ELUCIDATING THE ROLE OF S1PR5 IN TUMOR-SPECIFIC T CELLS DURING IMMUNOTHERAPY OF PANCREATIC CANCER

NIH T35 Medical Student Summer Research Program in Infection and Immunity

Marissa Kaufman 8/21/2024

PANCREATIC DUCTAL ADENOCARCINOMA

-Metastatic in 80% of patients upon diagnosis

-Symptoms are generally veiled until late-stage disease

-5-year survival rate of 12%

-Surgery not an option in these patients

COMBINATION IMMUNOTHERAPY (AGONISTIC ANTI-CD40 + ANTI-PD-L1)� FOR PANCREATIC CANCER

Burrack et al., JI 2021

HOW DOES THE COMBINATION THERAPY WORK (OR NOT WORK) IN PDA?

10X Genomics

scRNA-seq

CD8 T cells in spleen and tumor

AGONISTIC ANTI-CD40 OR THE COMBINATION IMMUNOTHERAPY EXPANDS CYTOTOXIC CD8 T CELLS IN SPLEEN

Spleen, no terminal effector population in tumor (not shown)

GENE EXPRESSION IN CYTOTOXIC CD8+ T CELLS THAT EXPAND IN SPLEEN IN RESPONSE TO IMMUNOTHERAPY

SPLEEN

CD8+ T cell clusters, merged data all treatment groups combined

WHAT ARE S1PR1AND S1PR5?

• T-bet (Tbx21) and Zeb2 are required for S1PR5 in CD8 T cells (Evrard et al, JEM, 2022)

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• S1PR5 overexpression promotes CD8 T cells in spleen and lymph node (Evrard et al, JEM, 2022)

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• S1PR5 regulate their trafficking via a mechanism independent of S1P gradients unlike other S1P receptors

• Sphingosine-1-phosphate receptor 1 (S1PR1) is a protein that binds to a signaling molecule called sphingosine 1-phosphate (S1P) and a KLF2 target gene.

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• S1PR1 is a G-protein-coupled receptor (GPCR) that is encoded by the S1PR1 gene in humans

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• S1PR1 guides lymphocytes out of lymphoid organs into blood and lymph in response to the high concentration of S1P

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S1PR1

S1PR5

Is S1PR5 trapping cytotoxic CD8 T cells in spleen after immunotherapy?

Ultimately could we target it to promote CD8 T cell egress from spleen to tumor to improve immunotherapy responses?

ASSESSING S1PR5 EXPRESSION ON KPC+CBR+ MICE SPLEENS

Il15C

Untreated, Monotherapies, Dual, Triple

GATING STRATEGY FOR TUMOR-SPECIFIC CD8 T CELLS IN SPLEEN

S1PR5 FREQUENCY GATED ON TOTAL CD8+ T CELLS

S1PR5 FREQUENCY GATED ON TOTAL CD8+ TETRAMER+ T CELLS

INCREASED S1PR5 FREQUENCY IS SPECIFIC TO TETRAMER BINDING T CELLS

S1PR5 FREQUENCY GATED ON TOTAL CD8+ TUMOR SPECIFIC KLRG1+,CX3CR1+ T CELLS

S1PR5 FREQUENCY INCREASED WITH IMMUNOTHERAPY IN KLRG1+,CX3CR1+ T CELLS

S1PR1 FREQUENCY GATED ON TOTAL CD8+ T CELLS

S1PR1 FREQUENCY GATED ON TOTAL CD8+ T CELLS TETRAMER+ T CELLS

S1PR1IS INCREASED IN TUMOR SPECIFIC CYTOTOXIC T CELLS

S1PR1 FREQUENCY GATED ON TOTAL CD8+ TUMOR SPECIFIC KLRG1+,CX3CR1+ T CELLS

S1PR1 FREQUENCY INCREASED WITH IMMUNOTHERAPY IN KLRG1+,CX3CR1+ T CELLS

WHAT IS THE SPATIAL LOCALIZATION OF S1PR1+ AND/OR S1PR5+ CD8 T CELLS DURING IMMUNOTHERAPY?

CONTROL (UNTREATED) TUMOR

DAPI

S1PR1

CD8

CONTROL (UNTREATED) TUMOR

DAPI

S1PR1

CD8

TUMOR + (ag-CD40+aPDL-1+Il15C)

DAPI

S1PR1

CD8

Tumor edge

Tumor periphery boarder

CONCLUSIONS

• Immunotherapy (agonistic anti-CD40 primarily) drives S1PR1 and S1PR5 in cytotoxic CD8 T cells in spleen
• Addition of IL-15C may further increase these proteins
• Preliminary results support CD8+S1PR1+ T cells are rare and in periphery of tumor

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FUTURE (ONGOING WORK)

• IF stain for S1PR1+CD8+ cells in spleen
• IF stain for S1PR5+CD8+ cells in tumor and spleen
• Assess the frequency of S1PR5/S1PR1 via flow cytometry in tumor sections
• Knock out S1PR5 gene for adoptive cell transfer into KPC2a mouse model

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THANK YOU!!!!!

Thank you to Dr. Ingunn Stronmes, Eddie Cruz, and the rest of the Stromnes lab for their immense support and guidance throughout this summer. My experience with you has been invaluable for learning new techniques and progressing my research career!

Thank you to Dr. Daniel Mueller and Drew Keup for running the NIH T35 Summer Immunology program!