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

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

2 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.

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

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

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

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

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

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

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

10 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

11 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

12 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

13 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

14 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 Cancer Res. 67: *Stoitzner P, et al Cancer Immunol Immunother. 57: *Gerner MY, et al J Immunol. 181: *DiLillo DJ, et al J Immunol. 184: **Li et al J Immunol. 166:

15 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.

16 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

17 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

18 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

19 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

20 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

21 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

22 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

23 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

24 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

25 T cell Staining Panels Core Stain CD4 CD45-1

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

27 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

28 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

29 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

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

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

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

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

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

35 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 Immunity. 35: )

36 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.

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

38 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

39 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

40 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

41 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)

42 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

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

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

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

46 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

47 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*

48 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

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

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

51 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 +/

52 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 +/

53 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 +/

54 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 +/

55 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 +/

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

57 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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