Cytotoxic Lymphocyte Precursors Research Articles

Phase I Trial of a Personalized Peptide Vaccine for Patients Positive for Human Leukocyte Antigen–A24 With Recurrent or Progressive Glioblastoma Multiforme

Personalized selection of suitable peptides for patients could offer a novel approach to developing cancer vaccines that boost anticancer immunity. We present the results of a phase I trial of 14 kinds of vaccine candidates (ITK-1) in patients with recurrent or progressive glioblastoma multiforme (GBM). From January 2006 to January 2008, 12 patients from eight Japanese hospitals who were positive for human leukocyte antigen-A24, including 10 patients refractory to temozolomide (TMZ), were enrolled. The dose escalation trial included three dose groups (1, 3, and 5 mg) to determine the safety and tolerability of ITK-1 peptides. Immunologic response was monitored by measuring levels of cytotoxic T-lymphocyte precursors and peptide-specific immunoglobulin G. In another ITK-1 phase I trial for advanced prostate cancer, the vaccination schedule was skipped or discontinued in all three patients receiving the highest dose (5 mg/peptide) because of injection site reactions. This trial was therefore ended without enrollment for the highest dose, and data were analyzed by intention to treat. No serious adverse drug reactions were encountered, and treatment was well tolerated. The vaccine induced dose-dependent immune boosting. The recommended dose of ITK-1 peptides is 3 mg/peptide. Personalized vaccination with ITK-1 peptides could be recommended in further stages of clinical trials. The safety and increased frequency of immune boosting offers potential clinical benefits in cases of recurrent or progressive GBM, even in TMZ-refractory settings.

Preeclampsia is associated with increased cytotoxic T-cell capacity to paternal antigens

During an uncomplicated pregnancy the conceptus is a semiallogeneic entity in which rejection is prevented by suppression of the maternal immune system. We hypothesized that this suppression is disturbed in patients with preeclampsia and that a maternal immune response to fetal (foreign/paternal) antigens in the fetal-maternal interface may be responsible for local inflammation, with subsequent endothelial dysfunction and systemic disease. Blood samples were obtained from 14 women with preeclampsia (cases), 14 gestational-age and parity-matched women with uncomplicated pregnancies (controls), and their partners. We determined the partner-specific cytotoxic T-lymphocyte precursor frequency (CTLpf) and the CTLpf directed to unrelated partners with uncomplicated pregnancies. We measured the CTLpf in peripheral blood mononuclear cells (PBMCs) from cases and controls using limited-dilution assays. In addition, proliferation was tested in a mixed-lymphocyte culture (MLR). The partner-specific CTLpf was significantly higher in cases compared with controls (median, 183 [15-338] vs 67 [9-232] per million PBMCs, P = .02). In contrast, in women with uncomplicated pregnancies, the partner-specific CTLpf was down-regulated compared with the CTLpf directed to an unrelated partner who fathered uncomplicated pregnancies (P = .02). No difference was found in partner-specific MLR response between cases and controls. These results suggest that women with preeclampsia have a higher cytotoxic T-cell response to paternal antigens compared with pregnant controls. This insufficiently suppressed immune response may eventually lead to the development of preeclampsia.

Methods to measure vaccine immunity

The biggest triumph in vaccination is the eradication of smallpox. Ironically, no methods to measure vaccine immunity were set in place. Protection and immunity was achieved without detailed analysis nor knowledge of cellular or humoral immunity induction. Today, owing to strict health and safety issues of vaccine manufacturing, vaccines must meet higher standards of safety and biochemical characterization than they did in the past. This has led to the production of highly purified vaccines, and the identification and isolation of the antigens responsible for protection. A vaccine cannot move forward without the demonstration of effective immunity induced by the regime. Moreover, contrary to the vaccination strategy that eradicated smallpox and other acute viral infections, several modern vaccines aim at the eradication of established chronic diseases such as chronic viral infections or cancer. As the pathogens or cancer antigens are generally intracellular, modern vaccines can rely less effectively on the neutralizing properties of antibody responses that cannot cross the cell membrane, and depend predominantly on T-cell-based recognition of affected cells for their elimination. Thus, new technologies have to be developed to measure vaccine immunity. Are we going round and round in circles? Are we making it harder for ourselves? Is vaccine development getting too hard? Why do we have to demonstrate immunity, if we can show protection? No regulatory agency will take a vaccine forward that is not highly purified and can demonstrate immunity induction. Several methods of measuring humoral and cellular immunity have been required to be developed to meet this criteria. Some methods include ELISA, cytotoxic T-lymphocyte (CTL) assay, CTL precursor frequency assay, T-cell proliferation assays, carboxyfluorescein diacetate succinimidyl ester assays, intracellular and extracellular cytokine production by cells in culture using either ELISA or multiplex and flow cytometry, polyfunctional T-cell assays, ELISpot and MHC class I/II tetramers. Thus, there is an enormous amount of information and reagents available for guiding vaccine and immunotherapeutics development. This special issue on ‘Methods to measure vaccine immunity’ focuses on a number of recent and promising approaches used to measure immunity induced following vaccination. Topics included in this special focus issue are as follows: Gene-expression profiling in vaccine therapy and immunotherapy for cancer [1]. The use of microarrays is described in order to understand the mechanism by which tumors are rejected; Antibody-profiling technologies for studying humoral responses to infectious agents [2]. Protein microarrays are used to evaluate antibody responses to thousands of antigens at one time. The luciferase immunoprecipitation system overcomes some drawbacks of the conventional protein microarray assay. These new technologies offer a new tool for understanding humoral immunity induction to proteins; Vaccine-induced antibody responses in patients with carcinoma [3]. A nice overview of current methods utilized for measuring antibody responses and for assessing their antitumor efficacy in the context of clinical trials; Methods to measure T-cell responses [4]. A mini review on recent methods used to measure T-cell immunity, such as the use of CTL, CTL precursor frequency assays, carboxyfluorescein diacetate succinimidyl ester, flow cytometric approaches, spectrophotometric approaches and use of ovalbumin-specific CD8+ T cells from OTI and OTII T-cell receptor tansgenic mice; New flow cytometric assays for monitoring cell-mediated cytotoxicity [5]. A comparison of IFN-γ ELISpot, granzyme B ELISpot and conventional 51Cr-release assays is given. A detailed analysis of the use of flow cytometric methods to measure T-cell immunity induction is demonstrated; Evaluation of cellular immune responses in cancer vaccine recipients [6]. Measurement of cellular immunity is addressed using NY-ESO-1 as an example and techniques such as delayed-type hypersensitivity, measurement of T cells from peripheral blood, use of overlapping peptides to measure CD4 and CD8 T cells, and the role of Treg cells in vaccine immunity induction are described; Development and application of phosphoflow as a tool for immunomonitoring [7]. This article describes the new flow cytometry technology, phosphoflow for measuring multiple intracellular signaling molecules in the immune system at a single-cell level for lymphocyte immune monitoring; Surface plasmon resonance for vaccine design and efficacy studies [8]. An outline of how surface plasmon resonance biosensors are emerging to understand how serum is used to monitor antibody immunity; The challenges of assessing infant vaccine responses in resource-poor settings [9]. A very nice and comprehensive article on methods to measure vaccine immune responses where only poor settings are available. There is a vast amount of information already available to promote a greater understanding into the role of specific immune induction to a vaccination regime. The use of newer immune monitoring tools as described in this special focus issue will serve to improve immune monitoring and determine the effectiveness of vaccine strategies for the treatment or immunotherapeutic approaches to diseases.

Detection and ex vivo expansion of dormant anti-viral CTL precursors isolated from recipients of unrelated umbilical cord blood transplant (UCBT). (145.31)

Abstract Two potentially fatal viruses ,CMV and adenovirus can be detected 2-4 weeks after UCBT. Infection may trigger in vivo priming of the infused na ve T cells despite immunosuppressive (IS)medications. We hypothesized that 1) threshold numbers of CTL precursors can be identified that may influence outcome 2) CTL precursors may reside below the level of detection during IS. Methods: Monocytes infected with an adenovirus vector encoding CMV pp65 (Ad5f35pp65) were used as APC in the presence of IL7. Antiviral CTLp was quantitated by IFNg ELISPOT among 1x10e5 responders after overlapping peptide pool stimulation. Results: 8 infected patients at a median age of 9.9 years have been studied before day 100 . Only 1/3 patients with adenovirus exhibited viremia while all 5 patients with CMV had viremia. In freshly drawn blood, 1/8 patients had any detectable reactivity. However, after 9-12 days of stimulation we could enumerate CTLp in 7/8 patients. For adenovirus, the median hexon-specific response was 52 SFC, and the penton-specific CTLp frequency was 13 SFC. CMV pp65-specific response was 6.7 SFC. No responses were found against CMV EI-1 demonstrating critical role for antigenic restimulation. All patients survive beyond 6 months. Conclusion: To our knowledge these are the first data demonstrating that dormant virus-specific CTLp are present already in the first 3 months in infected UCBT recipients. Although these rare events are below detection ex vivo expansion permits quantitation.

Cleavage of transaldolase by granzyme B causes the loss of enzymatic activity with retention of antigenicity for multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS resulting from a progressive loss of oligodendrocytes. Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes of the brain, and postmortem sections show concurrent loss of myelin basic protein and TAL from sites of demyelination. Infiltrating CD8(+) CTLs are thought to play a key role in oligodendrocyte cell death. Cleavage by granzyme B (GrB) is predictive for autoantigenicity of self-proteins, thereby further implicating CTL-induced death in the initiation and propagation of autoimmunity. The precursor frequency and CTL activity of HLA-A2-restricted TAL 168-176-specific CD8(+) T cells is increased in MS patients. In this paper, we show that TAL, but not myelin basic protein, is specifically cleaved by human GrB. The recognition site of GrB that resulted in the cleavage of a dominant TAL fragment was mapped to a VVAD motif at aa residue 27 by N-terminal sequencing and confirmed by site-directed mutagenesis. The major C-terminal GrB cleavage product, residues 28-337, had no enzymatic activity but retained the antigenicity of full-length TAL, effectively stimulating the proliferation and CTL activity of PBMCs and of CD8(+) T cell lines from patients with MS. Sera of MS patients exhibited similar binding affinity to wild-type and GrB-cleaved TAL. Because GrB mediates the killing of target cells and cleavage by GrB is predictive of autoantigen status of self proteins, GrB-cleaved TAL-specific T cell-mediated cytotoxicity may contribute to the progressive destruction of oligodendrocytes in patients with MS.

Immunological evaluation of personalized peptide vaccination monotherapy in patients with castration‐resistant prostate cancer

We previously reported that personalized peptide vaccine (PPV) therapy in combination with leutenizing hormone-releasing hormone (LH-RH) analog and estramustine phosphate in certain cases is safe and capable of inducing both immune responses and clinical responses for metastatic castration-resistant prostate cancer (CRPC) patients. In the present study, PPV monotherapy was given to CRPC patients. Twenty-three patients with metastatic CRPC were treated with PPV without any additional treatment modalities, including LH-RH analogs. Samples were analyzed for peptide-specific cytotoxic T-lymphocyte (CTL) precursor analysis and peptide-reactive IgG. Toxicity and immunological and clinical responses were assessed on a three-monthly basis. Seventeen patients were available for immunological and clinical evaluation. The vaccines were well tolerated, with grade 3 erythema at injection sites in only one patient. Augmentation of CTL or IgG responses to at least one of the peptides was observed in six of 17 (35%) and 15 of 17 (88%) patients tested, respectively. Among 57 peptides used, 9 and 36 peptides induced CTL and IgG responses, respectively. Delayed-type hypersensitivity reaction was observed in eight of 17 patients. More than 30% prostate-specific antigen (PSA) decline was observed in four of 17 patients. Of these, one patient achieved a complete PSA response and another patient showed a partial PSA response with profound shrinking of lymph node metastases and prostate. The overall median survival time was 24 months (range, 5-37 months). These results suggest that PPV monotherapy appears to be safe and capable of inducing peptide-specific immune responses and clinical responses in CRPC patients. This trial was registered with University Hospital Medical Information Network (UMIN) number R000003339.

Independent Regulation of Chemokine Responsiveness and Cytolytic Function versus CD8+ T Cell Expansion by Dendritic Cells

The ability of cancer vaccines to induce tumor-specific CD8+ T cells in the circulation of cancer patients has been shown to poorly correlate with their clinical effectiveness. In this study, we report that although Ags presented by different types of mature dendritic cells (DCs) are similarly effective in inducing CD8+ T cell expansion, the acquisition of CTL function and peripheral-type chemokine receptors, CCR5 and CXCR3, requires Ag presentation by a select type of DCs. Both "standard" DCs (matured in the presence of PGE2) and type 1-polarized DCs (DC1s) (matured in the presence of IFNs and TLR ligands, which prevent DCs "exhaustion") are similarly effective in inducing CD8+ T cell expansion and acquisition of CD45RO+IL-7R+IL-15R+ phenotype. However, granzyme B expression, acquisition of CTL activity, and peripheral tissue-type chemokine responsiveness are features exclusively exhibited by CD8+ T cells activated by DC1s. This advantage of DC1s was observed in polyclonally activated naive and memory CD8(+) T cells and in blood-isolated melanoma-specific CTL precursors. Our data help to explain the dissociation between the ability of cancer vaccines to induce high numbers of tumor-specific CD8+ T cells in the blood of cancer patients and their ability to promote clinical responses, providing for new strategies of cancer immunotherapy.

Relapse of Leukemia with Loss of Mismatched HLA Due to Uniparentaldisomy Follwing Haploidentical Hematopoietic Transplantation.

Abstract 2457Poster Board II-434 Introduction:Down-regulation or loss of human leukocyte antigen (HLA) expression can lead to impaired T-cell recognition and a blunted immune response to malignant tumors. We investigated HLA expression on leukemic cells derived from patients at the time of diagnosis and relapse after HLA haploidentical hematopoietic stem cell transplantation (HSCT) using flow cytometry with locus-specific antibodies. We hypothesized that the loss of HLA haplotype caused leukemic cells to escape immunosurveillance and consequently led to relapse of the disease. Materials and methods:The CD13+/34+ leukemic blasts were sorted by flow cytometry from bone marrow cells at the time of diagnosis and at the time of relapse. Genomic DNA was extracted from leukemic cells by fluorescence-activated cell-sorter as well as phytohemagglutinin-stimulated patient-derived T cells and subjected to single-nucleotide polymorphism (SNP) array analysis using GeneChip NspI arrays (Affymetrix, Tokyo, Japan). Allele-specific copy number was detected using Copy Number Analyser for GeneChip® software. The frequencies of cytotoxic T lymphocyte precursor (CTLp) specific for the recipient mismatched HLA molecules were analyzed using a standard limiting dilution assay. Allo-HLA-restricted CTL clones were isolated by standard limiting dilution and expanded for cytotoxicity assay against mismatched HLA transduced HLA class I-deficient 721.221 B-LCLs. Results:Two of three relapsed patients after HLA haploidentical HSCT demonstrated loss of HLA alleles on leukemic cells at the time of relapse and this loss was limited to the mismatched alleles in both patients. However, none of seven relapsed patients experienced haplotype loss following HLA matched HSCT. SNP array analyses of sorted leukemic cells at the time of diagnosis and at the time of relapse further revealed the copy number-neutral loss of heterozygosity, namely acquired uniparental disomy (UPD) on the short arm of chromosome 6, resulting in the total loss of the mismatched HLA haplotype. Recipient alloantigen-specific cytotoxic T-cell clones were generated from the donor that did not recognize the leukemic cells at the time of relapse, whereas those cells taken at diagnosis were recognized and efficiently killed. In one of the patients, we sought to determine if the number of CTLp had changed during the post-transplant course. A limiting dilution analysis with a split-well 51Cr-release assay was carried out to compare the CTLp frequencies specific for the mismatched antigens between the recipient and donor. Surprisingly, the CTLp frequencies reactive with recipient T cell blasts in CD8+ T cells obtained around Days 100, 180, and 300 (4 months before relapse) were undetectable, while the CTLp frequency obtained at Day 520 (1 month after the third DLI or 2 weeks after remission confirmed by bone marrow aspirate) was 8.6 × 10-5 [95% confidence interval (CI), 1.49 × 10-6 – 5.0 × 10-5]. The CTLp frequency in the donor CD8+ cells was 4.3 × 10-5 (95%CI, 7.2 × 10-5 – 2.5 × 10-5), which was close to that obtained after DLI in the recipient. Conclusions:These results suggest that the cytotoxic T lymphocyte response to mismatched HLA alleles can eradicate leukemic cells; however, escape from immunosurveillance by the loss of mismatched HLA alleles using UPD may be involved in relapse after haploidentical HSCT. Disclosures:No relevant conflicts of interest to declare.

Haploidentical Stem Cell Transplantation: High Doses of Alloreactive-T Cell Depleted Donor Lymphocytes Administered Post-Transplant Decrease Infections and Improve Survival without Causing Severe Gvhd.

Abstract 512Delayed immune reconstitution following intensive T cell depletion of the stem cell graft is the main complication limiting broad utilization of haplo-mismatched donors for stem cell transplantion (SCT). Indeed, it results in frequent and rapidly lethal infectious events. The ability to accelerate immune reconstitution following haplo-SCT would provide a unique opportunity to transplant the large number of patients who cannot find an HLA-matched donor. We present results of our Phase I clinical trial of haploidentical allogeneic SCT followed by an “add-back of donor T cells to accelerate immune reconstitution” (ATIR). This donor lymphocyte infusion (DLI) underwent photodynamic depletion (PD) of host-reactive T cells using dibromorhodamine as photosensitizer (Kiadis Pharma). Nineteen patients (11 M, 8 F) with very high-risk hematologic malignancies (mostly refractory or relapsed acute myeloid leukemia (10) and myelodysplastic syndromes (4), and refractory ALL (1), CLL (2), CML (1) and NHL (1)) entered the trial. Median age at SCT was 54 years (range: 19-62). HLA compatibility was 3/6 in 6 pts, 4/6 in 12 pts and 5/6 (DR mismatch) in 1 pt. Increasing doses of PD-treated donor cells (ATIR: 1×104 to 5.0 ×106 CD3+ cells/kg) were administered on day 34±6 after transplant. In the ATIR, greater than 95% of CD4+CD25+ and CD8+CD25+ T cells as well as anti-host cytotoxic T lymphocyte precursors (CTLp) were depleted from DLIs. All stem cell grafts underwent in vitro immunomagnetic T cell depletion using CD34+ positive cell selection (Miltenyi). The myeloablative regimen consisted of TBI (1200 cGy), thiotepa (5 mg/kg) and fludarabine (200 mg/m2). No GVHD prophylaxis was administered. All patients showed complete donor chimerism and durable hematologic engraftment. Five patients developed grade II GVHD affecting skin (n = 5 pts), liver (2 pts) and gastrointestinal tract (1 pt) at a median of 101 days post-SCT. No patient developed grade III-IV acute GVHD. Chronic GVHD developed in 9 pts, mostly in those receiving higher T cell doses. Treatment of acute and chronic GVHD involved steroids, tacrolimus and mycophenolate mofetil in 3 patients, steroids and tacrolimus in 3 pts, and steroids only in 3 pts. GVHD responded rapidly to treatment since the median duration of total immunosuppressive therapy in each patient was 187 days (range: 61-319 d). All 7 patients in cohorts 1-3, who received 1.3×105 or less CD3+ cells/kg, developed infectious complications (100% of pts), with 5 lethal episodes in these 7 pts. In sharp contrast, only 6 (50%) of the following 12 patients (cohorts 4-7) receiving ATIR with the highest CD3+ cell doses (3.2×105 to 5.0×106 CD3+ cells/kg) developed infections (p <0.05), none resulting in a fatal event (p<0.001). Interestingly, CD3 lymphocytes recovered earlier in the last 2 cohorts (6 and 7) receiving 2-5×106 CD3+ cells/kg than in the first 5 cohorts (7.9×105 or less CD3+ cells/kg) (p<0.01). Eight patients died: 4 of relapsed leukemia (3 AML; 1 ALL) and 4 of infections. Overall treatment related mortality (TRM) is 27% at 2 years post-SCT, with a TRM of 0% in patients receiving the highest CD3+ cell doses (cohorts 4-7). The overall survival is 60% at 2 years (median f-up: 12.1 mo; 95% confidence interval at 2 years: 37-83%). The 12 patients in cohorts 4-7 receiving the higher CD3+ cell doses had an improved survival (82% at 2 yrs) over the 7 patients in cohorts 1-3 administered a lower CD3+ cell dose (14% at 2 yrs) (p<0.05). Our results indicate that the post-transplant infusion of an ATIR-PD treated DLI is feasible, results in accelerated T cell reconstitution, and decreases the incidence and severity of infections without inducing severe GVHD. These results suggest a clinical benefit for patients receiving the highest ATIR doses and form the basis of an international pivotal clinical trial to decrease TRM in patients undergoing haploidentical stem cell transplantation. Disclosures:Roy:Kiadis Pharma: Research Funding. Egeler:Kiadis Pharma: Employment.

Detection and Ex Vivo Expansion of Anti-Viral T Cells Isolated From Recipients of Unrelated Umbilical Cord Blood Transplant.

Abstract 2245Poster Board II-222 Background:Viral infection is commonly detected within the first weeks after umbilical cord blood transplant (UCBT), likely triggering T cell activation and in vivo priming of infused naïve T cells. This hypothesis is supported by the observation that despite the continuous exposure to immunosuppressive (IS) drugs, viremia may resolve even in the absence of antiviral agents. However, IS may delay or even prevent effective T cell priming and the development of protective immunity, therefore causing significant morbidity and mortality. Hypothesis and Objective:Our laboratory has a long standing interest in defining pre-requisites of protective immunity after UCBT. In this study we focused on two of the most common potentially fatal viruses, CMV and Adenovirus to assess antigen-specific immune reconstitution. We hypothesized that 1) threshold numbers of CTL precursors can be identified for each virus that will impact clinical outcome 2) there may be circulating CTL precursors even if below the level of detection in lymphopenic patients that receive IS drugs, however, ex vivo restimulation could lead to expansion and subsequent quantitation. Methods:We have established in vitro assays to test the impact of cytokines and new culture techniques to promote anti-viral precursor survival and expansion. Monocytes in bulk cultures, infected with replication incompetent adenovirus vectors encoding the pp65 CMV matrix protein (Ad5f35pp65), served as APC. Antiviral responses were quantitated by ELISPOT, utilizing computerized enumeration of interferon gamma (IFNg) producing spot forming cells (SFC) in response to overlapping peptide spanning immunodominant viral antigens. Hexon and penton for adenovirus, and pp65 and EI-1 for CMV. Results:There were 8 patients enrolled on this IRB approved study transplanted at a median age of 9.9 years (range 2-17). Blood samples was obtained at a median of 70 days (range 34-470). Only 1 of 3 patients with adenovirus infection ( stool x1, urine x1, respiratory tract x 1) had previous viremia while all five (5) patients with CMV had viremia. Only 1 of the 3 adeno patients had detectable SFC from freshly drawn peripheral blood, 11 SFC/1×10e5 cells against hexon and penton respectively. None of the 5 CMV+ patients had detectable pp65 or EI-1-specific T cell responses from freshly drawn blood. However, after 9-12 days of ex vivo culture, in the presence of IL7 (10ng/ml), we were able to enumerate significantly amplified CTL responses in 7 of the 8 patients, with one CMV viremic the exception. In the remaining patients the median adenovirus hexon-specific response was 52 SFC/1×10e5 cells (range 20-90). the median penton-specific CTLp frequency was 13 SFC/1×10e5 (range 1 - 25). The median CMV pp65-specific response was 6.7 SFC/1×10e5 (range 0-14) while no responses were recorded against EI-1 demonstrating the critical role of antigenic restimulation provided by the Ad5f35pp65 construct lacking IE. The amplified immune responses were highly restricted; in the CMV+ patients (all adeno-) we were able to detect SFC only against pp65 and to neither IE or penton/hexon, while in the adenovirus+/CMV- patients (n=-3) we were able to detect SFC solely in those wells that were stimulated by hexon and penton peptides. All patients have survived at a median of 220 days after transplant (range 81-532). Conclusion:To our knowledge these ongoing experiments are the first to demonstrate that dormant virus-specific immune responses are present already in the first 100 days in most UCBT recipients (7/8 in this dataset) if infected with either adenovirus or CMV despite failure to detect these extremely rare events by standard ELISPOT assays from freshly isolated blood. Moreover, these data suggests that anti-viral CTLp can be expanded ex vivo by antigen specific restimulation that lends support to further efforts aimed at developing adoptive anti-viral T cell therapies. Disclosures:No relevant conflicts of interest to declare.

Establishing CD8+ T Cell Immunity by Adoptive Transfer of Autologous, IL-15 Expanded, Anti-Tumor CTL with a Central/Effector Memory Phenotype Can Induce Objective Clinical Responses.

Abstract 782The goal of cellular immunotherapy is to build long-lasting anti-tumor immunologic “memory” in patients and reject tumors for a lifetime. Previously, we and others demonstrated that IL-15 promotes the generation of T cells with a central memory (CM) phenotype which have the capacity to persist and establish effective anti-tumor memory in vivo. Furthermore, it has been shown that CD83 delivers a CD80-dependent T cell stimulatory signal that allows T cells to be long-lived. Based on these findings, we developed a system to generate large numbers of long-lived antigen-specific CD8+ T cells with a memory phenotype. This in vitro culture system utilizes IL-15 and a standardized, renewable artificial antigen presenting cell (aAPC) which was produced by transducing CD80, CD83, and HLA-A*0201 to the human cell line, K562. This aAPC can uniquely support the priming and prolonged expansion of large numbers of antigen-specific CD8+ CTL which display a central/effector memory (CM/EM) phenotype, possess potent effector function, and can be maintained in vitro for >1 year without any feeder cells or cloning.We hypothesized that adoptive transfer of these CTL with a CM/EM phenotype should result in anti-tumor memory in humans even without lymphodepletion or high dose IL-2. For our “first-in-human” clinical study, we chose the melanoma antigen MART1 as a target antigen, since MART1-specific HLA-A*0201+-restricted precursor CTL are detectable in some melanoma patients and can be immunophenotyped pre-infusion. Autologous CD8+ T cells were stimulated weekly with peptide-pulsed human cell-based aAPC and expanded with low dose IL-2 and IL-15. After three weeks, polyclonal MART1 CTL were reinfused without additional lymphodepletion, chemotherapy, IL-2, or vaccination. Eight study participants have enrolled and received a total of 15 MART1 CTL infusions (31% MART1 multimer positivity, median). All but one subject received two reinfusions where the 2nd graft was produced from CD8+ T cells harvested two weeks after the 1st reinfusion. To date, ≥2×109 CTL with potent effector function and a CM/EM phenotype were successfully generated for all subjects. No dose limiting toxicities were observed at either Dose Level 1 (2×108/m2) or Dose Level 2 (2×109/m2). Clinical activity was observed with a response by RECIST criteria in 1 subject, which was confirmed by a negative PET/CT 100 days following the last CTL infusion. In addition, 1 patient experienced a mixed response, 1 had stable disease, 3 had progression, and 2 are currently on active therapy.Multimer staining showed that, immediately post infusion, the percentage of CD8+ T cells specific for MART1 temporarily increased in all subjects, with the highest (6.5%) observed in subject #7. In 4 subjects, sustained increases in the frequency of MART1 specific T cells by more than two-fold (range 2.0-10x) for ≥21 days were observed despite the fact that no exogenous cytokines or vaccination was administered. Moreover, an increase of detectable MART1 specific T cells which display a CM phenotype was observed in all evaluable subjects and was observed for ≥35 days in 6 of 8 subjects. In subject #2, the conversion of MART1 CTL immunophenotype from a naïve to a mixture of naïve/memory phenotypes was observed for more than 6 months. We identified 10 individual MART1 T cell clonotypes from peripheral CD45RA- memory T cells on day 21. Clonotypic TCR Vbeta CDR3 analysis revealed that CTL grafts contained 7 out of 10 of these clonotypes. Furthermore, 6 clonotypes persisted in the peripheral CD45RA- memory fraction on days 39, 67 and/or 132. In Subject #3, who showed a mixed clinical response, 5 individual MART1 T cell clonotypes were isolated from lung metastases. 4 out of 5 clones were included in the CTL grafts. This finding supports the possibility that infused CTL can traffic and localize to sites of disease. Intriguingly, in both subjects, we were able to identify MART1 CTL clonotypes that were not detectable in the CTL grafts but possibly emerged after CTL infusion, indicating that adoptive transfer of MART1-specific CTL may provoke a de novo antitumor response.Taken together, these results suggest that CM/EM MART1 CTL generated ex vivo using our cell-based artificial APC in the presence of IL-15 may persist in vivo and induce de novo anti-tumor responses. Further enhancement of anti-tumor activity may be achieved through vaccination, cytokine administration, and/or removal of cytokine sinks and inhibitory factors following appropriate lymphodepletion. Disclosures:No relevant conflicts of interest to declare.

Pretransplant Immune Parameters Associate With Islet Allograft Outcome

Extensive and informative immunological assessments of islet transplant recipients have been limited due, in part, to the smaller numbers of patients transplanted compared with solid organ trials. Limitations related to blood volume and the logistical challenges of collecting multiple posttransplant samples for drug levels as well as clinical and metabolic parameters also pose challenges. Several variables affect islet allograft outcome, including allo- and autoimmune responses to transplanted islets, β-cell mass and islet quality, efficacy of the immunosuppressive regimen and its effect on islet function, as well as glycemic control. In fact, much debate has centered on whether or not islet attrition is due to immune-mediated loss, death of overworked cells caused by insufficient mass, and/or diabetogenic effect of various immune intervention agents. In addition, many of these parameters may have differing effects based on the transplanted mass, with marginal mass transplants being more susceptible to immune-mediated or stress-induced loss. Recently, several studies have demonstrated immunological alterations in islet allograft patients. Posttransplant changes in lymphocytes and lymphocyte subsets were reported to be variable depending on the immune suppression used but have not yet been associated with graft status or specific clinical outcomes (1–4). Higher posttransplant cytotoxic T-cell precursor frequency against donor antigens was associated with poorer outcome at 6 months in recipients treated with sirolimus-based maintenance therapy but not tacrolimus–mycophenolate mofetil (5). In one study, cytokine production in allogeneic mixed lymphocyte reactions (MLRs) was skewed toward …

Deficient TNF-α and IFN-γ Production Correlates With Nondetectable Donor-Specific Cytotoxicity After Clinical Kidney Transplantation

We previously reported that no cytotoxic T-lymphocyte precursor frequencies (CTLpf) were found in 60% of patients on azathioprine or mycophenolate mofetil+Pred long after kidney transplantation. We questioned whether the absence of donor-specific CTLpf was associated with low levels of stimulatory Th1 (tumor necrosis factor [TNF]-alpha, interferon [IFN]-gamma) or high levels of regulatory Th2 (interleukin [IL]-4, IL-6, IL-10) cytokines. In this study, peripheral blood monocyte cells (PBMC) were stimulated with irradiated donor cells. After 7 days, cytokine production was determined by cytokine bead array, and CTLpf by limiting dilution assay. Patients with detectable CTLpf (> or =10/10(6) PBMC) had significantly higher levels of TNF-alpha (P=0.04) and IFN-gamma (P=0.02) than patients with nondetectable CTLpf (<10/10(6) PBMC). Donor-reactive IL-4, IL-6, and IL-10 production was comparable in both patient groups. Additionally, CTLpf was positively correlated with TNF-alpha (rs=0.54, P=0.0003) and IFN-gamma (rs=0.64, P<0.0001) production. The absence of donor-specific CTLp after transplantation correlates with low levels of stimulatory cytokines, not with elevated levels of regulatory cytokines.

Unrelated donor marrow transplantation in children: transplant policy and outcome in Leiden Paediatrics SCT-Centre

The transplant policy for unrelated donor (UD) BMT at Leiden Paediatrics' SCT-Centre consisted of the use of (1) fully HLA-matched donors or, if not available, HLA-class I matched and/or cytotoxic T-lymphocyte precursor (CTLp)-negative donors and (2) protective isolation of the recipient and antimicrobial suppression of his/her gut microflora to prevent infections and acute GVHD. Engraftment, GVHD, relapse in the case of malignancy and survival were studied retrospectively in 126 evaluable children, transplanted between 1988 and 2005. In addition to the effect of HLA-matching, that of other transplant-relevant variables on the outcome was also studied. Actuarial OS was 65% and the EFS was 59%, 13% graft failures occurred and 7.5% > or =grade II acute GVHD. HLA-class II mismatches combined with HLA-class I matches resulted in a superior OS of 92%, as did a negative vs positive CTLp test, that is, 65 vs 33%. Analysis of other variables showed a poorer OS in patients > or =10 yrs vs <10 yrs, that is, 54 vs 73%, and in male recipients of a female donor graft, that is, 53 vs 69% for other combinations. UD-BMT can be optimized by permitting HLA-class I-matched and/or CTLp-negative donors, and probably by choosing male donors for male recipients.

Allograft-Specific Cytokine Profiles Associate with Clinical Outcome After Islet Cell Transplantation

Islet cell transplantation can cure type 1 diabetes, but allograft rejection and recurrent autoimmunity may contribute to decreasing insulin independence over time. In this study we report the association of allograft-specific proliferative and cytokine profiles with clinical outcome. Peripheral blood mononuclear cells were obtained of 20 islet recipients. Cytokine values in mixed lymphocyte cultures (MLC) were determined using stimulator cells with graft-specific HLA class II. Qualitative and quantitative cytokine profiles were determined before and after islet transplantation, blinded from clinical outcome. Cytotoxic T Lymphocyte precursor (CTLp) assays were performed to determine HLA class I alloreactivity. Allograft-specific cytokine profiles were skewed toward a Th2 or regulatory (Treg) phenotype after transplantation in insulin-independent, but not in insulin-requiring recipients. IFNgamma/IL10 ratio and MLC proliferation decreased after transplantation in insulin-independent recipients (p = 0.006 and p = 0.01, respectively). Production of the Treg cytokine IL10 inversely correlated with proliferation in alloreactive MLC (p = 0.008) and CTLp (p = 0.005). Production of IL10 combined with low-MLC reactivity associated significantly with insulin independence. The significant correlation between allograft-specific cytokine profiles and clinical outcome may reflect the induction of immune regulation in successfully transplanted recipients. Islet donor-specific IL10 production correlates with low alloreactivity and superior islet function.

Aurora-A kinase: a novel target of cellular immunotherapy for leukemia

AbstractAurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9–amino-acid epitope (Aur-A207-215: YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A–specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201–restricted manner. Importantly, Aur-A–specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A207-215 epitope–specific CTL precursors are present in peripheral blood of HLA-A*0201–positive and HLA-A*2402–positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.

Induction of human tumor‐associated differentially expressed gene‐12 (TADG‐12/TMPRSS3)‐specific cytotoxic T lymphocytes in human lymphocyte antigen‐A2.1–positive healthy donors and patients with advanced ovarian cancer

Tumor-associated differentially expressed gene-12 (TADG-12) is a serine protease recently found highly differentially expressed in epithelial ovarian cancer. The goal of this study was to identify potential immunogenic peptides derived from TADG-12 for immunotherapy of ovarian carcinoma. A bioinformatics approach (ie, the BIMAS algorithm, National Institutes of Health, http://bimas.dcrt.nih.gov/molbio/hla_bind) was used to identify multiple immunogenic peptides derived from TADG-12 that bind to human leukocyte antigen-A2.1 and elicit peptide-specific human cytotoxic T lymphocyte (CTL) responses in healthy individuals and in patients with advanced stage ovarian cancer. CD8+ CTL populations generated against 5 TADG-12-derived peptides were consistently able to induce lysis of autologous peptide-loaded target cells above background. Importantly, TADG-12 YLPKSWTIQV peptide-specific CTLs from healthy donors and ovarian cancer patients were found to effectively kill ovarian cancer cells naturally expressing TADG-12. Cytotoxicity was significantly inhibited by anti-human lymphocyte antigen (HLA)-A2.1 (BB7-2) and anti-HLA class I (W6 of 32) monoclonal antibodies, whereas natural killer-sensitive K562 cells were not lysed. TADG-12 YLPKSWTIQV peptide-specific CTL precursor frequency was low in peripheral blood leukocytes of normal donors and ovarian cancer patients, as determined by interferon-gamma production in enzyme-linked immunosorbent spot-forming cell assays. Intracellular cytokine expression measured by flow cytometry after OKT-3 monoclonal antibody stimulation showed a type 1 cytokine profile in YLPKSWTIQV peptide-specific CTLs. The TADG-12 YLPKSWTIQV peptide is an immunogenic epitope in ovarian tumors and may represent an attractive target for immunotherapy of ovarian cancer. These data may pave the way for TADG-12 peptide-derived cell-based therapy, including dendritic cell immunotherapy, for the vaccination of ovarian cancer patients harboring chemotherapy-resistant or residual disease.

Risk factors for development of neurologic disease after experimental exposure to equine herpesvirus-1 in horses

To identify risk factors associated with development of clinical neurologic signs in horses exposed to equine herpesvirus-1 (EHV-1). 36 adult horses. Blood samples collected before and after challenge inoculation with nonneuropathogenic or neuropathogenic EHV-1 were analyzed for leukocyte-associated viremia, serum neutralizing antibody, and EHV-1-specific cytotoxic T-lymphocyte precursors (CTLPs). Associations between variables and neurologic disease and correlations between age category or breed and development of neurologic disease were examined. 9 horses developed CNS signs (ataxia, hind limb paresis or paralysis, bladder atony, or recumbency). Neurologic deficits were correlated with infection by a neuropathogenic strain of EHV-1, age>20 years, high postexposure viremic load, and low preexposure concentration of CTLPs. No significant correlations were observed between preinfection titers or horse breed and postinfection development of neurologic signs. Horses with high concentrations of preexisting CTLPs, regardless of age, strain of virus, or titer, were more likely to control the magnitude of postinfection leukocyte-associated viremia and subsequent development of neurologic disease; therefore, CTLPs appear to be a critical requirement for protective immunity against EHV-1-induced myeloencephalopathy. The importance of achieving immunity related to high concentrations of vaccine-induced CTLPs in horses at high risk for exposure to neuropathogenic strains of EHV-1 is indicated.

Aurora-a Kinase: A Novel Target of Cellular Immunotherapy for Human Leukemia

[Purpose] Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family, and the Aur-A gene is located at chromosome 20q13. Aur-A is mainly expressed in the G2/M phase of the cell cycle and regulates mitotic cell division in normal cells. Aur-A is expressed exclusively in testis in normal tissues, but is abundantly expressed in various kinds of cancer including hematological malignancies. Its overexpression usually accompanies chromosomal abnormality related with the poor prognosis. Furthermore, the silencing of Aur-A gene has disrupted the proliferation of cancer cells and augmented their susceptibility to anti-cancer agents. These data strongly suggest that Aur-A is one of the crucial oncognes and a rational target for cancer treatment. Thus, we postulated that Aur-A might be an ideal target for cancer immunotherapy, and attempted to verify the feasibility of cellular immunotherapy for leukemia targeting Aur-A.[Methods] Nine-mer peptides derived from Aur-A which were algorithmically predicted to bind to HLA-A*0201 molecule were synthesized and assessed their binding affinities by the stabilization assay with T2 (HLA-A*0201) cells. Aur-A-specific cytotoxic T lymphocytes (CTLs) were generated by stimulation of CD8+ T cells with Aur-A peptide-pulsed autologous dendritic cells. Cytotoxicity of Aur-A-specific CTLs was defined by standard 51Cr-release assay. HLA class I restriction was examined with anti-HLA class I antibody and target-specificity of Aur-A-specific CTLs was examined by cold target inhibition assay with 51Cr-labeled HLA-A*0201-positive leukemia cell lines in combination with Aur-A peptide-loaded 51Cr-unlabeled HLA-A*0201-positive lymphoblastoid cell lines. The expressions of Aur-A mRNA and Aur-A protein in leukemia cells and normal cells were assessed by semi-quantitative real-time PCR and Western blotting. The frequencies of Aur-A-specific CTL precursors in the peripheral blood of healthy individuals and patients with leukemia were measured by tetramer staining.[Results] We established an HLAA* 0201-restricted and Aur-A207-215 peptide (YLILEYAPL)-specific CTL line (designated as AUR-1) by repeated stimulations of CD8+ T cells with Aur-A207-215 peptide-pulsed peripheral blood mononuclear cells from an HLA-A*0201-positive healthy individual. AUR-1 exerted cytotoxicity to various kinds of leukemia cell lines in an HLA-A*0201- restricted fashion. AUR-1 could discriminate freshly isolated leukemia cells from normal cells and normal mitotic cells, according to the expression levels of Aur-A mRNA. By using freshly isolated leukemia cells from CML patients' bone marrow mononuclear cells (BMMCs), we further investigated whether AUR-1 could lyse the leukemic progenitor cells. The CD34+CD38low fraction of CML BMMCs which include leukemic stem cells but not the normal counter part cells overexpressed Aur-A mRNA. Additionally AUR-1 could lyse CD34+ CML BMMCs, but not normal CD34+ cells. Finally, by using tetramer assay, Aur-A207-215-specific CTL precursors were detected more frequently in the peripheral blood of HLA-A*0201-positive patients with leukemia than that of healthy individuals.[Conclusion] This is the first report that Aur-A-specific CTLs can exert the cytotoxicity against leukemic stem cells but not normal hematopoietic stem cells. Results in this study suggests that the cellular immunotherapy targeting Aur-A is a promising strategy for the treatment of human leukemia.

Ex-Vivo Acquisition of Central Memory Phenotype Is Critical for Tolerance Induction by Donor Anti-3rd party CD8 T Cells in Allogeneic Bone Marrow Transplantation.

The robust immunity remaining after reduced intensity conditioning represents a major barrier to durable engraftment of MHC disparate T cell depleted BM transplants (TDBMT). One approach to address this challenge could be afforded by using donor CD8 CTLs, endowed with potent veto activity (the ability of a cell to specifically suppress CTL precursors directed against its antigens, while sparing other CTLs capable of affording protection against infections and malignancies). However, their use is limited due to their marked GVH reactivity. This problem can be addressed effectively by stimulation of donor CD8 T cells against 3rd party stimulators under exogenous IL2 deprivation. The anti-3rd party veto CTLs support engraftment of TDBMT without causing GVHD. Nevertheless, although these cells displayed highly efficient veto activity in vitro, they were less efficient in vivo, requiring administration in conjunction with Rapamycin. In our study we found, using in vivo imaging, that veto CTLs display poor homing to the host lymph nodes (LNs). Therefore, we hypothesized that the relatively low efficiency of veto CTLs in vivo could be attributed to their failure to co-localize with the host T cells (HTCs) at the LNs, during the critical time at which the HTCs are triggered against donor antigens. To enhance LNs homing we generated anti-3rd party CD8 T cells under culture conditions favoring the acquisition of central memory (Tcm) phenotype (CD44highCD62Lhigh). In order to monitor CTLs or Tcm cells adoptively transferred into irradiated recipients of BM allografts, we used (Host x Donor)F1 cells co-expressing host and donor MHC that can be distinguished by FACS. When CD62Lhigh Tcm cells, expanded in the presence of IL15, were administered, the number of cells located at the host LNs two days post transplant was 40-fold higher compared to that found for veto CTLs expanded in the presence of IL2, which exhibit a CD62Llow effector phenotype (p<0.05). Moreover, the total number of Tcm cells harvested from various organs was increased by 9 folds between days 2 to 6 post BMT (p<0.05) in sharp contrast to the CTLs which displayed a non significant proliferation, indicating that Tcm cells possess superior proliferative capabilities. In addition, the Tcm cells exhibited marked durable persistence in-vivo when tested 100 days post transplant, comprising 17%±9% of the total CD8 T cell compartment following infusion of 1x106 cells. When evaluated in a stringent mouse model for T cell mediated BM allograft rejection, Tcm cells displayed marked tolerizing activity. In this model, 3x106 Balb-Nude BM is transplanted into lethally irradiated C3H hosts that were previously inoculated with 1.25x104 HTC (a number sufficient to induce BM rejection and lethality). Thus, administration of 5x106 anti-3rd party Tcm cells derived from (C3H x BALB)F1 mice, with no further immunosuppression, led to overall survival of 83% (19/23) at 100 days post BMT, while administration of 107 anti-3rd party CTLs (bearing an effector phenotype) could only afford survival of 19% (3/16) (p<0.05) in the absence of Rapamycin. To evaluate the risk of GVHD, allogeneic Balb/c derived anti-3rd party Tcm cells or naive CD8 T cells were administered in conjunction with 5x106 Balb/c-Nude BM cells into lethally irradiated C3H hosts. As expected, naive CD8 cells caused lethal GVHD and only 6% (1/16) of the mice which received 2x106 naive cells survived 100 days post BMT. In contrast, the mice receiving Tcm cells displayed 83% survival (10/12 or 5/6 for 2x106 or 5x106 Tcm cells, respectively) with no signs of GVHD. Furthermore, when evaluated in the graft rejection model, 5x106 fully allogeneic donor Tcm cells induced survival, accompanied with complete donor chimerism at 100 days post transplant in 73% (11/15) of the recipients. The similar efficacy of overcoming rejection displayed by both fully allogeneic and F1 derived Tcm cells is in accordance with a veto mechanism, as opposed to potential ablation of host resistance by donor alloreactive clones. Collectively, the ability of anti-3rd party CD8 Tcm cells to expand and persist in vivo without GVHD, their marked LN homing and their capacity to overcome rejection of TDBMT in the absence of further immunosuppression, strongly suggest that these cells hold marked potential for tolerance induction in allogeneic hematopoietic stem cell transplantation.