Aparna Krishnan

Longitudinal Assessment of Cytomegalovirus (CMV)—Specific Immune Responses in Liver Transplant Recipients at High Risk for Late CMV Disease

Cytomegalovirus (CMV)-seronegative recipients (R(-)) of a liver transplant from CMV-positive donors (D(+)) are at high risk for developing late CMV disease after discontinuation of antiviral prophylaxis. Levels of viremia and CMV-specific interferon (IFN)- gamma -producing CD4(+) and IFN- gamma -producing CD8(+) T cell responses were prospectively measured from discontinuation of antiviral prophylaxis until 1 year after transplantation in 17 consecutive D(+)/R(-) patients. CMV loads of >1000 copies/mL were strongly associated with CMV disease in the 6 symptomatic patients. Despite immunosuppression, broadly diverse T cells specific for CMV lysate or peptide libraries spanning pp65 and immediate early (IE) 1 immunodominant CMV antigens developed in all patients. A vigorous CD8(+) T cell response to pp65 and IE1 antigens characterized the D(+)/R(-) cohort. Unexpectedly, none of these responses were predictive of CMV disease or viremia. No significant lymphopenia or functional impairment of CMV-specific T cells was detected in the symptomatic patients, whose morbidity was resolved after antiviral treatment while measurable CMV immunity was maintained during the 1-year observation period.

Programmed death-1 expression in liver transplant recipients as a prognostic indicator of cytomegalovirus disease

Immunological parameters that distinguish solid-organ transplant (SOT) recipients at risk for life-threatening cytomegalovirus (CMV) disease are being actively pursued to aid posttransplant management. A candidate marker is programmed death (PD)-1 receptor, whose overexpression has been associated with disease progression during persistent viral infections. To determine whether levels of this negative regulator of T cell activity are altered in SOT recipients with symptoms of CMV disease, a comparative PD-1 expression analysis was done in healthy, CMV-positive individuals and in liver transplant recipients. PD-1 levels were measured among the total population of CD8(+) and CD8(+) T cells binding to CMV-specific major histocompatibility complex class I tetramers. Minimal PD-1 expression was found in the healthy, CMV-positive cohort, and symptomatic SOT recipients had significantly higher PD-1 levels. PD-1 up-regulation was significantly associated with incipient and overt CMV disease and with viremia. Our findings suggest that PD-1 could be developed as a prognostic tool to predict CMV disease and guide therapeutic interventions.

Functional comparison of T cells recognizing cytomegalovirus pp65 and intermediate-early antigen polypeptides in hematopoietic stem-cell transplant and solid organ transplant recipients

The functional status of cytotoxic T lymphocyte (CTL) populations recognizing cytomegalovirus intermediate-early antigen (IE1) and pp65 polypeptides was investigated in peripheral blood mononuclear cells from hematopoietic stem-cell transplant (HSCT) and solid organ transplant recipients. Combined flow-based CD107a/b degranulation/mobilization and intracellular cytokine (ICC) assays using peptide libraries as antigens indicated that a significantly higher proportion of pp65-specific CTLs were in a more mature functional state, compared with IE1-specific CTLs. Degranulation/multiple cytokine ICC assays also indicated that a significantly higher proportion of pp65-specific than IE1-specific CTLs secreted both interferon- gamma and tumor necrosis factor- alpha and possessed greater cytotoxic potential. These results support our earlier findings of functional differences between CTLs recognizing individual epitopes within the IE1 and pp65 antigens in healthy donors and HSCT recipients and extend them to a broader array of human leukocyte antigen-restricted responses to those antigens. We also provide evidence of a relationship between cytotoxic function and the ability of cytomegalovirus-specific CTLs to secrete multiple cytokines.

Programmed death-1 receptor and interleukin-10 in liver transplant recipients at high risk for late cytomegalovirus disease

A. Krishnan, W. Zhou, S.F. Lacey, A.P. Limaye, D.J. Diamond, C. La Rosa. Programmed death‐1 receptor and interleukin‐10 in liver transplant recipients at high risk for late cytomegalovirus disease.
Transpl Infect Dis 2010: 12: 363–370. All rights reserved

Primary response against cytomegalovirus during antiviral prophylaxis with valganciclovir, in solid organ transplant recipients

Antiviral prophylaxis has proved successful for prevention of cytomegalovirus (CMV) disease in solid organ transplant (SOT) patients; though emerging data suggest that antiviral agents interfere with immunity, and may inhibit immune priming. In this context, we investigated levels and phenotype of primary CMV‐specific immune responses that developed during antiviral prophylaxis in a cohort of CMV seronegative recipients (R−) of a SOT from a seropositive donor (D+). We longitudinally monitored CMV viral load, antibodies and levels of the negative immuno‐modulator IL‐10. PBMC were stimulated with CMV‐specific peptide libraries to measure CD137 activation marker on CMV‐specific T‐cells and levels of PD‐1 receptor, which is over expressed on exhausted T‐cells. Unexpectedly, the majority (13/18) of D+R− patients who developed a primary CMV response showed early post‐transplant CMV‐specific responses, though levels of PD‐1 on CMV‐specific T‐cells remained elevated throughout prophylaxis. A strong inverse association was found between levels of plasma IL‐10 and CMV‐specific cellular immune responses. Our study suggests that during prophylaxis, subclinical CMV infection might have occurred in the D+R− patients, and primary CMV‐specific responses were detected early post‐transplant when levels of plasma IL‐10 were low. Extended prophylaxis or antiviral treatment did not appear to suppress CMV‐specific antibodies or T‐cells, which, however, showed exhaustion phenotypes.

A novel approach to evaluate the immunogenicity of viral antigens of clinical importance in HLA transgenic murine models.

Transgenic (Tg) mice expressing HLA class I alleles and lacking murine MHC class I represent a useful model for the pre-clinical evaluation of human vaccines, which focus on induction of CD8(+) T-cell responses. We have developed a platform to be used in Tg mice for exploring the immunogenicity of T-cell targets, whose immunologic epitopes have yet to be defined. To test the attributes of the evaluation system in the context of an important human pathogen, we have explored multiple antigens from cytomegalovirus (CMV). A panel of recombinant modified vaccinia Ankara (MVA) vectors, expressing various CMV proteins (CMV-MVA) was used to immunize HLA-A*0201, B*0702 and A*1101 Tg mice. Immune splenocytes were in vitro stimulated (IVS) either using syngeneic lipo-polysaccharide activated lymphoblasts or Tg HLA-I matched human EBV-transformed B-lymphoblastoid cells (LCL), both loaded with peptide libraries, encompassing the CMV protein under investigation. IVS performed with peptide library loaded lymphoblasts failed to provide a reliable stimulation. In contrast, the usage of LCL as antigen presenting cells (APC) of CMV peptide libraries resulted in a consistent and specific amplification of the Tg T-cell response in animals immunized with CMV-MVAs. The LCL IVS method reliably allowed defining the immunogenicity and immunodominant CD8(+) T-cell regions of uncharacterized CMV antigens. The combination of CMV-MVA vectors, unbiased pools of CMV-specific peptide libraries presented by Tg HLA-I matched LCL constitutes a valid tool for the pre-clinical evaluation of model candidate vaccines. This convenient method could find application to investigate the immunogenicity profile of cancer antigens or proteins from infectious human pathogens.

Characterization of immunologic properties of a second HLA-A2 epitope from a granule protease in CML patients and HLA-A2 transgenic mice

The serine proteases, neutrophil elastase (HNE) and proteinase 3 (PR3), are aberrantly expressed in human myeloid leukemias. T-cell responses to these proteins have been correlated with remission in patients with chronic myeloid leukemia (CML). Human PR3/HNE-specific CD8(+) T cells predominantly recognize a nonameric HLA-A2-restricted T-cell epitope called PR1 which is conserved in both Ags. However, CML patients have CD8(+) T cells in peripheral blood recognizing an additional HLA-A2 epitope termed PR2. To assess immunologic properties of these Ags, novel recombinant vaccinia viruses (rVV) expressing PR3 and HNE were evaluated in HLA-A2 transgenic (Tg) mice (HHDII). Immunization of HHDII mice with rVV-PR3 elicited a robust PR3-specific CD8(+) T-cell response dominated by recognition of PR2, with minimal recognition of the PR1 epitope. This result was unexpected, because the PR2 peptide has been reported to bind poorly to HLA. To account for these findings, we proposed that HHDII mice negatively selected PR1-specific T cells because of the presence of this epitope within murine PR3 and HNE, leading to immunodominance of PR2-specific responses. PR2-specific splenocytes are cytotoxic to targets expressing naturally processed PR3, though PR1-specific splenocytes are not. We conclude that PR2 represents a functional T-cell epitope recognized in mice and human leukemia patients. These studies are registered at www.clinicaltrials.gov as NCT00716911.

Autologous stem cell transplant (ASCT) for AIDS-related lymphoma (ARL)

High-dose therapy with ASCT is an established therapy for relapsed Non-Hodgkins (NHL) and Hodgkins lymphoma (HL). Randomized trials have shown a benefit for this approach when compared to standard dose salvage therapy for HIV-negative NHL. Since these first trials, transplant-related mortality (TRM) has decreased due to the use of peripheral stem cells and improved supportive care. Concomitantly, the treatment of HIV infection has also improved. Highly active antiretroviral therapy (HAART) has improved hematologic and immune function in HIV positive patients. In addition, with the use of HAART and prophylactic antibiotics, the incidence of opportunistic infections has greatly decreased. This improvement in control of OIs and immunologic function in HIV infected patients due to HAART set the platform for the use of ASCT in patients with high-risk ARL. Herein we report the long-term follow-up of 32 patients with ARL who underwent ASCT at the City of Hope Cancer Center between 1998 and 2007. Median age at ASCT was 42 years. Histologies included Diffuse large cell n = 16, Burkitts n = 9, Anaplastic large cell n = 2, HL n = 5. The conditioning regimen consisted of CBV (carmustine 450 mg/m2, cyclophosphamide (CY) 100 mg/kg, VP16 60 mg/kg) in 28 patients and FTBI 1200 cGY/CY 100 mg/kg/VP16 60 mg/kg in 4 patients, All patients engrafted at a median of 10 days to an ANC >500 (range 5–19 days). One patient died of regimen-related cardiac toxicity. Other regimen-related toxicities included grade 3–4 hepatic toxicity n = 3, interstitial pneumonitis n = 2. OIs included PCP pneumonia in 2 patients who were not compliant with prophylaxis, CMV infection n = 3, VZV n = 2. One case of treatment related myelodysplasia was seen and the patient ultimately died of myelodysplasia while in remission from his ARL. Median HIV viral load at ASCT was 5726 copies/ml, with 25 patients having an undetectable viral load. Median CD4 count at ASCT was 156 (range 25–1064), which rose to 420 (range 95–1164) at 2-year follow-up. Only 10 patients had an undetectable VL at 2 years. Three patients who were in remission were lost to follow-up after 4 years. Median f/u for the entire group is 47 (range 0.7–104) months. Two-year overall survival is 80 percent (95% CI 66–89) and progression-free survival (PFS) is 81 percent (95% CI 67–90). In conclusion, this large single institution series of ASCT in ARL demonstrates that the procedure has low transplant-related mortality and can lead to long-term remission without deleterious effects on the underlying HIV infection.

244. Development and Qualification of Assays for Human Embryonic Stem Cell (hESC)-Derived Cardiomyocytes Intended for Clinical Use

Stem cell therapy holds the promise for numerous degenerative diseases and injuries. Human embryonic stem cells (hESCs) are the starting cell population for many of these potential therapies since they are capable of seemingly indefinite proliferation in the pluripotent state and have the ability to differentiate into all cell types found in the adult. It is vital that the cellular therapeutics derived from these highly proliferative cells are well characterized prior to clinical use and the assays used to characterize these cellular products are well developed. At the Center of Biomedicine and Genetics (CBG), we have manufactured under cGMP numerous hESC-differentiated products including neuron stem cells (NSC), neuronal progenitor cells (NPC), retinal pigment epithelium (RPE) cells, dopaminergic neurons and cardiomyocytes intended for pre-clinical and early phase clinical studies. We have developed a systematic approach to characterize hESC-derived cell products using well developed technologies such as real-time quantitative polymerase chain reaction (RT-qPCR) and flow cytometry. We have standardized the development and qualification of each assay which involves the selection and banking of positive and negative controls, selection and banking of critical reagents, determination of assay conditions, qualification of assay, generation of assay qualification report and standard operating procedure (SOP). In addition, we have established a general guideline to assist the selection of appropriate markers for different products. Here we describe the application of this systematic approach to develop and qualify identity and purity assays for hESC-derived cardiomyocytes produced through hESC differentiation procedure in suspension culture. Selection of markers for the characterization of cardiomyocyte products will be discussed, and development and qualification of the assays will be reported.

445. cGMP Compliant Production for Human Embryonic Stem Cell Derived Retinal Pigment Epithelial Cells on a Synthetic Substrate for the Treatment of Non-Neovascular Age-Related Macular Degeneration for Phase I Clinical Study

Non-neovascular age-related macular degeneration (dry AMD) is a leading cause of irreversible vision loss and is associated with retinal pigment epithelium (RPE) dystrophy. The RPE monolayer is integral for maintenance of healthy photoreceptors. Many studies have shown that AMD can be recovered by cellular therapy through transplantation of RPEs into the sub-retinal space to re-establish RPE functionality and halt neurological degeneration. Here we present a cGMP compliant manufacturing process to producing transplantable RPE cells on a synthetic substrate for clinical phase I study.