression in allogeneic reactions. Given the role of ALDH as the rate-limiting step in retinoic acid (RA) biosynthesis and the crucial contributions of RA to both immune tolerance and CD4+ T cell function, pharmacologic manipulation of this pathway may open a new avenue for modulating alloreactivity after alloBMT.

Dendritic cells with lymphocyte-stimulating activity differentiate from human CD133 positive precursors

AbstractCD133 is a hallmark of primitive myeloid progenitors. We have addressed whether human cord blood cells selected for CD133 can generate dendritic cells, and Langerhans cells in particular, in conditions that promote that generation from CD34+ progenitors. Transforming growth factor-β1 (TGF-β1) and anti–TGF-β1 antibody, respectively, were added in some experiments. With TGF-β, monocytoid cells were recognized after 7 days. Immunophenotypically immature dendritic cells were present at day 14. After 4 more days, the cells expressed CD54, CD80, CD83, and CD86 and were potent stimulators in mixed lymphocyte reaction; part of the cells expressed CD1a and langerin, but not Birbeck granules. Without TGF-β, only a small fraction of cells acquired a dendritic shape and expressed the maturation-related antigens, and lymphocytes were poorly stimulated. With anti–TGF-β, the cell growth was greatly hampered, CD54 and langerin were never expressed, and lymphocytes were stimulated weakly. In conclusion, CD133+ progenitors can give rise in vitro, through definite steps, to mature, immunostimulatory dendritic cells with molecular features of Langerhans cells, although without Birbeck granules. Addition of TGF-β1 helps to stimulate cell growth and promotes the acquisition of mature immunophenotypical and functional features. Neither langerin nor Birbeck granules proved indispensable for lymphocyte stimulation.

In vivo effects of calcitriol on phenotypic and functional properties of dendritic cells.

Dendritic cells (DCs) play a central role in the initiation and expansion of T cell mediated immune responses with potential immunotherapy application. The compounds which have the ability to induce immunomodulatory effects on DCs may be employed for the treatment of immunopathologic conditions such as autoimmune diseases. The aim of this study was to investigate the in vivo effects of calcitriol (active form of vitamin D3) on DCs. 0.1 microgram calcitriol was injected intra-peritoneally into C57BL/6 mice every other day within 3 weeks, and spleen DCs were extracted by magnetic beads. The phenotypic and functional properties of DCs were studied by flow cytometry and mixed lymphocyte reaction (MLR), respectively. The expression of CD86 and MHC II, as maturation markers and costimulatory molecules were significantly decreased (p=0.028 and p=0.047, respectively) while CD11b expression, as a marker of mice myeloid DCs which mostly induces Th2 cytokine profile, was significantly increased (p=0.011). Allogeneic T cell stimulation in MLR was also significantly inhibited in comparison with the control groups (p<0.05). Our data indicate that in vivo calcitriol administration inhibits maturation and activation of DCs in the same manner as in vitro conditions.

Generation and characterization of chicken bone marrow‐derived dendritic cells

Dendritic cells (DCs) are bone marrow-derived professional antigen-presenting cells. The in vitro generation of DCs from either bone marrow or blood is routine in mammals. Their distinct morphology and phenotype and their unique ability to stimulate naïve T cells are used to define DCs. In this study, chicken bone marrow cells were cultured in the presence of recombinant chicken granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant chicken interleukin-4 (IL-4) for 7 days. The cultured population showed the typical morphology of DCs, with the surface phenotype of major histocompatibility complex (MHC) class II(+) (high), CD11c(+) (high), CD40(+) (moderate), CD1.1(+) (moderate), CD86(+) (low), CD83(-) and DEC-205(-). Upon maturation with lipopolysaccharide (LPS) or CD40L, surface expression of CD40, CD1.1, CD86, CD83 and DEC-205 was greatly increased. Endocytosis and phagocytosis were assessed by fluorescein isothiocyanate (FITC)-dextran uptake and fluorescent bead uptake, respectively, and both decreased after stimulation. Non-stimulated chicken bone marrow-derived DCs (chBM-DCs) stimulated both allogeneic and syngeneic peripheral blood lymphocytes (PBLs) to proliferate in a mixed lymphocyte reaction (MLR). LPS- or CD40L-stimulated chBM-DCs were more effective T-cell stimulators in MLR than non-stimulated chBM-DCs. Cultured chBM-DCs could be matured to a T helper type 1 (Th1)-promoting phenotype by LPS or CD40L stimulation, as determined by mRNA expression levels of Th1 and Th2 cytokines. We have therefore cultured functional chBM-DCs in a non-mammalian species for the first time.

Activation of human B cells by the agonist CD40 antibody CP-870,893 and augmentation with simultaneous toll-like receptor 9 stimulation

BackgroundCD40 activation of antigen presenting cells (APC) such as dendritic cells (DC) and B cells plays an important role in immunological licensing of T cell immunity. Agonist CD40 antibodies have been previously shown in murine models to activate APC and enhance tumor immunity; in humans, CD40-activated DC and B cells induce tumor-specific T cells in vitro. Although clinical translation of these findings for patients with cancer has been previously limited due to the lack of a suitable and available drug, promising clinical results are now emerging from phase I studies of the agonist CD40 monoclonal antibody CP-870,893. The most prominent pharmacodynamic effect of CP-870,893 infusion is peripheral B cell modulation, but direct evidence of CP-870,893-mediated B cell activation and the potential impact on T cell reactivity has not been reported, despite increasing evidence that B cells, like DC, regulate cellular immunity.MethodsPurified total CD19+ B cells, CD19+ CD27+ memory, or CD19+ CD27neg subsets from peripheral blood were stimulated in vitro with CP-870,893, in the presence or absence of the toll like receptor 9 (TLR9) ligand CpG oligodeoxynucleotide (ODN). B cell surface molecule expression and cytokine secretion were evaluated using flow cytometry. Activated B cells were used as stimulators in mixed lymphocyte reactions to evaluate their ability to induce allogeneic T cell responses.ResultsIncubation with CP-870,893 activated B cells, including both memory and naïve B cells, as demonstrated by upregulation of CD86, CD70, CD40, and MHC class I and II. CP-870,893-activated B cells induced T cell proliferation and T cell secretion of effector cytokines including IFN-gamma and IL-2. These effects were increased by TLR9 co-stimulation via a CpG ODN identical in sequence to a well-studied clinical grade reagent.ConclusionThe CD40 mAb CP-870,893 activates both memory and naïve B cells and triggers their T cell stimulatory capacity. Simultaneous TLR9 ligation augments the effect of CP-870,893 alone. These results provide further rationale for combining CD40 and TLR9 activation using available clinical reagents in strategies of novel tumor immunotherapy.

Interleukin-21: An Interleukin-2 Dependent Player in Rejection Processes

Interleukin (IL)-21 is the most recently described cytokine that signals via the common cytokine receptor (gammac), is produced by activated CD4+ T-cells, and regulates expansion and effector function of CD8+ T-cells. To explore the actions of IL-21 with other gammac-dependent cytokines in alloreactivity, mRNA expression of IL-21, IL-21R alpha-chain, and IL-2 proliferation and cytotoxicity was measured after stimulation in mixed lymphocyte reactions. Additionally, IL-21 and IL-21R alpha-chain expression was studied in biopsies of heart transplant patients. Analysis of mRNA expression levels of allostimulated T-cells showed a 10-fold induction of IL-21 and IL-21R alpha-chain. Interestingly, induction of IL-21 was highly dependent on IL-2 (as in the presence of anti-IL-2, anti-IL-2R alpha-chain, and the immunosuppressive drugs cyclosporine A, tacrolimus, and rapamycin) the transcription of IL-21 was almost completely inhibited, whereas in the presence of exogenous IL-2 the mRNA expression of IL-21 was even more upregulated. IL-21 functioned as a costimulator for IL-2 to augment proliferation and cytotoxic responses, while blockade of the IL-2 route abrogated these functions of IL-21. Blockade of the IL-21 route by anti-IL-21R alpha-chain monoclonal antibodies inhibited the proliferation of alloactivated T-cells. Also, in vivo alloreactivity was associated with IL-21/IL-21R alpha-chain expression. After heart transplantation, the highest intragraft IL-21, IL-21R alpha-chain, and IL-2 mRNA expression levels were measured during acute rejection (P<0.001, P=0.01, P=0.03). IL-21 is a critical cytokine for IL-2 dependent immune processes. Blockade of the IL-21 pathway may provide a new perspective for the treatment of allogeneic responses in patients after transplantation.

Improvement of Selective Depletion of Alloreactive T Cells.

Severe T cell depletion required for allogeneic hematopoietic stem cell transplantation from haplo-identical donors results in poor immune reconstitution and leads to high rates of mortality from infections, and relapse. One approach to overcome this problem is to infuse T cells depleted of alloreactivity. Selective depletion (SD) of alloreactive T cells is achieved by elimination of activated T cells after ex-vivo stimulation with recipient cells. To determine optimum selective depletion conditions, we have investigated the factors that modify alloreactivity of T cells.Methods: Alloreactivity was measured by one-way mixed lymphocyte reaction (MLR) using 3H thymidine uptake. PBMCs were used as responders and either irradiated expanded T cells (expT) or dendritic cells (DCs) as stimulators. T cells were expanded using anti-CD3 coated beads. DCs were generated from monocytes by GM-CSF and IL-4 stimulation. Selective depletion was performed by co-incubation of responder and stimulator cells for 72 hours and depletion of activated cells by an immunotoxin, LMB-2 (Anti-Tac (Fv)-PE-38), which was added to the culture at 24 and 48 hours. Effectiveness of the depletion was tested by a secondary MLR utilizing the original stimulator cells and unrelated third part cells.Results: Expansion of T cells has resulted in increase of HLA-DR, CD80 and CD86 expression compared to resting T cells (52.5% vs. 6%, 20.9% vs. 0.9%, and 32.9% vs. 20.9%, respectively), resulting in better stimulation in MLR (6505 cpm vs 1620 cpm). In one-way MLR using either PBMCs or CD25 depleted PBMCs as responders and expanded T cells and DCs as targets, with or without anti-CD28 in the culture media. DCs were better stimulators than expT cells (6636 vs. 4308). However, most dramatic effect was seen when anti-CD28 was added to the culture, increasing response to both expT cells and to a lesser extent DCs (40,169 and 19,303). Removal of CD25 positive cells also improved alloreactivity in all culture conditions (6636 in expT, 16,644 in DC, 57,363 in expT+CD28, and 30,943 in DC+CD28). To better define the effect of the target, we have performed Vbeta repertoire analysis of responding cells after expT cell, DC and expT cell+anti-CD28 stimulation. Flow cytometry revealed expansion of discrete Vbeta families, in addition to shared ones. We have then performed selective depletion using PBMCs or CD 25 depleted PBMCs as stimulators and expT cells, expT cell+anti-CD28, and DCs as stimulators. Residual alloreactivity after expT cell stimulation against original stimulators, DCs and third party cells were 7%, 147% and 99% respectively. Interestingly, after SD utilizing DCs as stimulators, there was substantial residual activity against expT cells (69%). When SD was performed using expT cells as stimulators with anti-CD28, combined with CD25 depletion, the depletion against both original stimulators and DCs was improved (2% and 54%, respectively).Conclusion: Depletion of regulatory T cells, and co-stimulation with ant-CD28 improves alloreactivity and selective depletion. Whether improvement in in-vitro selective depletion will result in better clinical outcome will be tested in a clinical trial.

Modification of B-CLL Cells Via Infection with a Replication-Defective MVA Virus Encoding Three Costimulatory Molecules: A Potential Approach to Tumor Cell Immunotherapy of B-CLL.

Chronic lymphocytic leukemia of B-cell origin (B-CLL) is a lymphoproliferative disorder characterized by the accumulation of slowly dividing, apoptosis-defective, clonal CD5+ B cells that typically involve the bone marrow, lymph nodes, spleen and blood of affected individuals. New therapeutic approaches are necessary to be investigated for the treatment of B-CLL. Several characteristics of the neoplastic B cells make B-CLL a good candidate for immunotherapy; evidence exists that B-CLL cells can be recognized by autologous anti-tumor T cells in an MHC-restricted manner. B-CLL cells, however, are themselves ineffective antigen-presenting cells mainly because of their inadequate costimulatory capacity. We have investigated the ability of in vitro manipulated B-CLL cells that express a triad of costimulatory molecules, designated TRICOM, to stimulate effective anti-tumor T-cell responses and their potential as cancer vaccines. Delivery of transgenes to the B-CLL cells could be accomplished through the use of a modified vaccinia virus strain Ankara designated MVA. This is a highly attenuated, non-replicative virus that has been demonstrated to be safe and well tolerated in both animals and humans. Here we report the use of a recombinant MVA vector to deliver the simultaneous expression of three human costimulatory molecules (B7-1, ICAM-1, LFA-3) on the surface of B-CLL cells. Infection was conducted using various multiplicity-of-infection (MOI) of MVA-TRICOM and the efficiency of the vector to enhance the expression of the encoded transgenes was evaluated by FACS analysis. The infection of B-CLL cells with MVA-TRICOM markedly increased the expression of B7-1, ICAM-1 and LFA-3 on the cell surface. The ability of the MVA-TRICOM-infected B-CLL cells to activate specific T cells was subsequently analyzed in proliferation assays, cytotoxic, and cytokine production assays. Proliferation of both autologous and allogeneic T cells was observed when MVA-TRICOM-infected B-CLL cells were used as stimulators in mixed lymphocyte reactions. Autologous CTLs were then generated in vitro using MVA-TRICOM-infected B-CLL cells as APC; these CTLs were capable of lysing uninfected autologous as well as allogeneic B-CLL cells. From our results we conclude that MVA-TRICOM-infected B-CLL cells can be used to induce B-CLL-specific CTLs that, in turn, can mediate the lysis of unmodified B-CLL cells. Our findings have implications for a potential use of MVA-TRICOM-infected B-CLL cells as tumor vaccines in the immunotherapy of B-CLL.

A simple method to optimize peripheral blood mononuclear cell preparation from cynomolgus monkeys and improve mixed lymphocyte reactions

Introduction: The standard Ficoll–Hypaque method used to isolate peripheral blood mononuclear cells (PBMC) gives very variable results when used with cynomolgus monkey blood. We have improved the method by using special cell processing tubes (CP-tubes), originally developed for clinical use. Methods: Blood samples were collected from cynomolgus monkeys and processed for PBMC preparation using either the classical Ficoll–Hypaque method or CP-tubes following various centrifugation protocols. The preparations were compared according to their cellular content as well as their response in the mixed lymphocyte reaction (MLR). Results: Good PBMC separation was achieved in >90% of samples by centrifugation of blood samples in CP-tubes for 40–45 min at 1650× g and 20 °C. For the remaining samples, poor PBMC separation was probably due to low corpuscular hemoglobin concentrations (<28 g/dl), but this could be rectified by using one to two additional centrifugations. The PBMC preparations thus obtained showed lower red blood cell (RBC) and polymorphonuclear (PMN) cell contamination, reacted well to mitogen and showed improved MLR stimulation indices vs. standard Ficoll–Hypaque-PBMC-derived preparations. The inhibitory effect of Cyclosporine-A (CsA) was within the low nanomolar range with both methods. Discussion: These results demonstrate that the use of CP-tubes is a practical way of improving PBMC separation and MLR responses with cynomolgus monkey blood.

Absence of humoral and cellular alloreactivity in baboons sensitized to pig antigens.

to study whether sensitization to pig antigens results in humoral and/or cellular sensitization to alloantigens in baboons, and thus increases the risks of organ allotransplantation after xenotransplantation. Serum from baboons that were naive (n = 4), sensitized to Gal alpha 1,3Gal (Gal) antigens (n = 2), or sensitized to Gal + non-Gal pig antigens (n = 2) were tested by flow cytometry for the presence of immunoglobulin G (IgG) and IgM antibodies that bind to pig or baboon peripheral blood mononuclear cells (PBMC). Two allosensitized baboons were used as positive controls. The same 10 sera were tested in a complement-mediated cytotoxicity assay to detect cytotoxic antibodies against pig, allo and self-PBMC. The T-cell responses of the same baboons to allogeneic and pig PBMC stimulators in mixed lymphocyte reaction (MLR) were studied. All baboon sera contained cytotoxic antibodies that bound to pig PBMC. Binding and cytotoxicity were higher in xenosensitized baboons, particularly in those sensitized to Gal + non-Gal antigens (P < 0.001). None of the naive or xenosensitized baboon sera bound to baboon PBMC. Serum from allosensitized baboons showed anti-baboon IgG and IgM binding, but there was no increase in binding to pig PBMC or in cytotoxicity to pig cells. The MLR response to pig stimulators in baboons sensitized to non-Gal pig antigens was greater than that of naive or Gal-sensitized baboons (P < 0.001), but there was no increase in the response to baboon cells. In baboons, no in vitro evidence that a previous pig xenograft might endanger the outcome of a subsequent allograft was documented.

Immunotherapeutic Effects of CTLA4Ig Fusion Protein on Murine EAE and GVHD

Background: CTLA4 (CD152), which is expressed on the surface of T cells following activation, has a much higher affinity for B7 molecules comparing to CD28, and is a negative regulator of T cell activation. In contrast to stimulating and agonistic capabilities of monoclonal antibodies specific to CTLA-4, CTLA4Ig fusion protein appears to act as CD28 antagonist and inhibits in vitro and in vivo T cell priming in variety of immunological conditions. We've set out to confirm whether inhibition of the CD28-B7 costimulatory response using a soluble form of human CTLA4Ig fusion protein would lead to persistent inhibition of alloreactive T cell activation. Methods: We have used CHO-dhfr cell-line to produce CTLA4Ig fusion protein. After serum free culture of transfected cell line we purified this recombinant molecule by using protein A column. To confirm characterization of fusion protein, we carried out a series of Western blot, SDS-PAGE and silver staining analyses. We have also investigated the efficacy of CTLA4Ig in vitro such as mixed lymphocyte reaction (MLR) & cytotoxic T lymphocyte (CTL) response and in vivo such as experimental autoimmune encephalomyelitis (EAE), graft versus host disease (GVHD) and skin-graft whether this fusion protein could inhibit alloreactive T cell activation and lead to immunosuppression of activated T cell. Results: In vitro assay, CTLA4Ig fusion protein inhibited immune response in T cell-specific manner: 1) Human CTLA4Ig inhibited allogeneic stimulation in murine MLR; 2) CTLA4Ig prevented the specific killing activity of CTL. In vivo assay, human CTLA4Ig revealed the capacities to induce alloantigen-specific hyporesponsiveness in mouse model: 1) GVHD was efficiently blocked by dose-dependent manner; 2) Clinical score of EAE was significantly decreased compared to nomal control; 3) The time of skin-graft rejection was not different between CTLA4Ig treated and control group. Conclusion: Human CTLA4Ig suppress the T cell-mediated immune response and efficiently inhibit the EAE, GVHD in mouse model. The mechanism of T cell suppression by human CTLA4Ig fusion protein may be originated from the suppression of activity of cytotoxic T cell. Human CTLA4Ig could not suppress the rejection in mouse skin-graft, this finding suggests that other mechanism except the suppression of cytotoxic T cell may exist on the suppression of graft rejection. (Immune Network 2003;3(4):302-309)

Poor yield of dendritic cell precursors from untreated pediatric cancer.

We have studied the generation of dendritic cells (DC) in vitro from healthy children and children with newly diagnosed cancer. Peripheral blood derived adherent cells were harvested and cultured in the presence of granulocyte-macrophage colony-stimulating-factor (GM-CSF) and interleukin-4 (IL-4). Differentiated DC were characterized morphologically and analyzed by flow cytometry and allogenic mixed lymphocyte reaction (MLR). The numbers of adherent cells were two-fold higher in healthy children than in those with malignant tumors: 1.5 x 10(5)/ml of blood (mean) versus 0.7 x 10(5)/ml, respectively (p = 0.025). No significant differences were found in the cell survival or yield after the in vitro cultivation of adherent cells. Cytological examination of cultured cells showed that they were similar to DC in adults, being large, irregularly shaped, with several thin membrane protrusions, and bean-shaped nuclei. Differentiated DC from healthy controls expressed CD86 and HLA-DR, but did not express monocyte markers CD14 and CD64 (FcgammaRI). The phenotype of DC from cancer patients was otherwise similar, except that a substantial proportion (24-85%) continued to express CD64 (p = 0.001). DC derived both from cancer patients and controls were strong stimulators in allogeneic MLR. We conclude that functionally capable DC can be generated in vitro from blood-derived adherent cells in children, but children with untreated cancer yield lower numbers of DC than healthy children. The continued expression of CD64 on DC derived from cancer patients may indicate that adherent cells from cancer patients are more resistant to signals inducing differentiation into DC.

Stimulation of autologous proliferative and cytotoxic T-cell responses by “leukemic dendritic cells” derived from blast cells in acute myeloid leukemia

AbstractEffective presentation of tumor antigens is fundamental to strategies aimed at enrolling the immune system in eradication of residual disease after conventional treatments. Myeloid malignancies provide a unique opportunity to derive dendritic cells (DCs), functioning antigen-presenting cells, from the malignant cells themselves. These may then co-express leukemic antigens together with appropriate secondary signals and be used to generate a specific, antileukemic immune response. In this study, blasts from 40 patients with acute myeloid leukemia (AML) were cultured with combinations of granulocyte-macrophage colony-stimulating factor, interleukin 4, and tumor necrosis factor α, and development to DCs was assessed. After culture, cells from 24 samples exhibited morphological and immunophenotypic features of DCs, including expression of major histocompatibility complex class II, CD1a, CD83, and CD86, and were potent stimulators in an allogeneic mixed lymphocyte reaction (MLR). Stimulation of autologous T-cell responses was assessed by the proliferative response of autologous T cells to the leukemic DCs and by demonstration of the induction of specific, autologous, antileukemic cytotoxicity. Of 17 samples, 11 were effective stimulators in the autologous MLR, and low, but consistent, autologous, antileukemic cytotoxicity was induced in 8 of 11 cases (mean, 27%; range, 17%-37%). This study indicates that cells with enhanced antigen-presenting ability can be generated from AML blasts, that these cells can effectively prime autologous cytotoxic T cells in vitro, and that they may be used as potential vaccines in the immunotherapy of AML.

Identification of a new subset of cells exhibiting dendritic phenotypes in patients affected by breast proliferative disorders

We report that a subset of circulating cells reacting with a monoclonal antibody raised against a protein marker is significantly increased in the peripheral blood of women carrying benign or malignant breast diseases, particularly in patients under 55 years of age with ductal mammary carcinomas. These cells were statistically (confidence level of 99%) less represented in a control population including healthy women or women carrying carcinomas of origin other than breast. Double staining analysis showed that they harbor markers of dendritic cells and exhibit endo- cytic activity, as determined by their ability to internalize FITC-dextran particles. Their dendritic morphology was further demonstrated by electron microscopy of sorted antibody-positive cells. However, expression of surface molecules, such as CD34 and CD14, usually not present in differentiated populations of dendritic cells was also observed. Adherent cells of patients with breast ductal carcinoma including mostly cells of this new subset were efficient stimulators of mixed lymphocyte reaction, attaining maximal stimulatory activity attained after TNFα treatment. In conclusion, we have shown that a subset of cells characterized by a phenotype suggestive of a yet undescribed stage of maturation of the dendritic cell lineage is accumulated in the blood of patients affected by breast proliferative disorders.