Peptide-pulsed DC Research Articles

MAGEA1 and hTERT Peptide Treatment Improves the Potency of The Dendritic Cell- Cytotoxic T Lymphocytes (DC-CTL) Immunotherapy in DAC Treated Acute Myeloid Leukemia.

Background: Acute myeloid leukemia (AML) is a type of heterogenous malignant hematological disorder. Recently developed immunotherapies such as chimeric antigen receptor T cell (CAR-T) do not demonstrated promising therapeutic results due to the off-target effect. The Dendritic cell-cytotoxic T lymphocyte adoptive immunotherapy (DC-CTL) is one of the recently developed immunotherapies. One of the reasons that DC-CTL does not work well in AML is the lack of antigens with high binding affinity, high antigen presentation potency, and the specificity to AML cells.Methods: DAC was used to treat AML cells to find overexpressed CTAs upon DAC treatment. The overexpression was confirmed at both mRNA and protein level by realtime PCR and western blotting. Peptides was designed by using the NetMHCpan database and EPIP based on the out-screened protein sequences. The peptides were then used to pulse DC-CTL coculture in vitro and tested the cytotoxicity of CTLs in vitro and their cancer inhibition potency in vivo.Results: Two cancer testis antigen (CTA) proteins, MAGEA1 and hTERT, was up-regulated in DAC treated AML cells. DC cells pulsed by the antigen peptides designed based on the sequence of these two proteins demonstrated increased potency to stimulate CTL cells in terms of cytokines secretion. These cytokines included IFN-γ, IL-6, and TNF-α. Moreover, enhanced in vitro cytotoxicity was found in CTL cells treated with peptide pulsed DC cells. AML progress was inhibited by CTA peptides pulsed DC-CTL in a mouse AML model.Conclusions: MAGEA1 and hTERT could possibly serve as specific tumor antigens upon DAC treatment, providing potential targets for the development of immunotherapies for AML in the future.

Vaccination with poly(IC:LC) and peptide-pulsed autologous dendritic cells in patients with pancreatic cancer

BackgroundDendritic cells (DCs) enhance the quality of anti-tumor immune response in patients with cancer. Thus, we posit that DC-based immunotherapy, in conjunction with toll-like receptor (TLR)-3 agonist poly-ICLC, is a promising approach for harnessing immunity against metastatic or locally advanced unresectable pancreatic cancer (PC).MethodsWe generated autologous DCs from the peripheral blood of HLA-A2+ patients with PC. DCs were pulsed with three distinct A2-restricted peptides: 1) human telomerase reverse transcriptase (hTERT, TERT572Y), 2) carcinoembryonic antigen (CEA; Cap1-6D), and 3) survivin (SRV.A2). Patients received four intradermal injections of 1 × 107 peptide-pulsed DC vaccines every 2 weeks (Day 0, 14, 28, and 42). Concurrently, patients received intramuscular administration of Poly-ICLC at 30 μg/Kg on vaccination days (i.e., day 0, 14, 28, and 42), as well as on days 3, 17, 21, 31, 37, and 45. Our key objective was to assess safety and feasibility. The effect of DC vaccination on immune response was measured at each DC injection time point by enumerating the phenotype and function of patient T cells.ResultsTwelve patients underwent apheresis: nine patients with metastatic disease, and three patients with locally advanced unresectable disease. Vaccines were successfully manufactured from all individuals. We found that this treatment was well-tolerated, with the most common symptoms being fatigue and/or self-limiting flu-like symptoms. Among the eight patients who underwent imaging on day 56, four patients experienced stable disease while four patients had disease progression. The median overall survival was 7.7 months. One patient survived for 28 months post leukapheresis. MHC class I –tetramer analysis before and after vaccination revealed effective generation of antigen-specific T cells in three patients with stable disease.ConclusionVaccination with peptide-pulsed DCs in combination with poly-ICLC is safe and induces a measurable tumor specific T cell population in patients with advanced PC.Trial registrationNCT01410968; Name of registry: clinicaltrials.gov; Date of registration: 08/04/2011).

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity.

Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4+ and CD8+ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8+ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8+ T cell epitope enhance subdominant pathogen-specific CD8+ T cell responses. More specifically, CD8+ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8+ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.

Dendritic cell–based immunotherapy targeting Wilms’ tumor 1 in patients with recurrent malignant glioma

Dendritic cell (DC)-based vaccination is considered a potentially effective therapy against advanced cancer. The authors conducted a Phase I study to investigate the safety and immunomonitoring of Wilms' tumor 1 (WT1)-pulsed DC vaccination therapy for patients with relapsed malignant glioma. WT1-pulsed and/or autologous tumor lysate-pulsed DC vaccination therapy was performed in patients with relapsed malignant gliomas. Approximately 1 × 10(7) to 2 × 10(7) pulsed DCs loaded with WT1 peptide antigen and/or tumor lysate were intradermally injected into the axillary areas with OK-432, a streptococcal preparation, at 2-week intervals for at least 5-7 sessions (1 course) during an individual chemotherapy regimen. Ten patients (3 men, 7 women; age range 24-64 years [median 39 years]) with the following tumors were enrolled: glioblastoma (6), anaplastic astrocytoma (2), anaplastic oligoastrocytoma (1), and anaplastic oligodendroglioma (1). Modified WT1 peptide-pulsed DC vaccine was administered to 7 patients, tumor lysate-pulsed DC vaccine to 2 patients, and both tumor lysate-pulsed and WT1-pulsed DC vaccine to 1 patient. The clinical response was stable disease in 5 patients with WT1-pulsed DC vaccination. In 2 of 5 patients with stable disease, neurological findings improved, and MR images showed tumor shrinkage. No serious adverse events occurred except Grade 1-2 erythema at the injection sites. WT1 tetramer analysis detected WT1-reactive cytotoxic T cells after vaccination in patients treated with WT1-pulsed therapy. Positivity for skin reaction at the injection sites was 80% (8 of 10 patients) after the first session, and positivity remained for these 8 patients after the final session. This study of WT1-pulsed DC vaccination therapy demonstrated safety, immunogenicity, and feasibility in the management of relapsed malignant gliomas.

Amplification Ex Vivo and Cytocidal Activity of Leukemia Tumor-Associated Antigen-Specific Cytotoxic T Lymphocytes

To explore the feasibility of amplifying the leukemia tumor associated antigens-specific cytotoxic T lymphocytes (TAA-CTL) ex vivo and to evaluate the cytotoxicity of TAA-CTL. The peripheral blood mononuclear cells were enriched by density gradient centrifugtion; TAA-CTL were generated by stimulation of PBMNC with peptide-pulsed DC at an effector-to-target ratio of 10:1; immunophenotype of TAA-CTL was analyzed by flow cytometry; cytotoxicity assay was used to evaluate the cytotoxic activity of TAA-CTL against peptide-pulsed autologous target cells (PHA-Blasts). TAA-CTL expanded from volunteer showed a mean expansion of 3.81±1.61, the phenotyping of the TAA-CTL was predominantly CD3+ (97.22±0.71)% with varying content of CD4+ (41.47±27.08)% and CD8+ (56.40±11.15)% T cells, it also contained few nature killer cells (0.50±0.31)% and rare residual B cells (0.14±0.20)%; the subpopulations of TAA-CTL and CTL were not statisticaly significantly different in the proportion (P>0.05); the detection of intracellular cytokines after stimulation with peptide showed that the secretion rates of IFN-γ and TNF-α in CD8+ TAA-CTL were (27.67±2.21)% and (34.2±0.71)%, while the secretion rates were (21.6±2.55)% and (9.97±3.44)% in CD4+ TAA-CTL. Compared with the CD8+ TAA-CTL group, the secretion rates of IFN-γ and TNF-α were (1.36±0.04)% and (5.58±0.03)% in CD8+ CTL, the rates of IFN-γ and TNF-α were (0.91±0.06)% and (1.60±0.07)% in CD4+ CTL. The secretions of IFN-γ and TNF-α in CTL were both significantly lower than those in TAA-CTL (P<0.01); the specific killing efficiency of the TAA-CTL against TAA-pulesd target cells were (77.00±1.00)%, (67.40±3.60)%, (60.55±2.45)% and (26.85±5.25)%, when the effecto-target ratios were 40:1, 20:1, 10:1 and 5:1, and there was negligible lysis of TAA-CTL for PHA-blast (P<0.01). TAA-CTL can be successfully induced and generated ex vivo from the healthy volunteer peripheral blood, and the TAA-CTL possess a specific killing activity.

Age-associated increase of IL-6 dampens anti-tumor immune responses through attenuating Th1 differentiation of tumor-specific CD4 T cells (TUM7P.928)

Abstract With age immune function progressively decreases and pro-inflammatory status is concomitantly developed. We focused on the impact of this inflammatory environment on tumor-specific CD4 T cells. We demonstrated that in contrast to tumor elimination in young hosts, adoptive transfer of OVA-specific young CD4 T cells and vaccination with OVA peptide-pulsed DC failed to induce efficient regression of OVA-expressing tumor cells in aged hosts. This was consistent with the result that in aged mice generation of tumor-specific Th1 cells was significantly attenuated. Interestingly, a blockade of IL-6 signaling partially restored Th1 differentiation of donor CD4 T cells and elicited tumor regression in aged hosts, but did not alter their expansion, IL-2 or IL-17A production. Furthermore, in vitro analyses revealed that IL-6 directly acted on CD4 T cells to attenuate Th1 differentiation. IL-6 blockade also restored the defect in an ability of tumor-specific CD4 T cells to promote anti-tumor CD8 T cells induction in aged mice. These results were confirmed by aged IL-6-deficient mice. However, the Th1-mediated help for tumor-specific CD8 T cells was not observed in aged mice when IFN-γ-deficient CD4 T cells were transferred into anti-IL-6 Ab-treated mice. These results imply that systemic increase of IL-6 is an extrinsic factor counteracting tumor-specific Th1 differentiation, and is responsible for the age-associated defect in mounting protective anti-tumor immunity in aged individuals.

Abstract SY32-03: Promoting tissue-specific antitumor immunity

Abstract Studies have shown that tissue specific T cells exist in the peripheral T cell repertoire in an ‘ignorant’ or naïve state. Using our mouse models, we have shown that tissue specific T cells can be activated in a variety of ways to induce autoimmunity or tumor immunity. We have examined different strategies to promote anti-tumor responses including vaccination, DC stimulation and transfer of tumor specific T cell populations. Several new studies have found the following results. 1) Peptide pulsed DC vaccines, are superior to peptide vaccines. 2) Transfer of tissue specific T cells have improved efficacy when stimulated with DCs pulsed with antigen in mouse models. 3) Human tumor infiltrating lymphocyte (TIL) cultures stimulated with DCs and anti-CD3 show noteable shift in various markers, supporting the possibility of improved effector function in vivo. Furthermore, lessons can be learned from chronic viral infection models, since the presence of sustained tumor antigens may mimic some of the same evasion strategies. With this rationale, we have shown that IL-7 can ‘cure’ mice of chronic viral infections as well as promote anti-tumor immunity. Further recent studies have shown that NK cells inhibit CD8 responses and importantly, chronic viral infections cannot be established in the absence of NK cells.

Dendritic Cell-Based Vaccines Positively Impact Natural Killer and Regulatory T Cells in Hepatocellular Carcinoma Patients

Immunotherapy of cancer must promote antitumor effector cells for tumor eradication as well as counteract immunoregulatory mechanisms which inhibit effectors. Immunologic therapies of cancer are showing promise, including dendritic cell-(DC-) based strategies. DC are highly malleable antigen-presenting cells which can promote potent antitumor immunity as well as tolerance, depending on the environmental signals received. Previously, we tested a peptide-pulsed DC vaccine to promote Alpha-fetoprotein (AFP-) specific anti-tumor immunity in patients with hepatocellular carcinoma (HCC), and reported on the CD8+ T cell responses induced by this vaccine and the clinical trial results. Here, we show that the peptide-loaded DC enhanced NK cell activation and decreased regulatory T cells (Treg) frequencies in vaccinated HCC patients. We also extend these data by testing several forms of DC vaccines in vitro to determine the impact of antigen loading and maturation signals on both NK cells and Treg from healthy donors and HCC patients.

MHC-I-restricted melanoma antigen specific TCR-engineered human CD4+ T cells exhibit multifunctional effector and helper responses, in vitro

MHC class I-restricted human melanoma epitope MART-1(27-35) specific TCR-engineered CD4+CD25- T cells synthesize Th1 type cytokines and exhibit cytolytic effector function upon cognate stimulation. A detailed characterization of such TCR-engineered CD4+CD25- T cells now reveals that they are multifunctional. For example, they undergo multiple rounds of division, synthesize cytokines (IFN-gamma, TNF-alpha, IL-2, and MIP1ss), lyse target cells, and "help" the expansion of the MART-1(27-35) specific CD8+ T cells when stimulated by the MART-1(27-35) peptide pulsed DC. Multiparametric analyses reveal that a single TCR-engineered CD4+ T cell can perform as many as five different functions. Nearly 100% MART-1(27-35) specific TCR expressing CD4+ T cells can be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies.

Abstract 1925: MHC class I-restricted MART-127-35 specific TCR engineered human CD4+ and CD8+ T cells exhibit polyfunctional properties

Abstract The “polyfunctional” property of antigen specific T cells has turned out to be an important element in successful host immune responses in AIDS and in malignancies. We have shown that human CD4+ CD25- T cells engineered to express MHC Class I-restricted MART-127-35 epitope specific TCR show cytolytic activity, secret Th1 type cytokines. We have now carried out an extended analysis of the functional dimension of MART-127-35 epitope specific TCR engineered CD4+ and CD8+ T cells. Freshly isolated circulating CD4+ and CD8+ T cells, transduced with MART-127-35 epitope specific DMF5 TCR containing retrovirus, were tested for proliferative, cytokine secretory and for cytolytic capacity (polyfunctionality analyzed with intracytoplasmic immunofluorescence staining for IL-2, IFN-γ, TNF-α, MIP-1β and CD107a using the SPICE software). The TCR engineered CD4+ T cells were also tested for their potential for “helping” cytolytic T cells response by CD8+ T cells in in vitro CTL generation cultures. The TCR-engineered CD4+ as well as CD8+ T cells were found to be polyfunctional. For example, they underwent multiple rounds of division following stimulation by peptide pulsed dendritic cells and by melanoma cells, synthesized multiple cytokines (IFN-γ, TNF-α, IL-2, MIP1ß) and lysed target cells. The TCR engineered CD4+ T cells also amplified the CD8+ CTL burst size in vitro. Multiparametric analyses revealed that a single TCR-engineered CD4+ T cell could perform as many as five different functions. Nearly 100% MART-127-35 specific TCR expressing CD4+ as well as CD8+ T cells could be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR-transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1925.

DNA vaccination with T‐cell epitopes encoded within Ab molecules induces high‐avidity anti‐tumor CD8+ T cells

Stimulation of high-avidity CTL responses is essential for effective anti-tumor and anti-viral vaccines. In this study we have demonstrated that a DNA vaccine incorporating CTL epitopes within an Ab molecule results in high-avidity T-cell responses to both foreign and self epitopes. The avidity and frequency was superior to peptide, peptide-pulsed DC vaccines or a DNA vaccine incorporating the epitope within the native Ag. The DNA Ab vaccine was superior to an identical protein vaccine that can only cross-present, indicating a role for direct presentation by the DNA vaccine. However, the avidity of CTL responses was significantly reduced in Fc receptor gamma knockout mice or if the Fc region was removed suggesting that cross presentation of Ag via Fc receptor was also important in the induction of high-avidity CTL. These results suggest that generation of high-avidity CTL responses by the DNA vaccine is related to its ability to both directly present and cross-present the epitope. High-avidity responses were capable of efficient anti-tumor activity in vitro and in vivo. This study demonstrates a vaccine strategy to generate high-avidity CTL responses that can be used in anti-tumor and anti-viral vaccine settings.

Antigen‐loaded ER microsomes from APC induce potent immune responses against viral infection

Although matured DC are capable of inducing effective primary and secondary immune responses in vivo, it is difficult to control the maturation and antigen loading in vitro. In this study, we show that ER-enriched microsomal membranes (microsomes) isolated from DC contain more peptide-receptive MHC I and II molecules than, and a similar level of costimulatory molecules to, their parental DC. After loading with defined antigenic peptides, the microsomes deliver antigenic peptide-MHC complexes (pMHC) to both CD4 and CD8 T cells effectively in vivo. The peptide-loaded microsomes accumulate in peripheral lymphoid organs and induce stronger immune responses than peptide-pulsed DC. The microsomal vaccines protect against acute viral infection. Our data demonstrate that peptide-MHC complexes armed microsomes from DC can be an important alternative to DC-based vaccines for protection from viral infection.

HLA typing demands for peptide-based anti-cancer vaccine.

Immunological treatment of cancer has made some very promising advances during the last years. Anti-cancer vaccination using peptides or peptide-pulsed dendritic cells and adoptive transfer of in vitro generated, epitope-specific T cells depend on a well-fitting interaction of HLA molecule and epitope. Accurate HLA-typing is a key factor for successful anti-cancer vaccination. No comprehensive data and no suggestion exist on the HLA-typing in this setting. We performed a systematic review of PubMed analyzing HLA-typing data in cancer vaccination trials over the last 4 years (2004-2007). Then, using the SYFPEITHI database, we calculated the peptide binding prediction of the eight most often used HLA-A*0201 binding epitopes. Finally, high-resolution typing [by sequence-specific primers (SSP)] data of a HLA-A*02 or HLA-A*24 positive population in Berlin, Germany, were analyzed. Forty-five cancer vaccination trials with 764 patients were included. Eighteen studies were performed in the USA, 13 in Europe, 12 in Asia (mainly Japan), and two in Australia. Most common diseases targeted were melanoma, prostate cancer, colorectal cancer, renal cell cancer, and breast cancer. The trials tested protocols using peptide plus adjuvants without DC or protocols using peptide-pulsed DC. In 38 trials (84%) HLA-A2 positive patients were vaccinated, in 11 studies (24%) HLA-A24 positive patients were vaccinated. Nineteen studies with 291 patients (38%) presented the HLA type as four-digit code (high-resolution), 26 studies with 473 patients (62%) presented the HLA-type in a low-resolution code. The method of HLA determination was given in six out of 45 trials (13%). Using the SYFPEITHI database we calculated the peptide binding prediction of the eight most often used HLA-A*0201 binding tumor antigen-derived epitopes for binding to HLA-A*0203. While the epitopes had a binding score of 17-28 for HLA-A*0201, the score for binding to HLA-A*0203 was zero in seven out of eight tested peptides. Only for one peptide the score was eight. Finally, we analyzed high-resolution data of HLA-A*02 and HLA-A*24 positive patients in Berlin, Germany. We found the HLA-A*0201 allele and HLA-A*2402 allele in 95%, respectively. HLA-A*0201 and HLA-A*2402 are most commonly used for peptide based vaccine in cancer. Data on HLA-typing given in the included cancer vaccine manuscripts are fractional. Only 13% report the method of HLA typing and most HLA types are given as low-resolution code. Looking at the binding of specific peptides to both the alleles, it is important to perform high-resolution typing. Further suggestions for immunogenetic laboratories and clinical tumor immunologists regarding HLA-typing for cancer vaccine trials and adoptive T cell transfer approaches are discussed.

Fetal/neonatal ethanol exposure irreversibly alters dendritic cell function in adult mice

Children born to alcoholic mothers are known to have increased incidence and severity of infections.We have developed a model of fetal/neonatal alcohol exposure (FAE) in which female mice are maintained on 10% EtOH in the drinking water during breeding and gestation, and then on 12% EtOH during lactation.Pups have successfully been weaned and showed no significant difference in weights from controls through 12 weeks of age.These pups have been studied at 20 d (weaning) and 12 weeks (young adult) for DC function.Splenic DC numbers did not change. FAE decreased the ability of these DC to stimulate allogeneic T cells,changes that persisted following cessation of EtOH exposure.In addition, antigen‐specific Foxp3+ regulatory T cells were induced following antigen presentation by FAE DC at a higher rate than by control DC. IL‐10 production by OTII T cells increased and IL‐2 production decreased following stimulation with OVA peptide‐pulsed FAE DC relative to control DC.These results indicate that mice born to EtOH‐consuming mothers have deficiencies in DC function that are persistent following cessation of EtOH exposure, and provide a mechanism, which could at least partially explain the increased incidence and severity of infection in children of alcoholic mothers.

Human Major Histocompatibility Complex (MHC) Class I Molecules with Disulfide Traps Secure Disease-related Antigenic Peptides and Exclude Competitor Peptides

The ongoing discovery of disease-associated epitopes detected by CD8 T cells greatly facilitates peptide-based vaccine approaches and the construction of multimeric soluble recombinant proteins (e.g. tetramers) for isolation and enumeration of antigen-specific CD8 T cells. Related to these outcomes of epitope discovery is the recent demonstration that MHC class I/peptide complexes can be expressed as single chain trimers (SCTs) with peptide, beta(2)m and heavy chain connected by linkers to form a single polypeptide chain. Studies using clinically relevant mouse models of human disease have shown that SCTs expressed by DNA vaccination are potent stimulators of cytotoxic T lymphocytes. Their vaccine efficacy has been attributed to the fact that SCTs contain a preprocessed and preloaded peptide that is stably displayed on the cell surface. Although SCTs of HLA class I/peptide complexes have been previously reported, they have not been characterized for biochemical stability or susceptibility to exogenous peptide binding. Here we demonstrate that human SCTs remain almost exclusively intact when expressed in cells and can incorporate a disulfide trap that dramatically excludes the binding of exogenous peptides. The mechanistic and practical applications of these findings for vaccine development and T cell isolation/enumeration are discussed.

Transplanted Donor CD8 TN Convert to TM in Severely Lymphopenic HCT Recipients and Are Distinguishable from Bona Fide Donor CD8 TM.

During the past several years, it has been appreciated that lymphoid cells expand under conditions of lymphopenia. T cell replete hematopoietic cell transplants (HCT) can therefore potentially provide both immediate and long-term immune function. Recent studies have reported that naïve CD8 cells (TN) undergo a phenotypic and functional conversion to memory CD8 cells (TN→TM) during homeostatic expansion. We proposed that transplanted antigen-specific CD8 TN can convert to memory-like cells post-HCT and subsequently provide immune function equivalent to that by infused memory CD8 cells (TM). A syngeneic murine HCT model was therefore utilized to investigate the homeostatic expansion, phenotype, and effector function of TM and TN populations following transplant. The CD8 TM population was generated in vitro from OT-I-Rag1−/− spleen cells cultured with rmIL-2 and OVA peptide followed by rmIL-15. Resultant OT-I TM (>99%) exhibited a central memory (CD44+/Ly6C+) phenotype. The OT-I TN population (CD44lo/Ly6Clo) was positively selected from OT-I mice using Miltenyi CD8 beads (>99% OT-I). Either 1.5×106 TM or TN, with 2×106 T cell-depleted B6 BM, were transplanted into 9.0-Gy conditioned syngeneic recipients. Both CFSE-labeled TM and TN proliferated almost immediately post-transplant. By day 7, TN had undergone slightly more divisions than TM. Between days 8 and 10, TN exhibited greater levels of BrudU incorporation compared to TM. Homeostatic expansion by CD8 TM lasted approximately 2 weeks whereas expansion of CD8 TN continued for another week and ultimately reached greater overall numbers. Cell surface analysis of transplanted populations demonstrated that CD8 TM retained their memory phenotype >7 months post-HCT. In contrast, the initially naive phenotype of transplanted TN began to resemble a memory phenotype by 14 days post-HCT and by 6 weeks exhibited such a phenotype. At this time, the transplanted TN→TM generated ex vivo lytic activity which was indistinguishable from transplanted TM. Interestingly, when challenged in vivo with peptide-pulsed DC >6 weeks post-HCT, OT-I cells in HCT recipients of TM and TN displayed differences in their ability to undergo antigen-driven expansion. The transplanted TM expanded following challenge, reaching maximal levels 8 days later, then contracted to a set-point higher than pre-challenge levels. In contrast, the transplanted TN→TM showed considerably less expansion post-challenge. These findings demonstrate that the conditions for the homeostatic expansion of donor TN and TM are present in recipients immediately following ablative conditioning and transplant. Moreover, expansion conditions appear to favor TN which reached higher homeostatic numbers. Although transplanted TN converted to a memory phenotype and demonstrated ex vivo lytic activity equivalent to transplanted TM, both memory populations did not undergo equivalent expansion following in vivo antigen exposure. These findings challenge the notion that antigen-independent TN→TM populations are functionally indistinguishable from bona fide TM cells. We propose that on a per-cell basis, transplanted TN→TM may function indistinguishably from transplanted TM. However, their initial period of rapid proliferation immediately following infusion may have diminished the ability of TN→TM to subsequently expand following in vivo antigen challenge.

An important role of Tyk2 in APC function of dendritic cells for priming CD8+ T cells producing IFN‐γ

Tyrosine kinase 2 (Tyk2) contributes to the signals triggered by IL-12 for IFN-gamma production by NK cells and T cells. We found in this study that Tyk2-deficient (-/-) mice showed increased susceptibility at the early stage after an i.p. infection with Listeria monocytogenes, accompanied by impaired IFN-gamma production. The numbers of both MHC class Ib (H2-M3)- or MHC class Ia (Kb)-restricted CD8+ T cells producing IFN-gamma and exhibiting cytotoxicity were significantly decreased in Tyk2-/- mice after infection with L. monocytogenes. Using an adoptive transfer system of OT-I cells expressing OVA(257-264)/Kb-specific TCR into Tyk2-/- mice followed by challenge with recombinant L. monocytogenes expressing OVA, we found that the defective Tyk2 signaling in the host environment was at least partially responsible for the impaired CD8+ T cytotoxic-1 (Tc1) cell responses in Tyk2-/- mice following the infection. Adoptive transfer with MHC class Ib- or MHC class Ia-binding peptide-pulsed BM-derived DC from Tyk2-/- mice induced lower levels of the Ag-specific CD8+ Tc1 cells producing IFN-gamma. These results suggest that Tyk2 signaling is also important for DC function in the induction of MHC class Ia- and class Ib-restricted CD8+ Tc1 cells following L. monocytogenes infection.

A cohort pilot study on therapeutic vaccination of advanced melanoma patients with dendritic cells loaded with multiple peptides or electroporated with mRNA

18011 Background: Therapeutic vaccination of melanoma patients with tumor specific antigenic peptides has been associated with increased frequency of anti-vaccine CTL precursor frequencies in peripheral blood and objective tumor regression in a low percentage of patients. Methods: We investigated the safety and activity of two autologous dendritic cell-based vaccines in 2 sequential cohorts of advanced melanoma patients. Patients first underwent a leucapheresis. Following enrichment in a semi-closed culture system using Cell Factories (Nunc), adherent cells were cultured in RPMI 1640 medium supplemented with 1% heat-inactivated autologous plasma, 500U/ml IL-4 and 1000 U/ml GM-CSF for 6 days and cryo-preserved. Upon thawing, DC were pulsed with 8 peptides or electroporated with synthetic mRNA encoding 7 antigens. Dendritic cell vaccines were administered by intradermal and subcutaneous biweekly injections for a total of 6 times (= cycle 1). Thereafter vaccination was repeated every 6w until clinical deterioration or patient refusal. Frequencies of blood anti-vaccine CTL were estimated by in vitro restimulation in limiting dilution conditions followed by detection with tetramer assay. Results: Fifteen patients were recruited (9M/6F; median age was 49 y, range 28–81). Seven HLA-A2 patients were treated with peptide pulsed DC (cohort A) and 7 patients (4 HLA-A2 and 3 HLA-A1) with mRNA electroporated DC (cohort B). One patient was sequentially treated with both vaccines. Five patients in cohort A and 3 patients in cohort B completed the first treatment cycle of 6 bi-weekly vaccinations (cycle 1 is ongoing in 3 patients of cohort B). Toxicity was limited to grade 1 or 2 local skin reactions at the vaccine administration sites in all patients. In cohort B one patient qualified for stable disease and 2 other patients with progressive disease displayed regression of individual metastases. A &gt;10-fold increase in CTL precursor frequency was observed in 2 out of 3 patients of cohort A that have been analyzed at present. Conclusions: Vaccination of melanoma patients with peptide pulsed or mRNA electroporated autologous DC vaccines was found to be feasible and safe. Updated results will be presented at the meeting. [Table: see text]

Identification of an HLA‐A24‐Restricted OY‐TES‐1 Epitope Recognized by Cytotoxic T‐Cells

OY-TES-1 was identified as a human homologue of the mouse, guinea pig, and pig proacrosin binding protein sp32 precursor. Differential expression levels of OY-TES-1 mRNA between testis and other normal tissues, and its expression in cancers indicated that OY-TES-1 would be classified as a cancer/testis antigen and considered to be a candidate of target antigen for cancer immunotherapy. In this study, we showed identification of HLA-A24-binding OY-TES-1 peptide, TES(401-409) (KTPFVSPLL) recognized by CD8 T-cells. Purified CD8 T-cells from healthy donors stimulated in vitro with the peptide-pulsed autologous DC and PBMC produced IFNgamma in response to the peptide-pulsed PBMC and showed cytotoxicity against the peptide-pulsed autologous EBV-B specifically. Furthermore, cytotoxicity was also observed against an OY-TES-1 mRNA-expressing tumor line, LK79. The retention time of the fraction in HPLC of the acid eluate from LK79 cells that showed positive sensitization against autologous EBV-B cells in recognition by CD8 CTL was the same as that of the fraction of the TES(401-409) peptide itself, suggesting that the TES(401-409) was a naturally processed peptide on LK79.

MHC class I‐associated presentation of exogenous peptides is not only enhanced but also prolonged by linking with a C‐terminal Lys‐Asp‐Glu‐Leu endoplasmic reticulum retrieval signal

Vaccination with antigenic peptide-pulsed antigen-presenting cells (APC) represents an attractive approach for therapy for cancer and diseases caused by intracellular infections. It has been suggested that sufficient stable MHC/peptide complexes on the surface of APC might play an important role in the generation of antitumor and antiviral immunity in vivo. In this study, we observed that exogenous peptides that were artificially fused with an endoplasmic reticulum (ER) retrieval signal, a C-terminal Lys-Asp-Glu-Leu sequence, could be efficiently presented by intracellular MHC class I molecules in a TAP- and proteasome-independent, but brefeldin A-sensitive manner. The APC retained the capacity to display surface MHC/peptide complexes for a prolonged period. In addition, our results show that vaccination with DC bearing our fusion peptides induced greatly enhanced specific CTL response, and resulted in significant inhibition of tumor growth. Thus, the ER retrieval signal modification can be regarded as a novel method for targeting exogenous peptides into the intracellular MHC class I presentation pathway, and may improve the clinical utility of vaccines based on synthetic peptide pulsed DC.