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A strategy for uncovering germline variants altering anti-tumor CD8 T cell response
Vijay Kumar Ulaganathan1, Martina H Vasileva2
1Klinik für Dermatologie & Allergology, Universitätsmedizin Göttingen, Göttingen 37075, Germany; Institut für Multiple Sklerose Forschung, Neuroimmunologie, Universitätsmedizin Göttingen, Göttingen 37075, Germany; University of Lorraine, NGERE Unit, Faculté de Médecine, 9 Avenue de La Forêt de Haye, Vandoeuvre-lès-Nancy 54505, France.
Abstract:
Among many factors known to alter the outcomes of T cell receptor (TCR)-induced proximal signaling, the role of human germline variants in dictating the individuality of the anti-tumor CD8 T cell response has remained challenging to address. Here, we describe a convenient strategy for molecular and functional characterization of phosphotyrosine-altering non-synonymous single nucleotide variations (pTyr-SNVs) that directly impact TCR-induced proximal phosphotyrosine motif-based signaling pathways. We devise an experimental co-cultivation set-up comprising a C57BL/6 mouse-derived metastatic melanoma cell line engineered to constitutively present ovalbumin (OVA) antigens and retrovirally engineered syngeneic major histocompatibility complex (MHC) Class I restricted OVA TCR-transgenic CD8 T cells (OT-I). Using the synthetic version of pTyr-SNV rs1178800678-G/T-encoding integrin alpha 4 (ITGA4) p.S1027I variant as a prototype, we show that under identical TCR stimulation conditions, genetically determined membrane-proximal immunoreceptor tyrosin activation motif (ITAM) results in increased tyrosine phosphorylation of 70 kDa zeta-chain-associated protein (ZAP70) and the levels of cytotoxic effector molecule granzyme B (GZMB), which in turn result in enhanced cytotoxic activity against metastatic melanoma cell line. This strategy paves the way for rapid molecular and functional characterization of anti-tumor immune response-linked germline pTyr-SNVs so as to improve our understanding of the genetic basis of individual-to-individual differences in anti-tumor CD8 T cell response.
Insights
Human germline variants influence CD8 T cell responses against cancer. A new strategy characterizes these variants, revealing how ITGA4 variants enhance T cell anti-tumor activity by increasing ZAP70 phosphorylation and granzyme B levels.
Area of Science:
- Immunology
- Genetics
- Cancer Research
Background:
- Individual differences in anti-tumor CD8 T cell responses are influenced by genetic factors.
- Understanding the impact of human germline variants on T cell receptor (TCR)-induced signaling is crucial but challenging.
Purpose of the Study:
- To develop a strategy for characterizing phosphotyrosine-altering single nucleotide variations (pTyr-SNVs) impacting TCR signaling.
- To investigate the functional consequences of specific germline variants on anti-tumor CD8 T cell activity.
Main Methods:
- Established a co-cultivation system with engineered melanoma cells and OT-I CD8 T cells.
- Utilized a synthetic pTyr-SNV (rs1178800678-G/T in ITGA4) as a prototype for characterization.
- Assessed tyrosine phosphorylation of ZAP70 and granzyme B (GZMB) levels.
Main Results:
- The ITGA4 p.S1027I variant led to increased ZAP70 tyrosine phosphorylation under identical TCR stimulation.
- Elevated GZMB levels and enhanced cytotoxic activity against melanoma cells were observed with the variant.
- Demonstrated a direct link between a specific germline variant and enhanced T cell effector function.
Conclusions:
- The developed strategy enables rapid molecular and functional assessment of germline pTyr-SNVs.
- Genetic variations in ITGA4 can significantly enhance anti-tumor CD8 T cell responses.
- This research provides insights into the genetic basis of individual variability in anti-tumor immunity.
