Expression Of HLA Class Research Articles

Combined Genetic Inactivation of β2-Microglobulin and CD58 Reveals Frequent Escape from Immune Recognition in Diffuse Large B Cell Lymphoma

We report that diffuse large B cell lymphoma (DLBCL) commonly fails to express cell-surface molecules necessary for the recognition of tumor cells by immune-effector cells. In 29% of cases, mutations and deletions inactivate the β2-Microglobulin gene, thus preventing the cell-surface expression of the HLA class-I (HLA-I) complex that is necessary for recognition by CD8(+) cytotoxic Tcells. In 21% of cases, analogous lesionsinvolve the CD58 gene, which encodes a molecule involved in T and natural killer cell-mediated responses. In addition to gene inactivation, alternative mechanisms lead to aberrant expression of HLA-I and CD58 in >60% of DLBCL. These two events are significantly associated in this disease, suggesting that they are coselected during lymphomagenesis for their combined role in escape from immune-surveillance.

Towards Phase 2/3 Trials for Epstein - Barr Virus (EBV)-Associated Malignancies,

Abstract 4043 Background:About 30% of lymphomas arising in immunocompetent individuals as well as >90% of undifferentiated nasopharyngeal carcinomas (NPCs) carry the EBV genome and express four of about 90 potential viral antigens (type 2 latency). We have targeted two of these antigens (LMP1 and LMP2) in clinical trials for the treatment of multiply-relapsed and refractory lymphoma and NPC. LMP-specific T cells had minimal short or long-term toxicities and produced tumor responses in over 70% patients with relapsed lymphoma and complete response in over 60%. In patients with NPC it was more difficult to manufacture LMP-specific T cells. 15/34 (45%) of infused patients had tumor responses including 5 CRs, which were predominantly observed in patients with locoregional disease (4/5).LMP-specific T cells were activated using an initial stimulation of patient PBMCs with autologous dendritic cells (DCs) transduced with an adenoviral vector expressing LMP1 and LMP2 (Ad5f35-LMP1-IRES-LMP2). Nine days later and weekly thereafter responder T cells were restimulated with autologous EBV-transformed B lymphoblastoid cell lines (LCLs) overexpressing LMP1 and LMP2 from the same Ad vector until sufficient cells for the dose, QC and research were expanded. This manufacturing strategy presents several critical barriers to its transition to pivotal phase 2 and 3 clinical trials. First, the live virus (EBV) and viral vectors (adenovirus) used in our current manufacturing process will significantly increase the regulatory hurdles. Second, autologous LCL lines required as an antigen-presenting cell (APC) from lymphoma patients are difficult to establish now that Rituximab is used as standard therapy for lymphoma. Third, T cells specific for adenovirus antigens, derived from vector (virion proteins) and EBV antigens expressed in the LCL, but not in type 2 latency tumors can dominate the cultures; a particular problem in patients with NPC. Fourth, the CTL manufacturing is complex and takes a minimum of 12 weeks. We have developed a new manufacturing strategy that addresses these problems. Methods:To manufacture type 2 latency antigen-specific T cells without using adenovirus or EBV, PBMCs are stimulated with overlapping peptide libraries (20 amino acids overlapping by 15) spanning the entire protein sequences of LMP1, LMP2 and EBNA1 in the presence of cytokines. By day 9, the T cells require restimulation, but B cells and monocytes that can present antigens effectively are depleted from the responding population. Therefore we used as APCs, autologous activated T cells (T-APCs) that express HLA class I and class II antigens pulsed with the same pepmixes together with an HLA-negative cell line (K562) expressing costimulatory molecules to prevent anergy induction. Results:This combination of antigen and antigen-presenting cells produces antigen-specific T cell expansion without reducing the T cell repertoire, which remains broad with subsequent stimulations. Competition between peptides within the libraries ensures that the avidity of the responding T cells remains high. By eliminating the requirement for EBV-LCLs we have reduced the minimum generation time from 10 weeks to ∼30 days. There were no detectable differences in the phenotype or function of these T cells, but the specificity was more focused on relevant antigens. In patients with relapsed NPC the frequency of LMP/EBNA1-specific T cells was increased in the presence of blocking antibodies to PD-L1. This phenomenon was not found in healthy donors, suggesting that type 2 latency antigen-specific T cells might be anergic in NPC patients. Conclusions:We have now developed a strategy that eliminates live virus and viral vectors from the manufacturing process, eliminates the requirement for robust patient B cells, eliminates the reactivation of adenovirus-specific T cells and irrelevant EBV-specific T cells, increases the frequency of LMP-specific T cells in the cultures and adds EBNA1 as an additional target antigen. The clinical equivalency of these T cells in patients with NPC and lymphoma remains to be tested in clinical trials. Disclosures:No relevant conflicts of interest to declare.

Human Mesenchymal Stem Cells Are Susceptible to Lysis by CD8+ T Cells and NK Cells

There is growing interest in the use of mesenchymal stem cells (MSCs) to improve the outcome of organ transplantation. The immunogenicity of MSCs is, however, unclear and is important for the efficacy of MSC therapy and for potential sensitization against donor antigens. We investigated the susceptibility of autologous and allogeneic MSCs for lysis by CD8(+) T-lymphocytes and NK cells in a kidney transplant setting. MSCs were derived from adipose tissue of human kidney donors and were CD90(+), CD105(+), CD166(+), and HLA class I(+). They showed differentiation ability and immunosuppressive capacity. Lysis of MSCs by peripheral blood mononuclear cells (PBMCs), FACS-sorted CD8(+) T cells, and NK cells was measured by europium release assay. Allogeneic MSCs were susceptible for lysis by cytotoxic CD8(+) T cells and NK cells, while autologous MSCs were lysed by NK cells only. NK cell-mediated lysis was inversely correlated with the expression of HLA class I on MSCs. Lysis of autologous MSCs was not dependent on culturing of MSCs in FBS, and MSCs in suspension as well as adherent to plastic were lysed by NK cells. Pretransplant recipient PBMCs did not lyse donor MSCs, but PBMCs isolated 3, 6, and 12 months after transplantation showed increasing lysing ability. After 12 months, CD8(+) T-cell-mediated lysis of donor MSCs persisted, indicating there was no evidence for desensitization against donor MSCs. Lysis of MSCs is important to take into account when MSCs are considered for clinical application. Our results suggest that the HLA background of MSCs and timing of MSC administration are important for the efficacy of MSC therapy.

HLA Antigens Shed from the Surface of Synthetic or Naturally Occurred Platelet-Derived Microparticles During Storage of Platelet Concentrate

The demand for standard platelet concentrates (PCs) has continued to increase in the recent years. Infusible platelet membranes (IPM) prepared from new or outdated human platelets have been developed as an alternative to standard PCs, with the additional advantage of long shelf life and increased viral safety. Reduction of HLA antigens on the IPM has been assigned as one of the probable advantages of this product. In re-examining this issue, we studied the existence of HLA class I on the surface of IPM microparticles. In comparison we also surveyed HLA expression on the surface of the naturally occurred platelet-derived microparticles (nPMPs) during 7days storage. Intended for producing IPM, PCs obtained from Iranian blood transfusion organization were lysed; virally inactivated with wet heat in the presence of a heat stabilizer and then sonicated. IPMs were separated using centrifugation and liquid-stored in 4°C. The expression of HLA class I antigens was surveyed using flow cytometry technique. HLA molecules were present on the microparticles. Shedding of HLA antigens was demonstrated from the surface of the both liquid-stored IPM and nPMPs during storage. Storage of IPM in 4°C was accompanied with significant reduction of HLA molecules. It seemed that achievement of HLA-free IPM could be impossible unless chloroquine treated platelets were used to prepare these microvesicles.

The fundamentals of neutrophil antigen and antibody investigations

Background The neutrophil population makes up the largest proportion (40–70%) of white blood cells and is crucial to our innate immune response. An unexpectedly significant proportion of neutrophils (∼28%) is located in the pulmonary circulation. This is an immune system front line wherein neutrophils guard an important interface between the external environment and the body. Neutrophils migrate to sites of infection and/or inflammation, where they phagocytose and destroy foreign microorganisms and mediate inflammation and eventual healing. Therefore, a decrease in neutrophil numbers often results in increased susceptibility to infection. Sustained or transient immune neutropenia occurs when neutrophil‐reactive alloantibodies or autoantibodies destroy neutrophils, or significantly compromise their function. Neutrophil‐reactive antibodies are capable of activating the neutrophil’s microbicidal arsenal with resultant damage to the surrounding tissue.Antigens and Antibodies There are currently eight well characterized human neutrophil antigens (HNA): HNA‐1a, HNA‐1b, HNA‐1c, HNA‐2a, HNA‐3a, HNA‐3b, HNA‐4a and HNA‐5a. Only HNA‐1a, HNA‐1b and HNA‐1c are specific to neutrophils, whilst the others are also expressed on other cell types. It is important to remember that neutrophils also express HLA Class I antigens, and that there is some evidence that adequately activated neutrophils may express HLA Class II antigens. This provides an interface between the innate and adaptive immune response. Therefore, both HNA and HLA Class I and II antibodies are capable of reacting with neutrophils in vivo and in vitro.Routine Antigen and Antibody Investigation Antigen genotyping protocols are now available for HNA‐1a, HNA‐1b, HNA‐1c, HNA‐3a, HNA‐3b, HNA‐4a and HNA‐5a. The serological examination of neutrophils is reliant on harvesting a representative, pure population of neutrophils, which is difficult because neutrophils are short lived (6–8 h) and fragile. The International Granulocyte Immunobiology Workshop recommends the use of both the granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT), complemented by the monoclonal antibody immobilization of granulocyte antigen (MAIGA) assay. The GIFT uses paraformaldehyde‐fixed neutrophils and detects the binding of antibodies to surface HNA and HLA antigens. Serological reaction in the GAT is reliant on viable neutrophils. It demonstrates the ability of antibodies to sensitize neutrophils and stimulate them to undergo chemotaxis towards other sensitized neutrophils, thus forming microscopic agglutinates. The MAIGA is useful in confirming antibody specificities as it employs monoclonal antibodies to known HNA epitopes. To screen for neutrophil‐reactive antibodies, serum or plasma is exposed to a screening panel of neutrophils in the GIFT and GAT. To differentiate between HNA and HLA antibodies, the simple strategy of using platelet pools to absorb out HLA antibodies can be used. Specificity is determined using the GIFT and GAT, and a phenotyped and genotyped neutrophil panel and confirmed with the MAIGA.Application The combination of the GIFT, GAT and MAIGA was originally developed for the reliable investigation of immune neutropenias. However, in the last decade these techniques have become fundamental to the investigation of immune mediated TRALI where neutrophils are a key target and effector cell.

Indoleamine 2,3 dioxygenase (IDO): a pivotal counter-regulatory switch at sites of inflammation

Indoleamine 2,3 dioxygenase (IDO): a pivotal counter-regulatory switch at sites of inflammation

Increased Frequencies of Myelin Oligodendrocyte Glycoprotein/MHC Class II-Binding CD4 Cells in Patients with Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune disease characterized by infiltration of pathogenic immune cells in the CNS resulting in destruction of the myelin sheath and surrounding axons. We and others have previously measured the frequency of human myelin-reactive T cells in peripheral blood. Using T cell cloning techniques, a modest increase in the frequency of myelin-reactive T cells in patients as compared with control subjects was observed. In this study, we investigated whether myelin oligodendrocyte glycoprotein (MOG)-specific T cells could be detected and their frequency was measured using DRB1*0401/MOG(97-109(107E-S)) tetramers in MS subjects and healthy controls expressing HLA class II DRB1*0401. We defined the optimal culture conditions for expansion of MOG-reactive T cells upon MOG peptide stimulation of PMBCs. MOG(97-109)-reactive CD4(+) T cells, isolated with DRB1*0401/MOG(97-109) tetramers, and after a short-term culture of PMBCs with MOG(97-109) peptides, were detected more frequently from patients with MS as compared with healthy controls. T cell clones from single cell cloning of DRB1*0401/MOG(97-109(107E-S)) tetramer(+) cells confirmed that these T cell clones were responsive to both the native and the substituted MOG peptide. These data indicate that autoantigen-specific T cells can be detected and enumerated from the blood of subjects using class II tetramers, and the frequency of MOG(97-109)-reactive T cells is greater in patients with MS as compared with healthy controls.

Human Leukocyte Antigen Class II Transgenic Mouse Model Unmasks the Significant Extrahepatic Pathology in Toxic Shock Syndrome

Among the exotoxins produced by Staphylococcus aureus and Streptococcus pyogenes, the superantigens (SAgs) are the most potent T-cell activators known to date. SAgs are implicated in several serious diseases including toxic shock syndrome (TSS), Kawasaki disease, and sepsis. However, the immunopathogenesis of TSS and other diseases involving SAgs are still not completely understood. The commonly used conventional laboratory mouse strains do not respond robustly to SAgs in vivo. Therefore, they must be artificially rendered susceptible to TSS by using sensitizing agents such as d-galactosamine (d-galN), which skews the disease exclusively to the liver and, hence, is not representative of the disease in humans. SAg-induced TSS was characterized using transgenic mice expressing HLA class II molecules that are extremely susceptible to TSS without d-galN. HLA-DR3 transgenic mice recapitulated TSS in humans with extensive multiple-organ inflammation affecting the lung, liver, kidneys, heart, and small intestines. Heavy infiltration with T lymphocytes (both CD4(+) and CD8+), neutrophils, and macrophages was noted. In particular, the pathologic changes in the small intestines were extensive and accompanied by significantly altered absorptive functions of the enterocytes. In contrast to massive liver failure alone in the d-galN sensitization model of TSS, findings of the present study suggest that gut dysfunction might be a key pathogenic event that leads to high morbidity and mortality in humans with TSS.

Role of HLA class II genes in susceptibility and resistance to multiple sclerosis: Studies using HLA transgenic mice

Multiple sclerosis (MS), an inflammatory and demyelinating autoimmune disease of CNS has both, a genetic and an environmental predisposition. Among all the genetic factors associated with MS susceptibility, HLA class II haplotypes such as DR2/DQ6, DR3/DQ2, and DR4/DQ8 show the strongest association. Although a direct role of HLA-DR alleles in MS have been confirmed, it has been difficult to understand the contribution of HLA-DQ alleles in disease pathogenesis, due to strong linkage disequilibrium. Population studies have indicated that DQ alleles may play a modulatory role in the progression of MS. To better understand the mechanism by which HLA-DR and -DQ genes contribute to susceptibility and resistance to MS, we utilized single and double transgenic mice expressing HLA class II gene(s) lacking endogenous mouse class II genes. HLA class II transgenic mice have helped us in identifying immunodominant epitopes of PLP in context of various HLA-DR and -DQ molecules. We have shown that HLA-DR3 transgenic mice were susceptible to PLP 91–110 induced experimental autoimmune encephalomyelitis (EAE), while DQ6 (DQB1*0601) and DQ8 (DQB1*0302) transgenic mice were resistant. Surprisingly DQ6/DR3 double transgenic mice were resistant while DQ8/DR3 mice showed higher disease incidence and severity than DR3 mice. The protective effect of DQ6 in DQ6/DR3 mice was mediated by IFN γ, while the disease exacerbating effect of DQ8 molecule was mediated by IL-17. Further, we have observed that myelin-specific antibodies play an important role in PLP 91–110 induced EAE in HLA-DR3DQ8 transgenic mice. Based on these observations, we hypothesize that epistatic interaction between HLA-DR and -DQ genes play an important role in predisposition to MS and our HLA transgenic mouse model provides a novel tool to study the effect of linkage disequilibrium in MS.

Prognostic impact of anticancer immune responses: an introduction

Cancer cells escape innate and adaptive immune responses by selection of non-immunogenic tumor cell variants (immunoediting) or by active suppression of the immune response (immunosubversion). One common strategy that employs tumor cells to elude a T-cell-mediated immune response is the downregulation or loss of expression of HLA class I molecules, often associated with an induction of the surface expression of HLA class II antigen, nonclassical HLA class I molecules, and/or NK cell-activating ligands such as MICA, MICB, or ULBP. These changes in the surface characteristics of tumor cells result from the selection pressure exerted by immune cells from the innate and adaptive compartment. The tumor antigen characteristics are "immunoedited" in the course of the disease, resulting in the survival of tumor variants with defective antigen presentation, mostly at the level of HLA class I.

Cancer immunotherapy: Progress and challenges in the clinical setting

Cancer immunologists have made enormous efforts to prove that innate and adaptive immune cells recognize tumor cells and induce tumor rejection in experimental animal models. These observations provided the rationale to study the role of the immune system in the control of tumor growth in cancer patients and to develop immunotherapeutic strategies to treat patients with cancers. Several lines of evidence suggest that anti-tumor immune responses may correlate with better clinical outcome in patients with cancers. Among them is the correlation between the presence of tumor-infiltrating T cells and the improved clinical outcome for patients with solid tumors [1–3]. In addition, a number of immunotherapies, such as high-dose IL-2 [4] and TA-specific monoclonal antibodies (mAb) [5], have provided long-term clinical benefits to a minority of cancer patients. Most importantly, the recent approval by the US FDA of the mAb ipilimumab directed against the co-inhibitory molecule CTLA-4 for patients with unresectable or metastatic melanoma represents a major breakthrough for mAb-based therapies in oncology practice [6]. The successful outcome of the randomized phase III clinical trial with ipilumumab has provided the much-needed incontrovertible clinical evidence that in humans, as in experimental animal models, the host’s immune system can control tumor growth. Furthermore, it has infused a considerable amount of optimism among tumor immunologists and clinical oncologists about the clinical potential of immunotherapy for the treatment of advanced cancers. However, there are also many examples of spontaneous or vaccine-induced TA-specific T- and B-cell immune responses that do not correlate with improved clinical status [7–9]. This discrepancy between immune and clinical responses underlines the need to better dissect the molecular and cellular events leading to tumor rejection in humans. Such an endeavor has greatly benefited from the molecular identification of TA expressed by human tumor cells, which are recognized by T cells and antibodies [5, 10, 11]. As a result, TA-specific immunotherapies have been implemented in clinical trials with molecularly defined cancer vaccines, TA-specific mAb and adoptive transfer of TA-specific T cells. Novel generations of cancer vaccines with molecularly defined TAs and potent adjuvants like toll-like receptor ligands appear to stimulate strong TA-specific T-cell responses but have shown evidence of clinical benefits in only a minority of patients with advanced cancer [7, 8, 12]. The adoptive transfer of TA-specific T cells remains technically challenging and the promising data obtained in terms of objective clinical responses and durability of responses from small monocentric clinical trials will need to be further confirmed in large multicenter clinical trials [13]. TA-specific mAb are clinically effective in a number of hematological malignancies and solid tumors and are routinely used in the clinic [5]. We now have a better understanding of the multiple mechanisms of tumor-induced immune escape, which are likely to cause the failure of the spontaneous or vaccine-induced immune responses to promote tumor regression in humans. In the tumor microenvironment, a number of negative regulators dampens anti-tumor immune responses and/or their therapeutic efficacy, including the production of cytokines (like TGF-β or IL-10), suppressive cells (regulatory T cells, myelosuppressive dendritic cells), defective antigen presentation by tumor cells (HLA or tumor antigen loss, antigen processing machinery defects), amino-acid catabolizing enzymes (indoleamine-2-3dioxygenase, arginase) and co-inhibitory pathways (like CTLA-4/CD28, PD-1/PD-L1) [14–17]. As a consequence, a number of therapies to specifically target these pathways are being developed to enhance TA-specific immune responses and to increase the likelihood of clinical benefits. In this article, commissioned to recognize National Cancer Survivors Day (the first Sunday in June each year, 5 June in 2011, see www.ncsdf.org), we will comment on the successes of immunotherapy of cancer in the clinical setting. In addition, we will discuss the challenges to optimize the use of cancer immunotherapies in the clinic.

HLA antigen and NK cell activating ligand expression in malignant cells: a story of loss or acquisition

Malignant transformation of cells is often associated with changes in classical and non-classical HLA class I antigen, HLA class II antigen as well as NK cell activating ligand (NKCAL) expression. These changes are believed to play a role in the clinical course of the disease since these molecules are critical to the interactions between tumor cells and components of both innate and adaptive immune system. For some time, it has been assumed that alterations in the expression profile of HLA antigens and NKCAL on malignant cells represented loss of classical HLA class I antigen and induction of HLA class II antigen, non-classical HLA class I antigen and/or NKCAL expression. In contrast to these assumptions, experimental evidence suggests that in some cases dysplastic and malignant cells can acquire classical HLA class I antigen expression and/or lose the ability to express HLA class II antigens. In light of the latter findings as well as of the revival of the cancer immune surveillance theory, a reevaluation of the interpretation of changes in HLA antigen and NKCAL expression in malignant lesions is warranted. In this article, we first briefly describe the conventional types of changes in HLA antigen and NKCAL expression that have been identified in malignant cells to date. Second, we discuss the evidence indicating that, in at least some cell types, classical HLA class I antigen expression can be acquired and/or the ability to express HLA class II antigens is lost. Third, we review the available evidence for the role of immune selective pressure in the generation of malignant lesions with changes in HLA antigen expression. This information contributes to our understanding of the role of the immune system in the control of tumor development and to the optimization of the design of immunotherapeutic strategies for the treatment of cancer.

Immunization with Recombinant HLA Classes I and II, HIV-1 gp140, and SIV p27 Elicits Protection against Heterologous SHIV Infection in Rhesus Macaques

Major histocompatibility complex (MHC) molecules expressed on the surface of human immunodeficiency virus (HIV) are potential targets for neutralizing antibodies. Since MHC molecules are polymorphic, nonself MHC can also be immunogenic. We have used combinations of novel recombinant HLA class I and II and HIV/simian immunodeficiency virus (SIV) antigens, all linked to dextran, to investigate whether they can elicit protective immunity against heterologous simian/human immunodeficiency virus (SHIV) challenge in rhesus macaques. Three groups of animals were immunized with HLA (group 1, n = 8), trimeric YU2 HIV type 1 (HIV-1) gp140 and SIV p27 (HIV/SIV antigens; group 2, n = 8), or HLA plus HIV/SIV antigens (group 3, n = 8), all with Hsp70 and TiterMax Gold adjuvant. Another group (group 4, n = 6) received the same vaccine as group 3 without TiterMax Gold. Two of eight macaques in group 3 were completely protected against intravenous challenge with 18 50% animal infective doses (AID(50)) of SHIV-SF162P4/C grown in human cells expressing HLA class I and II lineages represented in the vaccine, while the remaining six macaques showed decreased viral loads compared to those in unimmunized animals. Complement-dependent neutralizing activity in serum and high levels of anti-HLA antibodies were elicited in groups 1 and 3, and both were inversely correlated with the plasma viral load at 2 weeks postchallenge. Antibody-mediated protection was strongly supported by the fact that transfer of pooled serum from the two challenged but uninfected animals protected two naïve animals against repeated low-dose challenge with the same SHIV stock. This study demonstrates that immunization with recombinant HLA in combination with HIV-1 antigens might be developed into an alternative strategy for a future AIDS vaccine.

Cytotoxic T Lymphocytes Efficiently Recognize Human Colon Cancer Stem-Like Cells

Cancer stem-like cells (CSCs) and tumor-initiating cells (TICs) are a small population of cancer cells that share three properties: tumor initiating ability, self-renewal, and differentiation. These properties suggest that CSCs/TICs are essential for tumor maintenance, recurrence, and distant metastasis. Here, we show that cytotoxic T lymphocytes (CTLs) specific for the tumor-associated antigen CEP55 can efficiently recognize colon CSCs/TICs both in vitro and in vivo. Using Hoechst 33342 dye staining, we isolated CSCs/TICs as side population (SP) cells from colon cancer cell lines SW480, HT29, and HCT15. The SP cells expressed high levels of the stem cell markers SOX2, POU5F1, LGR5, and ALDH1A1 and showed resistance to chemotherapeutic agents such as irinotecan or etoposide.To evaluate the susceptibility of SP cells to CTLs, we used CTL clone 41, which is specific for the CEP55-derived antigenic peptide Cep55/c10orf3_193 (10) (VYVKGLLAKI). The SP cells expressed HLA class I and CEP55 at the same level as the main population cells. The SP cells were susceptible to CTL clone 41 at the same level as main population cells. Furthermore, adoptive transfer of CTL clone 41 inhibited tumor growth of SW480 SP cells in vivo. These observations suggest that Cep55/c10orf3_193(10) peptide-based cancer vaccine therapy or adoptive cell transfer of the CTL clone is a possible approach for targeting chemotherapy-resistant colon CSCs/TICs.

Leukocyte infiltrate in gastrointestinal adenocarcinomas is strongly associated with tumor microsatellite instability but not with tumor immunogenicity.

To analyze the correlation of genomic instability with leukocyte infiltrate in gastrointestinal carcinomas (GIACs) and with tumor immunogenicity, e.g., HLA class I cell surface expression defects and galectin-3 and PDL-1 expression. Lymphocyte and macrophage infiltrations were immunohistochemically studied in HLA class I negative GIACs with sporadic high-level microsatellite instability (MSI-H) or microsatellite stability (MSS). Tumors with MSI-H were associated with the following: dense infiltration (CD45, P<0.001); cytotoxic CD8-positive lymphocytes (P<0.001); and a complete absence of HLA class I cell surface expression, due to inactivating β2-microglobulin (β2-m) mutation in 50% of cases. In contrast, HLA class I negative tumors with MSS were significantly associated with fewer CD8-positive lymphocytes. There was no association between microsatellite instability and other molecular features of the tumor cells, including expression of galectin-3. Finally, macrophage infiltrate in the tumors was not correlated with microsatellite instability or HLA class I cell surface expression (CD64, P=0.63; CD163, P=0.51). Microsatellite instability appears to be the most important factor determining the composition, density, and localization of leukocyte infiltrate, which is independent of other molecular features such expression of HLA class I cells, galectin-3, or programmed death ligand-1. Accordingly, the strong intratumoral CD8+ T infiltration of MSI-H tumors may be produced by elevated levels of specific inflammatory chemokines in the tumor microenvironment.

RNA Interference of Human Papillomavirus Type 16 E7 Increases HLA Class I Antigen Expression in HaCaT-E7 Cells

High-risk human papillomaviruses (HPVs) are the major causative agents of cervical cancer. The E7 protein of high-risk HPV disturbs cell cycle control and down-regulates components of the antigen presentation pathway, suggesting an ideal target for development of the immunotherapy in HPV-positive cervical cancers. We previously reported that HPV16 E7 could down-regulate cell-surface HLA class I antigen accompanying decreased expression of transporter associated with antigen processing 1 (TAP-1). The purpose of this study was to determine whether knockdown of HPV16 E7 could up-regulate surface HLA class I antigen expression in HPV16 E7 expressing HaCaT cells (HaCaT-E7). An E7-specific small interfering RNA (siRNA) was transfected into the HaCaT-E7 cells, and the expression of HPV16 E7 was measured by real-time reverse transcriptase polymerase chain reaction and Western blot. With the use of flow cytometry analysis, the levels of cell surface HLA class I antigen and intracellular TAP-1 expression were detected. It was found that transfection of HPV16 E7-siRNA reduced HPV16 E7 expression as measured on messenger RNA and protein levels. The flow cytometry analysis showed that, compared with mock transfection, a statistically significant increase of approximately 75% in surface HLA class I levels was observed in HaCaT-E7 cells at 72 hours after transfection of E7 siRNA. Moreover, he knockdown of E7 in HaCaT-E7 cells could result in an increase of intracellular TAP-1 expression, which is essential for the expression of HLA class I at cell surface. Our study showed that the knockdown of HPV16 E7 could increase cell surface HLA class I antigen expression in HaCaT-E7 cells. In addition, for HPV-positive human cervical cancer, our observations indicate that the HPV E7 gene is a target of choice.

Loss of HLA Class-I Expression In Leukemic Cells That Relapsed After HLA-Matched and -Mismatched SCT

AbstractAbstract 2541 Background:The loss or down-regulation of HLA class-I antigens often occurs in solid tumors, but it is rarely demonstrated in leukemic cells. This phenomenon may explain why leukemic cells are immunogenic to some degree with allogeneic hematopoietic stem cell transplantation (allo-SCT) or donor leukocyte infusion, because HLA class-I antigens on leukemic cells are thought to present minor histocompatibility antigens (mHAs) and leukemia-associated antigens (LAAs) to donor T cells to elicit anti-leukemic cytotoxic T-cell (CTL) responses. Recent analyses of leukemic cells that relapsed after HLA-haploidentical SCT revealed that the leukemic cells lose their unshared HLA haplotype expression as a result of acquired uniparental disomy (UPD) on the short arm of chromosome 6 (6p). The loss of HLA from leukemic cells may occur after transplantation from HLA-identical donors if CTLs specific to mHAs or LAAs play a substantial role in the eradication of leukemic cells. This study evaluated this hypothesis by investigating HLA class-I expression on leukemic cells from patients at both the time of diagnosis and relapse after allo-SCT from HLA matched and mismatched donors. Objectives/Methods:Leukemic cells were obtained from five patients with myeloid leukemia (1 CML, 4 AML) both at diagnosis and relapse after allo-SCT. HLA class-I expression on leukemic cells was determined by flow cytometry using monoclonal Abs specific for the HLA-A allele. SCT donors were an HLA-matched sibling, an HLA-A locus-mismatched mother, an HLA-B and C locus-mismatched father, and 2 HLA-matched unrelated donors. The copy number-neutral loss of heterozygosity on 6p in leukemic cells was analyzed by a single-nucleotide polymorphism (SNP) array in patients after haploidentical SCT. The origin of the patients' HLA alleles (paternal or maternal) was determined by family studies on HLA. The post-transplantation donor T-cell responses against the leukemic cells were analyzed with an IFN-γ secretion assay. Results:HLA-A expression of leukemic cells obtained at relapse after allo-SCT was down-regulated in all 5 patients compared to that of leukemic cell obtained at diagnosis. The patient possessing HLA-A2/A11 (Case 1) was the recipient of BM possessing HLA-A24/A11 showed that 66% of leukemic cells at relapse were deficient in HLA-A2 expression. Leukemic cells restored HLA-A2 expression when this patient relapsed after the 2nd SCT using HLA-A2-matched cord blood, but at lower level in comparison to the level at diagnosis (MFI at diagnosis 177 vs. MFI at relapse 101). In vitro, cell-surface HLA-A2 expression was completely restored when leukemic cells at the 2nd relapse were treated in culture with IFN-γ (200 U/mL) for 48 hr. HLA-A2 expression in the shared haplotype (A0206-B3902-Cw0702-DR0405) was unexpectedly missing in 12% of the relapsed leukemic cells in another patient (Case 2) with HLA-2 loci (HLA-B and -C)-mismatched BM (A3101-B5601-Cw0401-DR0901), and this proportion increased to 83% at relapse after 2nd allo-SCT from the same donor (Figure). IFN-γ failed to restore HLA-A2 expression (MFI without IFN-γ 32 vs. MFI with IFN-γ 10) in vitro, and the genomic DNA extracted from leukemic cells at relapse showed a UPD of 6p. Donor-derived T-cells stimulated with leukemia cells from case 2 at diagnosis in vitro secreted IFN-γ in response to leukemia cells at diagnosis better than leukemia cells at relapse (1.67% vs. 0.85%). Conclusion:Loss of HLA-class I antigen occurs frequently in myeloid leukemia cells at relapse after allo-SCT regardless of the HLA disparities. T-cell attacks specific to the major as well as the mHAs, and LAAs may favor proliferation of leukemic cells deficient in the expression of HLA class-I. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

HLA Class I and EBV Positive Classical Hodgkin Lymphoma In the Chinese Population

AbstractAbstract 2688In Caucasian populations, the tumor cells of Epstein Barr virus (EBV)-positive classical Hodgkin Lymphomas (cHL) patients more frequently express HLA class I and HLA class II molecules compared to EBV-negative cHL patients. Since HRS cells also express other components of the antigen presenting pathway it can be anticipated that HRS cells in EBV-positive cHL should be able to present EBV derived antigenic peptides. In on our previous genotyping study we demonstrated that the HLA-A*02 allele was associated with a reduced and the HLA-A*01 allele with an increased risk of developing EBV-positive cHL. Intuitively, this can be explained by the generally acknowledged lack of HLA-A*01 restricted immune responses to latent EBV peptides. In this study, we analyzed HLA class I expression and the HLA association in relation to EBV in Asian cHL patients.Formalin-fixed and paraffin embedded (FFPE) tissue blocks were available for 145 cHL patients and from 79 controls from 5 hospitals from the Northern part of China. Hematoxylin & Eosin-stained sections were used to reclassify the histological subtypes according to the WHO classification. EBV status was determined by EBER in situ hybridization. Membranous expression of HLA class I was detected by immunohistochemistry using antibodies against HLA class I (HC-10) and ß2-microglobulin. DNA was isolated from FFPE samples to detect the HLA-A*02 allele by quantitative PCR. 23 cHL cases and 5 controls were excluded due to poor DNA quality.Positive EBV status was observed in 40% (58/145) of the Chinese cHL patients. As expected, the percentage of EBV-positive cases was much higher in the mixed cellularity subtype (71%) than in the nodular sclerosis subtype (16%; p<0.001). Expression of HLA class I was observed in 79% of the EBV-positive cHL patients and in 30% of the EBV-negative patients (p<0.001) consistent with the previous findings in the Dutch population. The HLA-A*02 allele was detected in 71% of the EBV-positive cHL and in 64% of the EBV-negative cHL patients (NS). In the controls HLA-A*02 was observed in 67% of the cases.In this Chinese population, the tumor cells of EBV-positive cHL more frequently retained membranous HLA class I expression, similar to the Caucasian populations. The inverse correlation between presence of the HLA-A*02 allele and positive EBV status, as observed in Caucasians, is not present in these northern Chinese cHL patients. Differences in ethnic background might explain discrepancies in HLA-A association with EBV-positive cHL in different populations. Disclosures:No relevant conflicts of interest to declare.

Efficient Generation of HLA-A24-Restricted WT1-Specific Cytotoxic T Lymphocytes Using Gene-Engineered Artificial Antigen-Presenting Cells

AbstractAbstract 2101 Background and Purpose:Recent success associated with adoptive transfer of antitumor T cells in lymphodepleted patients suggests the potential of adoptive immunotherapy to have a significant clinical impact. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we previously reported a culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL) from HLA-A2-positive melanoma patients by using K562-based artificial antigen-presenting cells (aAPCs). In the present study, we have applied the culture system to HLA-A24, one of the most common HLA class I antigen in the Asian population, and examined whether HLA-A2402-restricted WT1-specific CTL can be generated using aAPCs. Methods:HLA-A2402–positive peripheral blood mononuclear cells (PBMC) were obtained from healthy donors (n=4) and cancer patients (n=10). To establish antigen-specific T cells, CD8+ T cells were purified by positive selection using a magnetic beads method (Miltenyi Biotec). aAPCs were pulsed with an HLA-A2402 restricted, modified 9-mer WT1 immunodominant peptide (CYTWNQMNL). aAPCs were then irradiated with 200 Gy and added to purified CD8+ T cells at a ratio of 1:20 in 96-well plates in RPMI1640 supplemented with 10% human AB serum. Between stimulations, IL-2 (10 U/ml) and IL-15 (10ng/ml) (both from Peprotech) were added to the cultures. Results:Flow cytometry (FACS) analysis confirmed that aAPCs stably expressed transduced HLA class I, CD80 and CD83 molecules. aAPC did not express HLA class II molecules, CD40, CD154, or CD86. Following 3–4 rounds of weekly stimulation with peptide-pulsed aAPCs, WT1 peptide-specific CD8+ T cells were evaluated by a tetramer staining. The percentage of tetramer-positive cells was 0.082±0.0075% before stimulation. It increased to 0.865±0.528% (10.5 fold increase) and 1.89±1.12% (23.0 fold increase) following the third and the forth stimulation, respectively. There was no marked difference in magnitude of increase between healthy donors and cancer patients. However, when CD8+ T cells from patients vaccinated with WT1-peptide pulsed dendritic cells were stimulated with WT1-peptide-pulsed aAPCs, the percentage of tetramer-positive cells was significantly higher (11.72±6.46%) following the third stimulation. CD8+ T cells stimulated with WT1-peptide-pulsed aAPCs were negative for both A0201/WT1 and A2402/CMV tetramers, confirming HLA restriction and antigen specificity. FACS analysis revealed that WT1-specific CTL expanded with WT1-peptide-pulsed aAPCs expressed a memory phenotype. Furthermore, these CTL demonstrated cytotoxicity against CIR-A2402 target cells pulsed with a WT1 peptide in an LDH release assay. Unpulsed or HIV peptide-pulsed target cells were not lysed. Conclusions:These results demonstrated that HLA-A24-restricted WT1 specific CTLs with a memory phenotype can be generated ex vivo using peptide-pulsed gene-engineered aAPCs within a short period of time for clinical use. Disclosures:No relevant conflicts of interest to declare.

Stem Cells Derived from Human Amniotic Fluid Contribute to Acute Kidney Injury Recovery

Stem cells isolated from human amniotic fluid are gaining attention with regard to their therapeutic potential. In this work, we investigated whether these cells contribute to tubular regeneration after experimental acute kidney injury. Cells expressing stem cell markers with multidifferentiative potential were isolated from human amniotic fluid. The regenerative potential of human amniotic fluid stem cells was compared with that of bone marrow-derived human mesenchymal stem cells. We found that the intravenous injection of 3.5 × 10(5) human amniotic fluid stem cells into nonimmune-competent mice with glycerol-induced acute kidney injury was followed by rapid normalization of renal function compared with injection of mesenchymal stem cells. Both stem cell types showed enhanced tubular cell proliferation and reduced apoptosis. Mesenchymal stem cells were more efficient in inducing proliferation than amniotic fluid-derived stem cells, which, in contrast, were more antiapoptotic. Both cell types were found to accumulate within the peritubular capillaries and the interstitium, but amniotic fluid stem cells were more persistent than mesenchymal stem cells. In vitro experiments demonstrated that the two cell types produced different cytokines and growth factors, suggesting that a combination of different mediators is involved in their biological actions. These results suggest that the amniotic fluid-derived stem cells may improve renal regeneration in acute kidney injury, but they are not more effective than mesenchymal stem cells.