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-2 d mice has been linked to the locus encoding H-2L d 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-2 d 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  Survival was significantly impaired in the absence of ERAAP ERAAP: endoplasmic reticulum aminopeptidase associated with antigen processing – heterozygous/wt – deficient Flow cytometry PCR

9. T. gondii hybridomas Tachyzoites: rapidly growing T. gondii Immunization of resistant BALB/c (H-2 d ) 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

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-2L d or H-2K d MHC class I molecules

11. T. gondii hybridomas  Response of the hybridomas to T. gondii infected APCs expressing H-2L d but not to those expressing H-2K d MHC class I molecules  H-2L d and a T. gondii-derived peptide is necessary for hybridoma activation

12. 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-2L d fibroblasts with cDNA Incubation with CTgEZ.4 T cell hybridomas The five most positive had 100% identity to the dense granule protein 6 (GRA6)

13. Hybridoma stimulation Transfection of H-2L d 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

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

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

16. Fractation by HPLC BMDM: bone marrow–derived macrophages; BMDC: bone marrow–derived dendritic cells HPLC: high-performance liquid chromatography; GRA6: dense granule protein 6 Fractation of extracts of GRA6-transfected H-2L d 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-2L d

17. Monitoring of CD8 + T cells Orally infection of mice with T. gondii cysts Incubation of spleen and brain cells with H-2L d 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-2L d 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- 2L d 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-2 d mice

22. Immunization with HF10 BMDC: bone marrow–derived dendritic cells Immunization of B10.D2 H-2 d mice with BMDCs pulsed with HF10 or YL9  all control mice succumbed to infection within 12 d  all HF10-immunized mice survived

23. Immunization with HF10 BMDC: bone marrow–derived dendritic cells Immunization of C57BL/6 H-2 b mice with BMDCs pulsed with HF10 or YL9  no protection from T. gondii  protection from disease was MHC restricted

24. Immunization with HF10 BMDC: bone marrow–derived dendritic cells Depletion of CD8 + cells of B10.D2 H-2 d 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-2 d mice

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

26. 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-2L d complexes.

27. Processing and generation of HF10–H-2L d complexes BMDM: bone marrow–derived macrophages Transduction of H-2L d 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-2L d complex

28. Processing and generation of HF10–H-2L d 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

29. Processing and generation of HF10–H-2L d 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

30. Processing and generation of HF10–H-2L d 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

31. Processing and generation of HF10–H-2L d 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-2L d 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-2L d Successfull immunization of mice with HF10 against T. gondii Protection from disease was MHC restricted ERAAP-deficient APCs can`t generate the HF10–H-2L d complexes and can`t elicit HF10-specific CD8 + T cell response

33. Thank you for your attention