Generation of Cytomegalovirus (CMV)–Specific CD4 T Cell Lines Devoid of Alloreactivity, by Use of a Mixture of CMV–Phosphoprotein 65 Peptides for Reconstitution of the T Helper Repertoire

Pira, Giuseppina Li; Bottone, Laura; Ivaldi, Federico; Tagliamacco, Augusto; Fiordoro, Stefano; Ricciardi, Annamaria; Barbano, G; Manca, Fabrizio

doi:10.1086/427040

Cited by 24 publications

(34 citation statements)

References 61 publications

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“…In addition, pp65-specific killing was similar to that demonstrated in T cell lines expanded by repetitive specific stimulation with pp65-transgenic APC. Comparable results were also described by Rauser et al after expansion of selected pp65-specific T cells with autologous PBMC [15]. We also monitored antigen-specific T cells with MHC class I tetramers during the generation process.…”

Section: Discussionsupporting

confidence: 74%

“…Li Pira and Rauser et al generated pp65-specific T cells by repetitively stimulating them with a mixture of previously identified T cell epitopes. This strategy cuts the total number of peptides, but still depends on the knowledge of relevant epitopes [14,15]. For single proteins like pp65, a variety of data on relevant epitopes allow the design of a peptide mixture containing the most relevant candidates.…”

Section: Discussionmentioning

confidence: 99%

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HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

Hammer

Meyer²,

Brestrich³

et al. 2005

Eur. J. Immunol.

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Adoptive immunotherapy with antigen-specific T cells has been successfully used to treat certain infectious diseases and cancers. Although more patients may profit from T cell therapy, its more frequent use is restricted by limitations in current T cell generation strategies. The most commonly applied peptide-based approaches rely on the knowledge of relevant epitopes. Therefore, T cells cannot be generated for diseases with unknown epitopes or for patients with unfavorable HLA types. We developed a peptide-based approach for HLA type-independent generation of specific T cells against various proteins. It is based on short-time stimulation with peptide libraries that cover most CD4 + and CD8 + T cell epitopes of given proteins. The procedure requires no prior knowledge of epitopes because libraries are synthesized solely on the basis of the protein's amino acid sequence. Stimulation is followed by immunomagnetic selection of activated IFN-c-secreting cells and nonspecific expansion. To evaluate the protocol, we generated autologous T cells specific for a well-characterized antigen, the human cytomegalovirus phosphoprotein 65 (pp65). Generated T cell lines consisted of pp65-specific CD4 + and CD8 + lymphocytes that displayed antigen-specific killing and proliferation. The protocol combines the biosafety of peptide-based approaches with HLA type independence and may help to advance adoptive immunotherapy in the future.

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Section: Discussionsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

Hammer

Meyer²,

Brestrich³

et al. 2005

Eur. J. Immunol.

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“…However, since we started this trial, a recent report has shown that certain CMV peptide pools can be used to raise CMV-specific T cells from different individuals, irrespective of the HLA class haplotype. 40 In conclusion, this paper describes the first use of adoptive immunotherapy for CMV in haploidentical stem-cell transplants and first ever use of adoptive immunotherapy for Aspergillus infection. We achieved rapid pathogen-specific immune recovery associated with significantly reduced rates of CMV reactivation and galactomannan in serum.…”

Section: Discussionmentioning

confidence: 99%

Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation

Perruccio

Tosti

Burchielli

et al. 2005

Blood

346

277

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Aspergillus and cytomegalovirus are major causes of morbidity/mortality after haploidentical hematopoietic transplantation. The high degree of mismatching makes cell immunotherapy impossible as it would result in lethal graft-versus-host disease (GvHD). We generated large numbers of donor T-cell clones specific for Aspergillus or cytomegalovirus antigens. We identified clones potentially responsible for causing GvHD by screening them for cross-reactivity against recipient mononuclear cells. Nonrecipient reactive, pathogen-specific clones were infused soon after transplantation. They were CD4 ؉ and produced high levels of interferon-␥ and low levels of interleukin-10. In 46 control transplant recipients who did not receive adoptive therapy, spontaneous pathogen-specific T cells occurred in low frequency 9 to 12 months after transplantation and displayed a nonprotective low interferon-␥/high interleukin-10 production phenotype.

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“…Pentamers were from Proimmune, Oxford, United Kingdom. Protein antigens, derived from different opportunistic pathogens or whole inactivated pathogen bodies, have been described previously (6,7,8,9). Pathogens used as whole bodies or pathogens from which proteins were derived included the following: Candida albicans, Aspergillus fumigatus, Aspergillus niger, Cryptococcus neoformans, Toxoplasma gondii, Mycobacterium bovis ("Mycobacterium tuberculosis var.…”

Section: Methodsmentioning

confidence: 99%

“…), cytomegalovirus (CMV), and human immunodeficiency virus type 1 (HIV-1). Peptides were synthesized by INBIOS, Naples, Italy, or by JPT, Berlin, Germany (6,7,8,9). Final antigen concentrations were 10 g/ml for proteins and 1 g/ml for peptides.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Antigen-Specific T-Cell Responses with a Miniaturized and Automated Method

Pira

Ivaldi

Dentone

et al. 2008

Clin Vaccine Immunol

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The evaluation of antigen-specific T-cell responses is helpful for both research and clinical settings. Several techniques can enumerate antigen-responsive T cells or measure their products, but they require remarkable amounts of peripheral blood mononuclear cells (PBMCs). Since screening numerous antigens or testing samples from pediatric or lymphopenic patients is hampered in clinical practice, we refined a miniaturized, high-throughput assay for T-cell immunity. Antigens and cells in 10-l volumes were dispensed into 1,536-well culture plates precoated with anti-gamma interferon (anti-IFN-␥) antibodies. After being cultured, the wells were developed by enzyme-linked immunosorbent assay for bound cytokine. Miniaturization and automation allowed quantitation of antigen-specific responses on 10 4 PBMCs. This method was applied for epitope mapping of mycobacterial antigens and was used in the clinic to evaluate T-cell immunity to relevant opportunistic pathogens by using small blood samples. A comparison with conventional methods showed similar sensitivity. Therefore, current flow cytometric methods that provide information on frequency and phenotype of specific T cells can be complemented by this assay that provides extensive information on cytokine concentrations and profiles and requires 20-to 50-fold fewer PBMCs than other analytical methods.Numerous methods are currently available to enumerate antigen-specific T cells and to measure their functions, most of them being based on flow cytometric analysis. Such methods have been compared and critically discussed in recent reviews (4, 17). A common feature of these methods is the requirement, on average, of 2 ϫ 10 5 to 5 ϫ 10 5 peripheral blood mononuclear cells (PBMCs) to test a single antigen. This imposes a limitation on the analytical antigenic breadth, which depends on the number of available PBMCs. In the case of lymphopenic subjects, the population that most frequently needs to be monitored for immunocompetence, this is particularly crucial. Also, the small volumes of pediatric blood samples may result in inadequate numbers of PBMCs for extended analysis of specific T-cell immunity. By the same token, screening of large peptide panels for epitope mapping on PBMCs from healthy donors (blood donors or vaccinees) is also limited to a few hundred peptides (16) instead of the thousands of peptides needed to cover several immunodominant proteins (14). Therefore, we felt the need to develop a miniaturized assay that makes use of limited numbers of PBMCs, thereby enhancing our screening power.A miniaturized assay based on 384-well plates instead of conventional 96-well plates or 5-ml tubes was developed in our laboratory and named cell-enzyme-linked immunosorbent assay (cell-ELISA) (8). This method was based on a previous observation that lymphocyte cultures can be run in 96-well plates in which the wells had been precoated with an anticytokine antibody. The cytokine secreted by antigen-specific T cells was captured by the solid-phase antibody, and the plate was de...

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Generation of Cytomegalovirus (CMV)–Specific CD4 T Cell Lines Devoid of Alloreactivity, by Use of a Mixture of CMV–Phosphoprotein 65 Peptides for Reconstitution of the T Helper Repertoire

Abstract: By use of a simple procedure, an immunodominant peptide library can generate T cell lines from allodonors, for the perspective reconstitution of the Th repertoire in immunocompromised hemopoietic stem-cell transplant recipients.

Cited by 24 publications

References 61 publications

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation

Evaluation of Antigen-Specific T-Cell Responses with a Miniaturized and Automated Method

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