centro biotecnologie avanzate

LABORATORY OF: Cellular Immunology
CONTACT PERSON: Dr.Giuseppina Li Pira
Phone +39 010 5737357 E-mail: lipira@email.it

Description of Laboratory and Expertise:

The Laboratory of Giusi Li Pira has developed a specific expertise in the dissection of the human T cell immunity specific for different pathogens. Tissue culture techniques, flow cytometry, molecular biology and high-throughput immunological analyses are used to define the fine specificity of cellular immunity mediated by CD4 and CD8 lymphocytes. This knowledge is currently applied to the diagnostic definition of immunocompetence in different patient cohorts. In addition to analytical work, methods for bulk expansion of antigen-specific T cells have also been developed, with the perspective of producing T cell lines for therapeutic applications, i.e. immunoreconstitution in immunocompromised patients.

Abstract of Activities:

Epitope mapping of relevant pathogens. We have selected opportunistic pathogens (viruses, bacteria and fungi) responsible for severe infections in immunocompromised patients. By using established methods for analysis of T-cell responses, or novel methods, developed in our laboratory for high-throughput screening, we can define the immunogenic proteins from these pathogens. By using panels of synthetic peptides, we can define immunodominant epitopes carried by stimulatory peptides.
Diagnostic applications of epitope mapping. By using the sequence information derived from epitope mapping, we produce peptide libraries as convenient antigenic preparations to challenge T lymphocytes in vitro. Lymphocytes are obtained from patients in whom it is desirable to evaluate immunocompetence (AIDS, transplanted, chemotherapy treated, pediatric patients, etc.).
Therapeutic potential of antigen specific T-cells. Immunoreconstitution with antigen specific T cells has already proved an effective therapeutic measure for immunocompromised patients. Nevertheless, it is not widely applied because of difficulties related to the expansion of specific T-cell lines, to the availability of GMP--grade antigens, to the requirement for a GMP facility. To overcome these problems, we apply our expertise in the generation of long-term T-cell lines, in the use of antigenic peptides approved for GMP use and in the implementation of novel methods for T-cell manipulation in sealed systems.

Detailed Research Activities:

We have applied numerous methods to estimate T lymphocyte activation in response to specific antigens, i.e., proliferation based on thymidine incorporation and CFSE staining, intracytoplasmic cytokine staining, cytokine secretion, cytolytic assays. Several new antigenic proteins have been identified on Aspergillus and CD4 T cells with these specifities have been expanded in vitro for further functional and phenotypic studies.

Cytomegalovirus has also been extensively studied, in order to identify novel peptide sequences recognized by CD4 T helper cells. Peptide libraries containing CD4 and CD8 epitopes have been constructed in order to produce antigenic cocktails that are now used to define cellular immunity in immunocompromised patients. In addition to this diagnostic application, peptide libraries can be used to expand T cells in vitro under GMP condition to produce therapeutic T cell lines, according to a phase I protocol approved by the Italian Health Institute, in collaboration with A. Biondi (Centro Ricerca M. Tettamanti, Clinica Pediatrica, Università Milano Bicocca, Ospedale San Gerardo, Monza) and M. Introna (City Hospital, Bergamo).

Efforts are being made to improve our screening capacity, considering that large numbers of proteins and peptides need to be analyzed with limited amounts of lymphocytes obtained from healthy donors or from patients. Therefore we applied the microarray concept to miniaturize the functional assays used to test lymphocyte activation. We transferred our lymphocyte cultures from 96 well to 384 and 1536 well plates, thereby reducing the culture volumes from 200 to 10 microliters. This high throughput approach proved effective for large scale screening and is based on the use of robotic liquid handling equipment adapted to cellular work. This methodology has also been the subject of a recent patent application. This robust platform has now been applied to collaborative studies, in which we distribute pre-formatted microplates containg antigens and anti-cytokine antibodies. Plates can be stored for months and can be used locally to dispense and culture T cells. After incubation, the plates are snap--frozen without processing and shipped back to our lab for further development. This approach will prove valuable for collaborative studies on vaccination trials in developing countries with limited resources and will permit accurate analysis of T cell immunity, which is generally neglected for technological limitations at the trial site.
A specific study is focused on the analysis of T cell responsiveness of relevant pathogens in cord blood samples.

In order to improve the technologies to produce therapeutic T cell lines, we have further developed methods for the selection and expansion of pathogen specific T cells. Similar methods can be applied in principle to the selection and expansion of tumor specific T cells. In particular, we have refined culture methods based on sealed systems, in which lymphocytes are manipulated starting from the blood sample using unbreached connections to transfer cells from one bag to the other. Plastic bags, in fact, can replace tubes and containers. Gas permeable teflon bags can replace culture flasks. These procedures are also aimed at increasing biosafety of manipulated cells and may Eventually provide a viable alternative to manipulations in GMP facilities. In order to improve culture efficiency and to mimic in vivo conditions, we are also developing 3D bioreactors (with R. Quarto, at ABC). At the present time we have several ongoing collaborations with other groups that make their GMP facilities available for joint projects and thus the development of a biobank of therapeutic T cell lines is a realistic development in the short term.

In addition to the development of biotechnologies that we would like to apply to human health, we are also involved in more basic research, in collaboration with other Italian and foreign groups, keeping in mind the central role the immune system plays in numerous diseases.
In particular, with the support of an EU grant, we are testing viral and retroviral vectors (coll. with G. Sutter, D. von Lear) that carry marker genes that make cells resistant to HIV infection. The targets of these potential gene therapies are T lymphocytes and we are using our broad panel of antigen-specific CD4 T cell lines to look at infection and transduction efficiency of the vectors. Antigen specific T cell lines infected or transfected with viral vectors, respectively, are prepared and collected to investigate the possible effect of these manipulations on gene expression profiles using a microarray approach (coll. with K. Willard-Gallo). Gaining this information and improving efficiency or selective targeting are important goals in a therapeutic perspective.

Our Unit is in strict collaboration with the Unit of Viral Immunology (F. Manca), with which technologies, reagents and equipment are shared.
An active collaboration is also ongoing with the Unit of Stem Cells (R. Quarto) on the role of mesenchymal stem cells as potential immunoregulators.

Finally, our Unit is coordinating a consortium of 22 European partners, including basic research, applied and clinical investigation groups on the theme of Adoptive Cellular Immunoreconstitution. The goal of the consortium is to submit a large application in the context of the 2008 EU FP7 calls for development and application of novel biotechnologies in the field of cellular immunotherapy.

Applications and Developments:

We envisage three major potential applications and developments:
1. Expansion of basic knowledge of human cellular immunity: this will help understand the phenotype and functions of T-cells specific for antigens carried by relevant pathogens
2. Development of diagnostic tools: better methods for detection of specific T cell immunity and more standardized antigenic preparation will facilitate the development of reliable clinical assays
3. Development of therapeutic applications: an improved knowledge of the methods to expand antigen specific T-cells in vitro will facilitate the development of biotechnologies for preparation of therapeutic T-cell lines for adoptive cellular immunoreconstitution.

Managed core facilities:

1. Flow cytometry (CyAn DAKO, FACScan BD)
2. BSL2 laboratory
3. Hot room and scintillation counters
4. High-throughput equipment for cellular immunity and immunochemistry (Multiprobe, Hydra II, Multidrop, 1536 wellplate scanner, etc)

Ongoing collaborations:

International
Gerrit Koopman, Biomed. Primate Res. Ctr, Rijswijk, NL. Primate models for HIV infection
Karen Willard Gallo, Univ. Libre Bruxelles, BE. Gene expression profiling of specific T cells
Gerd Sutter, P. Erlich Inst, Frankfurt, DE. Viral vectors for human T cell vaccination
Dorothee von Laer, Univ. Frankfurt, DE. Retroviral vectors for delivery of HIV resistance genes
Balbino Alarcon, Univ. Madrid, ES. Retroviral vectors for delivery of marker genes in T cells

Domestic
Giorgio Palù, Univ. Padova, IT. Antisense RNA encoding retroviral vectors
Giovanna Dal Pozzo, CNR Naples, IT. Phage display libraries for expression of antigenic epitopes
Piergiuseppe Deberardinis, CNR Naples, IT. Transfer of specific T cell receptor genes
Andrea Biondi, II Univ. Milan, IT. Generation of GMP grade therapeutic T cell lines

Local
Barbara Parodi, IST, Genoa, IT. Construction of a bank of antigen specific T cell lines
Paolo Strada, San Martino Hospital, Genoa, IT. Cellular immunity in cord blood lymphocytes
Fabrizio Manca, Viral Immunology, Gaslini Inst. - ABC Genoa. Epitope mapping and T cell immunity
Rodolfo Quarto, Stem cell Unit, ABC, Genoa. T lymphocyte – mesenchymal stem cell interactions

Most recent and significant publications:

Li Pira G, Ivaldi F, Dentone C, Righi E, Del Bono V, Viscoli C, Manca F. Miniaturization and automation for measuring antigen specific T-cell responses. 2008, Cytometry, in press

Dander E, Li Pira G, Biagi E, Perseghin P, Renoldi G, Gaipa G, Introna M, Marin V, Manca F, Biondi A, D’Amico G. Characterization of migratory activity and cytokine profile of helper and cytotoxic CMV-specific T cell lines expanded by a peptide library. 2007, Exp. Haematol. In press

Li Pira G, Ivaldi F, Bottone L, Manca F. High throughput functional microdissection of pathogen-specific T-cell immunity using antigen and lymphocyte arrays. J Immunol Methods. 2007,326,22-32

Li Pira G, Kern F, Gratama J, Roederer M, Manca F. Measurement of antigen specific immune responses: 2006 update. Cytometry B Clin Cytom. 2007,72,77-85

D'Apice L, Sartorius R, Caivano A, Mascolo D, Del Pozzo G, Di Mase DS, Ricca E, Li Pira G, Manca F, Malanga D, De Palma R, De Berardinis P. Comparative analysis of new innovative vaccine formulations based on the use of procaryotic display systems. Vaccine. 2007,25,1993-2000

Li Pira G, Ivaldi F, Bottone L, Koopman G, Manca F. Koopman G, . Helper function of cytolytic lymphocytes: switching roles in the immune response. Eur J Immunol. 2007,37,:66-77.

Li Pira G, Ivaldi F, Bottone L, Quarto R, Manca F. Human bone marrow stromal cells hamper specific interactions of CD4 and CD8 T lymphocytes with antigen-presenting cells. Hum Immunol. 2006,67,976-85

Li Pira G, Bottone L, Ivaldi F, Risso M, Tripodi G, Manca F. A sealed and unbreached system for purification, stimulation and expansion of CMV specific human CD4 and CD8 lymphocytes. Transfusion 2006, 46,2053-62

Li Pira G, Bottone L, Ivaldi F, Accolla R, Koopman G, De Palma R, Manca F. Human naive CD4 T cell clones specific for HIV env persist for years in vivo in the absence of antigenic challenge. J. AIDS, 2005,40,132-139

Kern F, Li Pira G, Gratama J, Manca F, Roederer M. Measuring antigen specific immune responses: understanding immunopathogenesis and improving diagnostics in infectious diseases, autoimmunity and cancer. Trends Immunol. 2005,9,477-484

Li Pira G, Bottone L, Ivaldi F, Barbano G, Manca F. Generation of Cytomegalovirus specific CD4 T cell lines devoid of alloreactivity using a cocktail of CMV-pp65 peptides for reconstitution of the Th repertoire. J. Infect. Dis., 2005,191,215-226

Patent

Method for determining antigen-specific T cell responses in high throughput format
06012034.2, June 2, 2006

Inventors: Fabrizio Manca, Giusi Li Pira

ABC - based grants, contracts, services