| Objective: This study combined second-generation sequencing technology,TCR immunome library sequencing technology,bioinformatics analysis software and ELISPOT experimental method to explore the possibility of immune versatility among heteroallelic patients by changing the single anchoring residues of the 9mer peptide and designing orthogonal experiments to change the amino acid residues in the non-CDR3 binding region,so as to provides a reference for the rapid screening of new antigens with strong immunogenicity.Methods: 1.Obtain patients’ cancer and paracancerous tissue,perform second generation sequencing,get HLA-I genotyping,screen patients whose HLA-I genotyping meets the experimental conditions.2.Screening of experimental 9mer peptide:(a)Pre-experimental group: replace the initial antigenic peptide with H,W and F with a single anchoring residue,replace position 2 and position 9 respectively,get the corresponding 9mer peptide into the experiment;(b)Perform 20 amino acid classification,design orthogonal experiments,write codes using Python software,get the sequences of the 9mer peptides corresponding to the nine amino acid substitutions,and query their affinity and stability parameters with several HLA allelic genotype molecules with the highest percentage in the population on Net MHCpan4.1 and Net MHCstab1.0;(c)according to the minimum conditions for experimental entry(IC50 value < 1000 nm,p-MHC expected half-life > 0.5h),the 9mer peptide with better immunogenicity in each group was screened into the experiment with reference to the affinity and stability prediction parameters.3.In vitro synthesis of the screened 9mer peptide and non-methylated Cp G adjuvant.4.Peripheral blood of patients was obtained,dendritic cells presenting the experimental 9mer peptide were induced and cultured in vitro,and peripheral blood lymphocytes of the same patients were stimulated with them,and the 9mer peptide was quantified by ELISPOT assay was performed to quantify the immune effect of each peptide.5.T-lymphocytes generated after stimulation by dendritic cells presenting the experimental 9mer peptides were sent to TCR immunome library for sequencing to quantify the clonal expansion and diversity of T-lymphocytes.Results: 1.The ELISPOT results of the four 9mer peptide sequences obtained from the pre-experimental group showed that amino acid substitution for anchor position 2 or 9 could enhance the immunogenicity of the 9mer peptide,and there was no significant difference in the effect of residue substitution on the immunogenicity of the 9mer peptide at the two sites.2.2676 9mer peptides were obtained from the experimental group,and the parameter prediction results showed that the 9mer peptide corresponding to amino acid substitution in group 1 had the strongest binding affinity with HLA-I molecule,and the 9mer peptide corresponding to amino acid substitution in groups 2 and 7 had the strongest binding stability with HLA-I molecule.3.14 9mer peptides were screened according to the experimental conditions,and the 9mer peptideloaded mature dendritic cells were successfully induced to stimulate the proliferation and differentiation of T lymphocytes by simulating the neoantigen presentation process in vitro.4.The ELISPOT results of the experimental groups were counted and set to T.The T values were normalized by Z-Score to obtain T ’ value,Scheirer-Ray-Hare test was performed,and the results of data analysis showed that: a)the immunogenicity of the 9mer peptide corresponding to the amino acid substitution mode in group 7 was the strongest,and the experimental results were not completely consistent with the results of the software predicted parameters(not consistent with the predicted results of the affinity parameters and partially consistent with the predicted results of the stability parameters);b)the immunogenicity of different amino acid residue substitution mode had a significant effect on the immunogenicity of the neoantigen(P = 0.00006 < 0.05),while patients with heteroallelic genes had no significant effect on the immunogenicity of the neoantigen(P = 0.89570 > 0.05),and there was no interaction between the effect of different amino acid residue substitution modes and patients with heteroallelic genes on the immunogenicity of the neoantigen(P = 0.31661 > 0.05).5.Further,the top 9mer peptide-stimulated T lymphocytes in the immunogenicity prediction ranking were sent for TCRβ chain immunohistochemical library sequencing,and One-Way ANOVA was performed sequentially on Clonality values,Shannon.Index,and the results showed that amino acid residue substitution in the non-CDR3 binding region had an effect on clonal expansion of T cells(P1 = 0.984 > 0.05)and the diversity of clonal populations(P2=0.770>0.05)did not have a significant effect.When the Clonality values and Shannon.Index were subjected to LSD multiple comparisons between the groups,there was no significant difference in the effect of different substitutions of amino acid residues in the non-CDR3 binding region on the clonal amplification of TCRs and the clonal diversity of TCRs.6.In-depth analysis of TCR sequencing results showed that TRBV and TRBJ dominance-taking gene families overlapped in all four patients,and there were two overlaps.The greatest increase in the diversity of V-J gene combination in T lymphocytes stimulated by 9mer peptide in group 7 and no change in the diversity of V-J gene combination in T lymphocytes stimulated by 9mer peptide in group 3.Conclusion: 1.Amino acid substitution of anchor position 2 or 9 enhances the immunogenicity of the 9mer peptides,and for 9mer peptides with the same amino acid residues in A pocket and F pocket,changing the anchor residues in positions 2 and 9affects their immunogenicity to approximately the same extent.2.Amino acid residue substitution in the non-CDR3 binding region had a significant effect on the immunogenicity of the neoantigen,in which the nine-peptide with hydrophobic amino acid residues,electroneutral hydrophilic amino acid residues,and charged hydrophilic amino acid residues substituted at positions 1,2,and 9,in that order,was the most immunogenic.3.For HLA gene heterozygotes,there is no significant individual variability in the immune effect of neoantigen stimulation in heteroallelic patients,which means that the neoantigen is immunologically universal among heteroallelic patients.4.Patient’s basal immune status may not be related to the immune efficacy of neoantigen.5.V-J gene rearrangement can be homogeneous among individuals with different HLA-I genotypes,and V-J gene rearrangement is individual-specific,with hydrophobic amino acid residues at position 1,position 2,and position 9,in that order,and charged hydrophilic amino acid residues at the nine-peptide stimulating T lymphocytes to undergo V-J gene rearrangement most strongly.6.Rapid screening of strongly immunogenic neoantigen can be assisted by substitution of amino acid residues in the non-CDR3 region. |
PDF Full Text Request