Exogenous antigen typically enters the major histocompatibility complex: MHC) class II system for processing and presentation to CD4+ T cells. Experiments conducted by Michael Bevan in the 1970s demonstrated that exogenous antigen can also enter the MHC class I system and cross-prime CD8+ T cells; the process by which antigen presentation occurs in this context is termed cross-presentation. We have found a CD8+ T cell epitope within hen egg-white lysozyme: HEL) that is presented on MHC class I via the cross-presentation pathway. The work in this thesis examines two issues: 1) CD8+ T cell cross-priming by subcutaneous immunization with exogenous HEL; and 2) cross-presentation of HEL to CD8+ T cell hybridomas by antigen presenting cells: APCs) from naïve and immunized mice. Non-obese diabetic: NOD) mice immunized with HEL in complete Freund's adjuvant: CFA) primed HEL 11-25-specific CD4+ T cells and HEL 20-35-specific CD8+ T cells; CD8+ T cell cross-priming was confirmed by several methods, including CD8+ T cell depletion, analysis of mice lacking CD8+ T cells and blockade of responses by MHC class I antibodies. Cross-priming by HEL-CFA immunization did not require CD4+ T cell help but was reduced in the absence of interferon-α/β: IFNα/β) or IFNγ signaling; Batf3-/- mice lacking CD8α+ dendritic cells: DCs) also showed reduced cross-priming. We examined the ability of draining lymph node: dLN) DCs to process and cross-present soluble HEL to CD8+ T cell hydridomas. DCs isolated from immunized, but not naïve, mice cross-presented soluble HEL ex vivo. Cross-presentation by dLN DCs was enhanced by IFNα/β, reduced by depletion of CD8α+ DCs and did not require the transporter associated with antigen processing: TAP). Naïve DCs treated with a variety of inflammatory stimuli, including IFNα/β and IFNγ, failed to cross-present soluble HEL. Finally, we assessed MHC class I and II presentation of HEL in liposomes targeted to early or late endosomal compartments. HEL in either early or late endosomal liposomes was presented on MHC class II; in contrast, only HEL in early endosomal liposomes was cross-presented on MHC class I. MHC class II presentation required nascent MHC molecules and endosomal acidification. Cross-presentation of HEL in early endosomal liposomes required endosomal acidification but not proteasomal activity, nascent MHC molecules or TAP. Blockade of endosomal acidification enabled cross-presentation of soluble HEL and HEL in late endosomal liposomes. Altogether, these data indicate that several forms of exogenous HEL enter the MHC class I pathway in vivo and in vitro. TAP is not necessary for HEL cross-presentation, implying that antigen processing occurs within endosomal compartments but not the cytosol; this feature distinguishes HEL from other cross-presented antigens, especially ovalbumin, and provides a model for investigating the intersection between the endosomal pathway and the MHC class I system.
","abstract_html":"<p>Exogenous antigen typically enters the major histocompatibility complex: MHC) class II system for processing and presentation to CD4+ T cells. Experiments conducted by Michael Bevan in the 1970s demonstrated that exogenous antigen can also enter the MHC class I system and cross-prime CD8+ T cells; the process by which antigen presentation occurs in this context is termed cross-presentation. We have found a CD8+ T cell epitope within hen egg-white lysozyme: HEL) that is presented on MHC class I via the cross-presentation pathway. The work in this thesis examines two issues: 1) CD8+ T cell cross-priming by subcutaneous immunization with exogenous HEL; and 2) cross-presentation of HEL to CD8+ T cell hybridomas by antigen presenting cells: APCs) from naïve and immunized mice. Non-obese diabetic: NOD) mice immunized with HEL in complete Freund's adjuvant: CFA) primed HEL 11-25-specific CD4+ T cells and HEL 20-35-specific CD8+ T cells; CD8+ T cell cross-priming was confirmed by several methods, including CD8+ T cell depletion, analysis of mice lacking CD8+ T cells and blockade of responses by MHC class I antibodies. Cross-priming by HEL-CFA immunization did not require CD4+ T cell help but was reduced in the absence of interferon-α/β: IFNα/β) or IFNγ signaling; Batf3-/- mice lacking CD8α+ dendritic cells: DCs) also showed reduced cross-priming. We examined the ability of draining lymph node: dLN) DCs to process and cross-present soluble HEL to CD8+ T cell hydridomas. DCs isolated from immunized, but not naïve, mice cross-presented soluble HEL ex vivo. Cross-presentation by dLN DCs was enhanced by IFNα/β, reduced by depletion of CD8α+ DCs and did not require the transporter associated with antigen processing: TAP). Naïve DCs treated with a variety of inflammatory stimuli, including IFNα/β and IFNγ, failed to cross-present soluble HEL. Finally, we assessed MHC class I and II presentation of HEL in liposomes targeted to early or late endosomal compartments. HEL in either early or late endosomal liposomes was presented on MHC class II; in contrast, only HEL in early endosomal liposomes was cross-presented on MHC class I. MHC class II presentation required nascent MHC molecules and endosomal acidification. Cross-presentation of HEL in early endosomal liposomes required endosomal acidification but not proteasomal activity, nascent MHC molecules or TAP. Blockade of endosomal acidification enabled cross-presentation of soluble HEL and HEL in late endosomal liposomes. Altogether, these data indicate that several forms of exogenous HEL enter the MHC class I pathway in vivo and in vitro. TAP is not necessary for HEL cross-presentation, implying that antigen processing occurs within endosomal compartments but not the cytosol; this feature distinguishes HEL from other cross-presented antigens, especially ovalbumin, and provides a model for investigating the intersection between the endosomal pathway and the MHC class I system.</p>","abstract_has_math":false,"creators":["Belizaire, Roger"],"institution":null,"degree_name":"Doctor of Philosophy (PhD)","degree_level":"Dissertation","degree_discipline":"Biology and Biomedical Sciences: Immunology","degree_department":null,"school":null,"contributors":["Emil Unanue"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2012,"date_issued":"2012-01-15T08:00:00Z","date_published":"2012-01-15T08:00:00Z","updated_at":"2026-07-24T06:12:01Z","subjects":["Immunology","Biology","Antigen presenting cell","Cross-presentation","Freund's adjuvant","Lysozyme","non-obese diabetic mouse"],"languages":["English (en)"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.7936/K76Q1V76"],"render_values":[{"text":"https://doi.org/10.7936/K76Q1V76","href":"https://doi.org/10.7936/K76Q1V76","code":true}]}]},"links":{"outbound_url":"https://openscholarship.wustl.edu/etd/552","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Emil Unanue"]},{"key":"dc:creator","label":"Author","values":["Belizaire, Roger"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2112-01-15T08:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biology and Biomedical Sciences: Immunology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Immunology","Biology","Antigen presenting cell","Cross-presentation","Freund's adjuvant","Lysozyme","non-obese diabetic mouse"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["English (en)"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://openscholarship.wustl.edu/etd/552"]},{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.7936/K76Q1V76"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Exogenous antigen typically enters the major histocompatibility complex: MHC) class II system for processing and presentation to CD4+ T cells. Experiments conducted by Michael Bevan in the 1970s demonstrated that exogenous antigen can also enter the MHC class I system and cross-prime CD8+ T cells; the process by which antigen presentation occurs in this context is termed cross-presentation. We have found a CD8+ T cell epitope within hen egg-white lysozyme: HEL) that is presented on MHC class I via the cross-presentation pathway. The work in this thesis examines two issues: 1) CD8+ T cell cross-priming by subcutaneous immunization with exogenous HEL; and 2) cross-presentation of HEL to CD8+ T cell hybridomas by antigen presenting cells: APCs) from naïve and immunized mice. Non-obese diabetic: NOD) mice immunized with HEL in complete Freund's adjuvant: CFA) primed HEL 11-25-specific CD4+ T cells and HEL 20-35-specific CD8+ T cells; CD8+ T cell cross-priming was confirmed by several methods, including CD8+ T cell depletion, analysis of mice lacking CD8+ T cells and blockade of responses by MHC class I antibodies. Cross-priming by HEL-CFA immunization did not require CD4+ T cell help but was reduced in the absence of interferon-α/β: IFNα/β) or IFNγ signaling; Batf3-/- mice lacking CD8α+ dendritic cells: DCs) also showed reduced cross-priming. We examined the ability of draining lymph node: dLN) DCs to process and cross-present soluble HEL to CD8+ T cell hydridomas. DCs isolated from immunized, but not naïve, mice cross-presented soluble HEL ex vivo. Cross-presentation by dLN DCs was enhanced by IFNα/β, reduced by depletion of CD8α+ DCs and did not require the transporter associated with antigen processing: TAP). Naïve DCs treated with a variety of inflammatory stimuli, including IFNα/β and IFNγ, failed to cross-present soluble HEL. Finally, we assessed MHC class I and II presentation of HEL in liposomes targeted to early or late endosomal compartments. HEL in either early or late endosomal liposomes was presented on MHC class II; in contrast, only HEL in early endosomal liposomes was cross-presented on MHC class I. MHC class II presentation required nascent MHC molecules and endosomal acidification. Cross-presentation of HEL in early endosomal liposomes required endosomal acidification but not proteasomal activity, nascent MHC molecules or TAP. Blockade of endosomal acidification enabled cross-presentation of soluble HEL and HEL in late endosomal liposomes. Altogether, these data indicate that several forms of exogenous HEL enter the MHC class I pathway in vivo and in vitro. TAP is not necessary for HEL cross-presentation, implying that antigen processing occurs within endosomal compartments but not the cytosol; this feature distinguishes HEL from other cross-presented antigens, especially ovalbumin, and provides a model for investigating the intersection between the endosomal pathway and the MHC class I system.
"]},{"key":"dc:title","label":"Title","values":["Processing of Exogenous Hen Egg-White Lysozyme for Presentation on Major Histocompatibility Complex Class I and II Molecules"]}]}],"canonical_facts":{"dc:contributor":["Emil Unanue"],"dc:creator":["Belizaire, Roger"],"dc:date.available":["2112-01-15T08:00:00Z"],"dc:description.abstract":["Exogenous antigen typically enters the major histocompatibility complex: MHC) class II system for processing and presentation to CD4+ T cells. Experiments conducted by Michael Bevan in the 1970s demonstrated that exogenous antigen can also enter the MHC class I system and cross-prime CD8+ T cells; the process by which antigen presentation occurs in this context is termed cross-presentation. We have found a CD8+ T cell epitope within hen egg-white lysozyme: HEL) that is presented on MHC class I via the cross-presentation pathway. The work in this thesis examines two issues: 1) CD8+ T cell cross-priming by subcutaneous immunization with exogenous HEL; and 2) cross-presentation of HEL to CD8+ T cell hybridomas by antigen presenting cells: APCs) from naïve and immunized mice. Non-obese diabetic: NOD) mice immunized with HEL in complete Freund's adjuvant: CFA) primed HEL 11-25-specific CD4+ T cells and HEL 20-35-specific CD8+ T cells; CD8+ T cell cross-priming was confirmed by several methods, including CD8+ T cell depletion, analysis of mice lacking CD8+ T cells and blockade of responses by MHC class I antibodies. Cross-priming by HEL-CFA immunization did not require CD4+ T cell help but was reduced in the absence of interferon-α/β: IFNα/β) or IFNγ signaling; Batf3-/- mice lacking CD8α+ dendritic cells: DCs) also showed reduced cross-priming. We examined the ability of draining lymph node: dLN) DCs to process and cross-present soluble HEL to CD8+ T cell hydridomas. DCs isolated from immunized, but not naïve, mice cross-presented soluble HEL ex vivo. Cross-presentation by dLN DCs was enhanced by IFNα/β, reduced by depletion of CD8α+ DCs and did not require the transporter associated with antigen processing: TAP). Naïve DCs treated with a variety of inflammatory stimuli, including IFNα/β and IFNγ, failed to cross-present soluble HEL. Finally, we assessed MHC class I and II presentation of HEL in liposomes targeted to early or late endosomal compartments. HEL in either early or late endosomal liposomes was presented on MHC class II; in contrast, only HEL in early endosomal liposomes was cross-presented on MHC class I. MHC class II presentation required nascent MHC molecules and endosomal acidification. Cross-presentation of HEL in early endosomal liposomes required endosomal acidification but not proteasomal activity, nascent MHC molecules or TAP. Blockade of endosomal acidification enabled cross-presentation of soluble HEL and HEL in late endosomal liposomes. Altogether, these data indicate that several forms of exogenous HEL enter the MHC class I pathway in vivo and in vitro. TAP is not necessary for HEL cross-presentation, implying that antigen processing occurs within endosomal compartments but not the cytosol; this feature distinguishes HEL from other cross-presented antigens, especially ovalbumin, and provides a model for investigating the intersection between the endosomal pathway and the MHC class I system.
"],"dc:identifier":["https://openscholarship.wustl.edu/etd/552"],"dc:identifier.doi":["https://doi.org/10.7936/K76Q1V76"],"dc:language":["English (en)"],"dc:subject":["Immunology","Biology","Antigen presenting cell","Cross-presentation","Freund's adjuvant","Lysozyme","non-obese diabetic mouse"],"dc:title":["Processing of Exogenous Hen Egg-White Lysozyme for Presentation on Major Histocompatibility Complex Class I and II Molecules"],"thesis:degree_discipline":["Biology and Biomedical Sciences: Immunology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T06:12:01Z"}