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Cellular Immune Responses to Allografts and Cytomegalovirus
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Today the immunosuppressive treatment is kept to a level were the incidence of acute rejection is below 20% within the first year after transplantation. As a consequence, a group of transplanted patients is over-immunosuppressed and at risk for infections. There is therefore an urgent need for tools which are able to determine the cellular immune response after organ transplantation. This knowledge would facilitate the task of prospectively opimize the immunosuppressive treatment and identify patients at risk of developing rejection episodes or infections.

To address this issue, a rat-kidney transplantation model for acute rejection was developed to study immune responses to allografts. Infiltrating lymphocytes were analysed using an in vitro culture system which allowed cells to propagate from the biopsies to culture medium. The numbers of outgrowing cells were correlated with morphological and immunohistochemical signs of rejection. When immunosuppressive treatment was administered for 2 and four days after acute rejection, histology did not reveal any improvement, however cellular propagation was reduced by 50 and 75%, respectively. Using the tissue culture technique in human transplanted kidney grafts, which was originally developed for the animal model, the number of propagated cells measured was profoundly higher in grafts with acute cellular rejection than from grafts in other groups. In some cases the number of propagated cells was better correlated with the clinical outcome than the diagnosis made by morphological evaluation. To determine immune responses to cytomegalovirus (CMV), we utilised Human Leukocyte Antigen (HLA) tetramer staining and stimulation of T cells with viral antigens. Both of these techniques independently detected CMV specific T cells in immunosuppressed and healthy individuals with latent or active infection. Although the frequency of CMV specific T cells did not differ between groups, there was a functional impairment in immunosuppressed patients as evidenced by reduced interferon-gamma production. In conclusion, these techniques can be used to determine the cellular immune response to allografts and cytomegalovirus and prove valuable for the optimization of immunosuppressive protocols and antiviral treatment.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2003. , p. 100
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1269
Keywords [en]
Immunology, Transplantation, Rejection, Monitoring, Flow Cytometry, MHC, Tetramer, CMV, infection
Keywords [sv]
Immunologi
National Category
Immunology in the medical area
Identifiers
URN: urn:nbn:se:uu:diva-3441ISBN: 91-554-5652-9 (print)OAI: oai:DiVA.org:uu-3441DiVA, id: diva2:162825
Public defence
2003-06-06, Fåhraeussalen, C5, Rudbecklaboratoriet, Uppsala, 13:15
Opponent
Supervisors
Available from: 2003-05-16 Created: 2003-05-16 Last updated: 2018-01-13Bibliographically approved
List of papers
1. Ex vivo propagation and characterization of lymphocytes from rejecting rat-kidney allografts
Open this publication in new window or tab >>Ex vivo propagation and characterization of lymphocytes from rejecting rat-kidney allografts
1999 In: Transplant Immunology, Vol. 7, p. 189-196Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90459 (URN)
Available from: 2003-05-16 Created: 2003-05-16Bibliographically approved
2. Quantification of lymphocytes propagating from rat-kidney allografts: a tool to monitor anti-rejection treatment
Open this publication in new window or tab >>Quantification of lymphocytes propagating from rat-kidney allografts: a tool to monitor anti-rejection treatment
Show others...
2002 In: Transplant Immunology, Vol. 10, p. 31-36Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90460 (URN)
Available from: 2003-05-16 Created: 2003-05-16Bibliographically approved
3. Lymphocyte propagation from biopsies of kidney allografts: Correlation to morphological diagnosis and clinical outcome
Open this publication in new window or tab >>Lymphocyte propagation from biopsies of kidney allografts: Correlation to morphological diagnosis and clinical outcome
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-90461 (URN)
Available from: 2003-05-16 Created: 2003-05-16 Last updated: 2010-01-13Bibliographically approved
4. Characterization of CMVpp65-specific CD8+ T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants
Open this publication in new window or tab >>Characterization of CMVpp65-specific CD8+ T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants
Show others...
2000 (English)In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 69, no 11, p. 2243-2250Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Cytomegalovirus (CMV) is a ubiquitous herpesvirus that infects 50-90% of individuals in different populations. After primary infection, the virus persists latently in myeloid cells under the control of specific T-cells. Reactivation of CMV infection may cause lethal organ dysfunction and is frequently seen in immunosuppressed individuals. CD8+ cytotoxic T-cells (CTL) have a primary role in suppressing CMV reactivation, and the dominating CTL response is directed against pp65.

METHODS: MHC tetramers, that is, complexes between HLA class I (or class II) molecules and antigenic peptides conjugated to fluorochromes allow the direct visualization of antigen-specific receptor-carrying T-cells using flow cytometry. We constructed a novel MHC tetramer for identification of CMVpp65-specific CD8+ T-cells using HLA-A2 molecules folded with the immunodominant NLVPMVATV peptide.

RESULTS: The A2/pp65 tetramer specifically stained CMV-directed T-cell lines, and sorted cells showed CMV-specific cytotoxicity. High proportions (0.1-9%) of the CD8+ T-cells were A2/pp65 tetramer+ in healthy HLA-A2+ CMV carriers and in immunosuppressed kidney transplant patients with latent infection. Patients with reactivated CMV infection exhibited up to 15% A2/pp65 tetramer+ cells, which seemed to correlate with CMV load over time. A2/pp65 tetramer+ cells expressed T-cell activation markers.

CONCLUSIONS: The construction of a novel A2/pp65 MHC tetramer enabled the design of a rapid and precise flow cytometric method allowing quantitative and qualitative analysis of CMV-specific T-cells. The number of A2/pp65 tetramer binding CTLs in blood may prove to be clinically relevant in assessing the immune response to CMV.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-90462 (URN)10868621 (PubMedID)
Available from: 2003-05-16 Created: 2003-05-16 Last updated: 2017-12-14Bibliographically approved
5. Cellular responses to cytomegalovirus in immunosuppressed patients: circulating CD8+ T cells recognizing CMVpp65 are present but display functional impairment
Open this publication in new window or tab >>Cellular responses to cytomegalovirus in immunosuppressed patients: circulating CD8+ T cells recognizing CMVpp65 are present but display functional impairment
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2003 (English)In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 132, no 1, p. 96-104Article in journal (Refereed) Published
Abstract [en]

The availability of tetrameric complexes of HLA class I molecules folded with immunodominant peptides makes it possible to utilize flow cytometry for rapid and highly specific visualization of virus specific CD8+ T cells. An alternate technique is to incubate whole blood with specific antigens and to subsequently detect and characterize responding T cells (e.g. by performing intracellular staining of interferon-gamma). By using an HLA-A2 tetramer construct folded with the same immunodominant CMV-peptide as that used for peptide pulsing, we monitored both the presence and functional capacity of CMV-specific CD8+ T cells. In addition T cell activation was assayed by determination of CD38 and CD69 expression. Twelve organ transplant patients and 31 healthy blood donors with latent CMV infection were investigated using CMV pp65 tetramer staining and intracellular staining of interferon-gamma after CMV pp65 peptide pulsing or CMV lysate pulsing. CMV-specific T cells were detected in similar absolute numbers as well as frequencies of T cells in the two groups investigated. However, the CMV-specific CD8+ T cells in immunosuppressed individuals showed a decreased functional response to the CMV-peptide, as evidenced by reduced interferon-gamma production when compared to healthy blood donors (19%; 42%, P < 0·005). In addition, CD38 expression was markedly higher in immunosuppressed patients compared to healthy blood donors (24%; 6%, P < 0·005). In a case report we demonstrate that reactivation of CMV can occur in an immunosuppressed patient with high number of CMV-specific T cells, but without functional capacity. Hence, these findings reflect impaired activation of cytotoxic T cells controlling latent CMV infection in immunosuppressed patients.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-90463 (URN)10.1046/j.1365-2249.2003.02098.x (DOI)12653843 (PubMedID)
Available from: 2003-05-16 Created: 2003-05-16 Last updated: 2018-02-28Bibliographically approved

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