Critical role for the Ly49 family of class I MHC receptors in adaptive natural killer cell responses

Abstract

Adaptive natural killer (NK) cell memory represents a new frontier in immunology. Work over the last decade has discovered and confirmed the existence of NK cells with antigen-specific memories, which had previously been considered a unique property of T and B cells. These findings have shown that antigen-specific NK cells gain their specificity without the use of RAG proteins, representing a novel mechanism for generating antigen specificity, but the details of this mechanism have remained a mystery. We have discovered that members of the Ly49 family of surface receptors are critically involved in both the sensitization and the challenge phases of an NK cell memory response, as is antigen presentation from their binding partner, the class I MHC. Moreover, we demonstrate that the Ly49-interacting component of a presented antigen dictates the specificity of the NK cell memory response, implicating Ly49 receptors themselves in antigen-specific recognition. Finally, we demonstrate that adaptive NK cell memories can protect against an otherwise lethal melanoma without T cell or B cell support. These findings offer insight into the mechanism behind NK cell antigen specificity and demonstrate the clinical potential of this adaptive immune cell.

Keywords: Ly49 receptors; adaptive memory; cancer vaccines; immune memory; natural killer cells.

Fig. 1.

Ly49I expression is required for adaptive, T cell- and B cell-independent memory. (A) Schematic of the experimental ear swelling test. (B and C) Ear swelling following hapten challenge of T cell- and B cell-deficient mice with the indicated Ly49 modulations. Short-term (B) and long-term (C) memory is indicated. (D) Ear swelling for mice challenged with either the sensitizing hapten (solid lines) or an irrelevant hapten (dotted lines). (E and F) H&E staining of infiltrating cells into ears taken at day 2 of the ear swelling response as in B. (Magnification: E, 20×.) Each point in F represents the median value obtained across seven fields in E. Horizontal bars represent the mean value of these medians. (G and H) Ear swelling results using the indicated peptide antigens. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

NK cells mediate Ly49-dependent adaptive immunity. (A and B) Ear swelling in Rag1^−/− (A) or CD8⁺ T cell-deficient (B) mice after depletion of NK cells (NK1.1) or all CD4⁺ cells (CD4). (C) Ear swelling in Rag1^−/− or WT mice after depletion of all Ly49C/I⁺ cells, or Ly49G⁺ cells as a control. (D) Ear swelling following IFNγ neutralization (using XMG1.2 antibody) in Rag1^−/− and WT mice. (E) Ear swelling in Rag1^−/− mice lacking perforin. Results are pooled from two experiments. (F and G) Ear sections collected as in Fig. 1 were stained with anti-NKp46 to reveal NK infiltration. (Magnification: G, 20×; G Inset, 60×.) Each point in F represents the sum count of stained cells across five fields. Horizontal bars represent the mean value of these sums. Arrowheads in G represent NK cells. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 3.

Ly49C/I are involved in direct antigen recognition. (A) Ear swelling following nondepleting antibody-mediated blockade of Ly49C/I or Ly49G during sensitization or challenge phases as indicated. All groups were sensitized and challenged with DNFB except the naive group, which was only challenged. (B) Ear swelling in Rag1^−/− or WT mice following challenge with either the MHC-I–restricted ovalbumin peptide SIINFEKL (Ova-I) or the MHC-II–restricted peptide ISQAVHAAHAEINEAGR (Ova-II). (C) Ear swelling in Rag1^−/− mice following challenge with the H-2D^b–restricted HPV-E7 peptide RAHYNIVTF. (D and E) Schematic demonstrating the hybrid peptide design (D) used to sensitize mice in a repeat of the ear swelling test (E). Mice were then challenged with the indicated peptide and examined for cross-reactivity with the hybrid peptide. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

NK memory can mediate conventional cancer vaccines. (A and B) Ear swelling in Rag1^−/− mice following sensitization with whole ovalbumin (Whole Ova) protein and challenge with the MHC-I–restricted fragment of whole ovalbumin, SIINFEKL (A), or variant or irrelevant peptides as in Fig. 3 (B). (C) Tumor growth in Rag1^−/− mice following vaccination with the indicated whole protein and flank challenge with 1 × 10⁵ B16F10 melanoma cells transgenically expressing whole ovalbumin or parental, non–antigen-expressing cells as a control. Results are pooled from two experiments. (D) Immunohistology of tumors at end point, performed and quantified as in Fig. 2. (Magnification: D, 20×.) (E) Statistical analysis for the tumor infiltration was performed by one-way ANOVA followed by a Bonferroni post hoc test comparing members of the same challenge group. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.