Fig 2: C1/C2 dimorphism determines the distance between peptide p8 and HLA-C.(A) Hydrogen bond between HLA-C position 77 and amide in terminal peptide bond in C*08:02-G12D-9mer and C*05:01-A18L-9mer structures. (B–D) Distance between HLA-C position 77 Cß and Ca of peptide p8 Ser of HLA-C*08:02-G12D-9mer (B), HLA-C*05:01-G12D-A18L (C), and both overlaid (D). (E) Distances between HLA-C position 77 Cß and Ca of peptide pO-1 in 12 HLA-C crystal structures. (F) Distance between Tyr 171 O? and peptide N-terminus of C*08:02 and C*05:01. (G) Distance between Tyr 171 O? and peptide N-terminus in 12 HLA-C crystal structures. (H) Distance between peptide p8 side chain and HLA-C position 77 Val side chain in structures of C*08:02 and C*05:01. (I) Distance between peptide pO-1 side chain and HLA-C position 76 Val side chain in 12 HLA-C structures. (J) Torsion angle of terminal peptide bond in 12 HLA-C structures. (K) Stimulation of Jurkat-TCR9a+ and Jurkat-TCR10+ by 221 cells expressing HLA-C*08:02, HLA-C*05:01, HLA-C*05:01-N77S, or HLA-C*05:01-K80N preloaded with G12D-9mer (left) or G12D-10mer (right). Means and standard errors of IL-2 concentration in culture supernatant measured by ELISA from three biological replicates are shown. Significance in (E), (G), (I), and (J) was measured using an unpaired t-test with Welch’s correction, **p<0.01, ****p<0.0001. Source data available in Figure 5—source data 1. Figure 5—source data 1.Raw distances for Figure 5E, G, I.Raw angles for 5J. ELISA readings for 5K.

Fig 3: C1 but not C2 HLA-C allotypes present peptides with C-terminal (pO) Ala.(A) Stabilization of HLA-C on TAP-deficient 221 cells expressing HLA-C*08:02 or HLA-C*05:01. (B) Data from (A) shown as fold median fluorescent intensity (MdFI) relative to no peptide (NP) from a minimum of four independent experiments. (C) Stimulation of TCR9a+ Jurkat cells by 221C*05:01 cells preloaded with KRAS peptides at indicated concentrations. Means and standard errors of IL-2 concentration in culture supernatant measured by ELISA from two biological replicates are shown. (D) Frequency of indicated residues at the C-terminus (pO) in peptides eluted from HLA-C*08:02 or HLA-C*05:01. (E) Frequency of pO Ala in peptides eluted from HLA-C*08:02 or HLA-C*05:01. (F) Volcano plot displaying pO amino acid frequency from 21 HLA-C allotypes. The C2/C1 ratio is shown for the average frequency of each amino acid. (G) The frequency of pO Ala from 21 HLA-C allotypes by C1/C2 status. Statistical significance was assessed by unpaired t-test with Welsh’s correction, *p<0.05, **p<0.001, ****p<0.0001. Source data available in Figure 2—source data 1. Peptide sequences and p9 frequency analysis are available in Figure 2—source data 2. Figure 2—source data 1.Normalized MdFI values for Figure 2B, ELISA readings for Figure 2C and amino acids frequencies for Figure 2D-G. Figure 2—source data 2.HLA-C Peptide sequences used for analysis.

Fig 4: The expression of IL-6 (a), IFN-γ (b), IL-2 (c), TNF-α (d), and IL-10 (e) in PBMCs stimulated by the double knockdown (KD) anti-CD19 CAR-T cell killing conditioned medium was significantly reduced, as detected by RT-PCR. **P < .01, ***P < .001, mean ± SEM.

Fig 5: Large residues at p8 diminish T cell recognition of C1 HLA-C.(A–F) Stimulation of Jurkat-TCR9a+ cells by 221C*08:02 cells preloaded with pO-1 substitutions of G12D-9mer (A, B) and A18L-9mer (D, E) at indicated concentrations. Each peptide was tested individually in two independent experiments and displayed by p8 sequence as indicated. (C, F) Data from (A, B) and (D, E) are summarized in (C) and (F), respectively, by pooling data by indicated p8 substitutions. Means and standard errors of IL-2 concentration in culture supernatant measured by ELISA are shown. Source data available in Figure 7—source data 1. Figure 7—source data 1.ELISA readings for Figure 7.