CMV-specific CTLs Research Articles

Ocular Outcomes after Treatment of Cytomegalovirus Retinitis Using Adoptive Immunotherapy with Cytomegalovirus-Specific Cytotoxic T Lymphocytes

To describe ocular outcomes in eyes with cytomegalovirus (CMV) retinitis treated with adoptive immunotherapy using systemic administration of CMV-specific cytotoxic Tlymphocytes (CMV-specific CTLs). Retrospective cohort study. Patients with active CMV retinitis evaluated at a tertiary care academic center. Treatment of CMV retinitis with standard-of-care therapy (systemic or intravitreal antivirals) or CMV-specific CTLs (with or without concurrent standard-of-care therapies). The electronic medical record was reviewed to determine baseline characteristics, treatment course, and ocular outcomes, including best-corrected visual acuity (BCVA), treatments administered (CMV-specific CTLs, systemic antivirals, intravitreal antivirals), resolution of CMV retinitis, any occurrence of immune recovery uveitis, cystoid macular edema, retinal detachment, or a combination thereof. Seven patients (3 of whom had bilateral disease [n= 10 eyes]) were treated with CMV-specific CTLs, whereas 20 patients (6 of whom had bilateral disease [n= 26 eyes]) received standard-of-care treatment. Indications for CMV-specific CTL therapy included persistent or progressive CMV retinitis (71.4% of patients); CMV UL54 or UL97 antiviral resistance mutations (42.9%); side effects or toxicity from antiviral agents (57.1%); patient intolerance to longstanding, frequent antiviral therapy for persistent retinitis (28.6%); or a combination thereof. Two patients (28.6%; 4 eyes [40%]) received CMV-specific CTL therapy without concurrent systemic or intravitreal antiviral therapy for active CMV retinitis, whereas 5 patients (71.4%; 6 eyes [60%]) continued to receive concurrent antiviral therapies. Resolution of CMV retinitis was achieved in 9 eyes (90%) treated with CMV-specific CTLs, with BCVA stabilizing (4 eyes [40%]) or improving (4 eyes [40%]) in 80% of eyes over an average follow-up of 33.4 months. Rates of immune recovery uveitis, new-onset cystoid macular edema, and retinal detachment were 0%, 10% (1 eye), and 20% (2 eyes), respectively. These outcomes compared favorably with a nonrandomized cohort of eyes treated with standard-of-care therapy alone, despite potentially worse baseline characteristics. CMV-specific CTL therapy may represent a novel monotherapy or adjunctive therapy, or both, for CMV retinitis, especially in eyes that are resistant, refractory, or intolerant of standard-of-care antiviral therapies. More generally, adoptive cell transfer and adoptive immunotherapy may have a role in refractory CMV retinitis. Larger prospective, randomized trials are necessary.

Antibody-mediated Recruitment of Viral-specific Effector Cells via Recombinant Antibody-MHC T cells Engagers

Abstract Our lab has developed a novel T cell engager (TCE) whereby a recombinant antibody fragment specific for a tumor-associated antigen (TAA) of choice is genetically fused to HLA-A2/peptide complex that presents a viral T cell epitope. The HLA-A2/peptide complex is designed to engage the TCR of a CD8+ T cell while the scFV or Fab redirects specificity towards a TAA, thus recruiting potent antiviral memory CTLs to attack tumor cells. We used the highly immunogenic cytomegalovirus (CMV) pp65-derived peptide as a target peptide for CTLs, taking into consideration the CMV prevalence of seropositive humans. Mesothelin was used as a TAA. We monitored the binding of TCEs to target cells demonstrating that their binding is dependent exclusively on the interaction of the targeting domain (scFv/Fab) of the fusion molecule with mesothelin, and is dose-dependent. Potency of TCEs was determined in vitro using co-cultures of CMV-specific CTLs and mesothelin-expressing human tumor cell lines. The TCEs specifically and potently mediated the killing of mesothelin-positive cells in TCE-dose-dependent matter. Moreover, a significant increase in CD25 expression and dose-dependent secretion of IL2 and IFNγ were observed in CTLs exposed to mesothelin-positive cells. The results represent a powerful new approach for immunotherapy. The MHC-based TCEs can mediate specific and efficient recruitment of viral-specific CTLs to kill tumor cells, adding an approach by which specific population of T cells can be engaged and recruited to the tumor site in a controllable manner in contrast to current CD3-based T cells engagers that cannot selectively target the specificity of interest. Such treatments can be very potent but with decreased toxicities involved.

Clonal Dynamics and Publicness of CMV-Specific TCR Repertoire after Allogeneic Stem Cell Transplantation

[Background] Cytomegalovirus (CMV) disease is a major complication after allogeneic stem cell transplantation (SCT). However, mechanisms of adoptive immunity against CMV are not fully elucidated. Recently, high-throughput next-generation sequencing (NGS) technology made it possible to shed light on the detailed and comprehensive landscape of T-cell receptor (TCR) repertoire. In this study, we analyzed TCR repertoire of CMV-specific cytotoxic T-cells (CMV-CTLs) in patients who suffered from CMV reactivation after SCT to clarify the diversity and dynamics of CMV-specific T-cell immunity. [Methods] We sequentially collected peripheral blood mononuclear cells from patients with HLA-A*24:02 who received SCT in our institution. Samples were collected weekly or every two weeks from their neutrophil engraftment until approximately 100 days after SCT. CMV reactivation was evaluated weekly with CMV antigenemia test. CD8 and CMV pp65 tetramer positive cells were sorted and unbiased next-generation sequencing-based analyses of TRBV/TRBJ gene segments were performed at the timing of CMV reactivation in 16 patients, and TRA gene segments were also analyzed in 10 patients. In addition, TCR beta repertoires after 2-4 weeks were analyzed in 12 patients. In the 12 patients, the dynamics of TCR repertoire diversity and proportional changes of each clone were assessed. We evaluated the diversity by Shannon-Weber index, and we defined TCR beta clonotypes found in two or more patients using the same TRBV/TRBJ gene segments and CDR3 amino acid sequences as public. This study was approved by the ethics committee of Tokyo Metropolitan Komagome Hospital. [Results] Among 16 patients, 11 received bone marrow, 3 received peripheral blood stem cells, and 2 received cord blood transplant. Underlying diseases were acute myeloid leukemia in 7, acute lymphoblastic leukemia in 7, and myelodysplastic syndromes in 2 patients. Median age at SCT was 50 years old (range: 20-71). Median duration from SCT to first CMV reactivation was 39 days (range: 16-55) and 7 patients (43.8%) were administered systemic corticosteroid at the time of reactivation (prednisolone 10-30mg/day). Median peripheral blood CMV-CTLs count at that time was 29.47/uL (range: 4.65-229.6). In most patients TCR beta repertoire of CMV-specific CTLs when CMV reactivated was highly skewed (median Shannon-Weber index was 1.44 [range: 0.542-3.164]). TCR alpha and beta were analyzed together in 10 patients and their diversity correlated well (p<0.001). Interestingly, 12 of 16 patients (75.0%) had at least one public TCR and, in all 12 patients with public TCR, the most frequent TCRs were public. Diversity of TCR repertoire was significantly lower in patients with public TCR compared with those in patients without (p=0.039). There was no obvious association between presence of public TCR and multiple events of CMV reactivation (p=0.57). When we evaluated clonal transition in 12 patients whose TCR beta repertoires were analyzed sequentially, the public clonotypes were continuously detected in 10 of 11 patients with public TCR and remained dominant in 9, while in one patient frequency of the most frequent public clone declined massively (from 50.25% among assigned reads to 6.63%). In addition, there were two patterns of subsequent clonal behavior in TCR repertoire of CMV-CTLs. In 10 patients, prevailing clones persisted and TCR repertoires of CMV-CTLs remained oligoclonal (Figure A). However, in other two patients, TCR repertoires of CMV-CTLs became more diverse (Shannon-Weber index at the time of reactivation and a few weeks after was 0.558/2.958 in one patient and 1.471/3.86 in another), major clones lost, and new private clones appeared afterwards (Figure B). Polyclonal pattern (TCR repertoire of CMV-CTLs was diverse at the time of reactivation or after a few weeks) was detected in 3 patients out of 16 patients, and the pattern was exclusively seen in patients who were administered corticosteroid when CMV reactivated (42.9% vs 0.0%, p=0.063). [Conclusion] TCR repertoire of CMV-CTLs at the time of CMV reactivation after SCT is highly oligoclonal and frequently shared among different patients, but can dynamically change in a short period in some patients. Functional analyses of the dominant TCRs to understand their reactivity against CMV epitope and elucidation of the clinical significance and developmental mechanisms of clone shift are strongly warranted. Figure Disclosures Kitaura: Repertoire Genesis Inc.: Employment. Suzuki:Repertoire Genesis Inc.: Equity Ownership.

Characterization of High-Avidity Cytomegalovirus-Specific T Cells with Differential Tetramer Binding Coappearing after Allogeneic Stem Cell Transplantation.

CMV reactivation is a major complication after allogeneic stem cell transplantation (SCT). Immune reconstitution of CMV-specific CTLs (CMV-CTLs) is essential for virus control. During CMV-CTL monitoring using mutated HLA/CMV tetramers selectively detecting high-avidity T cells, we observed coappearance of CMV-CTLs with low (CMV tetlow CTLs) and high tetramer binding (CMV tethigh CTLs) in 53/115 CMV IgG+ patients stem cell transplanted from CMV IgG+ donors. However, the relevance of these coappearing differentially tetramer binding ("dual") CMV-CTLs was unclear. In this study, we investigated the kinetics, properties, and clinical impact of coappearing CMV tetlow and tethigh CTLs after allogeneic SCT. Patients with dual CMV-CTLs had more CMV tethigh than tetlow CTLs. Chimerism analysis of isolated CMV tetlow and tethigh CTLs revealed their exclusive donor origin. CMV tetlow and tethigh CTLs had an identical effector memory CD45RA-CCR7- and CD45RA+CCR7- T cell distribution, equal differentiation, senescence, and exhaustion marker expression and were negative for regulatory CD8+ T cell markers. Isolated CMV tetlow and tethigh CTLs were equally sensitive to CMV peptides in IFN-γ release and cytotoxicity assays. However, CMV tethigh CTLs proliferated more in response to low CMV peptide concentrations than tetlow CTLs. TCR repertoire analysis revealed that CMV tetlow and tethigh CTLs use different TCRs. Finally, dual CMV-CTLs were not associated with CMV antigenemia. In conclusion, these data show for the first time, to our knowledge, that both CMV tetlow and tethigh CTLs are functional effector T cells differing by proliferation, numbers in peripheral blood, and probably by their precursors without increasing the CMV reactivation risk after allogeneic SCT.

Possible Impact of Cytomegalovirus-Specific CD8+ T Cells on Immune Reconstitution and Conversion to Complete Donor Chimerism after Allogeneic Stem Cell Transplantation

Complete donor chimerism is strongly associated with complete remission after allogeneic stem cell transplantation (allo-SCT) in patients with hematologic malignancies. Donor-derived allo-immune responses eliminate the residual host hematopoiesis and thereby mediate the conversion to complete donor chimerism. Recently, cytomegalovirus (CMV) reactivation was described to enhance overall T cell reconstitution, to increase graft-versus-host disease incidence, and to reduce the leukemia relapse risk. However, the link between CMV and allo-immune responses is still unclear. Here, we studied the relationship between CMV-specific immunity, overall T cell reconstitution, and residual host chimerism in 106 CMV-seropositive patients transplanted after reduced-intensity conditioning including antithymocyte globulin. In accordance with previous reports, the recovery of CMV-specific cytotoxic T cells (CMV-CTLs) was more frequent in CMV-seropositive recipients (R) transplanted from CMV-seropositive than from seronegative donors (D). However, once CMV-CTLs were detectable, the reconstitution of CMV-specific CTLs was comparable in CMV R+/D– and R+/D+ patients. CD3+ and CD8+ T cell reconstitution was significantly faster in patients with CMV-CTLs than in patients without CMV-CTLs both in the CMV R+/D– and R+/D+ setting. Moreover, CMV-CTL numbers correlated with CD3+ and CD8+ T cell numbers in both settings. Finally, presence of CMV-CTLs was associated with low host chimerism levels 3 months after allo-SCT. In conclusion, our data provide a first indication that CMV-CTLs in CMV-seropositive patients might trigger the reconstitution of T cells and allo-immune responses reflected by the conversion to complete donor chimerism.

HTLV-1 Tax-Specific CTL Epitope-Pulsed Dendritic Cell Therapy Reduces Proviral Load in Infected Rats with Immune Tolerance against Tax.

Adult T cell leukemia/lymphoma (ATL), a CD4+ T cell malignancy with a poor prognosis, is caused by human T cell leukemia virus type 1 (HTLV-1) infection. High proviral load (PVL) is a risk factor for the progression to ATL. We previously reported that some asymptomatic carriers had severely reduced functions of CTLs against HTLV-1 Tax, the major target Ag. Furthermore, the CTL responses tended to be inversely correlated with PVL, suggesting that weak HTLV-1-specific CTL responses may be involved in the elevation of PVL. Our previous animal studies indicated that oral HTLV-1 infection, the major route of infection, caused persistent infection with higher PVL in rats compared with other routes. In this study, we found that Tax-specific CD8+ T cells were present, but not functional, in orally infected rats as observed in some human asymptomatic carriers. Even in the infected rats with immune unresponsiveness against Tax, Tax-specific CTL epitope-pulsed dendritic cell (DC) therapy reduced the PVL and induced Tax-specific CD8+ T cells capable of proliferating and producing IFN-γ. Furthermore, we found that monocyte-derived DCs from most infected individuals still had the capacity to stimulate CMV-specific autologous CTLs in vitro, indicating that DC therapy may be applicable to most infected individuals. These data suggest that peptide-pulsed DC immunotherapy will be useful to induce functional HTLV-1-specific CTLs and decrease PVL in infected individuals with high PVL and impaired HTLV-1-specific CTL responses, thereby reducing the risk of the development of ATL.

Most Closely HLA-Matched Third Party Allogeneic CMV Specific T-Cells Generated Using the Cytokine Capture System for the Treatment of CMV Infections after Allogeneic Stem Cell Transplantation

Adoptive immunotherapy with ex vivo expanded virus-specific T-cell lines from transplant donors has emerged as a potentially curative approach for the treatment of drug-refractory viral infections complicating allogeneic hematopoietic cell transplants (HSCT). In these studies, viral-specific T cells are generated in compliance with current good manufacturing practice (cGMP) following repeated rounds of antigen-driven stimulation over a number of weeks. Such protocols are logistically and technically demanding, requiring access to specialized GMP units and are difficult to apply in urgent clinical settings.The cytokine-capture (gamma-catch) procedure is a fast and simple method that takes <30 hours to isolate polyclonal IFN-gamma-secreting CD8+ and CD4+ viral-specific T cells and can be performed outside a dedicated GMP facility. The cells are directly transfused to the patient without the requirement for ex vivo expansion. The use of allogeneic third party T-cell donors offers the option of selecting donors with high viral-specific T cell precursor frequencies and may serve as an alternative for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor.Here, we report the interim results of a Phase 2 study in 11 patients with persistent CMV infection after HSCT, despite optimum anti-viral therapy for at least 14 days. We established a bank of cryopreserved peripheral blood mononuclear cells (PBMCs) from 36 healthy donors. Eligible patients were treated with small numbers of CMV-specific T cells selected by cytokine capture from the most closely HLA-matched third party donor. Manufacture of third party derived CMV specific CTLs was performed at the MDACC Core Facility. Cryopreserved PBMCs were thawed, washed and diluted at 1 × 109 cells in 100 mL MACS GMP TexMACS Reagent (Miltenyi Biotec) for approximately 16 hours at 37°C. The product was stimulated with MACS® GMP Peptivator® HCMV pp65 for approximately 4 hours at 37°C, CO2. Enrichment of cytokine-secreting cells was performed using the cytokine secretion system and the CliniMACS device for immunomagnetic separation. CMV-specific T cells were transfused directly after isolation without any further in vitro expansion.To date, 11 HSCT recipients have received CMV-specific T-cells generated by gamma-catch. Of 11 patients, 2 had failed to respond to foscarnet, 12 to ganciclovir and 8 to both agents. One patient who had a complete response (CR) to his first CMV-specific T-cell infusion received a second infusion 166 days after the first dose for the treatment of CMV retinitis. The median number of infused CD3+IFN-gamma+ T-cells was 2598/kg (range, 61/kg-12717/kg). There were no immediate infusion-related adverse events. CMV-specific CTLs controlled infection in 9 of 11 evaluable infusions; 6 CR and 3 partial response (PR). The overall response and CR rates at 4 weeks were 77% (95%CI; 51%, 95%) and 54.5% (95%CI; 25.4%, 78.6%). Interestingly, the success of adoptive T-cell transfer was not related to the dose of infused T-cells. The median ratio of CD8+ IFN-gamma+ to CD4+ IFN-gamma+ T-cells was 2.7 (range, 0.17-13.4). Among 6 CR patients, 4 had a second episode of CMV infection at a median of 71 days (range, 61-355 days) after the first CTL infusion. Following infusion, there was expansion of both CD8+ and/or CD4+ CMV-specific T-cells, which persisted for a minimum of 1 month following infusion. Recurrence of CMV PCR after CTL infusion was associated with loss of CMV CTL response.Despite the use of third-party donors with HLA disparity, only one patient developed de novo graft versus host disease (GVHD) of the gastrointestinal tract (grade 1) 3 months after CTL infusion. Three patients with a history of acute GVHD prior to CTL infusion had flares 5-6 months after CTL infusions and responded to systemic steroid use.In summary, these interim results demonstrate the safety, feasibility and efficacy of immunotherapy with the most closely HLA-matched CMV specific CTLs generated by cytokine capture for the treatment of drug resistant CMV infection after HSCT patients. The clinical trial continues to enroll and an updated report of the study will be presented at the meeting. DisclosuresCiurea:Spectrum Pharmaceuticals: Other: Advisory Board; Cyto-Sen Therapeutics: Equity Ownership.

Recruitment of Oligoclonal Viral-Specific T cells to Kill Human Tumor Cells Using Single-Chain Antibody-Peptide-HLA Fusion Molecules.

Tumor progression is often associated with the development of diverse immune escape mechanisms. One of the main tumor escape mechanism is HLA loss, in which human solid tumors exhibit alterations in HLA expression. Moreover, tumors that present immunogenic peptides via class I MHC molecules are not susceptible to CTL-mediated lysis, because of the relatively low potency of the tumor-specific CLTs. Here, we present a novel cancer immunotherapy approach that overcomes these problems by using the high affinity and specificity of antitumor antibodies to recruit potent antiviral memory CTLs to attack tumor cells. We constructed a recombinant molecule by genetic fusion of a cytomegalovirus (CMV)-derived peptide pp65 (NLVPMVATV) to scHLA-A2 molecules that were genetically fused to a single-chain Fv Ab fragment specific for the tumor cell surface antigen mesothelin. This fully covalent fusion molecule was expressed in E. coli as inclusion bodies and refolded in vitro. The fusion molecules could specifically bind mesothelin-expressing cells and mediate their lysis by NLVPMVATV-specific HLA-A2-restricted human CTLs. More importantly, these molecules exhibited very potent antitumor activity in vivo in a nude mouse model bearing preestablished human tumor xenografts that were adoptively transferred along with human memory CTLs. These results represent a novel and powerful approach to immunotherapy for solid tumors, as demonstrated by the ability of the CMV-scHLA-A2-SS1(scFv) fusion molecule to mediate specific and efficient recruitment of CMV-specific CTLs to kill tumor cells.

Assessment of the effector function of CMV-specific CTLs isolated using MHC-multimers from granulocyte-colony stimulating factor mobilized peripheral blood.

BackgroundAdoptive transfer of CMV-specific T cells has shown promising results in preventing pathological effects caused by opportunistic CMV infection in immunocompromised patients following allogeneic hematopoietic stem cell transplantation. The majority of studies have used steady-state leukapheresis for CMV-reactive product manufacture, a collection obtained prior to or months after G-CSF mobilization, but the procurement of this additional sample is often not available in the unrelated donor setting. If the cellular product for adoptive immunotherapy could be generated from the same G-CSF mobilized collection, the problems associated with the additional harvest could be overcome. Despite the tolerogenic effects associated with G-CSF mobilization, recent studies described that CMV-primed T cells generated from mobilized donors remain functional.MethodsMHC-multimers are potent tools that allow the rapid production of antigen-specific CTLs. Therefore, in the present study we have assessed the feasibility and efficacy of CMV-specific CTL manufacture from G-CSF mobilized apheresis using MHC-multimers.ResultsCMV-specific CTLs can be efficiently isolated from G-CSF mobilized samples with Streptamers and are able to express activation markers and produce cytokines in response to antigenic stimulation. However, this anti-viral functionality is moderately reduced when compared to non-mobilized products.ConclusionsThe translation of Streptamer technology for the isolation of anti-viral CTLs from G-CSF mobilized PBMCs into clinical practice would widen the number of patients that could benefit from this therapeutic strategy, although our results need to be taken into consideration before the infusion of antigen-specific T cells obtained from G-CSF mobilized samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0515-z) contains supplementary material, which is available to authorized users.

Epitopes of CMVpp65 Co-Presented by Multiple Allelic Variants of HLA Class-I Antigens: Implications for Adoptive Immunotherapy for CMV Using Third Party Donor – Derived CMV Specific CTLs

Epitopes of CMVpp65 Co-Presented by Multiple Allelic Variants of HLA Class-I Antigens: Implications for Adoptive Immunotherapy for CMV Using Third Party Donor – Derived CMV Specific CTLs

A Phase I Dose Escalation Trial of Donor T Cells Sensitized with Pentadecapeptides of the CMV-pp65 Protein for the Treatment of CMV Infections Following Allogeneic Hematopoietic Stem Cell Transplants.

Abstract 2262Poster Board II-239We have developed a novel alternative method of generating donor-derived T-cell lines specific for CMV-pp65 peptides for use in adoptive immunotherapy. In this approach, 138 pentadecapeptides (15-mers), with 11 amino acid overlaps spanning the entire 561 amino acid sequence of the CMV protein pp65 were synthesized and manufactured under conditions ensuring their purity and sterility for application in clinical trials. A pool of the 138 pentadecapeptides is loaded onto monocyte-derived dendritic cells derived from seropositive donors and the same donor's T cells are sensitized under GMP conditions. In our preclinical studies, we have been able to generate CMV-pp65 specific T-cell lines from each seropositive donor tested, irrespective of HLA genotype. During the culture period of 21-28 days, populations of T cells specific for CMV-pp65 selectively expanded 200-300 fold while T cells reactive against major or minor alloantigens were depleted. Currently, 9 patients (pts) with persistent/refractory CMV antigenemia, one pt with CMV pneumonia diagnosed four weeks after treatment, have been treated using cells manufactured by this method on a clinical trial approved by the FDA. One pt was treated on a single patient IND prior to final FDA approval; 8 pts were enrolled onto the clinical trial - 3 pts at a T cell dose of 5×105/kg; 3 pts at dose level II of 1×106/kg and 2 pts at dose level III of 2×106/kg. We report here the results of the first 7 pts, (the 2 latter pts are too early in treatment to be included at time of abstract admission). CMV specific CTLs were generated from HLA-identical unrelated donors (3 pts) or from HLA-identical siblings (6 pts) for 2 pts who underwent non-myeloablative conventional and 7 pts who received myeloablative T-cell depleted allogeneic transplants. Pts were eligible if they had persistent CMV antigenemia despite treatment with antiviral drugs or had toxicities precluding further treatment with antiviral agents. Prior to infusion, the T cells were tested to be CMV specific by cytotoxicity, intracellular Interferon gamma (IFN-g) production and MHC-tetramer staining (if available). Cells were also assayed to establish lack of alloreactivity, microbiological sterility and low endotoxin levels. The cytotoxic T cells demonstrated cytolytic activity against peptide-loaded autologous PHA blasts, but exhibited no cytotoxicity against non-pulsed HLA-matched or pulsed HLA-mismatched target cells. The HLA-restriction and the specific pp65-derived epitopes of the CMV-specific T-cells were characterized prior to the infusion. CMV-specific frequencies of the CD8+ cells measured by intracellular IFN-g or MHC tetramers ranged from 2 -70%. Post infusion, an increase in the absolute lymphocyte count correlated with an increase in CMV-specific T-cell frequencies to levels as high as 12% of CD8+ cells. These persisted for at least 7 months (10% of CD8+ cells) following the infusion. Notably, the same pp65-derived epitopes and their HLA-restrictions, which characterized the pre-infusion CTLs were detected in the pt specimens post infusion. Freshly isolated T cells from the blood of HLA-A*0201 positive pts obtained 3 months post infusion showed significant lysis of CMV infected and peptide-pulsed MRC-5 fibroblasts, while non infected/pulsed fibroblasts and pulsed HLA-mismatched targets were not lysed. Three of the treated pts exhibited an HLA-A*0201 as well as HLA-B*0701 allele. In all three pts, epitope-specific T cells for the HLA-A*0201 restricted NLVPMVATV peptide and the B*0701 restricted RPHERNGFTV peptide were detected and monitored in pre and post infusion T-cell populations. All 7 pts who were treated for persistent CMV antigenemia tolerated treatment well. None developed early signs or symptoms of GvHD at the dose levels tested. Six of the 7 pts cleared CMV viremia by 2-4 weeks following the T-cell infusions. One of the pts died six weeks after the CTL infusion of respiratory failure despite clearing CMV from blood and bronchial aspirates. The one pt who remained viremic following the CTL infusion continued on oral Valgancyclovir and subsequently became CMV antigen negative. The clearance of CMV antigenemia was preceded by an initial spike of CMV antigenemia and/or CMV PCR in all pts. These results from the first 7 pts on this trial indicate that donor T cells sensitized with this pool of synthetic overlapping CMV pp65 15-mers are safe and clear CMV viremia resistant to standard therapy. Disclosures:No relevant conflicts of interest to declare.

A Modified Peptide Stimulation Method for Efficient Amplification of Cytomegalovirus (CMV)-Specific CTLs

CMV-specific immunity is essential for control of human cytomegalovirus (HCMV) infection. Stem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the ex vivo production of cells for adoptive transfer is labor intensive and expensive. We report here a modified peptide stimulation method using CMV-specific epitope peptides to stimulate PBMCs for generation of CMV-specific CTLs. This method permits efficient amplification of CMV-specific CTLs and provides a large number of cells for FACS analysis from a single blood sample. Significantly, it achieves high frequencies of tetramer staining of CD8+ T cells allowing the data of different individuals to be easily compared and sequentially evaluated. Thus, this approach expands and selects HLA-restricted CMV-pp65-reactive T-cell lines of high specificity for potential adoptive immunotherapy.

Expansion of cytomegalovirus-specific cytotoxic T lymphocytes after peptides stimulation

目的 研制一种扩增特异细胞毒性T淋巴细胞频数的方法.方法 用不同浓度的巨细胞病毒(CMV)特异的表位肽pp65刺激人外周血单个核细胞(PBMCs)使CMV特异CTLs在PBMCs中的百分比增高.用四聚体-PE和CD8-FITC双标记PBMCs中的CMV特异CTLs,然后用流式细胞仪分析.结果 这个方法能有效扩增CMV特异CTLs,使在外周血中占淋巴细胞百分比不到1%的CMV特异CTLs迅速增加到接近20%,占CD+8 T淋巴细胞的40%以上,并能从单份血样中获得大量特异CTLs用于流式分析.结论 肽刺激培养法简便、易操作,能有效扩增CMV特异CTLs,使不同人的数据更容易对比和连续的评价.该方法也为过继性免疫疗法治疗巨细胞病毒疾病打下了基础。

Identification of HLA-A33-restricted CMV pp65 epitopes as common targets for CD8 + CMV-specific cytotoxic T lymphocytes

To identify an immunodominant HLA-A33-restricted epitope within the CMV matrix phosphoprotein 65 (pp65) that could be used to elicit CMV-specific CTLs. A computer algorithm was used to identify pp65 peptides that were expected to bind to HLA-A33. The peptides were screened for their ability to reactivate memory T lymphocytes from CMV-seropositive HLA-A33 donors by using quantitative real-time RT-PCR. The most promising peptides were then tested for their ability to expand a CD8(+) population of HLA-A33 CTLs that produced interferon-gamma (IFN-gamma) and were cytotoxic to either peptide-loaded or CMV-infected target cells. Sixteen out of 250 peptides were selected using a computer algorithm and peptide stimulation by 8 out of the 16 induced a significant quantity of IFN-gamma mRNA transcript from CMV-seropositive HLA-A33 peripheral blood mononuclear cells. All the eight peptides induced consistent T-cell reactivation. One specifically, the peptide pp65(91-100) (SVNVHNPTGR), proved to be more active. T cells in vitro sensitized for 2 or 3 weeks with pp65(91-100) were cytotoxic to both HLA-A33 peptide-loaded and CMV-infected target cells. CMV pp65(91-100) is a new HLA-A33-restricted peptide that broadens the list of antigenic determinants that can be used for CMV adoptive immunotherapy.

Analysis of memory T lymphocyte activity following stimulation with overlapping HLA-A*2402, A*0101 and Cw*0402 restricted CMV pp65 peptides

The continuous efforts aimed at the identification of new immune epitopes across the MHC system has led to the discovery that more than one peptide may be restricted to the same HLA antigen and function as an immune determinant for that association. The aim of this study was to compare the ability of two overlapping peptides, the nonamer (9-mer) cytomegalovirus (CMV) pp65341–349 (QYDPVAALF) and the decamer (10-mer) CMV pp65341–350 (QYDPVAALFF), and the esadecamer (16-mer) peptide containing both the 9-mer and 10-mer sequences, CMV pp65340–355 (RQYDPVAALFFFDIDL), to stimulate and maintain over time a T cell immune reactivation by HLA-A*2402, A*0101, and Cw*0402 cells from CMV-seropositive subjects. The 9-mer, 10-mer, and 16-mer peptides effectively stimulated CTLs from HLA-A*2402, HLA-A*0101, and HLA-Cw*0402 CMV seropositive donors. This data confirms that both the 9-mer and the 10-mer peptides are promiscuous and are not restricted to a single HLA antigen. CMV pp65341–349 and CMV pp65341–350 have the ability to produce CMV-specific CTLs in subjects with several different HLA types, presenting a practical advantage over other peptides that are restricted only to a single HLA antigen, and thus being optimal for CMV adoptive immune therapy. Moreover, since the 16-mer peptide encompasses both the 9-mer and 10-mer peptides, it may be better than either of these peptides for CMV adoptive immune therapy.

The Use of Algorithms with a Novel Score Standardization Method for the Selection of Candidate Immunogenic HLA-Specific Peptides Led to the Identification of New Immunogenic HLA-A*0201 Cytomegalovirus Epitopes.

The identification of HLA class I restricted epitopes within immunogenic proteins that are capable of stimulating memory T lymphocytes is an important step for both clinical vaccine and adoptive immunotherapy. Despite the strong immunogenicity demonstrated by specific HLA restricted peptides, the peptide's affinity to a specific HLA antigen may vary among individuals. Therefore, it is important to identify more than one immune determinant for each HLA antigen of interest. Two computer algorithms, one by Parker and another by Rammensee, are widely used in immunogenetic studies in order to predict which peptides bind best to specific HLA molecules. The use of these algorithms represents the first step in screening proteins. Parker's algorithm ranks potential peptide according to the predictive half-time disassociation of peptide/MHC complexes. Rammensee's algorithm ranks the peptides based on the presence of primary and secondary HLA-binding anchor residues. Starting with the sequence of Cytomegalovirus (CMV) phosphorylated matrix protein 65 (pp65; SwissProt ID: P06725), we derived from each algorithm a list of 250 nonamer (9-mer) peptides predicted to bind to the HLA-A*0201 allele. Standardizing the two groups of scores, corresponding to the endpoints of the stated interval, by the following formula: [(peptide score - mean of scores)/ standard deviation of scores], we obtained pure values that are independent of the unit of measurement, therefore homogeneous and thus comparable. This allowed us to link each peptide to a pair of values, one for each algorithm. The values derived from standardized Parker scores (X axis) were plotted against the values derived from standardized Rammensee scores (Y axis). We represented final results by a graph. All peptides having positive values with both algorithms and included in the first quadrant of the dot-scatter graph (double positive quadrant (+/+)) were likely to be highly immunogenic epitopes. Those peptides were included in a list of potential peptides to screen. In the presence of homoschedasticity, we selected only peptides showing mean values greater than 1 (among those having both the mean value greater than 0 and the standard deviation value greater than 1). Nine out of the 250 peptides were thus chosen within the CMV pp65 and their immunogenicity analyzed by ex vivo stimulation of PBMC and measured at the transcriptional level using the production of IFN-γ mRNA. Four out of nine peptides stimulated PBMCs from all HLA-A*0201 restricted CMV-seropositive subjects tested (p values: peptide #5, 0.011032; peptide #7, 0.015225; peptide #8, 0.025221; and peptide #9, 0.007302). The intracellular IFN-γ protein production by ex vivo peptide stimulation ranged from 0.14 to 0.62% compared to 0.03% for non stimulated cells. Moreover, each of the four selected peptide, either alone or in pool, was able to reactivate in vitro a strong immune T cell memory response, as measured by cytokine protein release and target cell lysis. The identification of multiple HLA class I restricted epitopes will allow the simultaneous administration of peptides with the ability to produce CMV-specific CTLs in patients bearing the same HLA types. This represents an extraordinary advantage for CMV adoptive immune therapy or vaccination.

555. Langerhans Cells Modified by Gene Transfer of Full Length CMV pp65 Are More Potent Inducers of Cytolytic T Lymphocytes in a CD4-Dependent Manner Than Either CMV p495 Gene-Modified or p495 Peptide-Pulsed Langerhans Cells

The use of dendritic cells (DCs) for immunotherapy is often limited to defined Ags with known MHC restrictions. Investigators are pursuing a number of approaches to introduce whole Ag, however. These would increase the range of MHC alleles presenting immunogenic peptide Ags by allowing DCs to process and present epitopes tailored to their own MHC alleles. We addressed this issue by generating recall immune responses in vitro against human cytomegalovirus (HCMV) by seropositive donors. CD34+ HPCs were transduced twice on RetroNectin-coated plates by GalV-pseudotyped oncoretroviral vectors encoding either the full-length HCMV pp65 or the HLA-A*0201-restricted CMV pp65495-503 peptide (p495). Differentiation and maturation of CD34+ HPC-derived Langerhans-type dendritic cells (LCs) followed over the next 14 days. These pp65-transduced and p495-transduced LCs were compared with nontransduced but p495-pulsed LCs, for the generation of CMV-specific CTLs after two rounds of autologous T cell stimulation without additional cytokines. Stimulation by pp65-transduced LCs, p495-transduced LCs, and p495-pulsed LCs respectively increased p495/HLA-A*0201 tetramer positive T cells by 51.3±24.1, 33.8±20.9, and 10.4±10.7-fold (n=4 different donors, P<0.05). CMV specific CD8+ CTLs induced by pp65-transduced LCs, p495-transduced LCs, and p495-pulsed LCs achieved respective frequencies of 714.4±280.8, 582.6±218.6, and 231.0±102.5 spot-forming cells (SFC) per 105 input cells (n=4, P<0.01) against p495-pulsed A*0201 targets in ELISpot assays for IFN-g secretion. pp65-transduced LCs that could present both class I and II MHC-restricted viral epitopes also expanded IFN-g -secreting CD4+ T cells, whereas p495-transduced LCs did not. Stimulation by p495-pulsed-LCs was even associated with a decline in CD4+ T cell numbers. pp65-transduced LCs also stimulated greater lytic activity than did either p495-transduced LCs or p495-pulsed LCs, based on 51Cr release from p495-pulsed A*0201 targets (n=3, P<0.05). CD4+ T cells boosted the overall CTL response, insofar as CD4+ T cell depletion before each round of stimulation decreased the number of effector CD8+ T cells by 70% at d7 and 89% at d14. p495-transduced LCs and p495-pulsed LCs gave similar results. Finally, CMV tetramer positive cells express low levels of CD62L, CD45RA, CD27, and CCR7, consistent with an effector memory phenotype. DCs transduced with a retroviral vector encoding the full-length pp65 protein can thus expand potent CTL presumably directed against multiple epitopes in a CD4+ helper T cell-dependent manner. DCs transduced with retroviral vectors expressing full-length antigens in serum free media should therefore provide effective cell-based vaccines against viral pathogens and cancer.

An algorithm for evaluating human cytotoxic T lymphocyte responses to candidate AIDS vaccines.

Development of an effective vaccine against HIV-1 will likely require the induction of a broad array of immune responses, including virus-specific CTLs and neutralizing antibodies. One promising vaccine approach involves live recombinant canarypox (CP)-based vectors (ALVAC) containing multiple HIV-1 genes. In phase I clinical trials in HIV-1-seronegative volunteers, the cumulative rate of detection of HIV-1-specific CTLs has been as high as 60-70%. In the present study, the factors associated with CTL responsiveness were evaluated in a subset of vaccinees immunized with a CP vector expressing portions of the gag, pro, and env genes of HIV-1 (ALVAC-HIV). CTL responses were detected in one of seven examined. While the responding individual had both CD4+ and CD8+ CTLs directed at multiple HIV-1 antigens, this response was not detectable 1 year after the last vaccination. In-depth characterization of "CTL nonresponders" showed that nonresponsiveness was not associated with defects in antigen processing or presentation. A generalized defect in CTL responsiveness was ruled out by parallel assays to detect CMV-specific CTLs from these same volunteers. Furthermore, HIV-1-specific memory CTLs were not detectable by peptide stimulation or by a novel technique for flow cytometric visualization of Gag epitope-specific T lymphocytes while HIV-1-seropositive donors frequently had 0.1-3% of CD8+ cells stain positively for this epitope (SLYNTVATL). Taken together, these results suggest that the lack of detectable HIV-1 CTLs in these volunteers was not due to classic MHC-linked nonresponsiveness.