Introduction - Toxoplasma gondii

• Obligate intracellular parasite

• Infects a wide range of avian and mammalian species

• Host: cat; can be carried by mammals and birds

• Can cause severe disease in humans

• Toxoplasmosis can have fatal effects on a fetus

• T. gondii can exist as either rapidly growing tachyzoites or bradyzoites that reside in semidormant cysts

Introduction - Toxoplasma gondii

• NK cells, CD4+ T cells and CD8+ T cells produce cytokines against T. gondii

• CD8+ T cells are known to have a critical protective function

• Resistance to toxoplasmic encephalitis in H-2d mice has been linked to the locus encoding H-2Ld MHC class I

• The mechanisms and antigens that elicit the activation and expansion of T. gondii–specific CD8+ T cell populations are not understood

Introduction - Antigen processing

• CD8+ T cells recognize intracellular protein derived peptides presented by MHC class I

• Antigenic peptides: proteolysis in the cytoplasm, transport into the ER, further processing by the aminopeptidase ERAAP

• ERAAP is very important for shaping of peptides for MHC class I

Clinical and Experimental Immunology 2005

TCR: T cell receptor; TAP: transporter associated with antigen processing; ERAAP: endoplasmic reticulum aminopeptidase associated with antigen processing

Questions

What are the natural antigens for MHC class I

presentation and how are they processed?

Infection of ERAAP-deficient mice

• i.p. infection of resistant H-2d mice (B10.D2) with T. gondii tachyzoites– ERAAP deficient– ERAAP-heterozygous– wild-type

Tachyzoites: rapidly growing T. gondii; ERAAP: endoplasmic reticulum aminopeptidase associated with antigen processing

ERAAP-deficient mice are susceptible to T. gondii

Survival was significantly impaired in the absence of ERAAP

ERAAP: endoplasmic reticulum aminopeptidase associated with antigen processing

– heterozygous/wt– deficient

Flow cytometry

PCR

T. gondii hybridomas

Tachyzoites: rapidly growing T. gondii

• Immunization of resistant BALB/c (H-2d) mice with γ-irradiated tachyzoites

• CD8+ T cells and CD4+ T cells produced IFN-γ

• Expansion of the T. gondii–specific CD8+ T cell populations by restimulation in vitro

T. gondii hybridomas

• Hybridoma were created by fusion of the T. gondii–specific CD8+ T cells and a TCR αβ-negative fusion partner with inducible β-galactosidase

• Occupancy of the TCR can be assayed by measurement of intracellular lacZ activity

• T. gondii infection of APCs expressing H-2Ld or H-2Kd MHC class I molecules

T. gondii hybridomas

Response of the hybridomas to T. gondii infected APCs expressing H-2Ld but not to those expressing H-2Kd MHC class I molecules

H-2Ld and a T. gondii-derived peptide is necessary for hybridoma activation

Identification of the natural T. gondii antigen GRA6

GRA6: dense granule protein 6; secreted by T. gondii

• Preparation of a plasmid cDNA library with mRNA from T. gondii tachyzoites

• Transfection of H-2Ld fibroblasts with cDNA

• Incubation with CTgEZ.4 T cell hybridomas

• The five most positive had 100% identity to the dense granule protein 6 (GRA6)

Hybridoma stimulation• Transfection of H-2Ld fibroblasts with full-length GRA6 cDNA or a c-

terminal deletion construct

• Incubation with hybridoma

GRA6: dense granule protein 6; secreted by T. gondii

CTgEZ.4 hybridoma were stimulated with full-length but not with mutated GRA6

the antigenic epitope was located in the deleted residues

Hybridomas recognizethe HF10 decapeptide

• Systematically testing of all potential peptides

the decapeptide HPGSVNEFDF (HF10) was recognized by the hybridoma

Fractation by HPLC

BMDM: bone marrow–derived macrophages; BMDC: bone marrow–derived dendritic cellsHPLC: high-performance liquid chromatography; GRA6: dense granule protein 6

• Fractation of synthetic HF10 analogs by HPLC

• Testing of all fractions for hybridoma activation

Fractation by HPLC

BMDM: bone marrow–derived macrophages; BMDC: bone marrow–derived dendritic cellsHPLC: high-performance liquid chromatography; GRA6: dense granule protein 6

• Fractation of extracts of GRA6-transfected H-2Ld L fibroblasts and T. gondii–infected BMDMs and BMDCs by HPLC

• Testing of all fractions for hybridoma activation

HF10 was the naturally processed product of the GRA6 protein presented by H-2Ld

Monitoring of CD8+ T cells

• Orally infection of mice with T. gondii cysts

• Incubation of spleen and brain cells with H-2Ld MHC multimers (DimerX) loaded with HF10 or QL9 4 weeks after infection

• Staining with PE-coupled α–mouse IgG1

Monitoring of CD8+ T cells

• 5% of splenic CD8+ T cells • 24% of CD8+ brain T cells• Only for HF10 but not QL9 HF10–H-2Ld was a naturally processed ligand

recognized by CD8+ T cells during T. gondii infection.

Monitoring of CD8+ T cells

• Speceficity of CD8+ T cells for HF10–H-2Ld was unexpected because the T. gondii genome contains over 8,000 proteinencoding genes

• Assesment of the relative frequency of CD8+ T cells specific for HF10 versus other potential antigens among all IFN-γ-producing CD8+ T cells elicited by T. gondii

Monitoring of CD8+ cells

• i.p. infection of mice with T. gondii tachyzoites

• Splenic CD8+ T cells were stimulated with T. gondii-infected J774 macrophages 4 weeks after infection

• 18% produced IFNγ

Monitoring of CD8+ T cells

• i.p. infection of mice with T. gondii tachyzoites

• Stimulation of the CD8+ T cells with J774 macrophages loaded with the HF10 peptide

• 20% produced IFNγ

GRA6-derived HF10 is an immunodominant T. gondii antigen in H-2d mice

Immunization with HF10

BMDC: bone marrow–derived dendritic cells

• Immunization of B10.D2 H-2d mice with BMDCs pulsed with HF10 or YL9

all control mice succumbed to infection within 12 d all HF10-immunized mice survived

Immunization with HF10

BMDC: bone marrow–derived dendritic cells

• Immunization of C57BL/6 H-2b mice with BMDCs pulsed with HF10 or YL9

no protection from T. gondii protection from disease was MHC restricted

Immunization with HF10

BMDC: bone marrow–derived dendritic cells

• Depletion of CD8+ cells of B10.D2 H-2d mice • Immunization with BMDCs pulsed with HF10 or YL9• Infection of splenocytes and peritoneal cells with T. gondii (GFP+)

CD8+ cells were critical for protection HF10 was able to elicit a protective CD8+ T cell response during T.

gondii infection in H-2d mice

Processing and generation of HF10–H-2Ld complexes

Processing and generation of HF10–H-2Ld complexes

BMDM: bone marrow–derived macrophages

• Treatment of T. gondii infected BMDMs with the proteasome inhibitors epoxomicin or lactacystin

• Incubation with hybridoma

lower CTgEZ.4 hybridoma activation with inhibitor treatment Proteasomes were required for the generation of HF10–H-2Ld complexes.

Processing and generation of HF10–H-2Ld complexes

BMDM: bone marrow–derived macrophages

• Transduction of H-2Ld into TAP-deficient or wild-type C57BL/6 BMDMs

• Infection with T. gondii

TAP-deficient BMDMs failed to stimulate the CTgEZ.4 hybdroma TAP transport was essential for presentation of the HF10–H-2Ld complex

Processing and generation of HF10–H-2Ld complexes

BMDM: bone marrow–derived macrophages; BMDC: bone marrow–derived dendritic cells

• Infection of ERAAP-heterozygous or ERAAP-deficient BMDMs or BMDCs with T. gondii

• Incubation with the CTgEZ.4 hybridoma

cells from ERAAP-deficient mice were not able to activate the hybridoma

Processing and generation of HF10–H-2Ld complexes

BMDM: bone marrow–derived macrophages; BMDC: bone marrow–derived dendritic cells

• Incubation of ERAAP-heterozygous or ERAAP-deficient BMDMs or BMDCs presenting HF10 with the hybridoma

• Incubation with the CTgEZ.4 hybridoma

no differences in ERAAP-heterozygous or ERAAP-deficient cells for hybridoma activation

Processing and generation of HF10–H-2Ld complexes

BMDM: bone marrow–derived macrophages

• Extraction of naturally processed peptides from ERAAP-deficient and ERAAP-heterozygous infected BMDMs

two peaks of antigenic activity– fraction 34 could serve as precursors of HF10– fraction 32 was barely detected in extracts of ERAAP-deficient

cells

Processing and generation of HF10–H-2Ld complexes

• Measurement of the T. gondii–induced CD8+ T cell responses of ERAAP-deficient mice

less HF10-specific CD8+ T cells in ERAAP-deficient mice than in ERAAP-heterozygous mice

ERAAP-deficient APCs can`t generate the HF10–H-2Ld complexes and can`t elicit HF10-specific CD8+ T cell response

Summary

• ERAAP-deficient mice are susceptible to T. gondii

• GRA6 is the natural T. gondii antigen

• HF10 is the naturally processed product of the GRA6 protein presented by H-2Ld

• Successfull immunization of mice with HF10 against T. gondii

• Protection from disease was MHC restricted

• ERAAP-deficient APCs can`t generate the HF10–H-2Ld complexes and can`t elicit HF10-specific CD8+ T cell response

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