A CMV seronegative donor to avoid T-cell inflation?

Abstract The human cytomegalovirus (CMV) is an important cause for mortality and morbidity after allogeneic stem cell transplantation (allo SCT) especially in patients without a CMV-specific T cell response. CMV seropositive patients allografted with a CMV seronegative donor do not rise up specific CMV-T cell immunity posttransplant and are therefore at great risk for CMV viremia and disease. In a prospective manner, we analyzed blood samples of 38 HLA-A2+ patients with different haematological malignancies for CMV specific T cells. Methods: Frequency of T cells with reactivity against the pp65-peptide (NLVPMVATV) was assessed by ELISPOT assay in 38 patients at defined time points after allo SCT. In patients with high CMV specific T cell frequencies, the T cell phenotype was determined by flow cytometry. Surveillance of CMV viremia was carried out by routine PCR-technique or pp65 Ag staining. Results: In high-risk patients (donor-/recipient+) viremia was observed in 7/9 patients, 3/7 developed clinical severe CMV-disease. In this group, only one patient presented a weak CMV-specific T cell response in the first year after transplantation, although CMV-specific T cells were demonstrated in 5/9 patients before transplantation. In contrast, 64% (11/17) of the CMV seropositive patients having had a CMV seropositive donor showed CMV-specific T cells around day 30. Their T cell frequencies remained stable at a relative high level during the whole time of our investigation period and no CMV disease was observed. CMV seronegative patients allografted with either a CMV seronegative or seropositive donor also remained disease-free. CMV specific T cells were detectable in 2/7 patients of the d+/r− group. FACS analysis revealed that most of the responding cells were of the activated effector phenotype CD8+/CD45RA+/HLA-DR+ with a low expression of CCR7 and CD27. Conclusion: CMV-seropositive patients receiving graft from a seronegative donor are at a high risk and therefore prone for CMV-viremia and -disease. The development of CMV-specific T cells i.e. immune reconstitution in high risk patients remains delayed or is completely missing during the first year post transplant. In contrast, seropositive recipients grafted with a seropositive donor develop a durable T cell response within 3–4 weeks and show no viremia at all. New strategies as donor vaccination and adoptive T cell transfer are warranted to prevent CMV-disease in CMV seropositive patients for whom only a seronegative donor can be found.

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Generation of Cytomegalovirus pp65-Specific T Cells from Seronegative Donors.

Blood ◽

10.1182/blood.v110.11.4860.4860 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4860-4860

Author(s):

Mindy M. Stamer ◽

Kimberly Dunham ◽

Lei Bao ◽

Kenneth G. Lucas

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Interleukin 2 ◽

The Other ◽

Interleukin 12 ◽

51Cr Release ◽

Seronegative Donor ◽

Interleukin 7 ◽

Ifn Γ

Abstract Cytomegalovirus (CMV) can cause significant disease in allogeneic stem cell transplant (SCT) recipients. Adoptive transfer of donor derived CMV-specific cytotoxic T lymphocytes (CTL) is currently used for prophylaxis and treatment of CMV infections in allogeneic transplant recipients, but this is only available for patients whose donors are CMV seropositive. We developed a method to prime and expand CMVpp65-specific T cells from the peripheral blood mononuclear cells (PBMC) of CMV seronegative donors. Monocyte-derived dendritic cells isolated from four CMV seronegative donors were pulsed with CMVpp65 peptides for two hours prior to T cell stimulation. CMVpp65 peptides consisted of 15 mers with an 11 amino acid overlap. T cells obtained from CMV seronegative donors were primed every seven days for three weeks using CMVpp65 peptide pulsed dendritic cells. Interleukin-7 and interleukin-12 were initially added to the T cell cultures to promote CTL differentiation. After ten days, interleukin-2 together with interleukin-7 and interleukin-12 were added at three day intervals. Intracellular cytokine staining (ICS) and 51Cr-release assays were used to measure the activity and specificity of CMVpp65 primed CMV seronegative donor T cells seven to ten days after the last stimulation. A four color ICS analysis revealed that CMVpp65-specific IFN-γ-producing CD4+ (Th1) T cells were expanded from two of the four seronegative donors; one of these two donors had CMVpp65-specific killing as measured by 51Cr-release assay, the other displayed non-specific killing. One of the other two donors exhibited CMVpp65 killing but lacked IFN-γ production. PBMC isolated from a CMV seronegative donor three weeks post CMV vaccination also exhibited CMV-specific killing after only one week in vitro stimulation, with no CMV specificity pre-vaccination. In conclusion CMV-specific effector cells can be expanded from some CMV seronegative donors using CMVpp65 peptide pulsed dendritic cells, interleukin-2, interleukin-7, and interlukin-12. These cells may have the potential to be used in adoptive cell therapy to prevent or treat CMV infections in SCT recipients from CMV seronegative donors. An alternative strategy for patients with persistent CMV infection may be to use CTL derived from a vaccinated donor. Analysis of CMVpp65-specific T cells from additional seronegative donors is in progress to further optimize ex vivo expansion.

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Characterization of Human Immunodeficiency Virus Type 1 (HIV-1) Gag- and Gag Peptide-Specific CD4+ T-Cell Clones from an HIV-1-Seronegative Donor following In Vitro Immunization

Journal of Virology ◽

10.1128/jvi.76.14.6987-6999.2002 ◽

2002 ◽

Vol 76 (14) ◽

pp. 6987-6999 ◽

Cited By ~ 15

Author(s):

Sara Venturini ◽

Donald E. Mosier ◽

Dennis R. Burton ◽

Pascal Poignard

Keyword(s):

Immune Response ◽

T Cell ◽

T Cell Response ◽

T Cell Clones ◽

Seronegative Donor ◽

Cell Clones ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Substantial evidence argues that human immunodeficiency virus type 1 (HIV-1)-specific CD4+ T cells play an important role in the control of HIV-1 replication in infected individuals. Moreover, it is increasingly clear that an HIV vaccine should elicit potent cytotoxic lymphocyte and antibody responses that will likely require an efficient CD4+ T-cell response. Therefore, understanding and characterizing HIV-specific CD4+ T-cell responses is an important aim. Here we describe the generation of HIV-1 Gag- and Gag peptide-specific CD4+ T-cell clones from an HIV-1-seronegative donor by in vitro immunization with HIV-1 Gag peptides. The Gag peptides were able to induce a strong CD4+ T-cell immune response in peripheral blood mononuclear cells from the HIV-1-seronegative donor. Six Gag peptide-specific CD4+ T-cell clones were isolated and their epitopes were mapped. The region of p24 between amino acids 201 and 300 of Gag was defined as the immunodominant region of Gag. A new T helper epitope in the p6 protein of Gag was identified. Two clones were shown to recognize Gag peptides and processed Gag protein, while the other four clones reacted only to Gag peptides under the experimental conditions used. Functional analysis of the clones indicated that both Th1 and Th2 types of CD4+ T cells were obtained. One clone showed direct antigen-specific cytotoxic activity. These clones represent a valuable tool for understanding the cellular immune response to HIV-1, and the study provides new insights into the HIV-1-specific CD4+ T-cell response and the induction of an anti-Gag and -Gag peptide cellular primary immune response in vitro.

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282: Efficient Induction and Isolation of a Primary CMV-specific CD8+ T Cell Response from CMV Seronegative Donors for the Treatment of Serious CMV-Related Complications in CMV Seropositive Patients Transplanted with a CMV Seronegative Donor

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2007.12.292 ◽

2008 ◽

Vol 14 (2) ◽

pp. 104-105

Author(s):

I. Jedema ◽

M. van de Meent ◽

P.L.J. van der Heiden ◽

E.W.A. Marijt ◽

P. Meij ◽

...

Keyword(s):

T Cell ◽

Cell Response ◽

Cd8 T Cell ◽

T Cell Response ◽

Seronegative Donor ◽

Efficient Induction

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Electron microscope study of the secretory activity of epithelial cells in embryonic thymus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015945x ◽

1990 ◽

Vol 48 (3) ◽

pp. 382-383

Author(s):

H. Alasam

Keyword(s):

Cell Differentiation ◽

T Cell ◽

Epithelial Cells ◽

Electron Density ◽

Secretory Activity ◽

T Cell Differentiation ◽

High Electron ◽

Transmission Electron ◽

Medullary Epithelial Cells ◽

Thymic Factor

The possibility that intrathymic T-cell differentiation involves stem cell-lymphoid interactions in embryos led us to study the ultrastructure of epithelial cell in normal embryonic thymus. Studies in adult thymus showed that it produces several peptides that induce T-cell differentiation. Several of them have been chemically characterized, such as thymosin α 1, thymopoietin, thymic humoral factor or the serum thymic factor. It was suggested that most of these factors are secreted by populations of A and B-epithelial cells.Embryonic materials were obtained from inbred matings of Swiss Albino mice. Thymuses were disected from embryos 17 days old and prepared for transmission electron microscopy. Our studies showed that embryonic thymus at this stage contains undifferentiated and differentiated epithelial cells, large lymphoblasts, medium and few small lymphocytes (Fig. 5). No differences were found between cortical and medullary epithelial cells, in contrast to the findings of Van Vliet et al,. Epithelial cells were mostly of the A-type with low electron density in both cytoplasm and nucleus. However few B-type with high electron density were also found (Fig. 7).

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