Mean Fluorescence Intensity Values Research Articles

Successful Interim Outcomes of Bortezomib Desensitization on Highly Sensitized Deceased Donor Kidney Transplant Waitlist Patients

Highly sensitized (HS) patients seldom have chance to receive kidney transplant. To increase transplantation rate among those population, previous IV immunoglobulin and rituximab desensitization protocol showed good transplant conversion rate, but also carried moderate rate of antibody mediated rejection. Bortezomib, a proteasome inhibitor can control the production of anti-HLA antibodies through plasma cell apoptosis. A single center waitlist desensitization program was employed to facilitate successful kidney transplantation in HS patients group. We prospectively enrolled 23 patients from Jul 2010 to Dec 2013. Desensitization protocol included two doses of IVIG (2 g/kg), single dose of rituximab (375 mg/m 2 , max 500 mg), and 4 days of bortezomib (1.3 mg/m 2 on days 1,3,7 and 9). Anti-HLA class I and II antibodies were determined by Luminex solid phase bead assay at baseline and M2, M3 and M6. We investigated the difference of transplant conversion rate between desensitized HS patients and non-desensitized HS waitlist patients. Also we analyzed the difference of PRA % values and mean fluorescence intensity (MFI) between pre- and post-desensitization serum. Mean age of treatment group was 48.7±11.2 years old. Male to female ratio was 12:11. O+ blood type patients were 34.8% of patients. Mean duration of previous renal replacement therapy were 153.5±65.5 months. Baseline mean class I and II PRA % values were 86±15.3 and 66±34. Baseline class I and II MFI values were 13,893±6,147 and 12,274±6,891. After desensitization, kidney transplant were successfully performed in 34.8% of patients, compared to 13.8% transplant conversion rate among non-desentized HS controls ( p =0.001 by Log-Rank). Among KT recipients, median duration of waiting after desensitization was 3 months with interquartile range 2-8.1 months. Class I PRA % had showed decreased values until 3 months after desensitization. After 6 months, there were no differences between the class I PRA values between pre-desensitization samples and post-desensitization samples. There were no other differences except class I PRA % values. Desensitization protocol were generally well tolerated, with the completion rate of 92% patients. There were no serious adverse events, but moderate adverse event were in 46%, most commonly fever and diarrhea. Highly sensitized waitlist patients are manageable by desensitization protocol including bortezomib.

Homeobox Transcription Factor VentX Regulates Differentiation and Maturation of Human Dendritic Cells

Dendritic cells (DCs) are specialized antigen presentation cells that play critical roles in the initiation and regulation of immune responses. The molecular determinants of DC differentiation and maturation are target of extensive investigation. VentX is a human homeobox transcriptional factor that regulates proliferation and differentiation of hematopoietic cells. In the current study, we report that ablation of VentX expression in monocytes significantly impaired their differentiation into DCs. Conversely, overexpression of VentX in monocytic THP1 cells accelerated their differentiation toward DCs. We showed that VentX regulates DC differentiation, in part, through modulating IL6 expression. Clinically, we found that VentX expression was elevated in intestinal lamina propria DCs (LPDCs) of inflamed mucosa from inflammatory bowel disease patients. Knockdown experiments suggested that VentX is essential for the maturation of LPDCs. In addition, corticosteroid treatment markedly decreased VentX expression in LPDCs and enforced expression of VentX counteracted the effects of corticosteroid on DCs maturation. Our data suggest that VentX is a critical transcriptional regulator of DC differentiation and maturation, and a potential target of immune regulation and therapy.

A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry

Flow cytometry is a powerful technique capable of simultaneous multi-parametric analysis of heterogeneous cell populations for research and clinical applications. In recent years, the flow cytometer has been miniaturized and made portable for application in clinical- and resource-limited settings. The sample preparation procedure, i.e. labeling of cells with antibodies conjugated to fluorescent labels, is a time consuming (∼45 min) and labor-intensive procedure. Microfluidics provides enabling technologies to accomplish rapid and automated sample preparation. Using an integrated microfluidic device consisting of a labeling and washing module, we demonstrate a new protocol that can eliminate sample handling and accomplish sample and reagent metering, high-efficiency mixing, labeling and washing in rapid automated fashion. The labeling module consists of a long microfluidic channel with an integrated chaotic mixer. Samples and reagents are precisely metered into this device to accomplish rapid and high-efficiency mixing. The mixed sample and reagents are collected in a holding syringe and held for up to 8 min following which the mixture is introduced into an inertial washing module to obtain ‘analysis-ready’ samples. The washing module consists of a high aspect ratio channel capable of focusing cells to equilibrium positions close to the channel walls. By introducing the cells and labeling reagents in a narrow stream at the center of the channel flanked on both sides by a wash buffer, the elution of cells into the wash buffer away from the free unbound antibodies is accomplished. After initial calibration experiments to determine appropriate ‘holding time’ to allow antibody binding, both modules were used in conjunction to label MOLT-3 cells (T lymphoblast cell line) with three different antibodies simultaneously. Results confirm no significant difference in mean fluorescence intensity values for all three antibodies labels (p < 0.01) between the conventional procedure (45 min) and our microfluidic approach (12 min).

The interaction between C5a and sphingosine-1-phosphate in neutrophils for antineutrophil cytoplasmic antibody mediated activation.

IntroductionC5a plays an crucial role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. The current study further investigated the interaction between C5a and sphingosine-1-phosphate (S1P) in neutrophils for ANCA-mediated activation.MethodsThe plasma levels of S1P from 29 patients with ANCA-associated vasculitis (AAV) in active stage and in remission were tested by enzyme-linked immunosorbent assay (ELISA). The generation of S1P was tested in C5a-triggered neutrophils. The effect S1P receptor antagonist was tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA.ResultsThe plasma level of circulating S1P was significantly higher in patients with AAV with active disease compared with patients in remission (2034.2 ± 438.5 versus 1489.3 ± 547.4 nmol/L, P < 0.001). S1P can prime neutrophils for ANCA-induced respiratory burst and degranulation. Compared with non-triggered neutrophils, the mean fluorescence intensity (MFI) value for CD88 expression was up-regulated significantly in S1P-triggered neutrophils. S1P receptor antagonist decreased oxygen radical production in C5a primed neutrophils induced by ANCA-positive IgG from patients. Blocking S1P inhibited C5a-primed neutrophil migration.ConclusionsS1P triggered by C5a-primed neutrophils could further activate neutrophils. Blocking S1P could attenuate C5a-induced activation of neutrophils by ANCA. The interaction between S1P and C5a plays an important role in neutrophils for ANCA-mediated activation.

Early response roles for prolactin cortisol and circulating and cellular levels of heat shock proteins 72 and 90α in severe sepsis and SIRS.

Objective. To evaluate the early heat shock protein (HSP) and hormonal stress response of intensive care unit (ICU) patients with severe sepsis/septic shock (SS) or systemic inflammatory response syndrome (SIRS) compared to healthy subjects (H). Methods. Patients with early (first 48 hrs) SS (n = 29) or SIRS (n = 29) admitted to a university ICU and 16 H were enrolled in the study. Serum prolactin, cortisol, and plasma ACTH were determined using immunoassay analyzers. ELISA was used to evaluate extracellular HSPs (eHSP90α, eHSP72) and interleukins. Mean fluorescence intensity (MFI) values for intracellular HSPs (iHSP72, iHSP90α) were measured using 4-colour flow-cytometry. Results. Prolactin, cortisol, and eHSP90α levels were significantly increased in SS patients compared to SIRS and H (P < 0.003). ACTH and eHSP72 were significantly higher in SS and SIRS compared to H (P < 0.005). SS monocytes expressed lower iHSP72 MFI levels compared to H (P = 0.03). Prolactin was related with SAPS III and APACHE II scores and cortisol with eHSP90α, IL-6, and lactate (P < 0.05). In SS and SIRS eHSP90α was related with eHSP72, IL-6, and IL-10. Conclusion. Prolactin, apart from cortisol, may have a role in the acute stress response in severe sepsis. In this early-onset inflammatory process, cortisol relates to eHSP90α, monocytes suppress iHSP72, and plasma eHSP72 increases.

Impact of the Presence and Duration of Donor-Specific Antibodies on Renal Function

BackgroundAlthough anti-human leukocyte antigen (HLA) antibodies (DSA) is associated with graft loss, 3 things remain unclear: whether the duration and strength of DSA affect renal function; what mean fluorescence intensity (MFI) cut-off should be used; and whether the DSA effect is additive in case of multiple DSAs. MethodsA study was made of 63 patients who received living donor kidney transplants with clonal deletion protocol and were followed up for 18 months with reduced doses of immunosuppressants. DSA was tested for monthly, using Luminex Mixed and Single Antigen beads (One Lambda, Inc., Canoga Park, CA, USA). Decrease of estimated glomerular filtration rate (eGFR) was obtained at baseline and 18 months after transplantation. Association of renal damage and DSAs was compared using several DSA models with several MFI cut-offs. ResultsAdditive DSA models always showed better association with renal damage than comprehensive models. When calculating the DSA effect in additive models, “proxy-area under the curve” (AUC)—a triangular approximation of the actual AUC—showed better association with renal damage than did DSA duration (R2 = 0.105 vs 0.087). Adjusting for other factors, 27% of the variation of GFR change was explained by proxy-AUC. No significant change of association occurred if the MFI cut-off level changed from 1000 to 3000. ConclusionOur results support the association of DSA with development of longitudinal renal damage. The clinical interpretation may be similar at MFI cut-offs of 1000, 2000, and 3000. An additive DSA effect may be expected in patients with multiple DSAs. Our study suggests the importance of frequently checking for DSA and reducing their MFI value to minimize renal damage by the antibodies.

Predicting Cell Of Origin In Diffuse Large B-Cell Lymphoma By High Dimensional Flow Cytometry

IntroductionDiffuse Large B-cell Lymphoma (DLBCL) is the most common histologic subtype of non-Hodgkin Lymphoma (NHL). Patients with DLBCL can be divided into prognostic subgroups according to the cell of origin (COO) of the lymphomas that are characterized as the germinal centre B-cell-like (GCB) and the activated B-cell-like (ABC) subgroup (Alizadeh et al Nature 2000:403:503-511). While Gene Expression Profile (GEP) is the “gold standard” method for defining COO in DLBCL, to perform GEP routinely on clinical cases would incur both financial and logistical pressures that would be challenging to overcome in many centers. As a result, several groups have endeavored to devise immunohistochemical (IHC) staining panels that recapitulate GEP results, but have met with variable success. In contrast with the inherently subjective nature of IHC result interpretation and the limited number of IHC-stained cells that can be assessed visually, flow cytometry is a widely available,mature and robust methodology that achieves quantitative measurement of multiple simultaneous antibody staining parameters on tens to hundreds of thousands of individual cells in a matter of minutes. In this study, we sought to devise a flow cytometric method for determining COO in DLBCL from freshly disaggregated patient lymph node biopsy specimens. MethodsSix GCB cell lines (OCI-Ly1, SU-DHL-10, SU-DHL-4, SU-DHL-8, Karpas 422 and Pfeiffer) and 6 ABC cell lines (SU-DHL-9, NU-DUL-1, OCI-Ly3, HBL-1, MD903 and OCI-Ly10) were stained with eight cell surface markers (CD45, CD19, CD10, CD27, CD38, CD138, CD44 and CD34), two intracellular markers (BCL6 and IRF4), and a viability dye simultaneously using a single-tube, 11-color assay and analyzed on a 4-laser BD LSRFortessa flow cytometer. Antibodies were chosen to include known GCB (CD10, CD27, CD38 and BCL6) and ABC (CD138, CD44 and IRF4) markers based on prior studies. CD45 and CD19 were included to assist in identifying the malignant B cell population and CD34 was included as an investigational marker. FCS file data were analyzed using FlowJo software. ResultsEvaluation of mean fluorescence intensity (MFI) values and their corresponding standard deviations for 40,000-190,000 viable events for each sample revealed CD27 and BCL6 to be expressed at significantly higher levels in GCB cell lines, and CD44 and IRF4 to be significantly higher in ABC cell lines. CD10 and CD34 tended toward higher expression in GCB and ABC cell lines, respectively, but these differences did not reach statistical significance according to the Student's t-test. Analysis of all 10 fluorescence parameters simultaneously (i.e. 10-dimensional data projection) requiring more sophisticated bioinformatic tools is currently in progress. According to the serial univariate COO decision algorithm outlined by Hans et al (Blood 2004;103:275-282), our flow cytometry results correctly assigned 5 out of 6 GCB and 4 out of 6 ABC cell lines (cell line COO designations as defined by GEP). DiscussionIn this study, we assessed the performance of a flow cytometry-based assay for determining COO in DLBCL. Our results demonstrated that most GCB and ABC DLBCL cell lines can be correctly assigned with initial 10-color flow panel; however, some markers were highly informative while others were less or not informative at all in COO discrimination. Further development will be needed to improve the antibody panel design to the point they are able to replicate GEP results faithfully. Although flow cytometry cannot be performed on archival pathology material (i.e. formalin fixed, paraffin embedded tissue), most community and academic pathology departments have either their own flow cytometry instrumentation on site or established local referral centers and thus fresh tissue can usually be allocated for multidimensional flow analysis. Our results support the usefulness of flow cytometry for COO determination in DLBCL. However, further optimization of the panel design and testing on patient biopsy samples is needed. Disclosures:No relevant conflicts of interest to declare.

Adhesion Of Acute Myeloid Leukemia Blasts To E-Selectin In The Vascular Niche Enhances Their Survival By Mechanisms Such As Wnt Activation

BackgroundAdhesion within the bone marrow microenvironment enhances leukemia survival and chemoresistance. Both normal hematopoietic stem cells and cancer cells are known to express E-selectin ligands, and adhesion of colon carcinoma cells to E selectin activates survival pathways such as NFκB (Porquet et al., BMC Cancer 11:285, 2011). E-selectin within the bone marrow vascular niche induces proliferation of normal hematopoietic stem cells (HSC), and a selectin inhibitor enhances HSC quiescence and self-renewal (Winkler et al., Nat Med 18:1651, 2012). We therefore initiated a study of E-selectin ligand expression and function by acute myeloid leukemia (AML) blasts to elucidate the potential role of E-selectin in AML biology and chemotherapy resistance. MethodsPrimary AML blasts and leukemia stem cells (LSCs) (CD34+CD38-CD123+) obtained with informed consent from 40 patients were analyzed for E-selectin ligands by flow cytometry for binding of E-selectin-Fc chimera or by labeling with the HECA452 antibody. Primary AML blasts were engrafted in NODscid IL2Rgc-/- mice for studies of E-selectin inhibitors in combination with chemotherapy. Human Stem Cell Signaling, Leukemia, Apoptosis, and NFκB PCR Arrays (SA Biosciences) were used to analyze gene expression by quantitative RT-PCR after adhesion of primary AML patient blasts to E-selectin coated plates, compared to bovine serum albumin (BSA) coated plates. The activation of the Wnt pathway was studied by luciferase reporter assay. ResultsWe find that the majority of primary patient acute myeloid leukemia blasts and leukemia stem cells express an E-selectin ligand, as demonstrated by flow cytometry by binding of E-selectin-Fc chimera and by staining with HECA-452 antibody [that recognizes hematopoietic cell E-/L-selectin ligand (HCELL) and cutaneous lymphocyte antigen (CLA)], as well as by binding to E-selectin coated plates. Flow cytometry analysis reveals that the mean percent binding of E-selectin-Fc chimera is 28% ± 24% (SD) by AML blasts, the mean % staining by the HECA-452 antibody 51% ± 35% (SD). De novo patients tended to have smaller mean fluorescence intensity (MFI) values than relapsed/refractory patients, as follows: for blasts, de novo mean 1441 ± 1127 (SD) vs. relapsed/refractory 4488 ± 4920 (SD) (Wilcoxon p=0.024), and for LSCs, de novo mean 1578 ± 1560 (SD) vs. relapsed/refractory 6601 ± 8498 (SD) (Wilcoxon p=0.061), suggesting upregulation of expression of E-selectin ligand for relapsed as compared to newly diagnosed patients. The specific E-selectin small molecule inhibitor GMI-1271 is able to overcome adhesion mediated chemotherapy resistance of AML in vitro and reduce the leukemia burden of primary AML engrafted NODscid IL2Rgc-/- mice in combination with chemotherapy agents daunorubicin and cytarabine. Addition of GMI1271 to chemotherapy in this xenograft model reduced the spleen burden at 2 weeks post treatment from 17.1 ± 10.4 X 106 hCD45+ cells/spleen to 7.6 ± 5.8 (p=0.04).To assess the molecular mechanism by which adhesion to E-selectin might protect AML blasts, we first screened with quantitative RT-PCR arrays. We found that adhesion to E-selectin caused upregulation of members of the Wnt and sonic hedgehog pathways for primary AML patient blasts grown on E-selectin vs. BSA coated plates, as well as members of other pathways critical to leukemia such as GM-CSF and IL-3 receptors and Fos. We then confirmed adhesion to E-selectin by AML blasts from 4 different patients enhanced activity of Wnt target genes by Wnt reporter assays. The Wnt reporter assay demonstrated 2-3 fold enhanced activity of Wnt target genes for AML blasts on E-selectin as compared to those on BSA, which increased to 3.3-4.5 fold with addition of Wnt3a. The inhibitor GMI-1271 reduced Wnt activity to 1.4-2.5 fold, similar to XAV939, an inhibitor of the Wnt/β catenin pathway that reduced activity to 1.1-1.8 fold. Similar reduction of Wnt pathway gene expression by GMI-1271 was also observed by the quantitative RT-PCR assay. ConclusionThese data support a critical role for E-selectin, likely in the vascular bone marrow niche, that promotes survival of AML, that can be targeted with therapeutic intent, and suggests that GMI-1271 should be explored as a treatment for AML in combination with chemotherapy. Disclosures:Chien:GlycoMimetics, Inc.: Research Funding. Haq:GlycoMimetics, Inc.: Research Funding. Magnani:GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Becker:GlycoMimetics, Inc.: Research Funding.

20-P

Aim The purpose of this study was to compare the reactivity of antibody (Ab) to cross-reactive epitope groups (CREGs) detected in the LABScreen® Single Antigen (LSA) bead panel by comparing the mean fluorescence intensity (MFI) of a individual beads to the MFI of the same bead tested using LABScreen®Singles beads. Methods Patient samples with known Ab to CREGs (n=30) were selected based on class I (LSA1) and II (LSA2) LABScreen® Single Antigen bead results (One Lambda Inc); 5 of these sera demonstrated reactivity to Bw4. These sera were retested using LABScreen® Singles beads (One Lambda Inc); these beads are the same beads/alleles included in the Single Antigen bead panels but available individually. The Singles beads tested include: A∗02:01, A∗24:02, B∗07:02, B∗44:02, B∗57:01, B∗14:01, DQB1∗02:01/DQA1∗03:01, DQB1∗03:01/DQA1∗03:01, DRB1∗03:01, and DRB1∗04:01. Results The Singles bead results were comparable to the LSA1 and LSA2 panel MFI reactivity in 26 of the 30 sera. However, 4 of the 5 Bw4 sera demonstrated increased MFI values in the Singles beads vs the original LSA1 results. The Singles beads with this Bw4 epitope are B∗44:02 and B∗57:01.[figure1]Fig. 1: This figure demonstrates the increased MFI values in the Singles beads for this Bw4 reactive serum. Conclusions There was no increase in MFI reactivity in the LSA1 and LSA2 panels as compared to the Singles beads in most of the sera analysed in this study. Because 4 of the 5 Bw4 reactive sera showed an increased reactivity with the Singles beads, we propose that Bw4 Ab may be underestimated in the LSA1 bead panel. There does not appear to be a dilution effect on the MFI reactivity in other CREG antibodies included in this study.

For Anti-HLA-Specific Donor Antibodies Detection By Flow Cytometry Cytotoxic Crossmatches Comparison of Methods

Anti-HLA-specific donor antibodies induce rapid, irreversible destruction of the transplant (hyperacute rejection) that today happens rarely due to immunologic studies—prospective crossmatch—of patients awaiting the kidney graft. The usual approach for pretransplant donor/recipient evaluation is based on 2 methods: (1) the cytotoxic complement crossmatch (CDC) and (2) the flow cytometric crossmatch (FCX). The CDC crossmatch is positive when complement-fixing antibodies are present, an absolute contraindication to kidney transplantation. The more sensitive FCX-positive crossmatch detects low concentrations of unable to fix performed antibodies complement. It is an “index” of possible damage due to accelerated rejection. The target of our study was to develop a cytotoxic flow cytometry crossmatch (cFCX) that detected cytotoxic antibodies move sensitively than the traditional CDC method and also was less subjective and more standardized for interpretation studying sera from 23 patients; the cFCX showed the requested efficiency characteristics even in an emergency. In addition, the new method permited one to calculate a cutoff for positivity (average value of the negative control + 2 standard deviations), assuring an “objective” interpretation of the results that agreed with the CDC but was more sensitive and accurate allowing solution of ambiguous results for cases of “doubt”-positive CDC crossmatch. Furthermore, our aim was to correlate the effect of the strength of the anti-HLA antibodies determined by mean fluorescence intensity value of LabScreen Single Antigen beads with results of CDC, cFCX, and FCX methods.

Interpretation of HLA single antigen bead assays

The introduction of single antigen bead (SAB) assays for detection and quantitation of HLA antibodies has improved our ability to identify and manage allosensitized transplant candidates and recipients and to improve organ allocation, and was critical to the creation of national paired kidney exchanges. The principal limitations of the technology have been detailed in the literature and include artifacts resulting in non-specific background, variability, lack of standardization, and interpretive challenges. Accurate interpretation of SAB assays requires consideration of a number of factors, including identification of epitope reactivity patterns, mean fluorescence intensity (MFI) values, patient history, and appreciation of individual bead and assay nuances. The MFI value provides an estimate of relative HLA antibody levels although limited by saturation and epitope distribution effects. A better understanding of SAB assays and MFI values will be necessary to ensure appropriate application of these assays clinically and a higher quality of antibody data used in support of published clinical studies.

Influence of Test Technique on Sensitization Status of Patients on the Kidney Transplant Waiting List

The exquisitely sensitive single antigen bead (SAB) technique was shown to detect human leukocyte antigen (HLA) antibodies in sera of healthy male blood donors. Such false reactions can have an impact on critical decisions, especially with respect to the determination of unacceptable HLA-antigen mismatches in patients awaiting a kidney transplant. We tested pretransplant sera of 534 patients on the kidney waiting list using complement-dependent cytotoxicity (CDC), enzyme-linked immunosorbent assay (ELISA) and SAB in parallel. Evidence of HLA antibodies was obtained in 5% of patients using CDC, 14% using ELISA, and 81% using SAB. Among patients without history of an immunizing event, 77% showed evidence of HLA antibodies in SAB. In contrast 98% of these patients were negative in ELISA and CDC. In patients without an immunizing event, SAB-detected antibodies reacted not always weakly but with mean fluorescence intensity (MFI) values as high as 14 440. High-MFI-value antibodies were found in some of these patients with HLA specificities that are rather common in general population, consideration of which would lead to unjustified exclusion of potential kidney donors. False SAB reactions can be unveiled by testing with additional antibody assays. Denial of donor kidneys to recipients based on HLA-antibody specificities detected exclusively in the SAB assay is not advisable.

Differential Expression of the Ly49GB6, but Not the Ly49GBALB, Receptor Isoform during Natural Killer Cell Reconstitution after Hematopoietic Stem Cell Transplantation

Inhibitory natural killer (NK) cell receptors specific for major histocompatibility complex class I (MHC-I) molecules include Ly49 receptors in mice and killer immunoglobulin-like receptors (KIR) in humans. The “licensing” or “arming” models imply that engagement of these receptors to self MHC-I molecules during NK cell development educates NK cells to be more responsive to cancer and viral infection. We recently reported that hematopoietic stem cell transplantation (HSCT) induced rapid and preferential expansion of functionally competent Ly49G+, but not other Ly49 family, NK cells independent of NK cell licensing via Ly49–MHC-I interactions. We now extend these studies to evaluate expression of the two Ly49G receptor isoforms Ly49GB6 and Ly49GBALB, using mice with different MHC-I haplotypes that express one or both of the isoforms. NK cells from CB6F1 (H-2bxd) hybrid mice express two different alleles for Ly49G receptor, Ly49GB6 and Ly49GBALB. We found that CB6F1 mice had more Ly49GB6+ NK cells than Ly49BALB+ NK cells, and that only Ly49GB6+ NK cells increased in relative numbers and in Ly49G mean fluorescence intensity values after HSCT similar to the B6 parental strain. We further observed that Ly49G+ NK cells in BALB/c (H-2d) and BALB.B (H-2b) mice, which have the same background genes, recover slowly after HSCT, in contrast to Ly49G+ NK cells in B6 (H-2b) recipients. The difference in expression of Ly49GB6 relative to Ly49GBALB was linked to differences in the activity of the Pro1 promoter between the two alleles. Thus, we conclude that the Ly49GB6 receptor dominates Ly49G expression on NK cells after HSCT in strains in which that allele is expressed. The data suggest that Ly49 allelic polymorphism within a particular Ly49 family member can differentially affect NK cell recovery after HSCT depending on the background genes of the recipient, not on the MHC-I haplotype.

Expression of CD200 and CD200R regulatory molecules on the CD83+ monocyte-derived dendritic cells generated from patients with laryngeal cancer

CD200 molecule may play a role in local tumor invasion and augmenting the metastatic capacity of squamous cell carcinoma. The objective of the study was to assess by means of flow cytometry the expression of CD200 and its receptor, CD200R, on CD83+ monocyte-derived dendritic cells (Mo-DCs), pulsed or not with autologous tumor cell lysates (aTCL) in patients who suffer from laryngeal carcinoma in comparison to healthy donors. The median value of CD200 mean fluorescence intensity (MFI) on the Mo-DCs pulsed with aTCL of the patients with laryngeal cancer was 61.94 and was statistically significantly higher than on the unpulsed Mo-DCs of these patients (24.81) and healthy donors (16.63), p = 0.0034 and p = 0.0004, respectively. Median MFI score of CD200R in specimen derived from patients with laryngeal cancer was 259.31 on Mo-DCs pulsed with aTCL, while in unpulsed Mo-DCs was 86.74 (p = 0.0035) and on the Mo-DCs from control group it was 67.51 (p = 0.0004). The obtained results showed a relation between the presence of laryngeal cancer and the expression of CD200 and CD200R molecules on the CD83+ Mo-DCs pulsed with autologous cancer cell lysates. This analysis may have implications for setting new therapeutic options for cancer immunotherapy in the future.

Abstract 459: PD-L1 expression in breast cancer cell lines after IFNγ treatment varies significantly between molecular subtypes.

Abstract Background The molecular subtypes of breast cancer described by Charles Perou provides greater insight into the underlying biology of different breast cancers. An important aspect of this biology is the ability of a particular breast cancer to evade the host immune response through activation of immunosuppressive pathways. Understanding the relationship between molecular subtypes and potential for immune evasion is important in determining which breast tumors can be targeted by immunomodulatory treatments. We focused specifically on the surface expression of the programmed death ligand 1 (PDL1) on breast cancer cell lines within different molecular subtypes. Binding of PD1 on T cells by its ligand PDL1 causes an inhibitory cascade resulting in T cell anergy and apoptosis. Expression of PDL1 on tumor cells creates an immunosuppressive environment reducing the number of tumor specific cytotoxic T cells. A potent inducer of PDL1 expression is interferon γ (IFN γ), which is frequently present in the tumor microenvironment. We sought to study differences in PDL1 expression between luminal (MCF7, AU565), basal A (BT20, HCC1143), and basal B (MDA231, HCC38) breast cancer cell lines provided by CK Zhang at ATCC. Methods PDL1 protein expression was analyzed in an ATCC assay ready breast cancer cell panel. Surface and intracellular PDL1 expression +/- IFN γ for 48hrs was measured by flow cytometry (BD Biosciences) on a LSRII with mean fluorescence intensity (MFI). The cells were treated with 50 μM of epigallocatechin gallate (EGCG) (Sigma), a STAT1 inhibitor, to explore the role of STAT1 signaling in the expression of PDL1. Levels of intracellular phosphoSTAT1 were also measured by flow (Beckman Coulter) in these experiments. Results Basal cells had the greatest baseline MFI values (mean 1817) vs luminal (mean 0). All cells increased PDL1 expression with IFN γ, but the basal B cells demonstrated the largest absolute increases by far (HCC38 MFI =13948 vs. MCF7 MFI =764). This was in part due to greater total PDL1 expression, but also a larger proportion of total expressed PDL1 localizing to the surface in comparison to the other molecular subtypes. Finally, treatment with EGCG was able to significantly inhibit expression of PDL1 and lowered levels of pSTAT1 especially in the HCC38 cell lines. Conclusion Our data suggests that basal type breast cancer cells (especially basal B) express greater levels of PDL1 both constitutively and in response to IFN γ. It appears that the STAT1 pathway is involved in mediating this effect in response to IFN γ. Prospective knowledge of a breast tumor's ability to phenotypically express PDL1 based on molecular subtype will allow for better selection of patients for future anti-PDL1 antibody clinical trials. Citation Format: Kimberly Luddy, Hatem Soliman. PD-L1 expression in breast cancer cell lines after IFNγ treatment varies significantly between molecular subtypes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 459. doi:10.1158/1538-7445.AM2013-459

1001-LB

Aim Determination of anti-HLA antibodies by fluorescence techniques is now the standard of practice. The antibodies are typically detected via Luminex® technology with a detected anti-HLA antibody assigned a mean fluorescence intensity (MFI) based on its abundance and binding capacity in the patient serum. The goal is to use the anti-HLA antibodies for a “virtual crossmatch” for (1) better patient-donor matching in organ transplantation, (2) for determination of appropriate platelet donors for those with chemotherapy-associated or poststem cell transplantation-associated thrombocytopenia, and (3) for determining whether allelespecific antibodies may be present which can interfere with hematopoietic stem cell transplantation (HSCT). The assay is more sensitive than the previously utilized CDC (complement-dependent-cell-cytotoxicity) assay, with the unintended risk that a patient may be inappropriately predicted as incompatible. The overall aim of the study was to compare the results of a CDC crossmatch with the donor-specific anti-HLA antibody (DSA) MFI values in order to determine which DSA (anti-HLA-A, anti-HLA-B, anti-HLA-Cw, anti-HLA-DR, anti-HLADR51, anti-HLA-DR52, anti-HLA-DR53, and anti-HLA-DQ) are likely meaningful at which MFI values. Method Patients and potential donors underwent HLA-typing by routine CDC assay with OneLambda® typing trays and by reverse SSO using OneLambda® reagents. Donor T and B lymphocytes underwent crossmatches with patient serum utilizing standard CDC assays in microtiter trays. Patient anti-HLA antibodies were determined using OneLambda® reagents via Luminex® analysis. CDC crossmatches are reported as negative (1 and 2) or positive (4, 6, and 8). DSA MFI were plotted versus the CDC crossmatch results. Statistical analysis was performed utilizing Student t test and Microsoft Excel. Data were also analyzed via contingency table for determining specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV). Results In the organ transplant setting, we compared the MFI of DSA with crossmatch results determined by the CDC technique. MFI values less than 1000 correlated well with having a negative CDC crossmatch, but values >1000 correlated poorly (specificity 51%, PPV 38%). For > 3000 MFI, specificity = 63%, PPV = 43%. For >10,000 MFI, specificity = 84%, PPV = 63%. NPV for: 1000 = 100%; 3000 = 97%; 10,000 = 98%. These data indicate the use of 1000 MFI as the cutoff for positive detection of DSA will result in prediction of an unsuitable donor-patient virtual match ∼62% of the time, improving to ∼ 37% for MFI of >10,000, but there is the risk for allowing for positive crossmatches in those proceeding forward. Additionally, the newer fluorescence-based assays allow for detection of anti-HLA antibodies at the allele-level of discrimination. Summarizing the presented data with data not shown: anti-HLA antibodies can be divided into (1) those which bind to a cell surface and activate complement, and thereby may be detrimental to endothelial cells in transplanted organs and (2) those which can bind to a cell surface, e.g., platelets, and directly exert a detrimental effect (without need for complement activation), likely by targeting the cells for removal by the reticuloendothelial system. These results indicate that better techniques need to be developed for determination of truly detrimental DSA, in order to better discriminate appropriate donor-recipient pairing for organ transplantation. Finally, since anti-HLA antibodies have been shown to result in “resistance to engraftment” in HSCT, the specific identification of anti-HLA antibodies directed against specific HLA allele products can allow for better HSCT donor selection, or the need for anti-HLA antibody reduction treatment prior to transplantation.

Case Report: Binding of a Clinically Relevant Human Leukocyte Antigen–DQα–Specific Antibody in a Kidney Graft Recipient is Inhibited by Donor-Type Human Leukocyte Antigen–DQβ Chain

In this case report, we have found what may be an immunization with donor-specific human leukocyte antigen (HLA)-DQα in combination with recipient-specific HLA-DQβ. A renal allograft recipient who did not comply with immunosuppressive therapy during pregnancy had graft failure 23 months posttransplantation with biopsy-proven humoral and cellular rejection. Sera were tested in a Luminex-based single-antigen bead assay. We compared Luminex reactivity with the degree of eplet mismatching between the recipient's own HLA-DQ chains and the HLA-DQ chains bound to the Luminex beads. Eplet calculations were done with the HLAMatchmaker. HLA-DQ similarities were compared further by dissimilarity scoring in HistoCheck. We observed that Luminex beads with donor-type HLA-DQα and HLA-DQβ bound less antibody than beads with donor-type HLA-DQα combined with recipient HLA-DQβ. In HLAMatchmaker, we identified all eplet mismatches between donor and recipient HLA-DQ. Next, we counted how many of these eplets were represented on the various Luminex beads. We found that antibody binding to the bead increased with the number of such mismatches for HLA-DQα. Surprisingly, antibody binding decreased as the number of eplet mismatches for HLA-DQβ increased, from a mean fluorescence intensity (MFI) value of 18,800 for no mismatched eplets to approximately 10,000 for 12 mismatched eplets. These findings were confirmed by comparing antibody binding with the structural dissimilarity score between the recipient HLA-DQ type and the HLA-DQ bound to the Luminex beads. In this patient, clinically relevant antibodies bound strongly to donor-like HLA-DQα chains when combined with recipient-like HLA-DQβ. HLA-DQβ chains more similar to those of the donor reduced the binding of donor- specific HLA-DQα antibody.

Systematic Comparison of Four Cell- and Luminex-Based Methods for Assessment of Complement-Activating HLA Antibodies

Efforts to increase the specificity and sensitivity of human leukocyte antigen (HLA) antibody detection assays recently led to the establishment of two novel Luminex bead-based assays to detect complement-activating antibodies by the assessment of complement products C1q or C4d. Here, we present a systematic comparison of the four methods, complement-dependent lymphocytotoxicity (CDC) and C1q-, C4d-, and IgG-Luminex, to assess or predict the complement-binding capability of HLA IgG antibodies. Forty-five sera of highly immunized patients have been assessed by in-house modified C1q- and C4d-Luminex assays and compared with standard CDC and IgG-Luminex. Antibody specificities assigned by the C1q- and C4d-Luminex assay revealed an excellent concordance of 94% and 97% for HLA class I and II, respectively. Complement-fixing HLA class II antibodies were found less frequently among IgG antibodies compared with class I. Both C1q- and C4d-Luminex detected, on average, three times more specificities than CDC. Although we found a high correlation of mean fluorescence intensity values between C1q- and C4d-Luminex assays, IgG mean fluorescence intensity was not a suitable surrogate marker for the prediction of complement binding. C1q- and C4d-Luminex assays are characterized by an increased sensitivity and specificity compared with CDC, the current standard in detecting complement-fixing HLA antibodies. Pretransplantation risk assessment for transplantation but also posttransplantation monitoring are important applications for both assays to improve overall allograft survival.

Issue highlights—March 2013

Issue highlights—March 2013

Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs). An indirect intracellular staining (ICS) method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI) values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.