Dendritic cell subsets and CD4 T cell immunity in Melanoma. Ben Wylie 1 st year PhD Candidate

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Dendritic cell subsets and CD4 T cell immunity in Melanoma Ben Wylie 1 st year PhD Candidate

Melanoma Melanoma is the 4 th most common cancer in Australia. Current treatment options are ineffective resulting in a poor prognosis. Novel T cell based therapies have been shown to significantly prolong survival in some patients.

T cell Immunity Dendritic cells (DCs) are the master regulators of T cell immunity. Adapted from Lake and Robinson. 2005. Nat Revs. Cancer. 5(5): 397-405

T cell Immunity Dendritic cells (DCs) are the master regulators of T cell immunity. Adapted from Lake and Robinson. 2005. Nat Revs. Cancer. 5(5): 397-405

T cell Immunity Dendritic cells (DCs) are the master regulators of T cell immunity. Adapted from Lake and Robinson. 2005. Nat Revs. Cancer. 5(5): 397-405

T cell Immunity Dendritic cells (DCs) are the master regulators of T cell immunity. Adapted from Lake and Robinson. 2005. Nat Revs. Cancer. 5(5): 397-405

T cell Immunity Dendritic cells (DCs) are the master regulators of T cell immunity. Adapted from Lake and Robinson. 2005. Nat Revs. Cancer. 5(5): 397-405

Tumour Immunity DCs acquire melanoma antigens and traffic to skin draining lymph nodes to present antigen. Adapted from Munz et al. 2005. J Exp. Med. 202: 203-207

CD4 + T cell Response Presentation of cognate antigen primes a T cell response leading to differentiation and proliferation of CD4 + T cells. Th0

CD4 + T cell Response Presentation of cognate antigen primes a T cell response leading to differentiation and proliferation of CD4 + T cells. Th0 Th1 Th2 Treg

CD4 + T cell Response Presentation of cognate antigen primes a T cell response leading to differentiation and proliferation of CD4 + T cells. Th0 Th1 Th2 Treg

CD4 + T cell Response Presentation of cognate antigen primes a T cell response leading to differentiation and proliferation of CD4 + T cells. Th0? Th1? Th2? Treg

CD4 + T cell Subsets Different subsets of CD4 + T cells have distinct cytokine profiles and function. Th Subtype Cytokines/Markers Th1 Th2 Th9 Th17 Th22 Cytotoxic CD4 + Treg IFNy IL-4, IL-5, IL-13 IL-9 IL-17A, IL-17F, IL-21 IL-22 CD107a, Granzyme B Foxp3, CD25, IL-10

CD4 + T cell Response in Melanoma Multiple groups using the OVA model system have reported that the CD4 T cell compartment remains ignorant to melanoma-derived antigen.* OT-II (CD4) T cells reported to be 100X less senitive to OVA antigen than OT-I (CD8) T cells.** *Preynat-Seauve O, et al. 2007. Cancer Res. 67:5009-16 *Stoitzner P, et al. 2008. Cancer Immunol Immunother. 57:1665-73 *Gerner MY, et al. 2008. J Immunol. 181:155-64 *DiLillo DJ, et al. 2010. J Immunol. 184:4006-16 **Li et al. 2001. J Immunol. 166: 6099-6103

B16.gD Melanoma Model gd-specific TCR transgenic mouse MHC Class II restricted for the glycoprotein D (gd) epitope of HSV CD4 + T cells recognise and proliferate in response to gd B16 melanoma cell line transduced to express the HSV-derived gd epitope and a GFP reporter gene.

B16.gD Melanoma Model gd-specific TCR transgenic mouse MHC Class II restricted for the glycoprotein D (gd) epitope of HSV CD4 + T cells recognise and proliferate in response to gd B16 melanoma cell line transduced to express the HSV-derived gd epitope and a GFP reporter gene. Parental Transduced GFP

B16.gD Melanoma Model Purified CD4 + T cells CFSE Label gdt-ii Mouse (CD45.1) B16.gD Tumour Bearing Mouse (CD45.2) gd Harvest tdln & Spleen

B16.gD Melanoma Model Purified CD4 + T cells CFSE Label gdt-ii Mouse (CD45.1) B16.gD Tumour Bearing Mouse (CD45.2) gd Harvest tdln & Spleen

B16.gD Melanoma Model Purified CD4 + T cells CFSE Label gdt-ii Mouse (CD45.1) B16.gD Tumour Bearing Mouse (CD45.2) gd Harvest tdln & Spleen

B16.gD Melanoma Model Purified CD4 + T cells CFSE Label gdt-ii Mouse (CD45.1) B16.gD Tumour Bearing Mouse (CD45.2) gd CD45-1 CD4 Harvest tdln & Spleen

Antigen Specific CD4 + T cell Proliferation Tumour Draining Node Spleen CFSE Dilution CFSE Dilution Inoculate B16-gD cutaneous I.V. 5 x 10 5 CFSE gdt-ii Harvest/Analysis Day -x Day 0 Day 2.5

Antigen Specific CD4 + T cell Proliferation Tumour Draining Node Spleen CFSE Dilution CFSE Dilution Inoculate B16-gD cutaneous I.V. 5 x 10 5 CFSE gdt-ii Harvest/Analysis Day -x Day 0 Day 2.5

CD4 + T cell Phenotyping Aim 1: Phenotyping the CD4 + T cell response generated to melanoma derived antigens. Th1 Th2 Th9 Th17 Th22 Cytotoxic Treg IFN-γ TNFα IL-4 IL-9 IL-17 IL-22 Granzyme B IL-10 FOXP3

Experimental Plan Harvest tumour draining lymph node cells and restimulate for intracellular cytokine staining. Adoptive transfer of antigen specific CD4 + T cells. PMA/Ionomycin Restim + BFA gd Harvest tdln 60 hours later 5 hours Stain for subset specific intracellular cytokines

T cell Staining Panels Core Stain CD4 CD45-1

T cell Staining Panels Th1/Th17/Th22 IFNy IL-17A IL-22 Core Stain CD4 CD45-1

T cell Staining Panels Th1/Th17/Th22 IFNy IL-17A IL-22 Core Stain CD4 CD45-1 Th2/Th9 IL-4 IL-13 IL-9

T cell Staining Panels Th1/Th17/Th22 IFNy IL-17A IL-22 Treg Foxp3 IL-10 CD25 Core Stain CD4 CD45-1 Th2/Th9 IL-4 IL-13 IL-9

T cell Staining Panels Th1/Th17/Th22 IFNy IL-17A IL-22 Treg Foxp3 IL-10 CD25 Core Stain CD4 CD45-1 Th2/Th9 IL-4 IL-13 IL-9 Cytotoxic Granzyme B CD107a CD25

RESULTS HSV Control Th1 Panel: gdt-ii response (white open) vs. endogenous (grey shaded) IFNγ IL-17α IL-22

RESULTS HSV Control Th2 Panel: gdt-ii response (white open) vs. endogenous (grey shaded) IL-4 IL-9 CD44

Cytotoxic Panel: RESULTS HSV Control gdt-ii response (white open) vs. endogenous (grey shaded) CD107a Granzyme B CD25

RESULTS HSV Control Treg Panel: gdt-ii response (white open) vs. endogenous (grey shaded) Endogenous Transferred 10.6 14.8 CD25 Foxp3 IL-10

Optimisation Panel Design Markers and colours Positive Controls In vitro T cell skewing Gating FMOs, isotypes or single stains

Aim 2: Dendritic Cell Subsets Determine the ability of dendritic cell subsets to drive unique and more effective T cell responses to melanoma. It has been shown recently that different DC subsets are able to promote unique T cell responses. Langerhans cells required for development of Th17 cells during Candida albicans infection.* *(Igyarto B. Z. 2011. Immunity. 35: 260-72)

Aim 2: Dendritic Cell Subsets Determine the ability of dendritic cell subsets to drive unique and more effective T cell responses to melanoma. Hypothesis: Different subsets of DCs may be able to drive more productive CD4 + T cell responses against melanoma.

Ex vivo Proliferation Assay Sort DC subsets from tumour bearing mice and coculture with specific CD4 + T cells ex vivo. B16.gD Tumour 60 hours @ 37 C T cell Transgenic Mouse gd Sorted DCs from tdlns CFSE labelled CD4 T cells Analyse CFSE Dilution

Mouse Dendritic Cell Subsets Mouse DC Phenotyping: Subtype Markers Location Plasmacytoid B220 + PDCA1 + Circulating Resident CD8 + CD4 - Lymph/Spleen Resident CD4 + CD8 - Lymph/Spleen Resident CD8 - CD4 - Lymph/Spleen Migratory CD11b + Dermis Migratory CD103 + Dermis Migratory CD326 + Epidermis

Mouse Dendritic Cell Subsets Mouse DC Phenotyping: Subtype Markers Location Plasmacytoid B220 + PDCA1 + Circulating Resident CD8 + CD4 - Lymph/Spleen Resident CD4 + CD8 - Lymph/Spleen Resident CD8 - CD4 - Lymph/Spleen Migratory CD11b + Dermis Migratory CD103 + Dermis Migratory CD326 + Epidermis

Mouse Dendritic Cell Subsets Mouse DC Phenotyping: Subtype Markers Location Plasmacytoid B220 + PDCA1 + Circulating Resident CD8 + CD4 - Lymph/Spleen Resident CD4 + CD8 - Lymph/Spleen Resident CD8 - CD4 - Lymph/Spleen Migratory CD11b + Dermis Migratory CD103 + Dermis Migratory CD326 + Epidermis

Mouse Dendritic Cell Subsets To further segregate DCs: XCR1 (cross-presenting DCs) Clec9A (cross-presenting DCs and pdcs) SIRPα (CD8 - resident and migratory DCs) DEC205 (CD8 + resident and migratory DCs) CD86 (co-stimulation marker)

Mouse Dendritic Cell Subsets To further segregate DCs: XCR1 (cross-presenting DCs) Clec9A (cross-presenting DCs and pdcs) SIRPα (CD8 - resident and migratory DCs) DEC205 (CD8 + resident and migratory DCs) CD86 (co-stimulation marker) CD86 SIRPα XCR1

Sorting DC Subsets Sort DC subsets from tumour bearing mice and coculture with specific CD4 + T cells ex vivo. Live Cells

Sorting DC Subsets Sort DC subsets from tumour bearing mice and coculture with specific CD4 + T cells ex vivo. Live Cells DCs

Sorting DC Subsets Sort DC subsets from tumour bearing mice and coculture with specific CD4 + T cells ex vivo. Live Cells DCs CD8a DCs

Sorting DC Subsets Sort DC subsets from tumour bearing mice and coculture with specific CD4 + T cells ex vivo. Live Cells DCs CD8a DCs CD103 DCs Langerhans Cells

Sorting DC Subsets Sort DC subsets from tumour bearing mice and coculture with specific CD4 + T cells ex vivo. Live Cells DCs CD8a DCs CD103 DCs CD11b DCs* Langerhans Cells CD11c DCs*

Mouse Dendritic Cell Subsets Mouse DC Phenotyping: Subtype Markers Location Plasmacytoid B220 + PDCA1 + Circulating Resident CD8 + CD4 - Lymph/Spleen Resident CD4 + CD8 - Lymph/Spleen Resident CD8 - CD4 - Lymph/Spleen Migratory CD11b + Dermis Migratory CD103 + Dermis Migratory CD326 + Epidermis

Ex vivo Proliferation DC subsets from tumour bearing mice co-cultured with antigen specific CD4 + T cells ex vivo.

Ex vivo Proliferation DC subsets from tumour bearing mice co-cultured with antigen specific CD4 + T cells ex vivo.

Recombinant Mouse Models Interrogate antigen presentation by DC subsets in vivo by using recombinant mice. Mouse/DC LCs CD11b + DC CD103 + DC CD8 + DC Migratory Migratory Migratory LNresident LNresident CD4 + DC CD11c.DTR - - - - - CCR7 o/o - - - + + Clec9A.dTR + + - - + hulangerin.dta - + + + + Batf3 o/o + + - + + CD11c.DTR B6 or IAE -/- Chimaeras +/- - - - -

Recombinant Mouse Models Interrogate antigen presentation by DC subsets in vivo by using recombinant mice. Mouse/DC LCs CD11b + DC CD103 + DC CD8 + DC Migratory Migratory Migratory LNresident LNresident CD4 + DC CD11c.DTR - - - - - CCR7 o/o - - - + + Clec9A.dTR + + - - + hulangerin.dta - + + + + Batf3 o/o + + - + + CD11c.DTR B6 or IAE -/- Chimaeras +/- - - - -

Recombinant Mouse Models Interrogate antigen presentation by DC subsets in vivo by using recombinant mice. Mouse/DC LCs CD11b + DC CD103 + DC CD8 + DC Migratory Migratory Migratory LNresident LNresident CD4 + DC CD11c.DTR - - - - - CCR7 o/o - - - + + Clec9A.dTR + + - - + hulangerin.dta - + + + + Batf3 o/o + + - + + CD11c.DTR B6 or IAE -/- Chimaeras +/- - - - -

Recombinant Mouse Models Interrogate antigen presentation by DC subsets in vivo by using recombinant mice. Mouse/DC LCs CD11b + DC CD103 + DC CD8 + DC Migratory Migratory Migratory LNresident LNresident CD4 + DC CD11c.DTR - - - - - CCR7 o/o - - - + + Clec9A.dTR + + - - + hulangerin.dta - + + + + Batf3 o/o + + - + + CD11c.DTR B6 or IAE -/- Chimaeras +/- - - - -

Recombinant Mouse Models Interrogate antigen presentation by DC subsets in vivo by using recombinant mice. Mouse/DC LCs CD11b + DC CD103 + DC CD8 + DC Migratory Migratory Migratory LNresident LNresident CD4 + DC CD11c.DTR - - - - - CCR7 o/o - - - + + Clec9A.dTR + + - - + hulangerin.dta - + + + + Batf3 o/o + + - + + CD11c.DTR B6 or IAE -/- Chimaeras +/- - - - -

Flow Cytometry Intracellular Cytokine Staining: T cell phenotyping Sorting: DC subsets Proliferation Assays: CFSE dilution

Acknowledgements Supervisors: Dr. Jason Waithman Prof. Prue Hart Group Members: Dr. Vanessa Fear Dr. Elke Seppanen Sarah Lacey Rachael Zemek This work is supported by grants from Cancer Australia and Cure Cancer Australia.

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