A strategy for uncovering germline variants altering anti-tumor CD8 T cell response

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.

Characterizing the Lassa Virus Envelope Glycoprotein Membrane Proximal External Region for Its Role in Fusogenicity

The membrane-proximal external region(MPER)of Lassa virus(LASV)glycoprotein complex(GPC)is critical in modulating its functionality.Till now,the high-resolution structure of the intact GPC,including MPER is not available.In this study,we used alanine substitution to scan all 16 residues located in LASV MPER.Western blotting and quantification fusion assay showed that the residues located at the C terminus of the HR2(M414 and L415)and N terminus of the MPER(K417 and Y419)are critical for GPC-mediated membrane fusion function.Furthermore,cell surface biotinylation experiments revealed that M414 A,K417 A and Y419 A expressed similar levels as WT,whereas L415 A mutant led to a reduction of mature GPC on the cell surface.Moreover,substitution of these residues with the similar residue such as M414 L,L415 I,K417 R and Y419 F would partly compensate the loss of the fusion activity caused by the alanine mutant in these sites.Results from this study showed that several key residues in the MPER region are indispensable to promote the conformational changes that drive fusion events and shed light on the structure analysis of LASV GPC and anti-LASV therapeutics.