Monitoring of cytomegalovirus-specific CD8+ T-cell response with major histocompatibility complex pentamers in kidney transplant recipients

Introduction

Cytomegalovirus (CMV) can reactivate causing serious clinical problems during immunosuppression. CMV-specific CD8⁺ T cells play an important role in the control of CMV reactivation. Using pentameric major histocompatibility complex (MHC) peptide complexes, we investigated cellular immune responses to CMV among healthy individuals and kidney transplantation recipients in Korea, which is an endemic area of CMV infection.

Materials and methods

Analysis of CMV-specific T cells was performed on 28 healthy individuals and 40 recipients who bore human leukocyte antigen (HLA)-A2 or -A24. CMV pp65 pentamer-binding cells incubated with various monoclonal antibodies were measured by four-color flow cytometry.

Results

Detectable levels of pentamer⁺ CD8⁺ T cells were present in 109/139 samples (78.4%) that stained with the A*02NLV-pentamer, while 15/67 samples (22.4%) stained with the A*24QYD-pentamer (P < .01). Among patients with HLA-A2, 22/24 (91.7%) samples showing positive CMV antigenemia revealed detectable pentamer⁺ CD8⁺ T cells, while 87/115 (75.7%) displaying negative CMV antigenemia had detectable pentamer⁺ CD8⁺ T cells (P = .04). There was no significant difference in percentages of pentamer⁺ CD8⁺ T cells between patients who did versus who did not experience episodes of CMV infection. The subpopulation of CMV-specific CD8⁺ T cells in transplantation recipients was evaluated using phenotypic markers; memory cells comprised the majority of the CMV-specific CD8⁺ T-cell population.

Conclusion

The A*02NLV-pentamer complex was useful to monitor CMV-specific T cells. However, MHC pentamer-based techniques did not provide a clear distinction between patients who are or are not at risk for CMV infection.     

Detection of vimentin-specific autoreactive CD8+ T cells in cardiac transplant patients

BACKGROUND: Evidence is emerging that autoimmunity can play a role in allograft rejection. Reports have described the presence of autoantibodies in transplant patients and CD4+ autoreactive T cells in rodent models of allograft rejection. The objective of this study was to seek evidence of CD8+ T-cell-mediated autoimmunity in the transplant setting. The author have previously observed autoimmunity to the non-polymorphic cytoskeletal protein vimentin in cardia transplant patients. In this study, vimentin antibody positive patients were screened for the presence of vimentin-specific self-major histocompatibility complex class I-restricted CD8+ T cells. METHODS: Two peptide sequences from vimentin that bound HLA-A*0201 were identified and fluorochrome-labeled A*0201 tetramers with each peptide were constructed to screen for vimentin-specific T cells. RESULTS: Tetramer-binding CD8+ T cells were detected in peripheral blood lymphocytes from two of six patients after expansion by in vitro stimulation with peptide. Tetramer-binding T cells produced interferon-gamma in an antigen-specific fashion. No autoreactive T cells specific for vimentin were detected after peptide stimulation of T cells from eight healthy A*0201-positive volunteers. CONCLUSIONS: This finding is the first evidence of CD8+ T-cell-mediated autoimmunity in human transplant patients.     

Characterization of cytotoxic function of CMV-pp65-specific CD8+ T-lymphocytes identified by HLA tetramers in recipients and donors of stem-cell transplants

BACKGROUND: Human cytomegalovirus (CMV) is a ubiquitous herpesvirus that is an important complication of bone marrow and allogeneic stem-cell transplant (HSCT). CD8 T-lymphocytes have an important role in immunity against CMV, but correlation between antigen-specific subpopulations of these cells and protection are still unclear. METHODS: Flow analysis with fluorescently-conjugated human leukocyte antigen (HLA) class I tetramers (Tet) was used to investigate levels of CMV-specific CD8 T-lymphocytes in peripheral blood monocyte cells (PBMC) samples from HSCT donors and recipients and their ability to produce interferon (IFN)-gamma on stimulation with either CMV antigenic peptide or nonspecific mitogenic stimulation. Chromium release assays were used to evaluate ex vivo CMV-specific cytotoxicity associated with the PBMC samples. RESULTS: Use of Tet in conjunction with fluorescently conjugated anti-T-cell receptor (TCR) beta-chain variable (Vbeta) monoclonal antibodies indicated that the Vbeta repertoires associated with Tet cells seen in two HSCT recipients were similar to the Vbeta repertoires of the Tet cells in their HSCT donors. Significant ex vivo cytotoxicity against peptide-loaded targets was measured from several recipient samples after transplant. However, PBMC from the HSCT donors, even when containing populations of CMV-specific Tet cells capable of secreting IFN-gamma in response to peptide stimulation, possessed no ex vivo CMV-specific cytotoxicity. CONCLUSIONS: We hypothesize that in the setting of the reconstituting immune system of HSCT recipients, CMV reactivation may stimulate a functional change in CMV-specific CD8 T-lymphocytes, rendering them able to directly lyse target cells presenting CMV antigens without in vitro stimulation. These findings have important implications for development of vaccines designed to induce protective cellular immunity to CMV in transplant recipients.     

Decreased circulating CD4+CD25highFoxp3+ T cells during acute rejection in liver transplant patients

Background

CD4⁺CD25^highFoxP3⁺ regulatory T (Treg) cells have been implicated to maintain immunologic tolerance. They have been investigated in acute renal allografts rejection episodes (ARE). This study was performed to examine the frequency of peripheral blood (PB) CD4⁺CD25^highFoxP3⁺ Treg cells among liver transplantation patients with prior benign end-stage liver diseases in relation to ARE.

Methods

This prospective analysis of 55 patients who underwent liver transplantation from 2004 to 2009 did not include prisoners either as donors or recipients. PB was obtained from liver transplant patients longitudinally: pretransplantation, posttransplantation within 1 year, and at the time of an episode of ARE to measure by flow cytometry circulating CD4⁺CD25^highFoxP3⁺ T cells. Blood samples were drawn during ARE with concomitant liver biopsies. The rejector group was defined in the 14/55 cases who suffered an ARE; in the other patients with stable liver function were classified as the nonrejector group. We compared the number of circulating CD4⁺CD25^highFoxP3⁺ T cells between the 2 groups.

Results

There was no difference in the levels of circulating CD4⁺CD25^highFoxP3⁺ T cells pretransplantation. Interestingly, circulating CD4⁺CD25^highFoxP3⁺ T cells were significantly lower among the rejector compared with the nonrejector (2.23 ± 0.54% vs 2.99 ± 0.86%; P < .01). Longitudinal analysis revealed circulating CD4⁺CD25^highFoxP3⁺ T cells of patients in the rejector group to be significantly lower during rejection than during quiescence (2.23 ± 0.54% vs 3.68 ± 0.70%; P < .0001). The frequency of circulating CD4⁺CD25^highFoxP3⁺ T cells negatively correlated with a Rejection Activity Index (r = −0.80; P < .01).

Conclusion

Monitoring peripheral CD4⁺CD25^highFoxP3⁺ T cell levels may be useful to evaluate the immune state, potentially acting as a sensitive marker for ARE diagnosis among liver transplantation patients. Moreover, they may contribute to the mechanisms of Treg-mediated acceptance of liver transplantations.     

Cross-reactivity of cytomegalovirus-specific CD8+ T cells to allo-major histocompatibility complex class I molecules

BACKGROUND: In transplantation settings, cytomegalovirus (CMV) infection is a common complication. CMV infection is associated with a higher incidence of graft rejection in solid organ transplantation and graft-versus-host disease in bone marrow transplantation. The underlying mechanism of this association could be the generation of CMV-specific CD8 T cells capable of cross-reacting with alloantigens present on graft and host, respectively. METHODS: Whereas as to date, no direct ex vivo analysis can be performed of the CD8 T-cell repertoire directed at allo-major histocompatibility complex (MHC) class I molecules, virus-specific cells can be readily enumerated by use of MHC-peptide tetrameric complexes. In this study, the authors used this technique to analyze potential overlapping CD8 T-cell repertoires between self-MHC-viral peptide and allo-MHC complexes by stimulating CMV-specific CD8 T cells with alloantigens. RESULTS.: The authors found that CMV-specific CD8 T cells are activated and proliferate on stimulation with alloantigens. CONCLUSIONS: Although these cells are cytotoxic against CMV-peptide pulsed target cells, no cytotoxicity of CMV-specific cells to alloantigens could be detected, inferring that there are other mechanisms of graft damage by alloantigen-stimulated virus-specific CTL.     

EBV-specific memory CD8+ T cell phenotype and function in stable solid organ transplant patients

Immune responses to EBV in immunosuppressed (IS) solid organ transplant (SOTx) recipients have not been well characterized. Here we evaluate the phenotype and function of EBV-specific CD8+ T cells in peripheral blood isolated from "stable" IS SOTx recipients. The EBV-specific CD8+ T cell memory subset distribution in the peripheral blood of patients was examined by flow cytometric analysis using HLA-A2 tetramers incorporating BMLF1 (lytic), and LMP2 and EBNA3A (latent)-derived peptides, in conjunction with mAbs against the CD45RO, CD45RA, and CD62L markers. The ability of CD8+ T cells to produce IFN-gamma in response to the same EBV-derived peptides was measured by ELISPOT assay. Patients and healthy normal donors exhibited similar anti-EBV CD8+ T cell frequencies and specificities against the EBV epitopes evaluated. When compared to healthy normal donors, an overall significant expansion of the CD8+ T cell "effector memory" (CD45RO+/CD62L-) pool, including that of EBV "latent" (LMP2 and EBNA3A)-specific CD8+ T cells was detected in IS SOTx patients. However, the patients' EBV-specific CD8+ T cells showed decreased IFN-gamma production to the EBV-peptide stimulation. These results indicate that the impairment of EBV-specific CD8+ T cell activity is not due to clonal depletion, but is mainly due to impaired functional activation.     

CD4dullCD8bright double-positive T-lymphocytes have a phenotype of granzyme Bpos CD8pos memory T-lymphocytes.

BACKGROUND: T-lymphocytes that co-express CD4 and CD8 antigens may be found in small percentages in the peripheral blood of healthy individuals, and have a CD4brightCD8dull phenotype. CD4dullCD8bright T-lymphocytes have been found only in temporal association with some viral infections. METHODS: Four-colour flow cytometric analysis of peripheral blood mononuclear cells from a renal transplant recipient with cytomegalovirus infection was performed. RESULTS: A small but clearly distinguishable subpopulation of CD4dullCD8bright double-positive T-lymphocytes was detected, that exhibited phenotypic characteristics of cytotoxic T-lymphocytes and were granzyme B positive. Furthermore, no naive cells appeared to be present within this subpopulation. CONCLUSIONS: CD4dullCD8bright double-positive T-lymphocytes are enriched for memory and effector cytotoxic T cells.     

Effect of hormone replacement therapy on CD4+ and CD8+ numbers, CD4+/CD8+ ratio, and immunoglobulin levels in hemodialysis patients

Uremia induces a suppression of the immune status. A large clinical literature suggests that estradiol (E2) plays a critical role in immune function. A large proportion of women hemodialysis patients faced early menopause and inadequate estrogen levels. The aim of the present study is to evaluate the effect of hormone replacement therapy on immune function in terms of CD4+ numbers (inducer/helper T cells), CD8+ numbers (cytotoxic/suppressor T cells), CD4+ / CD8+ ratio, and IgG, IgM, IgA levels in woman hemodialysis patients. In our study, 15 female hemodialysis patients (median age 32.6 range 24-45) were treated with triphasic estrogen/progesterone preparation (estradiol 2 mg for 10 days, and afterwards estradiol 2 mg+norethisterone 1 mg for another 10 days, and at the end estradiol 1 mg for 6 days) for 6 months. CD4+ numbers, CD8+ numbers, and IgG, IgA, and IgM levels were determined before and after HRT. The "paired-samples T" test was used for statistical analysis of pretreatment and posttreatment values. A significant increase was observed for CD4+ numbers (582 +/- 435 versus 637 +/- 445, p = 0.04) and CD4+/CD8+ ratio (1.4 +/- 0.16 to 2.4 +/- 0.3, p < 0.01) after hormone replacement therapy (HRT). Serum immunoglobulin levels were not changed significantly. In conclusion, in postmenopausal hemodialysis patients, HRT significantly increased CD4+ numbers and CD4+ / CD8+ ratio, but no effect was observed in IgM, IgG, and IgA levels. Long-term clinical effects of HRT on immune system should be investigated in dialysis patients with further studies.     

Prognostic value of cytotoxic T-lymphocytes and CD40 in biopsies with early renal allograft rejection

After renal transplantation, different immunological and non-immunological factors lead to long-term allograft deterioration. Acute rejection episodes are one risk factor for chronic renal allograft dysfunction (CRAD). Following the current Banff classification the histological grade in acute rejection episodes is of limited prognostic value, therefore, additional morphological surrogate markers would be helpful. We investigated the biopsies of 91 patients with early acute rejection episodes for the immunohistochemical expression of key molecules (perforin, granzyme B, TIA-1, CD40) in the T cell-mediated rejection process. Staining results were correlated to long-term allograft outcome. Patients with greater than 2% of granzyme B or greater than 25% of CD40-positive cells in the interstitial infiltrate showed significantly shorter allograft survival. Patients with a CD40-positive vascular rejection or greater than 2% of granzyme B-positive cells in the interstitial infiltrate were significantly correlated with an earlier onset of CRAD. Our findings provide potential morphological surrogate markers in biopsies with early acute rejection episodes after renal transplantation. These could become part of combined clinical and histological algorithms, allowing patient-specific risk estimation and customized therapy options to be made.     

Kinetic analysis reveals potency of CD4+ CD25bright+ regulatory T-cells in kidney transplant patients

Donor-specific hyporesponsiveness as occurs after allogeneic kidney transplantation may be mediated by repression of effector cells by a specific subset of T-cells: the CD4(+) CD25(bright+) FoxP3(+) regulatory T-cells (Tregs). Here, we examined the suppressive capacity of Tregs isolated from the leukafereses product of 6 kidney transplant recipients, by reconstituting Tregs to responder T-cells at several time-points after initiation of proliferation. We show that Tregs derived from kidney transplant patients potently restrain proliferation to donor-antigens and 3rd party-antigens in classic reconstitution assays (i.e. addition of Tregs at the start of the co-incubation). However, when Tregs were added 5 days after initiation of proliferation, they were still capable of suppressing proliferation to donor-antigens (by 38%) but no longer to 3rd party-antigens. Thus, we conclude that the potency of Tregs to suppress reactivity to specific antigens should be determined by reconstitution to ongoing reactions.     

Characterization of CMVpp65-specific CD8+ T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants

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.     

CD8+ T cells contribute to the development of transplant arteriosclerosis despite CD154 blockade

BACKGROUND: The CD40-CD154 receptor-ligand pair plays a critical role in allograft rejection by mediating the activation of endothelial cells, antigen-presenting cells, and T cells. Blockade of this interaction prevents acute allograft rejection and leads to prolonged allograft survival in numerous experimental models, but in most cases indefinite graft survival is not achieved due to evolving transplant arteriosclerosis. In this study, we have used a model of transplant arteriosclerosis to investigate whether CD4+ and CD8+ T cells are differentially affected by CD154 blockade. METHODS: BALB/c (H2d) aortic grafts were transplanted into C57BL/6 (H2b) recipients treated with anti-CD154 monoclonal antibody in the presence or absence of CD8+ T-cell depletion. Histology and morphometric measurements were performed on day 30 after transplantation. RESULTS: Only combined treatment with anti-CD154 and anti-CD8 monoclonal antibodies resulted in a significant reduction of intimal proliferation (33 +/-10% vs. 67+/-14%; untreated control). Administration of either antibody alone did not produce this effect. Thymectomy did not alter the degree of intimal proliferation observed in any of the treatment groups. CONCLUSIONS: Our data provide direct evidence that CD8+ T cells are not targeted effectively by CD154 blockade and that the transplant arteriosclerosis seen after CD154 blockade is not due to recent thymic emigrant T cells.