CMV-specific Cytotoxicity Research Articles

Comparative Study of the Influence of Proteasome Inhibitor MG132 and Ganciclovir on the Cytomegalovirus-Specific CD8+T-Cell Immune Response

Cytomegalovirus(CMV) reactivation causes immunopathy, graft malfunction, and even rejection. The traditional anti-CMV drug ganciclovir is not able to prevent reactivation of endogenous virus. Recent studies have found that proteasome inhibitor (PI) is able to suppress CMV replication. In this study we investigated the influence of proteasome inhibitor MG132 and ganciclovir on the CMV-specific CD8(+) T-cell immune response. We found that interferon-γ (IFN-γ) production in response to CMV-infected fibroblasts was reduced under the influence of MG132 in a dose-dependent manner. A marked reduction was observed at 0.5 μM. Likewise, CMV-specific cytotoxicity of CD8(+) T cells was decreased in the presence of MG132. In contrast, the traditional CMV replication inhibitor ganciclovir (10 μM) had no such effect. These findings might have important implications in reducing CMV-associated immunopathy by altering epitope generation through the application of selective proteasome inhibitors.

Generation of WT1-specific CTL from CMV stimulated CD8 cells (41.32)

Abstract WT1 responsive CTL were generated from CMVpp65 stimulated CD8 cells isolated from three CMV-seropositive HLA-A2 normal donors. CD8 selected T cells were stimulated with dendritic cells pulsed with CMVpp65 overlapping peptides. 10 days after the primary stimulation, CD8 T cells were restimulated with CMV pp65 peptide pulsed PBMC. The CMV-stimulated CTL exhibited CMV-specific but not WT1-specific cytotoxicity after the second stimulation as measured by 51CR release assay (Donor 1=54% and 7%, Donor 2=77% and 14%). However, all CMV-stimulated CTL stained with PE-RMFPNAPYL, a WT1A2 tetramer (Donor 1=2.51%, Donor 2=3.85%, Donor 3=0.49%). EBV-CTL generated from an HLA-A2 donor, and WT1 CTL generated from a non-HLA-A2 donor did not stain with the WT1A2 tetramer (0.02% and 0.01% respectively). Therefore, the WT1A2 tetramer was cross-reactive with pp65-CTL and exhibited HLA-A2 specificity. To enrich the CMV-CTL WT1A2 tetramer staining population, we PE-selected donor 1 PE-WT1 tetramer binding pp65 stimulated CTL. The WT1-selected cells were cloned by limiting dilution. 7 of 8 WT1-selected clones stained with the WT1A2 tetramer ranging from 0.89%-3.56%. WT1-selected clones showed three types of specific cytotoxicity: 1) against pp65-only (3 clones=22-34%), 2) against WT1-only (2 clones=43-44%), or 3) against both WT1 and CMV (1 clone=55% and 24% respectively). 2 of the 8 clones did not exhibit either WT1 or CMV specific cytotoxicity. Only 1 of the 2 clones that exhibit WT1 specific cytotoxicity exhibited WT1-specifc IFN-γ production, as measured by ELISPOT. All clones that displayed CMV-specific cytotoxicity also exhibited CMV-specific IFN-γ production. To our knowledge this is the first description of WT1 cross-reactive, CMV-specific CTL.

INFUSION OF CYTOMEGALOVIRUS SPECIFIC CYTOTOXIC T LYMPHOCYTES FROM A SERO-NEGATIVE DONOR CAN FACILITATE RESOLUTION OF INFECTION AND IMMUNE RECONSTITUTION

We report a stem cell transplant patient with a therapy-refractory cytomegalovirus (CMV) infection who received CMV-specific T cells from his sero-negative stem cell donor. This donor received the Towne strain CMV vaccine, and T cells were expanded using monocytes pulsed with pp65 overlapping peptides. CMV DNA decreased after the CTL infusion, and CMV-specific cytotoxicity increased. This strategy could be implemented in similar situations or with persistent viremia post-transplant.

Rapid Generation of CMV pp65-specific T cells for Immunotherapy

Adoptive immunotherapy with cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) has been shown to be an effective means of restoring cellular immunity to this virus and preventing CMV infection after allogeneic stem cell transplantation. Problems with current strategies include requirements for generating dendritic cells or other antigen presenting cells for stimulating CTL and the time needed for cell culture. The adherent cell fraction of peripheral blood mononuclear cells from 6 CMV seropositive donors were pulsed with pooled CMV pp65 peptides and incubated with nonadherent peripheral blood lymphocytes. CTL lacking specific cytotoxicity to pp65 were restimulated at day 10 of culture using peptide pulsed adherent cells. Of the 6 CMV seropositive donors tested, 5 had specific cytotoxicity to CMV pp65 (range 31% to 75%), with no alloreactivity. The resulting pp65-specific CTL consisted of a mixture of CD4 and CD8 cells, with 1% to 29% of CD8 cells and 0.5% to 10% CD4 cells making interferon-gamma (IFN-gamma) in response to pp65. The donor from whom we could not detect CMV-specific cytotoxicity had detectable CD4 and CD8 CMV pp65 CTL by intracellular cytokine analysis for IFN-gamma. Using this simplified strategy for expanding CMV pp65 CTL, adoptive immunotherapy with pp65-specific CTL could be made available in a more timely manner for patients who have persistent or therapy refractory CMV infections.

In vitro priming and expansion of cytomegalovirus-specific Th1 and Tc1 T cells from naive cord blood lymphocytes

Adoptive transfer of CMV-specific cytotoxic T cells (CTLs) expanded in vitro from memory donor T cells can reduce the incidence of CMV disease in allogeneic transplant recipients. However, this approach has been unavailable in the cord blood (CB) transplantation setting because CB T cells are antigen naive and biased toward Th2/Tc2 function. We developed a protocol to in vitro prime and expand CMV-specific CTLs from CB. T cells were primed with cytokines to trigger skewing toward Th1/Tc1 lineage before encountering monocyte and CD34+ progenitor-derived dendritic cells loaded with CMV antigen and its immune complex. CMV-pulsed cultures expanded significantly more over 4 to 6 weeks than CMV cultures despite identical cytokine milieu. T cells isolated from CMV+ cultures showed a preferential expansion of CD45RA-/RO+/CD27+ T cells compared to CMV- cultures. CMV-specific IFN-gamma- and TNF-alpha-producing CD4+ (Th1) and CD8+ (Tc1) T cells were enriched after 3 to 4 weeks and CMV-specific cytotoxicity developed 1 to 2 weeks later.

The Functional Activity of Genetically Engineered T Cell Receptor Transferred T Cells Is Highly Dependent on Pairing Properties of the Transferred TCR α and β Chains.

The genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T cell immunity towards viral infections and malignancies. Transfer of virus- or tumor-specific TCRs has demonstrated to endow T cells with redirected antigen specificity. We demonstrated redirected anti-leukemic reactivity of CMV specific T cells using gene transfer of minor histocompatibility antigen HA-2 specific TCRs. The HA-2-TCR-modified T cells exerted high cytolytic activity against HA-2 expressing target cells, including leukemic cells, and not against target cells negative for the HA-2 mHag. After cloning of the TCR-transferred T cells, we demonstrated that the HA-2-TCR cell surface expression, measured by HA-2-tetramer staining, was variable on the transduced T cell clones, and that the cytolytic capacity of the T cells correlated with the level of HA-2-TCR expression.Since we could demonstrate that this variation in HA-2-TCR expression was not due to differences in transgene expression, we investigated whether the endogenous TCRs influenced the expression of the introduced TCR. CMV-A2 specific T cells were isolated from peripheral blood and transduced with the HA-2-TCR. In control transduced CMV specific T cells we observed 5 different high affinity CMV specific TCRs. CMV specific T cells transduced with the HA-2-TCR that expressed predominantly the HA-2-TCR, expressed only one of these types of CMV-TCR, and in CMV specific T cells with low HA-2-TCR expression two different types of CMV-TCRs were found. These data indicated that the level of expression of the introduced TCR is strongly influenced by the endogenous TCR. To investigate whether this was due to differences in promotor activity of the endogenous and retrovirally introduced TCR, the three CMV-TCRs were characterized and transferred into unselected peripheral T cells. T cells transferred with the weak competitior CMV-TCR that was strongly downregulated in CMV specific T cells by introduction of the HA-2-TCR, showed low CMV specific cytotoxicity and no tetramer staining. In contrast, T cells transferred with the strong competitor CMV-TCR that was modestly downregulated in CMV specific T cells by introduction of the HA-2-TCR, revealed strong CMV specific cytotoxic activity and tetramer staining. These data demonstrate that the introduced and endogenous TCRs compete for cell surface expression, and that this competition is dependent on characteristics of the different TCRs and independent of whether the TCR is retrovirally introduced or naturally expressed. To investigate whether the cell surface expression of the different TCRs was determined by preferential pairing properties of the individual TCR chains, TCR α and β deficient Jurkat 76 cells were transduced with the three CMV-specific TCRs or with chimeric TCRs consisting of the TCR α chain of one TCR with the TCR β chain of another TCR. TCRαβ membrane expression revealed that TCRs with a strong competitor phenotype expressed higher levels of TCRαβ than the TCR that was a weak competitor. TCRαβ expression of Jurkat cells transduced with chimeric TCRs indicated that the expression level of the different TCRs was determined by the pairing properties of the individual TCR α and β chains and not by differences in protein expression. In conclusion these data demonstrated that introduced and endogenous TCRs compete for cell surface expression in favor of the TCR that has the highest intrinsic pairing properties.

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

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. 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. 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. 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.

Simultaneous Ex Vivo Expansion of Cytomegalovirus and Epstein-Barr Virus–Specific Cytotoxic T Lymphocytes Using B-Lymphoblastoid Cell Lines Expressing Cytomegalovirus pp65

AbstractCytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced lymphoproliferative disease are serious complications associated with allogeneic stem cell transplantation. Immunotherapy using ex vivo expanded, virus-specific cytotoxic T lymphocytes (CTL) has been explored and proven to be effective in therapeutic or prophylactic regimens for CMV and EBV infections. To generate CTL specific for both CMV and EBV, we engineered EBV-transformed B-lymphoblastoid cell lines (BLCL) to express CMV pp65 for use as antigen-presenting cells (APC). BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. Western blot analysis and immunocytochemistry confirmed the expression of pp65 in the transduced cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV seropositive donors were stimulated with autologous pp65-expressing BLCL weekly for 3 weeks. Chromium release assays showed that the resulting CTL cultures possessed specific cytotoxicity against EBV and CMV. Recombinant vaccinia viruses encoding individual CMV peptides were used to demonstrate that this CMV-specific cytotoxicity was specific for pp65. Assays on CD4- and CD8-depleted CTL fractions indicated that CD8+ CTL mediated the pp65-specific cytotoxicity. These CMV/EBV-specific CTL recognized CMV- and EBV-infected targets sharing HLA class I antigens, but not HLA mismatched targets. Our results demonstrate that BLCL can be used as APC to stimulate expansion of EBV- and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.

Simultaneous Ex Vivo Expansion of Cytomegalovirus and Epstein-Barr Virus–Specific Cytotoxic T Lymphocytes Using B-Lymphoblastoid Cell Lines Expressing Cytomegalovirus pp65

Cytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced lymphoproliferative disease are serious complications associated with allogeneic stem cell transplantation. Immunotherapy using ex vivo expanded, virus-specific cytotoxic T lymphocytes (CTL) has been explored and proven to be effective in therapeutic or prophylactic regimens for CMV and EBV infections. To generate CTL specific for both CMV and EBV, we engineered EBV-transformed B-lymphoblastoid cell lines (BLCL) to express CMV pp65 for use as antigen-presenting cells (APC). BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. Western blot analysis and immunocytochemistry confirmed the expression of pp65 in the transduced cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV seropositive donors were stimulated with autologous pp65-expressing BLCL weekly for 3 weeks. Chromium release assays showed that the resulting CTL cultures possessed specific cytotoxicity against EBV and CMV. Recombinant vaccinia viruses encoding individual CMV peptides were used to demonstrate that this CMV-specific cytotoxicity was specific for pp65. Assays on CD4- and CD8-depleted CTL fractions indicated that CD8+ CTL mediated the pp65-specific cytotoxicity. These CMV/EBV-specific CTL recognized CMV- and EBV-infected targets sharing HLA class I antigens, but not HLA mismatched targets. Our results demonstrate that BLCL can be used as APC to stimulate expansion of EBV- and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.

HLA-restricted cytotoxic T lymphocytes are an early immune response and important defense mechanism in cytomegalovirus infections.

Eighty-eight bone marrow transplant recipients were studied for development of cytomegalovirus (CMV) infections and associated cytotoxic lymphocyte responses. Sixty-one patients developed CMV infection that was diagnosed by virus isolation (18 patients), fourfold rises in serum antibodies (13), or both (30). Interstitial pneumonitis developed in 27 patients. Among patients tested during infection, HLA-restricted, CMV-specific cytotoxic T cell responses occurred in 25 patients, and non-restricted, non-T cell cytotoxic responses, or responses of undetermined cell type, occurred in 10 patients. Levels of CMV-specific cytotoxicity in infected and uninfected patients were compared in two ways. Eighty-nine percent of patients (32 of 36) with cytotoxicity greater than or equal to 15% lysis (50:1 effector-to-target-cell ratio) and 100% of patients (21 of 21) with sequential increases in cytotoxicity of greater than or equal to 15% lysis were infected. The median time of occurrence of CMV-specific cytotoxic responses preceded the median onset of viral shedding by 1.4 weeks, or rises in titers of serum antibody by 1.7 weeks, and of interstitial pneumonitis by 2.5 weeks. Immune competence, as demonstrated by survival from infection, was always associated with a CMV-specific cytotoxic response. The HLA-restricted cytotoxic T cell response appears to be important as an early host-defense mechanism and an early diagnostic evidence of infection.

Interleukin-2 enhances the depressed natural killer and cytomegalovirus-specific cytotoxic activities of lymphocytes from patients with the acquired immune deficiency syndrome.

The recently described acquired immune deficiency syndrome (AIDS) is characterized by the occurrence of severe opportunistic infections and an aggressive form of Kaposi's sarcoma. A variety of profound defects in cell-mediated immunity have been reported in association with the AIDS, including deficiencies in natural killer (NK) cell activity and cytomegalovirus (CMV)-specific cytotoxicity. In the present study, the in vitro effects of interleukin-2 (IL-2) and interferon beta (IFN Beta) on these abnormalities were examined to assess the potential use of these lymphokines in the immunotherapeutic treatment of this syndrome. The peripheral blood lymphocytes (PBL) from six male homosexuals with AIDS and an active CMV infection exhibited markedly depressed NK cell and CMV-specific cytotoxic lymphocyte responses compared with uninfected, heterosexual control subjects. Incubation of PBL with IFN Beta enhanced the NK cell activity and the CMV-specific cytotoxicity of only one of six and neither of two AIDS patients, respectively, while enhancing the NK cell activity of all six control subjects. In contrast, IL-2 dramatically enhanced both the NK cell and the CMV-specific cytotoxic lymphocyte activities of all of the patients. These results indicate that IL-2 can substantially potentiate the depressed cytotoxic effector functions of PBL from AIDS patients, while IFN Beta has little effect.