Human gene marker/therapy clinical protocols.
Explore the source record for details and available documents.
Clinical Protocols↗
Biomedical subjects
S R Riddell
Publications and source records attributed to S R Riddell.
Isolation of tyrosinase-specific CD8+ and CD4+ T cell clones from the peripheral blood of melanoma patients following in vitro stimulation with recombinant vaccinia virus.
The identification of Ags expressed by tumor cells and recognized by autologous T cells has led to the prospect of treating cancer by adoptive transfer of tumor-reactive T cells selected for Ag specificity. Tyrosinase is an Ag expressed by normal melanocytes as well as melanoma cells for which responses by autologous T cells have been detected. To evaluate the frequency with which tyrosinase-specific T cells can be isolated from melanoma patients for potential use in therapy, a recombinant vaccinia virus expressing tyrosinase was constructed for infection of autologous APCs that could be used to stimulate T cells reactive with this protein. Eight patients were studied, with peripheral blood serving as the source of both responder T cells and autologous APCs. Tyrosinase-specific CD8+ CTL clones were isolated from five of the eight patients with melanoma. The tyrosinase-specific CTL generated in this manner recognized autologous tumor cells as well as targets expressing the recombinant virus vector. CTL clones from three of the individuals were restricted to HLA-A28, -B8, and -B60, which have not previously been identified as alleles that can present immunogenic tyrosinase peptides. Tyrosinase-specific CD4+ T cell clones were isolated from six of the eight patients by stimulation with autologous APCs infected with recombinant vaccinia virus, and all these CD4+ clones were capable of recognizing autologous tumor cells. These studies demonstrate a high prevalence of CD4+ and CD8+ tyrosinase-specific responses in peripheral blood and support the feasibility of using peripheral blood to generate T cells for tumor therapy without the requirement for isolating T cells that have infiltrated tumor sites.
Animals↗
Cytomegalovirus selectively blocks antigen processing and presentation of its immediate-early gene product.
Recognition of virus-infected cells by CD8+ cytotoxic T lymphocytes requires that the viral proteins be processed into peptides, the derived peptides transported into the endoplasmic reticulum and inserted into the binding groove of a major histocompatibility complex class I molecule, and the antigenic complex exported to the cell surface. However, viral pathogens can disrupt this process and interfere with immune recognition. These mechanisms may be vital to large viruses such as human cytomegalovirus (CMV), which causes persistent infection despite producing over 200 potentially antigenic proteins during the sequential immediate-early, early and late phases of viral gene expression. Products of CMV early-phase gene expression can globally block class I presentation and prevent recognition of infected cells by cytotoxic T lymphocytes, but an essential viral transcription factor, the 72K principal immediate-early protein, is abundantly expressed before this blockade. However, only a few host CD8+ cytotoxic T lymphocytes specific for immediate-early protein are present in seropositive individuals, and these lyse CMV-infected cells poorly. Here we demonstrate selective abrogation of immediate-early peptide presentation by a CMV matrix protein with associated kinase activity and suggest that modification of a viral protein can result in limiting access to the processing machinery and evasion of cytotoxic-T-cell recognition.
Antigen Presentation↗
Human gene marker/therapy clinical protocols.
Explore the source record for details and available documents.
Clinical Protocols↗
T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients.
The introduction and expression of genes in somatic cells is an innovative therapy for correcting genetic deficiency diseases and augmenting immune function. A potential obstacle to gene therapy is the elimination of such gene-modified cells by an immune response to novel protein products of the introduced genes. We are conducting an immunotherapy trial in which individuals seropositive for human immunodeficiency virus (HIV) receive CD8+ HIV-specific cytotoxic T cells modified by retroviral transduction to express a gene permitting positive and negative selection. However, five of six subjects developed cytotoxic T-lymphocyte responses specific for the novel protein and eliminated the transduced cytotoxic T cells. The rejection of genetically modified cells by these immunocompromised hosts suggests that strategies to render gene-modified cells less susceptible to host immune surveillance will be required for successful gene therapy of immunocompetent hosts.
Antigen Presentation↗
Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.
BACKGROUND: Cytomegalovirus (CMV) disease in immunocompromised patients correlates with a deficiency of CD8+ cytotoxic T lymphocytes specific for CMV. We evaluated the safety and immunologic effects of immunotherapy with clones of these lymphocytes in recipients of allogeneic bone marrow transplants. METHODS: Clones of CD8+ cytotoxic T cells specific for CMV proteins were isolated from the blood of bone marrow donors. Fourteen patients each received four intravenous infusions of these clones from their donors beginning 30 to 40 days after marrow transplantation. The reconstitution of cellular immunity against CMV was monitored before and during the period of infusions and for up to 12 weeks after the final infusion. The rearranged genes encoding the T-cell receptor served as markers in evaluating the persistence of the transferred T cells. RESULTS: No toxic effects related to the infusions were observed. Cytotoxic T cells specific for CMV were reconstituted in all patients. In vitro measurements showed that cytotoxic activity against CMV was significantly increased (P < 0.001) after the infusions in 11 patients who were deficient in such activity before therapy. The level of activity achieved after the infusions was similar to that measured in the donors. Analysis of rearranged T-cell-receptor genes in T cells obtained from two recipients indicated that the transferred clones persisted for at least 12 weeks. Cytotoxic-T-cell activity declined in patients deficient in CD4+ T-helper cells specific for CMV, suggesting that helper-T-cell function is needed for the persistence of transferred CD8+ T cells. Neither CMV viremia nor CMV disease developed in any of the 14 patients. CONCLUSIONS: The transfer of CMV-specific clones of CD8+ T cells derived from the bone marrow donor is a safe and effective way to reconstitute cellular immunity against CMV after allogeneic marrow transplantation.
Adolescent↗
Cellular adoptive immunotherapy after bone marrow transplantation.
Explore the source record for details and available documents.
Animals↗
Principles for adoptive T cell therapy of human viral diseases.
The development of successful adoptive immunotherapy for human virus infections is predicated on an understanding of the effector cells and mechanisms essential for providing the host with a protective response to acute infection and the requirements for long-term in vivo survival of transferred cells that will be necessary to provide memory responses to persistent and latent viral infections. In this review, we discuss the results of recent studies examining the effector mechanisms mediated by virus-specific alpha beta + T cells and the strategies viruses have evolved to evade recognition by such T cells and/or to interfere with the expression of T cell effector functions. The evasion strategies employed by individual viruses can render T cell subsets or T cells of particular specificities less effective in eliminating virally infected cells, and consequently they are less desirable choices for use in adoptive therapy. Insights derived from described studies of the pathogenesis and immunobiology of virus infections have resulted in the development of clinical adoptive immunotherapy studies for infections with CMV, EBV, and HIV. Although the results from such studies are preliminary, the principle that virus-specific T cells can be successfully transferred and can mediate therapeutic efficacy in humans has already been affirmed. The use of recently developed methods, such as retroviral-mediated gene transfer, to genetically modify antigen-specific T cell clones provides a novel approach to overcome limitations and improve on the safety and efficacy observed in these initial studies, suggesting that more widespread use of adoptive transfer of specific T cells as a therapeutic regimen should be feasible in the near future.
Animals↗
Pathogenesis of cytomegalovirus pneumonia in immunocompromised hosts.
Cytomegalovirus (CMV) is capable of establishing persistent or latent infection after primary infections and reactivation in immunocompromised hosts. The understanding of the nature of this latency is incomplete. The acquisition of CMV-specific immune responses appears essential for limiting primary infection and maintenance of a clinical state of latency. Host immunity induced by CMV infection includes both cellular and humoral responses. Cell-mediated responses by both CD4+ (T helper) and CD8+ cytotoxic T lymphocytes (CTLs) are detected in CMV-seropositive individuals. Studies in animal models and in immunocompromised patients have implicated deficiencies in class I MHC-restricted, CD8+CMV-specific CTL responses in the progression of experimental and human CMV infection and pneumonia. Adoptive transfer of CD8+CMV-specific CTLs in immunodeficient marrow transplant recipients provides evidence that adoptive immunotherapy with T-cell clones of defined specificity and function can be used to safely and successfully restore immunity to a human pathogen.
Acquired Immunodeficiency Syndrome↗
A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes.
Herpes simplex virus (HSV) infection of human fibroblasts rapidly renders the cells resistant to lysis by HSV-specific CD8+ cytotoxic T lymphocytes (CTLs), which normally recognize cell surface major histocompatibility complex (MHC) class I proteins presenting viral peptides. Within 3 hr of infection with HSV, MHC class I protein complexes are retained in the endoplasmic reticulum (ER)/cis Golgi and show properties of complexes lacking antigenic peptide. The HSV immediate-early protein ICP47 is both necessary and sufficient to block transport of class I proteins and to inhibit lysis by CD8+ CTLs. The target for ICP47 is not known, but since ICP47 does not associate with membranes, it appears that ICP47 inhibits the production or stabilization of antigenic peptides or their translocation into the ER/cis Golgi. Thus, by expressing ICP47, HSV can evade detection by CD8+ T lymphocytes, perhaps explaining the predominance of CD4+ rather than CD8+ HSV-specific CTLs in vivo.
Amino Acid Sequence↗
Recovery of HLA-restricted cytomegalovirus (CMV)-specific T-cell responses after allogeneic bone marrow transplant: correlation with CMV disease and effect of ganciclovir prophylaxis.
Protection from cytomegalovirus (CMV) disease in immunocompromised hosts has been shown to correlate with recovery of the host virus-specific CD8+ T-cell response. The administration of ganciclovir to immunosuppressed transplant recipients as antiviral prophylaxis has reduced the early risk of CMV disease, but late disease is observed with increased frequency, suggesting that recovery of the CMV-specific T-cell responses necessary for protective immunity may be delayed in these patients. Therefore, we evaluated reconstitution of CMV-specific T-cell responses in 47 bone marrow transplant (BMT) recipients entered on a randomized placebo-controlled study of ganciclovir. The study drug was initiated at a mean of 24 days after BMT. At day 30 to 40, a minority of patients had recovery of T-cell immunity to CMV and the frequency of reconstitution was equivalent in patients randomized to ganciclovir or placebo. The failure of ganciclovir to effect early reconstitution may reflect the short duration of treatment. Early recovery was associated with the infusion of BM from a CMV seropositive donor (P = .07 for CD8+ cytotoxic T cell (CTL), P = .04 for CD4+ Th). Between day 40 and day 90, recovery of deficient CD8+ and CD4+ CMV-specific T-cell responses occurred in the majority of individuals that received placebo, but in a minority of ganciclovir recipients. Two cases of late-onset CMV disease occurred in ganciclovir recipients. In all patients, the presence of a CTL response to CMV conferred protection from subsequent CMV disease (P = .005), and these protective CTL responses are shown to be specific for structural virion proteins similar to the responses in immunocompetent CMV seropositive individuals. These data confirm the importance of CMV-specific T-cell responses and suggest that a delay in recovery of these responses as a result of ganciclovir prophylaxis may contribute to the occurrence of late CMV disease.
Adolescent↗
Genetic modification of T cell clones to improve the safety and efficacy of adoptive T cell therapy.
Our laboratory has developed methods to isolate human antigen-specific cytolytic CD8+ T cell clones and to expand such clones in vitro to numbers sufficient for T cell therapy of human diseases. Studies in immunocompromised bone marrow transplant patients at high risk for disease associated with cytomegalovirus have demonstrated that administration of more than 10(9) CD8+ T cell clones is safe and can effectively reconstitute a deficient human immune response. Our laboratory is applying this strategy of adoptive therapy to the treatment of human cancer, starting with the subset of patients with Hodgkin's disease who show expression of proteins encoded by the Epstein-Barr virus in their malignant Reed-Sternberg cells. The development of efficient systems such as retroviral vectors for the introduction of genes into primary cells has made it possible to consider overcoming some of the limitations of the effector T cells that normally mediate response to an antigen. Our laboratory is attempting to modify T cell clones by the introduction of genes before transfer as a means to improve the safety and/or efficacy of T cell therapy.
Antigens, Viral↗
Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes.
Primary cytomegalovirus (CMV) infection and reactivation of persistent CMV are associated with significant morbidity and mortality in immunocompromised individuals. Although recovery from CMV disease is correlated with the development of CMV-specific cytotoxic T lymphocytes (CTL), the major viral target antigens to which the response is directed are ill-defined, though they may comprise viral structural elements. We now identify the CMV matrix protein pp65 as a significant target antigen for CD8+ class I major histocompatibility complex (MHC)-restricted CMV-specific CTL derived from the peripheral blood of four of five latently infected individuals. CMV-specific CTL recognition of pp65 on target cells occurs prior to the onset of viral gene expression and persists throughout the duration of the replicative cycle. Recognition in the absence of viral gene expression suggests that abundant viral protein enters the normal trafficking pathway upon viral penetration and is readily made available to MHC molecules for presentation at the cell surface. Thus pp65 specific CTL may represent an important effector population for early control and limitation of CMV infection and disease. The observation that CMV-specific CTL can be induced in vitro using peptide fragments derived from pp65 supports the future use and manipulation of this and similar effector populations in a clinical setting.
Antigens, Viral↗
Therapeutic reconstitution of human viral immunity by adoptive transfer of cytotoxic T lymphocyte clones.
Explore the source record for details and available documents.
Animals↗
Selective reconstitution of CD8+ cytotoxic T lymphocyte responses in immunodeficient bone marrow transplant recipients by the adoptive transfer of T cell clones.
The adoptive transfer of T cells specific for antigens encoded by pathogens and tumors has been an effective treatment for infections and malignancies in animal models and is potentially applicable for infections occurring in immunodeficient bone marrow transplant recipients. This article reviews recent insights derived from studies of the immunobiology of human cytomegalovirus (CMV) infection in healthy and immunodeficient hosts and the development of adoptive immunotherapy as prophylaxis for CMV infection in recipients of allogeneic bone marrow transplants.
Animals↗
Alkali hydrolysis of recombinant proteins allows for the rapid identification of class I MHC-restricted CTL epitopes.
The characterization of the epitopes recognized by CTL provides insights into the nature of protective immune responses and facilitates the development of methods to enhance immunity to human pathogens. However, no easily applicable approach for CTL epitope identification has been developed. We present a rapid and efficient method for locating CTL epitopes within a protein. The gene encoding the protein of interest is inserted into an inducible prokaryotic expression vector. Random peptides are then generated by alkali digestion of intact or lysed Escherichia coli expressing the protein and assayed for the presence of the epitope by coating target cells for a standard CTL targeting assay. A large panel of clones containing serial 3'-deletions of the gene is then generated by exonuclease III digestion, and the expressed truncated proteins are similarly analyzed for the presence of the antigenic peptide. The epitope is located by determining the deletion points of clones expressing sequential truncations and differing in Ag expression. This technique was used to identify the H-2Ld-restricted nonamer in E. coli beta-galactosidase, with residues 876-884 representing the naturally processed epitope. To test the applicability of this method to other proteins, two genes from human CMV, an often fatal pathogen in immunocompromised individuals, were screened for HLA class I-restricted epitopes. An HLA-B18-restricted epitope from the CMV major immediate-early protein was found to lie between residues 378 and 389, and an HLA-B35-restricted epitope from the CMV pp65 matrix protein was characterized as residues 123 to 131. The results demonstrate that this technique can be used to rapidly identify CTL epitopes within a chosen protein and should be useful for assaying viral isolates or neoplasms for loss of epitopes after mutation and selection by host immune responses.
Amino Acid Sequence↗
CD8+ cytotoxic T cell therapy of cytomegalovirus and HIV infection.
The development of CD8+ cytotoxic T cell responses to viral pathogens is crucial for the prompt resolution of acute infections and for the control of viruses which persist in the host. Thus, cytomegalovirus often causes life threatening disease in immunosuppressed humans who fail to develop or maintain CD8+ cytotoxic T cells. Similarly, the loss of CD8+ cytotoxic T cell responses to HIV correlates with the development of AIDS. Recent investigations in the immunobiology of cytomegalovirus and HIV have resulted in the application of immunotherapeutic strategies designed to reconstitute or augment deficient CD8+ cytotoxic T cell responses to these human pathogens.
Acquired Immunodeficiency Syndrome↗
Selective interference with class I major histocompatibility complex presentation of the major immediate-early protein following infection with human cytomegalovirus.
Responses of cytotoxic T-cells (Tc) to human cytomegalovirus (CMV) represent the predominant mechanism by which hosts resist CMV infection. The CMV major immediate-early protein (IE) is present throughout the virus replicative cycle. Studies were performed to determine whether Tc specific for IE effectively lyse CMV-infected targets and are thus capable of providing protective immunity against infection. After in vitro stimulation of peripheral blood mononuclear cells with CMV-infected autologous fibroblasts, Tc specific for IE were not readily detectable in CMV-reactive polyclonal Tc lines. However, after stimulation of peripheral blood mononuclear cells with cells selectively expressing IE, weak but detectable IE-specific Tc responses were observed. The frequency of IE-specific Tc clones derived from cultures stimulated with IE-expressing cells was 50 to 100 times lower than the frequency of Tc clones specific for other CMV proteins isolated from cultures stimulated with CMV-infected cells. All of the IE-specific Tc clones, which efficiently lysed targets selectively expressing IE, demonstrated minimal lysis of CMV-infected fibroblasts, despite abundant IE expression in these target cells. In contrast to these results with IE, other viral proteins were efficiently presented during all phases of CMV infection. These data suggest that CMV has evolved a unique mechanism for selectively limiting the presentation of the potentially immunogenic IE protein, which may preclude IE-specific Tc from providing protective immunity to CMV infection.
Antigen-Presenting Cells↗