[Show abstract][Hide abstract] ABSTRACT: We have used flow cytometry to study the stability and peptide-binding capability of MHC class I (MHC-I) on the surface of normal C57BL/6 mouse T lymphoblasts. The MHC-I molecules on each cell are nearly evenly divided into two populations with mean half-life values of approximately 1 and 20 h. Our observations suggest that members of the later contain peptide bound with medium to high affinity. Cell surface MHC-I molecules capable of binding exogenous peptide (thus, "peptide-receptive") belong almost entirely to the less stable population. Before exogenous peptide can bind, MHC-I must undergo a change, probably loss of a very low affinity peptide. For MHC-I-K(b), we found that the maximum rate for binding of exogenous peptide corresponds to a t(1/2) value of 12 min. To maintain the 50:50 steady-state distribution of long- vs short-lived MHC-I molecules on the cell surface, approximately 20 short-lived molecules must be exported to the cell surface for each long-lived molecule.
Preview · Article · Dec 2001 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: With the aim of identifying natural killer (NK) activation receptors, we immunized BALB/c mice with (BALB/cxB6)F1 NK LAK cells and made B-cell hybridomas. These were screened for monoclonal antibody (MAb) reacting with an NK activation receptor by using an antibody-induced redirected lysis (AIRL) assay against FcR-bearing P815 targets. Four hybridomas, clones 1C10, 1F10, 2D10 and 4G4, were selected for further characterization. Protein G-purified MAbs from these clones activated both resting and IL-2 activated B6 or F1 NK cells in the AIRL assay. 1F10 MAb, but not the other three MAbs, could compete for the binding of anti-NK1.1 (PK136) MAb to F1 NK cells. The four MAbs were screened for their ability to bind to or activate NK cells from the mouse strains SJL/J, DBA/2, 129/J, C3H/J, and BALB.K. None showed activity except IC10, which could bind to and activate SJL/J NK cells. When members of the NKR-P1 family from both B6 mice (A, B, and C genes expressed) and SJL mice (only A and B genes expressed) were expressed in Jurkat cells and tested for their antibody reactivity, PK136 MAb was found to recognize B6 NKR-P1C and SJL/J NKR-P1B; IC10 MAb was found to recognize NKR-P1-A, -B and -C from B6, but not NKR-P1A or -B from SJL/J; and 1F10 MAb was found to react only with B6 NKR-P1C.
[Show abstract][Hide abstract] ABSTRACT: Exposure of primary T cell blasts to stress in the forms of heat, hydrogen peroxide, or high-density growth conditions resulted in a state of enhanced susceptibility to killing by syngeneic IL-2-activated NK cells or lymphokine-activated killer cells, but not to killing by CTL. Cytotoxicity was perforin mediated and was not due to decreased target expression of total MHC class I. The levels of stress used had little effect on cell viability. For thermal stress, sensitization increased with temperature, required a minimum exposure time, and disappeared when cells were given a long enough recovery time. Our data support a model that predicts that activated NK cells play a role in the immunosurveillance of nontransformed stressed cells in normal animals.
Preview · Article · Oct 2000 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: NK-mediated cytotoxicity involves two families of receptors: activating receptors that trigger lysis of the target cells being recognized and inhibitory receptors specific primarily for MHC I on the target cell surface that can override the activating signal. MHC I molecules on the cell surface can be classified into molecules made stable by the binding of peptide with high affinity or unstable molecules potentially capable of binding high affinity peptide (hence, peptide receptive) and being converted into stable molecules. It has been previously shown that the Ly-49A inhibitory receptor recognizes stable Dd molecules. We show in this study that the inhibitory receptor Ly-49CB6 recognizes peptide-receptive Kb molecules, but does not recognize Kb molecules once they have bound high affinity peptide.
Preview · Article · Dec 1999 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: The mouse NKR-P1 family includes at least three genes: NKR-P1A, -B, -C. Neither surface expression nor function of the NKR-P1B gene product has previously been shown. Here, we demonstrate that the SJL/J allele of the NKR-P1B gene product is expressed on SJL/J NK cells, and is recognized by PK136 mAb. Interestingly, the same mAb does not recognize the NKR-P1B gene product of C57BL/6. We have also generated a novel mAb, 1C10, that recognizes an activation receptor on SJL/J NK cells. Activation of the NKR-P1B receptor-inhibited 1C10 mAb induced redirected lysis and recruited SHP-1, indicating that NKR-P1B is an inhibitory receptor. Therefore, the mouse NKR-P1 gene family, like the Ly49 family, includes both activation and inhibitory receptors.
Preview · Article · Jun 1999 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: NK recognition and lysis of targets are mediated by activation receptor(s) whose effects may be over-ridden by inhibitory receptors recognizing class I MHC on the target. Incubation of normal lymphoblasts with a peptide that can bind to their class I MHC renders them sensitive to lysis by syngeneic NK cells. By binding to class I MHC, the peptide alters or masks the target structure recognized by an inhibitory NK receptor(s). This target structure is most likely an "empty" dimer of class I heavy chain and beta2m as opposed to a "full" class I trimer formed by binding of specific peptide that is recognized by CTL.
Preview · Article · Aug 1998 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: We have investigated the capacity of human MHC class I HLA-B gene products, HLA-B27, -B7 (fully human), and -B7kb (human-mouse hybrid consisting of the alpha1 and alpha2 domains of HLA-B7, and the alpha3 and cytoplasmic domains of mouse H-2Kb), expressed on mouse NK cells during ontogeny to influence NK recognition of otherwise syngeneic mouse target cells. Despite a high level of surface expression of the transgene (comparable to that of endogeneous H-2DbKb molecules), the direct killing of YAC-1 targets, and the killing of P815 targets in a redirected lysis assay, the NK effectors of these transgenic mice could not mediate hybrid resistance-like killing of nontransgenic C57BL/6 target cells either in vitro or in vivo. Splenocytes from B6-B27 mice could be used to generate CTL lines against a B27-binding peptide, implying that T cells restricted by HLA-B27 developed during ontogeny. NK cells from B6-B27 could lyse B6-B27 Con A lymphoblasts pulsed with Db-binding peptide but not B27-binding peptides. Taken together, our results show that these human HLA-B transgene products cannot function as class I MHC "self" elements for mouse NK cells, even when present throughout ontogeny.
Preview · Article · Jan 1998 · The Journal of Immunology