Changes In Antigen Expression Research Articles

Transient inhibition of lysosomal functions potentiates nucleic acid vaccines.

Nucleic acid vaccines have shown promising results in the clinic against infectious diseases and cancers. To robustly improve the vaccine efficacy and safety, we developed an approach to increase the intracellular stability of nucleic acids by transiently inhibiting lysosomal function in targeted tissues using sucrose. To achieve efficient and localized delivery of sucrose in animals, we designed a biomimetic lipid nanoparticle (LNP) to target the delivery of sucrose into mouse muscle cells. Using this approach, viral antigen expression in mouse muscle after DNA vaccination was substantially increased and prolonged without inducing local or systemic inflammation or toxicity. The same change in antigen expression would be achieved if the vaccine dose could be increased by 3,000 folds, which is experimentally and clinically impractical due to material restrictions and severe toxicity that will be induced by such a high dose of nucleic acids. The increase in antigen expression augmented the infiltration and activation of antigen-presenting cells, significantly improved vaccine-elicited humoral and T cell responses, and fully protected mice against the viral challenge at a low dose of vaccine. Based on these observations, we conclude that transient inhibition of lysosome function in target tissue by sucrose LNPs is a safe and potent approach to substantially improve nucleic acid-based vaccines.

Guidelines for the flow cytometric minimal residual disease monitoring in B-lineage acute lymphoblastic leukemia after CD19-directed immunotherapy

Multicolor flow cytometry is now routinely used in laboratory practice for the minimal residual disease (MRD) monitoring in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Wide application of CD19-directed immunotherapy leads to frequent loss of CD19 expression, that hampers significantly the flow cytometric MRD detection methodology. We developed an antibody panel and data analysis algorithm for multicolor flow cytometry, which is a reliable method for MRD detection in patients with BCP-ALL treated with CD19-directed therapy. We recommend a single-tube 11-color panel for MRD detection, which is adapted for the case of possible CD19 loss. Based on patterns of antigen expression changes and the relative expansion of normal CD19-negative BCPs, guidelines for multicolored flow cytometry data analysis and interpretation are established. The recommended approach is reliable tool for therapy response monitoring displaying the same effectiveness with the more laborious and costly molecular techniques.

Mechanisms of Resistance to Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs), with antibodies targeted against specific antigens linked to cytotoxic payloads, offer the opportunity for a more specific delivery of chemotherapy and other bioactive payloads to minimize side effects. First approved in the setting of HER2+ breast cancer, more recent ADCs have been developed for triple-negative breast cancer (TNBC) and, most recently, hormone receptor-positive (HR+) breast cancer. While antibody-drug conjugates have compared favorably against traditional chemotherapy in some settings, patients eventually progress on these therapies and require a change in treatment. Mechanisms to explain the resistance to ADCs are highly sought after, in hopes of developing next-line treatment options and expanding the therapeutic windows of existing therapies. These resistance mechanisms are categorized as follows: change in antigen expression, change in ADC processing and resistance, and efflux of the ADC payload. This paper reviews the recently published literature on these mechanisms as well as potential options to overcome these barriers.

Reliable Flow-Cytometric Approach for Minimal Residual Disease Monitoring in Patients with B-Cell Precursor Acute Lymphoblastic Leukemia after CD19-Targeted Therapy.

We aimed to develop an antibody panel and data analysis algorithm for multicolor flow cytometry (MFC), which is a reliable method for minimal residual disease (MRD) detection in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treated with CD19-directed therapy. The development of the approach, which was adapted for the case of possible CD19 loss, was based on the additional B-lineage marker expression data obtained from a study of primary BCP-ALL patients, an analysis of the immunophenotypic changes that occur during blinatumomab or CAR-T therapy, and an analysis of very early CD19-negative normal BCPs. We have developed a single-tube 11-color panel for MFC-MRD detection. CD22- and iCD79a-based primary B-lineage gating (preferably consecutive) was recommended. Based on patterns of antigen expression changes and the relative expansion of normal CD19-negative BCPs, guidelines for MFC data analysis and interpretation were established. The suggested approach was tested in comparison with the molecular techniques: IG/TR gene rearrangement detection by next-generation sequencing (NGS) and RQ-PCR for fusion-gene transcripts (FGTs). Qualitative concordance rates of 82.8% and 89.8% were obtained for NGS-MRD and FGT-MRD results, respectively. We have developed a sensitive and reliable approach that allows MFC-MRD monitoring after CD19-directed treatment, even in the case of possible CD19 loss.

Expression and regulation of Siglec-6 (CD327) on human mast cells and basophils

Mast cells (MC) and basophils are effector cells of allergic reactions and display a number of activation-linked cell surface antigens. Of these antigens, however, only a few are functionally relevant and specifically expressed in these cells. We sought to identify MC- and basophil-specific surface molecules and to study their cellular distribution and regulation during cytokine-induced and IgE-dependent activation. Multicolor flow cytometry was performed to recognize surface antigens and to determine changes in antigen expression upon activation. We identified Siglec-6 (CD327) as a differentially regulated surface antigen on human MC and basophils. In the bone marrow, Siglec-6 was expressed abundantly on MC in patients with mastocytosis and in reactive states, but it was not detected on other myeloid cells, with the exception of basophils and monocytes. In healthy individuals, allergic patients, and patients with chronic myeloid leukemia (CML), Siglec-6 was identified on CD203c+ blood basophils, a subset of CD19+ B lymphocytes, and few CD14+ monocytes, but not on other blood leukocytes. CML basophils expressed higher levels of Siglec-6 than normal basophils. IL-3 promoted Siglec-6 expression on normal and CML basophils, and stem cell factor increased the expression of Siglec-6 on tissue MC. Unexpectedly, IgE-dependent activation resulted in downregulation of Siglec-6 in IL-3-primed basophils, whereas in MC, IgE-dependent activation augmented stem cell factor-induced upregulation of Siglec-6. Siglec-6 is a dynamically regulated marker of MC and basophils. Activated MC and basophils exhibit unique Siglec-6 responses, including cytokine-dependent upregulation and unique, cell-specific, responses to IgE-receptor cross-linking.

Abstract 6004: Surveying surface antigen expression in multiple myeloma preclinical models

Abstract Multiple myeloma (MM) is a progressive hematological cancer with a 5-year survival rate of 53% (1). Novel therapeutic strategies are being developed to target specific MM surface antigens. Yet, changes in antigen expression through MM progression are poorly understood in the clinic and have not been well characterized in preclinical models. Here, we were interested in understanding how BCMA, CD38, CD138 and HLA-DR surface expression patterns may be affected as MM progresses in preclinical models. To do so, ARH-77 and RPMI-8226 cells were inoculated subcutaneously while MM.1S, MM.1R, U266 and NCI-H929 cells were dosed intravenously into NSG mice. Tumor xenograft or bone marrow, whole blood and spleen were processed for flow cytometry analysis. Antigen expression was measured at different stages of tumor progression as well as in vitro. Soluble BCMA levels were assessed in the mouse serum by ELISA. ARH-77 and RPMI-8226 subcutaneous xenografts show preferential growth when inoculated in PBS and 50% growth factor-reduced Matrigel, respectively. BCMA expression was reduced in both ARH-77 and RPMI-8226 xenografts compared to in vitro culture. CD38 and HLA-DR expression increased as ARH-77/PBS and RPMI-8226/Matrigel tumor volume progressed. All 4 antigens were detected in the bone marrow from MM.1S, MM.1R and U266-bearing mice, as early as 5-6 weeks post intravenous injection. In contrast, antigens were not detected in any tissue from mice injected with NCI-H929, likely indicating failed engraftment. At 5 weeks, antigens were detected in the whole blood and spleen in MM.1S-bearing mice only, suggesting organ invasion and a more advanced disease-stage compared to MM.1R or U266. Experiments are being carried out to test antigen expression in tissue at late-stage tumor progression as well as serum BCMA levels in the intravenous MM models. These data highlight antigen expression differences in MM cells when analyzed in mouse tissue compared to in vitro culture. Like the widely variable expression observed between patients (2, 3), BCMA and CD138 were differentially expressed in the mouse bone marrow between models. Our observations suggest that commonly targeted antigens in MM vary kinetically in vivo and can be measured by flow cytometry. The present findings also support the use of RPMI-8226 and MM.1S cell lines when screening for antigen-specific immunotherapies and combinatorial studies for MM treatment.

Thrombopoietin-Receptor Agonist Romiplostim Attenuates Hematopoietic System Injury By Promoting the Recovery of Early Hematopoiesis and the Hematopoietic Environment in the Bone Marrow and Spleen of Mice Exposed to Lethal Total Body γ-Irradiation

High-dose radiation exposure, usually caused by radiological accidents or nuclear disasters, can cause lead to acute radiation syndrome (ARS), which is a life-threatening condition. The International Atomic Energy Agency recommends the use of a combination of various cytokines or bone marrow (BM) transplant for treating high-dose exposure, depending on the degree of radiation exposure and ARS. Therefore, drugs that respond quickly, which can be used to treat a large number of radiation-associated casualties represent a good treatment option. However, certain hematopoietic cytokines, such as the granulocyte macrophage-colony stimulating factor and interleukin-3, which are effective against hematopoietic dysfunction and have been used in previous accidents, are not approved as pharmaceutical drugs in Japan. Although granulocyte-colony stimulating factor is only approved domestically for the treatment of ARS, it has been found that a single dose is insufficient to improve the survival rate of individuals exposed to lethal ionizing radiation. We have previously demonstrated that an effective protocol of drug therapy, based on approved pharmaceutical drugs in Japan, is the administration of romiplostim (RP), a thrombopoietin-receptor myeloproliferative leukemia virus proto-oncogene (c-mpl) agonist, at a dose of 50 μg/kg body weight/day for 3 consecutive days. This regimen achieved 100% survival at 30-days in 8-weekold female C57BL/6JJcl mice when administered immediately following exposure to a lethal dose (7 Gy) of 137Cs γ-rays. In the present study, we assessed the mechanisms by which RP ameliorated radiation-induced hematopoietic damage in mice after lethal total body γ-irradiation (TBI) by focusing on the recovery and regeneration of BM and spleen. Mice were divided into 4 groups: control group, RP group, TBI group, and TBI + RP group. On days 4, 10, 18, and 30 after TBI, morphological and histological changes of BM and spleen and antigen expression changes of cells isolated from both tissues were assessed. While the body weight of animals in the TBI group decreased significantly by day 18 compared to controls, that of mice receiving RP remained comparable. Similarly, on day 18, leukocyte, platelet, erythrocyte, and hemoglobin levels were the lowest in TBI mice. Conversely, these values markedly recovered in the TBI + RP group. Splenic atrophy was assessed by weighing the spleen after TBI, and it was observed that while the spleen contracted significantly following TBI, mice in the RP + TBI group showed a 3 to 4-fold increase in spleen weight and in cell numbers on day 18. Endogenous colonies, which are indicators of hematopoiesis, were also observed in the spleen after day 10. In addition, TBI induced severe myelosuppression that resulted in the loss of hematopoietic function. On day 18 after irradiation, while the BM exhibited obvious myeloablation, that of the RP + TBI treated mice was comparable to controls. Further, the number of BM cells in mice RP + TBI treated mice continued to increase till 30 days after TBI but did not attain pre-TBI levels. Additionally, analysis of cell-surface antigen expression in BM and spleen cells showed that, compared to the TBI group, RP administration promoted the recovery of hematopoietic stem cells, multipotent progenitor cells, and common lymphocyte precursor cells. In particular, a dramatic increase in the number of common myelo-erythroid progenitors were observed in the spleen on day 18 in the TBI + RP treated mice, accompanied with the disappearance of splenic cyst structures and acceleration of megakaryocyte hematopoiesis. These results suggest that thrombopoietin-receptor agonist RP promotes the recovery of early hematopoiesis and hematopoietic environment in both BM and spleen of lethal TBI mice, especially the promoting megakaryocytopoiesis in the spleen. As a result, treating lethally-irradiated mice with RP results in 100% survival. DisclosuresNo relevant conflicts of interest to declare.

Sézary syndrome and mycosis fungoides: An overview, including the role of immunophenotyping

This review discusses the definition and major categories of cutaneous T-cell lymphoma, Sézary syndrome and mycosis fungoides, and the role of immunophenotyping in their diagnosis. The following key points are raised: (a) Sézary syndrome and mycosis fungoides cells most often have a characteristic CD3+ CD4+ CD7- and/or CD26- immunophenotype. (b) This immunophenotype is not specific, but can assist in the distinction from non-neoplastic T cells and other subtypes of mature T-cell neoplasm. (c) However, small subsets of normal and reactive T-cells can have an overlapping immunophenotype, and can be distinguished by evaluating for additional changes in antigen expression.

Detection of Dysregulated Host Response By Flow Cytometry May Pre-Empt Early Diagnosis of Sepsis after Cardiac Surgery

INTRODUCTION Sepsis is a life-threatening organ dysfunction caused by dysregulated host response to infection. Early detection of sepsis is very crucial in its management, as there is an increase of 8% mortality for every hour delay in commencing therapy (Kumar et al., 2006). A wide variety of diagnostic techniques proposed, could not be clinically translated due to poor sensitivity & specificity, high levels of heterogeneity and complexity of assay preparation. Currently most clinical settings depend on Procalcitonin (PCT) and C-reactive protein (CRP) for diagnosis, which also lack sensitivity and specificity. The objective of our study was early detection of sepsis in patients undergoing cardiac surgery. Blood stream infection, confirmed by blood culture (the gold standard), requires large turnaround time and is less sensitive here due to prophylactic antibiotics. SOFA score overestimate the probability of sepsis due to the impaired cardiovascular parameters and inotrope support (Howitt et al., 2018). The Society of Thoracic Surgeons (STS) criteria to detect sepsis need positive blood culture to identify sepsis within the first 48 hours post-surgery. Since none of the above can cater early diagnosis, the most appropriate way is to target dysregulated host response. It has been reported that increased expression of CD64, on neutrophil surface (nCD64) is associated with proinflammatory response and down-regulation of HLA-DR expression on circulating monocytes (mHLA-DR) is associated with anti-inflammatory response in humans. Citing this interplay between pro and anti-inflammatory response in sepsis, we hypothesized that the relative expression of these antigens may detect dysregulated host immune response and thereby may provide a criterion for early diagnosis of sepsis MATERIALS AND METHODS A flowchart of the experimental steps is shown in Fig1. Adult patients, who underwent cardiac surgery, were selected for this double-blinded study after the approval from the appropriate Institutional Ethics Committee. The study was un-blinded after the initial set of experiments, with biochemical and clinical outcome of the patients. The data sets were analyzed (GraphPad Prism v8.1.1) and a p value of < 0.05 was considered statistically significant RESULTS Out of the total patients (n=33), 7 patients were diagnosed with suspected sepsis and 1 with proven sepsis as per STS criteria, substantiated by longer ICU and hospital stay (Table1). The optimized Flowcytometry panel and gating strategies is shown in Fig.2 The expression of nCD64, mHLA-DR and SI (Sepsis Index) in all patients before surgery did not show any statistically significant difference with that of healthy controls [Figure-3A]. At 24 hours post-surgery, all patients had significant up-regulation of nCD64 and down-regulation of mHLA-DR. A similar significant elevation was observed in CRP and PCT [Figure-3 B-D], but insignificant difference exists between sepsis and non-sepsis patients (p values in Table1). Therefore the diagnostic efficacy of all this measurements and scoring scheme, in identifying sepsis at 24 hours was poor (Table2) A useful diagnostic criterion is obtained by calculating the fold increase in nCD64 (I64) & SI (ISI) and fold decrease in mHLA-DR (DHLA) at 24 hour. It was observed that many patients had ISI≥10, due to I64 approximately 2 and DHLA 5. Based on this observation, a diagnostic criterion able to detect 'dysregulated host immune response' at 24 hour post-surgery is identified. The criterion is: 10 fold or more increase in SI combined with either ≤1.8 fold increase in nCD64 or ≤5.2 fold decrease in mHLA-DR. CONCLUSION The data obtained from this pilot study was analysed based on different criteria to identify the best possible way to detect the onset of sepsis post-cardiac surgery. The discriminative power of many tests to differentiate sepsis and SIRS is inadequate. We propose a combination of fold changes in antigen expression, which could so far, identify all sepsis patients, since the measurements detect the underlying biological mechanism, picking up both exacerbated proinflammatory response and immunoparalysis. The significance of the result is that the proposed diagnostic criteria could potentially pre-empt diagnosis of sepsis at 24 hours post-surgery, before the onset of any clinically identifiable symptoms of the disease. This needs to be substantiated by extending the study on a larger patient cohort. Disclosures Mony: BD Biosciences: Research Funding. Jain:BD Biosciences: Employment.

A pilot study of pembrolizumab in combination with decitabine and hypofractionated index lesion radiation in pediatric and young adult patients with relapsed and refractory solid tumors or lymphoma.

TPS25 Background: A potential limitation to the use of immune checkpoint inhibitors in younger patients with immune-resistant histologies is related to low tumor associated antigen expression and an immunosuppressive microenvironment. Low dose decitabine, as an inhibitor of DNA methyltransferase, can unrepress immunogenic cancer testes antigen expression, while hypofractionated radiation promotes infiltration of immune effector cells, stimulation of toll-like receptors on dendritic cells, and induction of type I interferons and other cytokines. As induced PD-L1 expression may also result, we hypothesize that decitabine, radiation, and the PD-1 inhibitor, pembrolizumab, will be synergistic. Here, we aim to verify the safety and preliminary activity of the combination. Correlative studies include an investigation of changes in antigen expression, the kinetics of immune effector and regulatory cell phenotype and function, and microbiome. Methods: Treatment program: Every 28 day cycle, patients receive 10 mg/m2 IV decitabine on days 1-5 and 2 mg/m2 (max dose 200 mg) IV pembrolizumab on day 8. Hypofractionated radiation (8 Gy x 3) to one or more lesion(s) is given on days 5-7 of the second cycle only. Adult patients may consent to optional biopsy of an irradiated lesion. Beyond cycle 3, patients may undergo elective surgery of one or more lesions. Patients remain on therapy for 27 cycles, absent progression by irRECIST or unacceptable toxicity. Patient eligibility: Patients 12 months to 40 years with available archived tissue may be considered, and must have adequate organ function including an absolute neutrophil count ≥ 750/mm3 and platelet count ≥ 75,000/mm3. Patients may have stable central nervous system metastases. Patients previously treated with radiotherapy may be eligible. Patients previously treated with PD-1 inhibitor therapy may be eligible, unless treated for an immune related adverse event. Current enrollment: This trial has been open to enrollment since February of 2018. No dose limiting toxicities have been observed in 2 patients evaluable for toxicity. Clinical trial information: NCT03445858.

No prognostic significance of immunophenotypic changes at the end of remission induction therapy in children with B-lineage acute lymphoblastic leukemia

Detection of aberrant antigen expression in acute lymphoblastic leukemia (ALL) by flow cytometric is proposed for the quantification of minimal residual disease (MRD). There are few studies that investigate the stability of the antigen expression in children with B lineage ALL at the end of remission induction therapy and determine its prognostic impact. Between 2010 and 2015, 691 bone marrow specimens of childhood ALL were sent at diagnosis for immunophenotypic characterization, and follow-up samples for MRD were analyzed on day 33. Of these, 155 patients with MRD more than or equal to 0.01% were eligible for the study. Immunophenotypic studies were performed by multiparametric flow cytometry using four-colour monoclonal antibody combinations. Overall, 86 of 155 (55.5%) cases showed phenotype shifts at least one marker. CD19 was the most stable markers. By contrast, CD20 was significantly different between diagnosis and day 33 in nearly one third of the cases. Shifts of antigen expression was not significantly associated with EFS, RFS or OS (P > 0.05). Multivariate analysis showed that WBC and BCR-ABL have independent prognostic value in childhood ALL. Changes in antigen expressions were commonly occurred at the end of induction and not associated with prognostic value in patients whose MRD were positive on day 33.

Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. DisclosuresNo relevant conflicts of interest to declare.

Changes in Antigen Expression in a Follow-up of Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma

Objectives:Diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) depends on specific immunophenotype of select antigens and their characteristic density. However, reports detailing how the expression of specific antigen changes over time of disease course are sparse. Methods: We identified 37 CLL/SLL with initial and follow-up samples studied by flow cytometry. Expression levels of a panel of antigens were compared between initial and follow-up samples from the same patients. Results: There is a significant decrease in the CD23 density and in the percentage of CD23 positive cells, and a decrease in the density of CD5 in the follow-up samples. In contrast, a significant increase in the density of HLA-DR antigen was observed in the follow-up. Conclusion: The expression of selected antigen changes in the course of CLL/SLL. These alterations do not impact the diagnosis of CLL/SLL based on the immunophenotype of follow-up samples and should not influence the ability to determine minimal residual disease by flow cytometry. However, they can potentially influence the responsiveness to the monoclonal antibody therapy and should be further systematically evaluated.

Effects of epithelial to mesenchymal transition on T cell targeting of melanoma cells.

Melanoma cells can switch phenotype in a manner similar to epithelial to mesenchymal transition (EMT). In this perspective article, we address the effects of such phenotype switching on T cell targeting of tumor cells. During the EMT-like switch in phenotype, a concomitant change in expression of multiple tumor antigens occurs. Melanoma cells undergoing EMT escape from killing by T cells specific for antigens whose expression is downregulated by this process. We discuss melanoma antigens whose expression is influenced by EMT. We assess the effect of changes in the expressed tumor antigen repertoire on T-cell mediated tumor recognition and killing. In addition to escape from T cell immunity via changes in antigen expression, mesenchymal-like melanoma cells are generally more resistant to classical chemotherapy and radiotherapy. However, we demonstrate that when targeting antigens whose expression is unaltered during EMT, the capacity of T cells to kill melanoma cell lines in vitro is not influenced by their phenotype. When considering immune therapies such as cancer vaccination, these data suggest escape from T cell killing due to phenotype switching in melanoma could potentially be avoided by careful selection of target antigen.

Increased expression of CD20 and CD45 and diminished expression of CD19 are features of follicular lymphoma

Increased expression of CD20 and CD45 and diminished expression of CD19 are features of follicular lymphoma Qingyan Liu,1 Linda S Weaver,1 David Liewehr,2 David Venzon,2 Maryalice Stetler-Stevenson,1 Constance M Yuan11Flow Cytometry Unit, Laboratory of Pathology, 2Biostatistics and Data Management Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, USAAbstract: Follicular lymphoma (FL) is a relatively common, well-characterized lymphoma with recognizable morphologic and immunophenotypic features. Nevertheless, close examination of its immunophenotypic profile by mean fluorescent intensity (MFI) and potential relationship between antigens, as well as histologic grade, has not been extensively assessed by flow cytometry immunophenotyping (FC). We examined the immunophenotypic profile, including heavy and light chain analysis, of 41 nodal FL cases with FC analysis and tissue diagnosis. Additionally, MFI of CD45, CD19, CD20, CD22, and CD10 were examined. The relationship between the antigen expression on FL cells and normal B cells in the same sample, and any association with each other or with histologic grade, were analyzed statistically. We observed brighter CD45 and CD20 expression in FL than normal B cells(P < 0.0001); dimmer CD19 in FL than normal B cells (P = 0.03); brighter CD20 in grade 2 than grade 1 histology (P = 0.0023). No correlation was observed between CD10 MFI and other antigens. Increased expression of CD20, CD45, and dim expression of CD19 are features of nodal FL. FC assessment of MFI detects subtle changes in antigen expression not entirely apparent by visual examination of dot plots or immunohistochemistry. MFI is a means of uncovering subtle but unique immunophenotypic features in both well-known, and less well-defined neoplastic hematolymphoid entities.Keywords: lymphoma, lymphoproliferative disorder, flow cytometry, mean fluorescent intensity, immunophenotyping, immunoglobulin heavy chain

The preparation of leukemia cell vaccine expressing BCG heat shock protein 70 and anti-leukemia effect in vitro

Gene-modified cell vaccines are the best way to achieve the immunotherapy for all types of acute leukemia. In this study, heat shock protein 70 (HSP70) gene of BCG was transfected into the acute leukemia cells and its anti-leukemia effect was further studied. Results showed that short-term culture of the leukemia cells exhibited increased number and no change in antigen expression. After HSP70 gene transfection, the yellow-green fluorescence on the leukemia cell surface was observed under a fluorescence microscope. The immunogenicity of HSP70-transfected cells exhibited that autologous lymphocytes proliferated significantly and secreted higher amount of IFN-γ, and cytotoxic T lymphocytes induced more beneficial anti-leukemia effects. These results suggested that gene transfection of BCG HSP70 could significantly enhance the immunogenicity of leukemia cells. It may be used as a suitable candidate gene-modified cell vaccine for cancer immunotherapy.

Glioma-associated antigens associated with prolonged survival in a phase I study of ICT-107 for patients with newly diagnosed glioblastoma.

2042 Background: Active immunotherapy offers the potential for high tumor-specific toxicity and sustained tumoricidal activity. We developed a dendritic cell (DC) immunotherapy to target specific glioma-associated antigens and induce a potent T cell response. Methods: In this study HLA-A2 or A1 positive patients were vaccinated with DC pulsed with MHC class I peptides from tumor associated antigens (TAA) expressed on gliomas. TAA epitopes included HER2, TRP-2, gp100, MAGE-1, IL13Rα2, and AIM-2. TRP-2, HER2 and AIM-2, are also overexpressed on glioblastoma multiforme (GBM) cancer stem cells. In this phase I trial, mononuclear cells isolated by leukapheresis from newly diagnosed GBM patients and differentiated into DC, were pulsed with TAA peptides, and administered intradermally three times at two week intervals. Results: 16 newly diagnosed GBM patients were enrolled since May 2007 with last follow up as of 1/28/2011. Vaccination was followed by adjuvant chemotherapy. TAA expression by qRT-PCR showed all patients expressed at least three TAA with 75% expressing all six, 87% expressing five and 94% expressing four. Correlations of increased PFS and quantitative expression of MAGE1 (p=0.03), AIM2 (p=0.003), gp100 (p=0.05) and HER2 (p=0.04) was observed. Variable changes in antigen expression were observed in the recurrent tumors. 7 of 15 tested (47%) had increased IFN gamma immune responses to a pool of the peptides. Immune responders showed a trend toward better survival. Six patients continue to show no tumor recurrence, three of whom have gone almost four years (43.5-49.8 m) and three more than 2.5 years (31.1-34.2 m). Median progression-free survival time was 16.9 months with median OS survival time not reached. At two years, the PFS rate was 43.75%; the two-year OS rate was 80.2%. Conclusions: Expression of four ICT-107 targeted antigens in the pre-vaccine tumors correlate with prolonged survival as measured by PFS and OS in newly diagnosed GBM patients. A randomized placebo controlled phase II trial is currently underway.

Cell adhesion molecule expression in human lens epithelial cells after glucocorticoid exposure

Abstract Purpose The aim of the study was to investigate changes in cell adhesion molecule expression in human lens epithelial cells subjected to glucocorticoids. Methods Human lens epithelial cells (HLEC) were exposed to dexamethasone for 24 hours. Cell morphology was examined by transmission electron microscopy (TEM), cell adhesion molecule expression was studied by western blot and immuno‐histochemistry. Results TEM showed multilayering of cells, vacuole formation and appearance of electron‐dense multivesicular bodies in HLEC exposed to 0.1, 1, 10 and 100 µM dexamethasone. Western blot and densitometry analysis revealed that α‐catenin, β‐catenin and GR expression were significantly decreased in dexamethasone exposed HLEC compared to unexposed cells. No significant change in N‐cadherin, γ‐catenin or vimentin expression between dexamethasone exposed and unexposed cells were present. Immunohistochemical analysis showed no significant change in antigen expression for any of the antigens examined. Conclusion The multilayering of HLEC seen with TEM, indicate defects in regulation of cell migration, which is supported by a decrease in cell adhesion molecule expression.

Modulation of antigen expression in B‐cell precursor acute lymphoblastic leukemia during induction therapy is partly transient: Evidence for a drug‐induced regulatory phenomenon. Results of the AIEOP‐BFM‐ALL‐FLOW‐MRD‐Study Group

Changes of antigen expression on residual blast cells of acute lymphoblastic leukemia (ALL) occur during induction treatment. Many markers used for phenotyping and minimal residual disease (MRD) monitoring are affected. Glucocorticoid (GC)-induced expression modulation has been causally suspected, however, subclone selection may also cause the phenomenon. We investigated this by following the phenotypic evolution of leukemic cells with flow cytometry from diagnosis to four time points during and after GC containing chemotherapy in the 20 (of 360 consecutive) B-cell precursor patients with ALL who had persistent MRD throughout. The early expression changes of CD10 and CD34 were reversible after stop of GC containing chemotherapy. Modulation of CD20 and CD45 occurred mostly during the GC phase, whereas CD11a also changed later on. Blast cells at diagnosis falling into gates designed according to "shifted" phenotypes from follow-up did not form clusters and were frequently less numerous than later on. Our data support the idea that drug-induced modulation rather than selection causes the phenomenon. The good message for MRD assessment is that modulation is transient in at least two (CD10 and CD34) of the five prominent antigens investigated and reverts to initial aberrant patterns after stop of GC therapy, whereas CD20 expression gains new aberrations exploitable for MRD detection.

Investigation on the Effect of Immune Selection on Resistance to Bactericidal Antibodies to Group B Meningococci In Vitro

The induction of resistance by immune selective pressure to bactericidal antibodies from humans immunized with Novartis recombinant meningococcal group B vaccines was assessed. Serum bactericidal antibody titers against selected bacteria were within assay variability through a selection event frequency of 1 in 10(-5). No change in antigen expression was observed by Western blotting.