Identification of a dominant CD4 T cell epitope in the membrane lipoprotein Tul4 from Francisella tularensis LVS

Valentino, Michael D.; Hensley, Lucinda L.; Skrombolas, Denise; McPherson, Pamela; Woolard, Matthew D.; Kawula, Thomas H.; Frelinger, Jeffrey A.; Frelinger, John G.

doi:10.1016/j.molimm.2009.01.008

Cited by 19 publications

(38 citation statements)

References 55 publications

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“…To further concentrate our identification on the appropriate T cell response, we elected to track antigen-specific CD4 + T cells, since these cells would directly recognize and respond to Francisella infected cells. Despite identified and predicted CD4 + and CD8 + T cell epitopes in Francisella vaccine strains (17–19), there has been no success in producing MHCI and MHCII tetramers to track Ftt antigen-specific cells in Francisella infected mice. To overcome this problem, we generated recombinant ATCC LVS, RML LVS, and SchuS4 strains ectopically expressing the well-characterized gp61-80 epitope from lymphocytic choriomenigitis virus (LCMV) fused to the Francisella protein, IglC.…”

Section: Resultsmentioning

confidence: 99%

Inclusion of Epitopes That Expand High-Avidity CD4+ T Cells Transforms Subprotective Vaccines to Efficacious Immunogens against Virulent Francisella tularensis

Roberts

Crane

Wehrly

et al. 2016

The Journal of Immunology

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T cells are the immunological cornerstone in host defense against infections by intracellular bacterial pathogens, such as virulent Francisella tularensis (Ftt). The general paucity of novel vaccines for Ftt over the past 60 years can, in part, be attributed to the poor understanding of immune parameters required to survive infection. Thus, we developed a strategy utilizing classical immunological tools to elucidate requirements for effective adaptive immune responses directed against Ftt. Following generation of various Francisella strains expressing well-characterized lymphocytic choriomeningitis virus (LCMV) epitopes, we found that survival correlated with persistence of antigen-specific CD4+ T cells. Function of these cells was confirmed in their ability to more effectively control Ftt replication in vitro. The importance of understanding the antigen-specific response was underscored by our observation that inclusion of an epitope which elicits high avidity CD4+ T cells converted a poorly protective vaccine to one that engenders 100% protection. Together, these data suggest that improved efficacy of current tularemia vaccine platforms will require targeting appropriate antigen-specific CD4+ T cell responses and that elucidation of Francisella epitopes that elicit high avidity CD4+ T cell responses, specifically in humans, will be required for successful vaccine development.

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Section: Resultsmentioning

confidence: 99%

Inclusion of Epitopes That Expand High-Avidity CD4+ T Cells Transforms Subprotective Vaccines to Efficacious Immunogens against Virulent Francisella tularensis

Roberts

Crane

Wehrly

et al. 2016

The Journal of Immunology

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“…The assay proceeded overnight at 37°C. Activation of PSA-HI hybridoma was detected using the β-galactosidase substrate X-gal and enumerating blue cells (Turner et al, 2001; Valentino et al, 2009; Valentino et al, 2011). …”

Section: Methodsmentioning

confidence: 99%

“…Recombinant proteins were isolated by lysing the bacterial pellets with 8M urea (pH8) either with or without addition of Nonidet P-40 detergent as indicated in the figure legends and purified over Ni-NTA column (Qiagen, Valencia, CA) in a high-throughput fashion using a BioRobot 3000 (Qiagen, Valencia, CA). Purified protein was coupled to tosylactivated M280 magnetic Dynabeads (Invitrogen, Carlsbad, CA) as described (Valentino et al, 2009; Valentino et al, 2011). To examine expression and purification procedures, an aliquot of each purified protein from all of the library expression plates (20 × 96 well plates) was blotted onto Hybond C membrane using a 96-well dot blot apparatus (Bio-Rad, Hercules, CA).…”

Section: Methodsmentioning

confidence: 99%

“…The T cell fusion partner BWZ.36/CD8 + , which can be used to make MHC class II and class I restricted hybridomas, was kindly provided by Dr. Nilabh Shastri and maintained as previously described (Sanderson and Shastri, 1994). The Tul4 specific hybridoma FT13 1E4B4 was previously characterized by our lab (Valentino et al, 2009). F. tularensis Live Vaccine Strain (LVS) was obtained from the CDC in Atlanta, GA.…”

Section: Methodsmentioning

confidence: 99%

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A broadly applicable approach to T cell epitope identification: Application to improving tumor associated epitopes and identifying epitopes in complex pathogens

Valentino

Abdul-Alim

Maben

et al. 2011

Journal of Immunological Methods

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Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens.

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“…S2A) (28). M12 cells were generated expressing I-A b tethered to the Ova peptide or to three distinct peptides bound to I-A b : the E641 peptide derived from the West Nile Virus Envelope protein (641-655), a peptide from the Francisella tularensis lipoprotein Tul4 (Ft 86-99), or the 2W peptide from I-Eα (52-68) (29)(30)(31). Although the OT-II CD4WT and CD4T cells responded to Ova, they failed to respond to the E641, Ft, or 2W:I-A b pMHC complexes.…”

Section: δBindmentioning

confidence: 99%

Functional evidence for TCR-intrinsic specificity for MHCII

Parrish

Deshpande

Vasic

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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How T cells become restricted to binding antigenic peptides within class I or class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) via clonotypic T-cell receptors (TCRs) remains debated. During development, if TCR-pMHC interactions exceed an affinity threshold, a signal is generated that positively selects the thymocyte to become a mature CD4 + or CD8 + T cell that can recognize foreign peptides within MHCII or MHCI, respectively. But whether TCRs possess an intrinsic, subthreshold specificity for MHC that facilitates sampling of the peptides within MHC during positive selection or T-cell activation is undefined. Here we asked if increasing the frequency of lymphocyte-specific protein tyrosine kinase (Lck)-associated CD4 molecules in T-cell hybridomas would allow for the detection of subthreshold TCR-MHC interactions. The reactivity of 10 distinct TCRs was assessed in response to selecting and nonselecting MHCII bearing cognate, null, or "shaved" peptides with alanine substitutions at known TCR contact residues: Three of the TCRs were selected on MHCII and have defined peptide specificity, two were selected on MHCI and have a known pMHC specificity, and five were generated in vitro without defined selecting or cognate pMHC. Our central finding is that IL-2 was made when each TCR interacted with selecting or nonselecting MHCII presenting shaved peptides. These responses were abrogated by anti-CD4 antibodies and mutagenesis of CD4. They were also inhibited by anti-MHC antibodies that block TCR-MHCII interactions. We interpret these data as functional evidence for TCR-intrinsic specificity for MHCII.TCR | MHC | restriction | CD4 | Lck P ositive and negative selection limit the αβT-cell repertoire to cells expressing clonotypic T-cell receptors (TCRs) that distinguish the antigenicity of peptides embedded within class I and class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) based on their source of origin (i.e., self or foreign) (1-4). Approximately 7.5% of CD4 + CD8+ doublepositive (DP) thymocytes express TCRs that interact with selfpMHC above an affinity threshold required for positive selection, whereas 7.5% cross a higher affinity threshold that mediates negative selection and the remaining TCRs fail to direct positive selection (5). The rules that restrict TCR recognition of antigenic peptides within MHCI or MHCII are unresolved.Two models have been proposed to explain MHC restriction. One posits that restriction is imposed by CD4 or CD8 during thymocyte development to eliminate TCRs that recognize non-MHC ligands (2, 6). Here, the CD4-and CD8-associated Src kinase, p56Lck [lymphocyte-specific protein tyrosine kinase (Lck)], is sequestered away from the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR-associated CD3δe, CD3γe, and CD3ζζ signaling modules. Positively selecting signals are then generated in thymocytes expressing TCRs that bind MHCII or MHCI together with CD4 or CD8, respectively, as this localizes Lck to the ITAMs. T...

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Identification of a dominant CD4 T cell epitope in the membrane lipoprotein Tul4 from Francisella tularensis LVS

Cited by 19 publications

References 55 publications

Inclusion of Epitopes That Expand High-Avidity CD4+ T Cells Transforms Subprotective Vaccines to Efficacious Immunogens against Virulent Francisella tularensis

Inclusion of Epitopes That Expand High-Avidity CD4+ T Cells Transforms Subprotective Vaccines to Efficacious Immunogens against Virulent Francisella tularensis

A broadly applicable approach to T cell epitope identification: Application to improving tumor associated epitopes and identifying epitopes in complex pathogens

Functional evidence for TCR-intrinsic specificity for MHCII

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