John T. Vaage

MICROBIAL COLONIZATION INFLUENCES COMPOSITION AND T-CELL RECEPTOR VBETA REPERTOIRE OF INTRAEPITHELIAL LYMPHOCYTES IN RAT INTESTINE

Studies in mice have shown that the composition of intestinal intraepithelial lymphocytes (IEL) may be markedly altered by gut microbial colonization. Such modulation was studied in a rat model by the use of germ‐free and conventionalized animals from which IEL from the small intestine were isolated and analysed by flow cytometry. Conventionalization caused expansion as well as phenotypic alterations of T‐cell receptor (TCR) α/β+ IEL in that the proportions of CD4+ and CD8αβ+TCRα/β+ cells were increased, while the double negative (CD4− CD8−) fraction was reduced. Microbial colonization also influenced the TCR Vβ repertoire of CD8+ IEL in that the proportions of Vβ8.2+ and Vβ10+ cells were increased, whereas Vβ8.5+ and Vβ16+ cells were relatively decreased. Moreover, conventionalization influenced the levels of TCR cell surface expression in the same Vβ subsets. Three‐colour flow‐cytometric analysis demonstrated that skewing of the Vβ repertoire was most pronounced in the CD8αα+ subset, although the numerical increase of IEL mainly included the CD8αβ+ subset. In contrast to IEL, the TCR Vβ repertoire in mesenteric lymph nodes was unchanged after intestinal colonization. These results confirm that TCRα/β+ IEL subpopulations respond dynamically to the microbial gut flora and suggest that their Vβ repertoire can be shaped by luminal microbial antigens.

Selective activation of cAMP-dependent protein kinase type I inhibits rat natural killer cell cytotoxicity.

The present study examines the expression and involvement of cAMP-dependent protein kinase (PKA) isozymes in cAMP-induced inhibition of natural killer (NK) cell-mediated cytotoxicity. Rat interleukin-2-activated NK cells express the PKA α-isoforms RIα, RIIα, and Cα and contain both PKA type I and type II. Prostaglandin E2, forskolin, and cAMP analogs all inhibit NK cell lysis of major histocompatibility complex class I mismatched allogeneic lymphocytes as well as of standard tumor target cells. Specific involvement of PKA in the cAMP-induced inhibition of NK cell cytotoxicity is demonstrated by the ability of a cAMP antagonist, (Rp)-8-Br-adenosine 3′,5′-cyclic monophosphorothioate, to reverse the inhibitory effect of complementary cAMP agonist (Sp)-8-Br-adenosine 3′,5′-cyclic monophosphorothioate. Furthermore, the use of cAMP analog pairs selective for either PKA isozyme (PKA type I or PKA type II), shows a preferential involvement of the PKA type I isozyme, indicating that PKA type I is necessary and sufficient to completely abolish killer activatory signaling leading to NK cell cytotoxicity. Finally, combined treatment with phorbol ester and ionomycin maintains NK cell cytotoxicity and eliminates the cAMP-mediated inhibition, demonstrating that protein kinase C and Ca2+-dependent events stimulate the cytolytic activity of NK cells at a site distal to the site of cAMP/PKA action.

The genes and gene organization of the Ly49 region of the rat natural killer cell gene complex

We here report the cDNA sequences of 11 new rat Ly49 genes with full and three with incomplete open reading frames. Although obtained from different inbred rat strains, these as well as six previously published cDNA represent non‐allelic genes matching different loci in the Brown Norway (BN) rat genome, which is predicted to contain 34 Ly49 loci distributed over the distal part of the NK cell gene complex. Some of the cloned genes appear to be mutated to non‐function in the BN genome, which harbors additional genes with full open reading frames, suggesting at least 26 non‐allelic functional Ly49 genes in the rat. Of the encoded receptors, 13 are predicted to be inhibitory, eight to be activating, whereas five may be both (‘bifunctional’). Phylogenetic analysis bears evidence of a highly dynamic genetic region, in which only the most distally localized Ly49 gene has a clear‐cut mouse ortholog. In phylograms, the majority of the genes cluster into three subgroups with the genes mapping together, defining three chromosomal regions that seem to have undergone recent expansions. When comparing the lectin‐like domains, the receptors form smaller subgroups, most containing at least one inhibitory and one activating or ‘bifunctional’ receptor, where close sequence similarities suggest recent homogenization events.

Rat NKp46 activates natural killer cell cytotoxicity and is associated with FcεRIγ and CD3ζ

NKp46 has been identified in the human, rat, mouse, monkey, and cattle. We have generated a monoclonal antibody, WEN23, against rat NKp46. By flow cytometry, NKp46 is expressed by all natural killer (NK) cells but not by T cells, B cells, granulocytes, monocytes, dendritic cells, or macrophages. Thus, NKp46/WEN23 is the first NK cell‐specific marker in the rat. In a redirected lysis assay, preincubation of the effector cells with WEN23 augmented lysis of the Fc receptor (FcR)+ murine tumor target cells, indicating that NKp46 is an activating NK cell receptor. Moreover, preincubation of the effector cells with WEN23 F(ab′)2 fragments reduced killing of target cells, confirming the activating function of NKp46 and indicating that the mouse tumor target cells express a ligand for rat NKp46. Lysis of FcR− mouse and human tumor target cells was reduced after incubation of effector cells with WEN23, suggesting that rat NKp46 recognizes a ligand that is conserved between primates and rodents. By Western blot and immunoprecipitation using WEN23, NKp46 is expressed as a monomer of ∼47 kDa in interleukin‐2‐activated NK cells. The immunoreceptor tyrosine‐based activation motif bearing adaptor proteins CD3ζ and the γ chain of FcRI for IgE (FcɛRIγ) with NKp46 from lysates of NK cells, indicating that rat NKp46 activates NK cell cytotoxicity by similar pathways as CD16.

Positive and negative MHC class I recognition by rat NK cells.

Summary: The prompt rejection of transplanted allogeneic lymphocytes by rat NK cells in non‐sensitized recipients (allogeneic lymphocyte cyto‐toxicity or ALC) is determined by MHC genes as well as by genes located in the NK complex. The same genetic control is found when NK alloreactivity is measured by an in vitro assay, and we have employed this assay to delineate the specificity of NK cells for the MHC. The MHC of the rat, RT1, contains class 1 genes situated on either side of the class Il/class III region. The majority of these class 1 genes are located in the RT1.C region and expressed class I products usually behave as non ‐classical (class Ib) molecules. They do not serve as restriction elements for the vast majority of conventional a/p T‐cells, in contrast to those class molecules encoded by one or more loci in the classical (doss la) region, RT1. A. However, NK cells appear to recognize the products of either class 1 region. Immunogenetic studies suggest that NK cells are inhibited by RT1.A molecules, whereas RT1.C region molecules may have a dual role in regulating NK cytolytic activity, i.e. they either inhibit or activate natural killing. Based on THESE premises, a model is proposed in which identification of a target as self or non‐self depends on different receptors for class 1 in single NK cells, interpreting coincident positive and negative signals from the various target class I molecules. The putative role of peptides presented by class I, the biological implications, and the evolution of the NK receptors and ether ligands are discussed.

Ly-49s3 Is a Promiscuous Activating Rat NK Cell Receptor for Nonclassical MHC Class I-Encoded Target Ligands

Previous studies of the rapid rejection of MHC-disparate lymphocytes in rats, named allogeneic lymphocyte cytotoxicity, have indicated that rat NK cells express activating receptors for nonclassical MHC class I allodeterminants from the RT1-C/E/M region. Using an expression cloning system that identifies activating receptors associated with the transmembrane adapter molecule DAP12, we have cloned a novel rat Ly-49 receptor that we have termed Ly-49 stimulatory receptor 3 (Ly-49s3). A newly generated anti-Ly-49s3 Ab, mAb DAR13, identified subpopulations of resting and IL-2-activated NK cells, but not T or B lymphocytes. Depletion of Ly-49s3-expressing NK cells drastically reduced alloreactivity in vitro, indicating that this subpopulation is responsible for a major part of the observed NK alloreactivity. DAR13-mediated blockade of Ly-49s3 inhibited killing of MHC-congenic target cells from the av1, n, lv1, and c haplotypes, but not from the u or b haplotypes. A putative ligand was mapped to the nonclassical MHC class I region (RT1-C/E/M) using intra-MHC recombinant strains. Relative numbers of Ly-49s3+ NK cells were reduced, and surface levels of Ly-49s3 were lower, in MHC congenic strains expressing the putative Ly-49s3 ligand(s). In conclusion, we have identified a novel Ly-49 receptor that triggers rat NK cell-mediated responses.

The Novel Inhibitory NKR-P1C Receptor and Ly49s3 Identify Two Complementary, Functionally Distinct NK Cell Subsets in Rats

The proximal region of the NK gene complex encodes the NKR-P1 family of killer cell lectin-like receptors which in mice bind members of the genetically linked C-type lectin-related family, while the distal region encodes Ly49 receptors for polymorphic MHC class I molecules. Although certain members of the NKR-P1 family are expressed by all NK cells, we have identified a novel inhibitory rat NKR-P1 molecule termed NKR-P1C that is selectively expressed by a Ly49-negative NK subset with unique functional characteristics. NKR-P1C+ NK cells efficiently lyse certain tumor target cells, secrete cytokines upon stimulation, and functionally recognize a nonpolymorphic ligand on Con A-activated lymphoblasts. However, they specifically fail to kill MHC-mismatched lymphoblast target cells. The NKR-P1C+ NK cell subset also appears earlier during development and shows a tissue distribution distinct from its complementary Ly49s3+ subset, which expresses a wide range of Ly49 receptors. These data suggest the existence of two major, functionally distinct populations of rat NK cells possessing very different killer cell lectin-like receptor repertoires.

Microbial colonization induces oligoclonal expansions of intraepithelial CD8 T cells in the gut

Two populations of CD8+ IEL generally express restricted, but apparently random and non‐overlapping TCR repertoires. Previous studies in mice suggested that this could be explained by a dual origin of CD8+ IEL, i.e. that CD8αβ+ IEL derive from a few peripheral CD8+ T cell lymphoblasts stimulated by microbial antigens in gut‐associated lymphoid tissue, whereas CD8αα+ IEL descend from an inefficient intestinal maturation pathway. We show here that the gut mucosa, instead, becomes seeded with surprisingly broad and generally non‐overlapping CD8 IEL repertoires and that oligoclonality is induced locally after microbial colonization. In germ‐free (GF) rats, both CD8αβ+ and CD8αα+ IEL displayed surprisingly diverse TCR Vβ repertoires, although β‐chain diversity tended to be somewhat restricted in the CD8αα+ subset. CDR3 length displays in individual Vβ‐Cβ and Vβ‐Jβ combinations generally revealed polyclonal distributions over 6–11 different lengths, similar to CD8+ lymph node T cells, and CDR3β sequencing provided further documentation of repertoire diversity. By contrast, in ex‐GF rats colonized with normal commensal microflora, both CD8αβ+ and CD8αα+ IEL displayed oligoclonal CDR3 length distributions for most of the Vβ genes analyzed. Our data suggest that microbial colonization induces apparently random clonal expansions of CD8αβ+ and CD8αα+ IEL locally in the gut.

Rat natural killer cell receptor systems and recognition of MHC class I molecules

Summary: Rat natural killer (NK) cells recognize MHC‐I molecules encoded by both the classical (RT1‐A) and non‐classical (RT1‐C/E/M) MHC class I (MHC‐I) regions. We have identified a receptor, the STOK2 antigen, which belongs to the Ly‐49 family of killer cell lectin‐like receptors, and we have localized the gene encoding it to the rat natural killer cell gene complex. We have also shown that it inhibits NK cytotoxicity when recognizing its cognate MHC‐I ligand RT1‐A1c on a target cell. This is the first inhibitory Ly‐49–MHC‐I interaction identified in the rat and highlights the great similarity between rat and mouse Ly‐49 receptors and their MHC ligands. However, the mode of rat NK‐cell recognition of target cells indicates that positive recognition of allo‐MHC determinants, especially those encoded by the RT1‐C/E/M region, is a prevalent feature. NK cells recruited to the peritoneum as a consequence of alloimmunization display positive recognition of allodeterminants. In one case, NK cells activated in this way have been shown to be specific for the immunizing, non‐classical class I molecule RT1‐Eu. These findings show that allospecific NK cells sometimes show features reminiscent of the adaptive immune response.

Regional Phenotypic Specialization of Intraepithelial Lymphocytes in the Rat Intestine Does Not Depend on Microbial Colonization

Recent studies in mice and humans have provided evidence for regional specialization of gut intraepithelial lymphocytes (IEL). Here the authors report striking regional variability in the composition of IEL in rat small and large intestine. Two‐colour immunofluorescence in situ analysis showed that the distribution of the CD3+ and CD3− IEL subpopulations varied, the proportion of T cells (CD3+) being higher in the ileum than in the jejunum and smallest in the colon. These differences were explained by variable numbers of the T‐cell receptor (TCR)α/β+ (both CD8+ and CD4+) but not the TCRγ/δ+ subset. Moreover, the various IEL subpopulations showed distinct intraepithelial distribution patterns with CD4+ and CD8αβ+ T cells situated near the lamina propria, while CD3− IEL were located preferentially towards the adluminal part of the epithelium. Regional phenotypic variation did not depend on intestinal colonization because analogous results were obtained in germ‐free rats. Conventionalization nevertheless caused a marked relative increase of small intestinal TCRα/β+ but not TCRγ/δ+ IEL. This increase was more sustained in the jejunum than ileum and eventually reduced the phenotypic IEL differences between the two sites. By contrast, microbial colonization of the colon induced only a transient increase of intraepithelial TCRα/β+ cells with no permanent phenotypic alterations. Both CD3+ and CD3− IEL contained subpopulations that expressed NKR‐P1 independent of intestinal colonization. These results demonstrate phenotypic specialization of IEL at different levels of the gut and suggest that the indigenous flora is not essential to this end.