Quantifying Capsid Peptide:MHC I Complexes Following Adeno-Associated Virus (AAV) Transduction

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3737-3737
Author(s):  
Gary C. Pien ◽  
Nicole C. Hasbrouck ◽  
Marcela V. Maus ◽  
Federico Mingozzi ◽  
Katherine A. High
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Presentation ◽  
Mhc Class I ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Factor Ix ◽  
Class I ◽  
Adeno Associated Virus

Abstract Recent data from a clinical trial of adeno-associated virus (AAV) 2-mediated hepatic gene transfer of factor IX into hemophilia B subjects demonstrated the presence of AAV2-capsid-specific CD8+ T cells and identified an immunodominant AAV2 epitope. The expansion and contraction of this CD8+ T cell population coincided with the rise and fall of an asymptomatic elevation in liver transaminases, and correlated with the expression and subsequent loss of factor IX expression. These observations suggest that CD8+ T cells directed against AAV capsid antigen clear the transduced hepatocytes. Indeed, others in our laboratory have directly shown the lysis of AAV-transduced hepatocytes by CD8+ T cells in vitro. However, the stoichiometry and kinetics of AAV capsid degradation and processing for MHC class I presentation are presently unknown. We now report the generation of soluble AAV capsid-specific T cell receptor (TCR) tetramers and their use in quantifying capsid peptide:MHC I complexes presented on cell surfaces. CD8+ T cell clones specific for AAV2-derived peptide 74 (VPQYGYLTL) presented in the context of HLA-B*0702 were isolated and used to generate TCR cDNA. This cDNA was transfected into CHO cells to produce soluble TCR α and β chain fusion proteins. The soluble TCR was biotinylated, then tetramerized to streptavidin-conjugated fluorochrome for use in flow cytometric analyses. Cell lines expressing HLA-B*0702 were loaded with cognate AAV2 peptide, a cross-reactive AAV1 peptide (IPQYGYLTL), or irrelevant HLA-B*0702 epitopes derived from HIV-1 gp120 (IPRRIRGGL) or EBV nuclear antigen (RPPIFIRRL). Staining with TCR-tetramers reveals that this reagent binds AAV-derived peptides specifically. In addition, this reagent exhibits HLA-specificity, as cognate peptide loaded onto cells which express alternate HLA alleles was not detected. These results demonstrate that TCR-tetramers retain their antigen and MHC context specificities. Capsid peptide titration experiments using HLA-B*0702-bearing JYA2B7 and SK-MES-1 cell lines reveal that TCR-tetramers exhibit a limit of detection of approximately 105 cell surface peptide:MHC I complexes. Efficiency of detection is estimated at 1 peptide:MHC I complex for every 1010 peptide molecules added to cell culture. Preliminary studies indicate that the number of presenting complexes wanes by 48 hours following peptide loading. In contrast, AAV1-mediated transduction experiments demonstrate that capsid peptide:MHC I complexes are detectable after 48 hrs. In conclusion, these results are in agreement with our central hypothesis that AAV capsid is degraded upon transduction and gains access to MHC Class I antigen presentation pathways. Studies with the TCR-tetramers will allow definition of kinetics and capsid dose-dependence of antigen presentation, defining parameters of cell vulnerability to host immune-mediated clearance of transduced cells.

scholarly journals Conditional silencing of H-2Dbclass I molecule expression on dendritic cells modulates the protective and pathogenic kinetics of virus-antigen specific CD8 T cell responses during Theiler’s Virus infection

2019 ◽  
Author(s):  
Zachariah P. Tritz ◽  
Robin C. Orozco ◽  
Courtney S. Malo ◽  
Lila T Yokanovich ◽  
Katayoun Ayasoufi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Presentation ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Class I ◽  
Viral Control ◽  
Cell Responses

ABSTRACTTheiler’s murine encephalomyelitis virus (TMEV) infection of the central nervous system is rapidly cleared in C57BL/6 mice by an anti-viral CD8 T cell response restricted by the MHC class I molecule, H-2Db. While the CD8 T cell response against neurotropic viruses is well characterized, the identity and function of the antigen presenting cell(s) involved in this process is(are) less well defined. To address this gap in knowledge, we developed a novel C57BL/6 H-2Dbconditional knockout mouse that expresses an H-2Dbtransgene in which the transmembrane domain locus is flanked by LoxP sites. We crossed these H-2DbLoxP mice with MHC class I-deficient mice expressing Cre-recombinase under either the CD11c or LysM promoter in order to silence H-2Dbrestricted antigen presentation predominantly in dendritic cells or macrophages, respectively. Upon challenge with intracranial TMEV infection, we observe that CD11c+ APCs are critical for early priming of CD8 T cells against the immunodominant TMEV peptide VP2121-130 presented in the context of the H-2Dbmolecule. This stands in stark contrast to later time points post TMEV infection where CD11c+ APCs appear dispensable for the activation of antigen-specific T cells; the functionality of these late-arising antiviral CD8 T cells is reflected in the restoration of viral control at later time points. These late-arising CD8 T cells also retain their capacity to induce blood-brain barrier disruption. In contrast, when H-2Dbrestricted antigen presentation was selectively silenced in LysM+ APCs there was no overt impact on the priming of Db:VP2121-130 epitope-specific CD8 T cells, although a modest reduction in immune cell entry into the CNS was observed. This work establishes a model system which enables critical dissection of MHC class I restricted antigen presentation to T cells, revealing cell specific and temporal features involved in the generation of antiviral CD8 T cell responses. Employing this novel system, we established CD11c+ cells as a pivotal driver of acute, but not later-arising, antiviral CD8 T cell responses against the TMEV immunodominant epitope VP2121-130, with functional implications both for T cell-mediated viral control and immunopathology.

scholarly journals An antigen-specific pathway for CD8 T cells across the blood-brain barrier

2007 ◽  
Vol 204 (9) ◽  
pp. 2023-2030 ◽  
Author(s):  
Ian Galea ◽  
Martine Bernardes-Silva ◽  
Penny A. Forse ◽  
Nico van Rooijen ◽  
Roland S. Liblau ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class I ◽  
Blood Brain Barrier ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Class I ◽  
Cerebral Endothelium ◽  
Cognate Antigen ◽  
The Brain

CD8 T cells are nature's foremost defense in encephalitis and brain tumors. Antigen-specific CD8 T cells need to enter the brain to exert their beneficial effects. On the other hand, traffic of CD8 T cells specific for neural antigen may trigger autoimmune diseases like multiple sclerosis. T cell traffic into the central nervous system is thought to occur when activated T cells cross the blood-brain barrier (BBB) regardless of their antigen specificity, but studies have focused on CD4 T cells. Here, we show that selective traffic of antigen-specific CD8 T cells into the brain occurs in vivo and is dependent on luminal expression of major histocompatibility complex (MHC) class I by cerebral endothelium. After intracerebral antigen injection, using a minimally invasive technique, transgenic CD8 T cells only infiltrated the brain when and where their cognate antigen was present. This was independent of antigen presentation by perivascular macrophages. Marked reduction of antigen-specific CD8 T cell infiltration was observed after intravenous injection of blocking anti–MHC class I antibody. These results expose a hitherto unappreciated route by which CD8 T cells home onto their cognate antigen behind the BBB: luminal MHC class I antigen presentation by cerebral endothelium to circulating CD8 T cells. This has implications for a variety of diseases in which antigen-specific CD8 T cell traffic into the brain is a beneficial or deleterious feature.

scholarly journals The natural immune response to inhaled soluble protein antigens involves major histocompatibility complex (MHC) class I-restricted CD8+ T cell-mediated but MHC class II-restricted CD4+ T cell-dependent immune deviation resulting in selective suppression of immunoglobulin E production.

1993 ◽  
Vol 178 (3) ◽  
pp. 889-899 ◽  
Author(s):  
C McMenamin ◽  
P G Holt
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class I ◽  
Mhc Class Ii ◽  
Cd8 T Cells ◽  
Immunoglobulin E ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Class I

The immunological basis for atopy is currently ascribed to an inherent bias in the CD4+ T cell response to nonreplicating antigens presented at mucosal surfaces, resulting in dominance of the T helper 2 (Th2) interleukin 4 (IL-4)-producing phenotype, which favors IgE production. In contrast, the "normal" response to such antigens involves a predominance of interferon gamma (IFN-gamma)-producing Th1 clones. This difference has been suggested to be the result of active selection in atopics for Th2 (and hence against Th1) clones at the time of initial antigen presentation. In the study below, we demonstrate that the natural immune response to inhaled protein antigens, particularly in animals expressing the low immunoglobulin E (IgE) responder phenotype, includes a major histocompatibility complex (MHC) class I-restricted CD8+ T cell component, the appearance of which is associated with active suppression of IgE antibody production. Thus, continued exposure of rats to aerosolized ovalbumin (OVA) antigen elicits a transient IgE response, that is terminated by the onset of a state of apparent "tolerance" to further challenge, and this tolerant state is transferable to naive animals with CD8+ T cells. Kinetic studies on in vitro T cell reactivity in these aerosol-exposed rats demonstrated biphasic CD4+ Th2 responses which terminated, together with IgE antibody production, and coincident with the appearance of MHC class I-restricted OVA-specific IFN-gamma-producing CD8+ T cells. However, the latter were not autonomous in vitro and required a source of exogenous IL-2 for initial activation, which in CD(8+)-enriched splenocyte cultures could be provided by small numbers of contaminating OVA-specific CD4+ T cells. This represents the first formal evidence for the induction of an MHC class I-restricted T cell response to natural mucosal exposure to an inert protein antigen, and is consistent with a growing literature demonstrating sensitization of MHC class I-restricted CD8+ T cells by deliberate immunization with soluble proteins. We suggest that crossregulation of MHC class II-restricted CD4+ T cells via cytokine signals generated in parallel CD8+ T cell responses represents a covert and potentially important selection pressure that can shape the nature of host responses to nonreplicating antigens presented at mucosal surfaces.

scholarly journals CD8 T Cell Recognition of Endogenously Expressed Epstein-Barr Virus Nuclear Antigen 1

2004 ◽  
Vol 199 (10) ◽  
pp. 1409-1420 ◽  
Author(s):  
Steven P. Lee ◽  
Jill M. Brooks ◽  
Hatim Al-Jarrah ◽  
Wendy A. Thomas ◽  
Tracey A. Haigh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Epstein Barr Virus ◽  
Cd8 T Cell ◽  
Class I ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Epstein Barr

The Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 contains a glycine-alanine repeat (GAr) domain that appears to protect the antigen from proteasomal breakdown and, as measured in cytotoxicity assays, from major histocompatibility complex (MHC) class I–restricted presentation to CD8+ T cells. This led to the concept of EBNA1 as an immunologically silent protein that although unique in being expressed in all EBV malignancies, could not be exploited as a CD8 target. Here, using CD8+ T cell clones to native EBNA1 epitopes upstream and downstream of the GAr domain and assaying recognition by interferon γ release, we show that the EBNA1 naturally expressed in EBV-transformed lymphoblastoid cell lines (LCLs) is in fact presented to CD8+ T cells via a proteasome/peptide transporter–dependent pathway. Furthermore, LCL recognition by such CD8+ T cells, although slightly lower than seen with paired lines expressing a GAr-deleted EBNA1 protein, leads to strong and specific inhibition of LCL outgrowth in vitro. Endogenously expressed EBNA1 is therefore accessible to the MHC class I pathway despite GAr-mediated stabilization of the mature protein. We infer that EBNA1-specific CD8+ T cells do play a role in control of EBV infection in vivo and might be exploitable in the control of EBV+ malignancies.

Identification of Novel MHC Class I and Class II Epitopes of WT1 Using a Peptide Library Screen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2764-2764 ◽  
Author(s):  
Katayoun Rezvani ◽  
Stephan Mielke ◽  
Yasemin Kilical ◽  
Matthias Grube ◽  
Hiroshi Fujiwara ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Class Ii ◽  
Peptide Library ◽  
Class I ◽  
Cell Responses

Abstract Although several HLA-A*0201-restricted immunodominant peptides from the leukemia-restricted protein WT-1 are characterized, T cell responses to peptide sequences binding to other common class I and II epitopes of WT-1 remain almost completely unexplored. A more comprehensive definition of the WT-1 antigen would extend peptide antigen vaccines to individuals lacking HLA-A*0201 and *2402 and improve vaccine potency by recruiting both CD4+ and CD8+ T cell responses. Here we used a WT1 peptide library to identify WT-1 peptide sequences inducing CD4+ and CD8+ T cell responses in normal individuals and patients with AML and other myeloid leukemias. Six cases were studied. The library consisted of 110 15mer peptides overlapping by 11aa covering the entire WT-1 protein in 21 pools. Monocytes were isolated by plastic adherence and pulsed with peptide pools for 3 hours. Autologous CD8+ and CD4+ T cells were then added. Pools of peptides were prepared in such a way that each peptide was represented in two different peptide pools, allowing the identification of the respective peptide by responses in the two corresponding pools. Cells were harvested for RNA extraction and reverse transcription. Real time PCR (RQ-PCR) was used to identify peptide-specific induction of IFN-γ and IL-2 in CD8+ and CD4+ T cells. The SYFPEITHI binding motif software was then used to predict the probable HLA restriction for the candidate epitopes. To confirm candidate peptide immunogenecity and HLA restriction, selected peptides were synthesized and tested individually. In addition to the known HLA-A*0201 peptides WT37, WT126, WT187 and WT235 we identified 20 new MHC class I and II epitopes of WT1. Four were restricted by more than one HLA allele, demonstrating the promiscuity of epitope binding. One epitope (VPGVAPTLV) was restricted to HLA-A*0201 and HLA-B*5101. One epitope (SGQFTGTAGACRYGP) was restricted by a class I HLA allele, namely HLA-*6801 and a class II HLA allele, DR*1501. Two epitopes (YGPFGPPPPSQASGQ and QKKFARSDELVRHHN) were restricted by multiple MHC class II alleles. The proliferative response of CD4+ and CD8+ T cells to candidate peptides was confirmed using CFSE labeling. We now plan to characterize the antileukemic effects of CD4+ and CD8+ T cells induced by these peptides with a view to designing broad-spectrum vaccines inducing leukemia-reactive T cells across a wide range of HLA types.

Conserved Amino Acid Sequences in Parvovirus B19 and Adeno-Associated Virus Stimulate Functionally Cross-Reactive CD8 T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3269-3269
Author(s):  
Samuel L. Murphy ◽  
Daniel J. Hui ◽  
Shyrie Edmonson ◽  
Katherine A. High
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Parvovirus B19 ◽  
Cell Epitope ◽  
Cd8 T Cell ◽  
Cross Reactivity ◽  
Factor Ix ◽  
T Cell Epitope ◽  
Adeno Associated Virus

Abstract Use of adeno-associated virus (AAV) to achieve liver-targeted gene transfer for the treatment of hemophilia B in human subjects resulted in the expression of therapeutic levels of Factor IX. However, expression of Factor IX was transient and receded to baseline levels over a period of several weeks. The loss of transgene expression was coincident with a rise in CD8+ T cells specific for sequences of the AAV capsid protein, a phenomenon never observed in small and large animal model studies of this treatment. In this study, we tested whether capsid sequences conserved between the pathogenic parvovirus B19 and AAV (~25% identity) could result in cross-reactive activation of capsid specific T-cells. Functional cross-reactivity could lead to an effective vaccination against AAV following exposure to B19 virus, a common infectious virus which was recently shown to elicit a strong CD8 T cell response that can persist for up to one year after exposure to the virus. Furthermore, B19 virus tropism is restricted to humans and this could explain the absence of this outcome in animal models. In order to test our hypothesis, Balb/C mice were immunized against the capsid protein of AAV serotypes 2 or 8 (AAV2 and AAV8). Isolation of splenocytes from mice immunized against AAV2 capsid was followed by ELISpot analysis that compared the response against the immunodominant AAV2 CD8 T cell epitope (VPQYGYLTL) to the response against the homologous B19 capsid epitope (PPQYAYLTV). Indeed cross-reactivity was observed: using ELISpots for IFN-g, incubation with the AAV epitope yielded 1866 spot-forming units (sfu) per 106 splenocytes (average of 3 determinations), and incubation with the B19 epitope yielded 537 sfu/106 splenocytes, while incubation with irrelevant peptide yielded <10 sfu/106 splenocytes (n=5 mice per group). This decreased but significant cross-reactive population of T cells was similarly observed following immunization against AAV8 capsid and ELISpot analysis, with an average of 2137 sfu/106 splenocytes in response to the immunodominant AAV8 CD8 T cell epitope (IPQYGYLTL) and an average of 486 sfu/106 splenocytes in response to the B19 epitope. These results were further substantiated by intracellular cytokine staining, which defined CD8+ T cells as the responding cell population and showed cross-reactivity at levels consistent with ELISpot analysis. To test whether such cross reactivity could be observed in humans, we identified several epitopes through bioinformatics which showed the potential to be cross-reactive in the context of specific HLA haplotypes. Human splenocytes from one B0702 anonymous donor and three B44 anonymous donors were subjected to three rounds of in vitro stimulation with the homologous AAV and B19 capsid peptide sequences. Subsequent ELISpot analysis showed that none of these expansions resulted in the selective expansion of cells specific for these peptides. We conclude from these studies that CD8+ T cells specific for parvovirus capsid sequences can be functionally cross-reactive with AAV capsid, but that further studies of human lymphocytes will be required to determine the significance of this finding for human gene transfer trials.

scholarly journals Self MHC class I–licensed NK cells enhance adaptive CD8 T-cell viral immunity

Blood ◽  
2011 ◽  
Vol 117 (19) ◽  
pp. 5133-5141 ◽  
Author(s):  
Michael D. Stadnisky ◽  
Xuefang Xie ◽  
Ebony R. Coats ◽  
Timothy N. Bullock ◽  
Michael G. Brown
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Class I ◽  
Inhibitory Receptor ◽  
Class I Mhc ◽  
Mhc I

AbstractMHC class I (MHC I) is essential to NK- and T-cell effector and surveillance functions. However, it is unknown whether MHC I polymorphism influences adaptive immunity through NK cells. Previously, we found that MHC I Dk, a cognate ligand for the Ly49G2 inhibitory receptor, was essential to NK control of murine (M)CMV infection. Here we assessed the significance of NK inhibitory receptor recognition of MCMV on CD8 T cells in genetically defined MHC I Dk disparate mice. We observed that Dk-licensed Ly49G2+ NK cells stabilized and then enhanced conventional dendritic cells (cDCs) recovery after infection. Furthermore, licensed NK support of cDC recovery was essential to enhance the tempo, magnitude, and effector activity of virus-specific CD8 T cells. Minimal cDC and CD8 T-cell number differences after low-dose MCMV in Dk disparate animals further implied that licensed NK recognition of MCMV imparted qualitative cDC changes to enhance CD8 T-cell priming.

Class I–unrestricted noncytotoxic anti–HTLV-I activity of CD8+ T cells

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1994-1995 ◽  
Author(s):  
Masako Moriuchi ◽  
Hiroyuki Moriuchi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Membrane Filter ◽  
Cd8 T Cell ◽  
Class I ◽  
Type I ◽  
Peripheral Blood Mononuclear ◽  
Permeable Membrane

Abstract Although it is widely believed that viral clearance is mediated principally by the destruction of infected cells by cytotoxic T cells, noncytolytic antiviral activity of CD8+ T cells may play a role in preventing the progression to disease in infections with immunodeficiency viruses and hepatitis B virus. We demonstrate here that (1) replication of human T-lymphotropic virus type I (HTLV-I) is more readily detected from CD8+ T-cell–depleted (CD8−) peripheral blood mononuclear cells (PBMCs) of healthy HTLV-I carriers than from unfractionated PBMCs, (2) cocultures of CD8− PBMCs with autologous or allogeneic CD8+ T cells suppressed HTLV-I replication, and (3) CD8+ T-cell anti-HTLV-I activity is not abrogated intrans-well cultures in which CD8+ cells are separated from CD8− PBMCs by a permeable membrane filter. These results suggest that class I-unrestricted noncytolytic anti–HTLV-I activity is mediated, at least in part by a soluble factor(s), and may play a role in the pathogenesis of HTLV-I infection.

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