Common Minor Histocompatibility Antigen Discovery Based upon Patient Clinical Outcomes and Genomic Data

.... Tetramer-Cytokine Flow Cytometry B lymphocytes from healthy donors expressing HLA-A0201 were immortalized using Epstein-Barr virus infection to generate EBV-LCLs. TRIM42 protein expression in each LCL was confirmed by western blot. Genomic DNA was isolated from each EBV-LCL, and PCR was used to amplify DNA flanking the T4A associated cSNP (rs9876490). Conventional Sanger sequencing was performed to determine the genotype at rs9876490 for each EBV-LCL. One EBV-LCL line had a genotype predicted to

...-PROTEAN Trans-Blot Module (Biorad, Hercules, CA) at 250 mA for 70 min and blocked with 5% milk for 1 hr prior to incubation with the anti-TRIM42 antibody (Abcam, Cambridge, MA) at 11000 dilution overnight at 4C. The next day, the membrane was washed for 30 min in TBS-0.1% Tween and then incubated with horse-radish peroxidase conjugated anti-rabbit IgG antibody for 1 hr and developed using the Amersham ECL Western Blotting Detection kit (GE Healthcare, Piscataway, NJ). The membrane was reprobed with

... not have a tetramer+ population was obtained from a patient 9 months post-transplant who was on systemic steroids for extensive cGvHD ( Figure 3D ). We did not identify any tetramer+ populations in an HLA-A2- control ( Figure 3E ), or 2 HLA-A2+, 3-month post-transplant samples from patients genetically predicted to be gIR- to GLYTYWSAGA ( Figure 3F, 3G ). The P12 peptide, renamed T4A, is derived from the alanine allele on cSNP rs9876490_C, which is contained in the protein TRIM42. 10.1371

... blots on whole cell lysates of normal human tissue (colon, heart, liver, skin, testis and PBMC), 4 human AML samples and Jurkat cells (positive control) are shown (4D). Western blotting of Jurkat cells reveals 2 bands of roughly 100 kDa (full length TRIM42) and 50 kDa (unknown protein product). The TRIM42 band is seen at various levels in all AML samples and faintly in PBMC. No TRIM42 protein is detected in the other human tissues. GAPDH expression (40 kDa) was used as a loading control. Subcellular fractionation was performed on AML blasts and isolated healthy donor granulocytes (4E). The fractions are labeled C, cytosolic; N, nuclear; M, membrane; Sk, cytoskeletal. No TRIM42 protein is observed in granulocytes, but the same bands seen in the Jurkat cell positive control are detected in the N and Sk fractions of the AML blasts. The allele frequencies and genotypes for rs9876490, the T4A associated cSNP, in the original ethnic populations used by the International HapMap Project (CEU, HCB, JPT, YRI

...). In the case of rs9876490 the T4A gIR+ frequency observed in our cohort () fits the predicted MRD SCT curve well. Because of concerns about immune-mediated toxicity from mHA-specific CTLs towards non-hematopoietic tissues, we evaluated TRIM42 tissue expression. We performed western blots to probe for TRIM42 protein in normal human tissues and AML samples ( Figure 4D ). Western blotting for TRIM42 in Jurkat cells, a positive control, revealed two bands of roughly 100 kDa and 50 kDa. The high MW

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Gene: TRIM42 - tripartite motif containing 42 - (NCBI)

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