Introduction - Toxoplasma gondii

2. 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

3. 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

4. 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

5. Clinical and Experimental Immunology 2005 TCR: T cell receptor; TAP: transporter associated with antigen processing; ERAAP: endoplasmic reticulum aminopeptidase associated with antigen processing

6. Questions What are the natural antigens for MHC class I presentation and how are they processed?

7. 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

8. ERAAP-deficient mice are susceptible to T. gondii Flow cytometry – heterozygous/wt – deficient PCR  Survival was significantly impaired in the absence of ERAAP ERAAP: endoplasmic reticulum aminopeptidase associated with antigen processing

9. T. gondii hybridomas • 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 Tachyzoites: rapidly growing T. gondii

10. 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

11. 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-derivedpeptide is necessary for hybridoma activation

12. Identification of the natural T. gondii antigen GRA6 • Preparation of a plasmid cDNA library with mRNA from T. gondiitachyzoites • 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) GRA6: dense granule protein 6; secreted by T. gondii

13. Hybridoma stimulation • Transfection of H-2Ld fibroblasts with full-length GRA6 cDNA or a c-terminal deletion construct • Incubation with hybridoma • CTgEZ.4 hybridoma were stimulated with full-length but not with mutated GRA6 • the antigenic epitope was located in the deleted residues GRA6: dense granule protein 6; secreted by T. gondii

14. Hybridomas recognizethe HF10 decapeptide • Systematically testing of all potential peptides •  the decapeptide HPGSVNEFDF (HF10) was recognized by the hybridoma

15. Fractation by HPLC • Fractation of synthetic HF10 analogs by HPLC • Testing of all fractions for hybridoma activation BMDM: bone marrow–derived macrophages; BMDC: bone marrow–derived dendritic cells HPLC: high-performance liquid chromatography; GRA6: dense granule protein 6

16. Fractation by HPLC • 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 BMDM: bone marrow–derived macrophages; BMDC: bone marrow–derived dendritic cells HPLC: high-performance liquid chromatography; GRA6: dense granule protein 6

17. 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

18. 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.

19. 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

20. 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

21. 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

22. Immunization with HF10 • 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 BMDC: bone marrow–derived dendritic cells

23. Immunization with HF10 • 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 BMDC: bone marrow–derived dendritic cells

24. Immunization with HF10 • 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 BMDC: bone marrow–derived dendritic cells

25. Processing and generation of HF10–H-2Ld complexes

26. Processing and generation of HF10–H-2Ld complexes • 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. BMDM: bone marrow–derived macrophages

27. Processing and generation of HF10–H-2Ld complexes • 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 BMDM: bone marrow–derived macrophages

28. Processing and generation of HF10–H-2Ld complexes • 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 BMDM: bone marrow–derived macrophages; BMDC: bone marrow–derived dendritic cells

29. Processing and generation of HF10–H-2Ld complexes • 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 BMDM: bone marrow–derived macrophages; BMDC: bone marrow–derived dendritic cells

30. Processing and generation of HF10–H-2Ld complexes • 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 BMDM: bone marrow–derived macrophages

31. 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

32. 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

33. Thank you for your attention