Natural killer (NK) cell subsets in the mouse. NK-1.1+/LGL-1+ cells restricted to lysing NK targets, whereas NK-1.1+/LGL-1- cells generate lymphokine-activated killer cells

Our laboratory has recently identified a novel Ag, LGL-1, that is expressed on a major population of mouse NK cells. Two color immunofluorescence analysis has demonstrated that spleen cells consist of two major subsets of NK cells. We have identified an NK-1.1+/LGL-1+ subset that consists of 50% of the total NK cells and an NK-1.1+/LGL-1- subset comprising the remaining 50%. Because numerous reports have identified NK cells as the major cell type mediating lymphokine-activated killing (LAK), the NK-1.1+/LGL-1+ and NK-1.1+/LGL-1- subsets were examined for their contribution toward LAK generation, as defined by their ability to lyse P815 tumor targets. Antibody plus C depletion experiments with the use of anti-LGL-1 indicated that LGL-1+ cells were not found on LAK precursor or effector cells. Two-color cell sorting experiments were also performed to separate freshly isolated NK-1.1+/LGL-1+ spleen cells from the NK-1.1+/LGL-1- subset. It was found that the vast majority of LAK activity (greater than 95%) is derived from the NK-1.1+/LGL-1- cells. Cell sorting of LAK effectors also demonstrated that the NK-1.1+/LGL-1- cells mediated the vast majority of lysis against P815 targets. Similar results were obtained when NK cell subsets were analyzed for their contribution toward ADCC. These findings may prove important in understanding and further elucidating the contribution of NK cells to the LAK phenomenon. Our data also indicates that subsets of NK cells exist that may function differently in response to stimulation by various lymphokines and cytokines.     

Allelic variation in the ectodomain of the inhibitory Ly-49G2 receptor alters its specificity for allogeneic and xenogeneic ligands

The Ly-49 multigene receptor family regulates mouse NK cell functions. A number of Ly-49 genes exhibit allelic variation, but the functional significance of allelic differences in extracellular domains of Ly-49 receptors regarding ligand specificity is largely unknown. Amino acid differences exist in the extracellular domains of the B6 and BALB/c allele products of the inhibitory Ly-49G receptor. We constructed chimeric Ly-49 receptors consisting of common cytoplasmic and transmembrane regions of the activating Ly-49W receptor fused with the ectodomains of the B6 and BALB/c alleles of Ly-49G. Expression of these chimeras in the RNK-16 rat NK cell line allowed us to study the specificity of inhibitory receptor ectodomains as they stimulated NK lytic activity. We found that the ectodomain of the BALB/c allele of Ly-49G recognizes both H-2D(d) and D(k) class I MHC alleles, whereas the ectodomain of the B6 allele of Ly-49G recognizes D(d), and not D(k). The specificity for D(k) as well as D(d) of the wild-type Ly-49G(BALB/c) allele product was confirmed with RNK-16 transfectants of this inhibitory receptor. Furthermore, the ectodomain of the Ly-49G(BALB/c) allele recognizes a distinct repertoire of xenogeneic ligands that only partially overlaps with that recognized by Ly-49G(B6). Our results indicate that allelic variation in Ly-49 extracellular domains can have functional significance by altering Ly-49 receptor specificity for mouse class I MHC and xenogeneic ligands.     

Chromosomal location of the Ly-49 (A1, YE1/48) multigene family. Genetic association with the NK 1.1 antigen

The Ly-49 (A1, YE1/48) Ag is a disulfide-linked dimer with 44-kDa subunits, and is expressed on the cell surface of rare T cell tumors of C57BL/6 origin. Although this Ag is undetectable by flow microfluorimetry analysis, normal cells have been shown to express the A1 Ag by immunoprecipitation experiments performed on surface radioiodinated spleen and thymus cells. We (J. Immunol. 143:1379, 1989) and others (J. Immunol. 142:1727, 1989) have recently isolated cDNA encoding the Ly-49 Ag. Southern blots with an Ly-49 cDNA probe revealed multiple bands, consistent with cross-hybridization to other members of a multigene family, and significant RFLP between the C57BL/6 and BALB/c strains. When the RFLP patterns displayed by other common laboratory strains as well as informative recombinant inbred strains were examined, the Ly-49 gene family displayed five RFLP patterns and the entire family was found to reside on a contiguous stretch of the distal portion of mouse chromosome 6, the same region to which the NK1.1 Ag has been mapped. Although tissue distribution studies and transfection analysis ruled out the possibility that Ly-49 was identical to NK1.1 Ag, approximately 20% of NK1.1 cells isolated from normal spleen coexpressed Ly-49 and all Ly-49+ cells were CD3-. Although spleen cells cultured in high doses of rIL-2 demonstrated similar coexpression of NK1.1 and Ly-49, approximately 10% of CD3+ cells coexpressed Ly-49. The chromosomal mapping data and the expression of the Ly-49 and NK1.1 Ag suggest that the NK1.1 Ag may be a member of the Ly-49 multigene family.     

The NKR-P1B gene product is an inhibitory receptor on SJL/J NK cells

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.     

Investigations into the nature of Igh-V region-restricted T cell interactions by using antibodies to antigens on methylcholanthrene-induced sarcomas. I. Analysis of an Igh-V-restricted suppressor-inducer factor

We have previously demonstrated the relationship between antigens on BALB/c methylcholanthrene (MC)-induced fibrosarcomas and T cell regulatory molecules by using a variety of antisera raised to these sarcomas in BALB/c and BALB/c X C57BL/6 (CB6F1) mice. One such pool of antiserum, a CB6F1 anti-CMS 4 (Pool XIV) serum, was used to investigate the nature of the T cell regulatory structures recognized by these antibodies. Pool XIV antiserum was capable of blocking the induction of feedback suppression by Ly-1 TsiF, an SRBC-specific suppressor T cell factor secreted by Ly-1+, 2- I-J+ T cells. Ly-1 TsiF induces suppression by interacting with an Ly-1+,2+ I-J+ T cell target. Successful interaction of Ly-1 TsiF with its target cell requires genetic homology between inducer and target cells at the variable region of the immunoglobulin heavy chain gene complex (Igh-V). The addition of Pool XIV antiserum to primary in vitro anti-SRBC cultures resulted in blocking the ability of Ly-1 TsiF from Igha (BALB/c) and Ighj (CBA/J) mice to induce suppression on syngeneic cells, whereas suppression induced by Ly-1 TsiF in Ighb (B6), Ighc (DBA/2), Ighd (A/J), and Ighe (AKR) mice are unaffected by addition of the Pool XIV antiserum. The ability of Pool XIV antiserum to block Ly-1 TsiF activity is linked to the Igh region, because Pool XIV antiserum can block Ly-1 TsiF from BALB/c (H-2d, Igha) and the Igh congenic B.C9 (H-2b, Igha) while not affecting Ly-1 TsiF activity on B6 (H-2b, Ighb) or its Igh congenic C.B20 (H-2d, Ighb). In CB6F1 animals, Pool XIV antiserum could block the ability of CB6F1 Ly-1 TsiF to suppress BALB/c spleen cells but not B6 spleen cells. Conversely, Pool XIV antiserum could block the ability of BALB/c Ly-1 TsiF to suppress CB6F1 spleen cells, whereas B6 Ly-1 TsiF showed normal suppressive activity in the presence of Pool XIV antiserum. In contrast, Pool XIV was capable of blocking the ability of Ly-1 TsiF from BALB/c into CB6F1 bone marrow chimeras (BMC) to suppress both BALB/c and B6 mice, whereas the activity of Ly-1 TsiF from B6 into CB6F1 BMC on BALB/c or B6 spleen cells was unaffected by the addition of Pool XIV antiserum. We then investigated the molecular nature of the molecule recognized by Pool XIV antiserum on the Ly-1 TsiF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of murine T cells by staphylococcal enterotoxin E: requirement of MHC class II molecules expressed on accessory cells and identification of V beta sequence of T cell receptors in T cells reactive to the toxin

We investigated a mechanism leading to activation of murine T cells by staphylococcal enterotoxin E (SEE). L cells transfected with I-Ab genes but not control L cells supported IL-2 production by SEE-induced C57BL/6 T lymphoblasts upon restimulation with SEE. mAb to I-Ab markedly inhibited the above response. Flow cytometric analyses showed that SEE-induced C57BL/6 T lymphoblasts are composed of both CD4+ T cells and CD8+ T cells, and that larger parts of them bore V beta 11 (40-75%). mAb to V beta 11 markedly inhibited the SEE-induced proliferative response and IL-2 production by T cells. Analysis of SEE-induced IL-2 production in spleen cells from various mouse strains showed that C57BL/6 and B10.A(4R) mice (I-E, not expressed; V beta 11+ T cells, normally generated) are highly responsive to SEE. In contrast, BALB/c, C3H/HeN, (C57BL/6 x BALB/c or C3H/HeN) F1 mice (I-E, normally expressed and V beta 11+ T cells, deleted), and SJL and C57L mice (V beta 11 genes, deleted) are weakly responsive to SEE. The results indicate that SEE activates mainly T cells bearing V beta 11 in physical association with MHC class II molecules expressed on AC. In addition, the results indicate that SEE activates both CD4+ T cells and CD8+ T cells.     

Monoclonal antibody 4H12 recognizes subsets of adherent-lymphokine activated killer cells and splenic natural killer cells from pregnant and neonatal mice

Using a new mAb, 4H12, that recognizes a plasma membrane-associated Ag on granulated metrial gland cells, we identified subsets of murine NK cells in spleen cell-derived adherent lymphokine activated killer cells and in the spleens of neonatal and pregnant mice. In spleen cell adherent-lymphokine-activated killer cultures, 4H12 Ag was detected on a small subset of cells after 7 days culture and expression increased with time to 70% of the cells after 21 days culture. 4H12+ cells were large (up to 70 microns) and granular. The Ag was also detected in the cytoplasmic granules of some, but not all 4H12 surface positive cells. Coexpression studies indicated 4H12+ cells were predominantly positive for the NK1.1 and ASGM1 Ag, negative for the MAC-1 and F4/80 Ag, and +/- for the LGL-1 and CD3 Ag. Subsets of 4H12+/-, LGL-1+/- exhibited morphologic characteristics restricted to specific phenotypic subsets. The 4H12-/LGL-1+ subset was shown to contain the smallest, least granular cells, whereas the 4H12+/LGL-1+/- subsets contained the largest and most granular cells. Although 4H12 expression was negligible in the spleens of normal adult mice, spleen cells of neonatal and pregnant mice contained subsets of NK1.1+ cells that coexpressed 4H12. The 4H12+/NK1.1+ and 4H12-/NK1.1+ subsets displayed differential levels of NK1.1 expression. 4H12+ NK cells were NK1.1 low to high, whereas 4H12- NK cells were NK1.1 high only. The functional significance of subsets of NK cells in IL-2 culture and in the spleens of neonatal and pregnant mice remains to be elucidated.     

Heterogeneity of natural killer cell subsets in NK-1.1+ and NK-1.1- inbred mouse strains and their progeny.

The 4LO3311 monoclonal antibody, a new NK-specific reagent recently produced in our laboratory, reacts with spleen cells of 11 mouse strains, most of which do not express the NK-1.1 alloantigen recognized by the PK136 mAb. Among positive strains, the susceptibility of spleen cells to the complement-dependent NK-inhibiting activity of the 4LO3311 mAb was variable but independent of the initial NK cell activity level of cells tested. This property was furthermore not modified after poly(I:C) stimulation. The susceptibility of spleen cells to the in vitro 4LO3311 mAb plus complement treatment is however influenced by the absolute number of 4LO3311+ cells as well as by the density of the corresponding alloantigen at the cell surface. Moreover, it was established that the strain-related variations observed also depended upon the relative size of the 4LO3311 cell subset within the lytic NK cell population. Indeed, when C3H (NK-1.1-4LO3311+) mice were inoculated with the 4LO3311 mAb, the lytic activity of their spleen cells was almost unaltered but 4LO3311-reactive cells were no longer detected in the spleen of treated animals and remaining NK cells were totally resistant to the in vitro 4LO3311 mAb plus complement treatment. These findings indicate that the 4LO3311 mAb identifies a subset rather than all NK cells, even in a NK-1.1- strain. Since a NK-1.1-unreactive cell subset was identified in NZB (NK-1.1+4LO3311-) mice inoculated with the PK136 mAb, the NK-1.1+ cell population is not necessarily responsible for all the splenic NK cell activity in all NK-1.1+ strains. In B6C3F1 hybrid mice, a relatively large subset of NK-1.1-4LO3311- cells was found in addition to those expressing the NK-1.1, the 4LO3311 alloantigen, or both. According to these results, NK cell heterogeneity should thus be taken as an evolving concept whose resolution appears more and more complex with the identification of new NK-specific reagents.     

Role of Ly-6 in lymphocyte activation. I. Characterization of a monoclonal antibody to a nonpolymorphic Ly-6 specificity

In studies with alloantisera and monoclonal antibodies (mAb) a number of antigenic determinants have been defined that are the products of the Ly-6 locus on murine chromosome 2 and that are expressed primarily on B and T lymphoid cells. It remains controversial whether these antigenic determinants are encoded by a single gene or a multigene complex. We have characterized a new rat mAb, D7, which recognizes a cell surface antigen whose expression on nonactivated peripheral lymphocytes varies from strain to strain. The phenotype of the staining profile, i.e., high or low percentage of D7-positive cells, mapped to the Ly-6 locus as assayed by strain distribution studies, RI lines, and Ly-6 congenic strains. The binding of D7 to Ly-6.1-positive strains could be inhibited by mAb directed to the Ly-6E.1 specificity, whereas D7 could inhibit the binding of mAb specific for Ly-6A.2 to cells from Ly-6.2-positive strains. Coprecipitation studies followed by Western blot analysis confirmed that D7 reacts with both Ly-6E.1- and Ly-6A.2-bearing molecules. The most likely explanation for these findings is that Ly-6A.2 and Ly-6E.1 represent allelic specificities. Further dissection of the complexity of the Ly-6 antigen system and determination of its possible functional importance in lymphocyte activation should be greatly facilitated by the availability of xenogeneic mAb that recognize framework determinants on multiple Ly-6 products.     

Functional natural killer T cells in experimental mouse strains, including NK1.1- strains.

Natural killer T (NKT) cells are a newly discovered subset of lymphocytes. It appears that this subset has potential as important regulators of immune responses. But because there are relatively few NKT cells in lymphoid organs and because of technical difficulties in detecting NKT cells in most mouse strains, the roles of NKT cells have not been fully identified and little attention has been paid to the roles of NKT cells in immunological experiments in which NK1.1- strains were used. To examine the existence of functional NKT cells in various strains of experimental mice, including NK1.1- strains, we utilized alpha-galactosylceramide (KRN7000) which is thought to react specifically with NKT cells. Indeed, we could confirm that early cytokine (IL-4 and IFN-gamma) secretion at 2 h after the injection of KRN7000 was dependent on NKT cells. With this in vivo system, we have successfully detected the presence of functional NKT cells in various mouse strains, including AKR/N, BALB/c, C3H/HeJ, C3H/HeN, C57BL/6, C.B-17, CBA/N, NC, NOD, SJL, W/Wv, aly/aly and aly/+. Notable increases of serum IL-4 were detected in W/Wv and aly/+ strains, and defective response of IFN-gamma in SJL mice and that of IL-4 in NOD mice were observed. This is the first report to show the functional significance of NKT cells in cytokine secretion in various mouse strains in response to a ligand for the T cell receptor of NKT cells.     

Expression of murine killer immunoglobulin-like receptor KIRL1 on CD1d-independent NK1.1<Superscript>+</Superscript> T cells

Three mouse killer immunoglobulin-like receptors (KIRs), namely, KIR3DL1, KIRL1, and KIRL2, have recently been identified in C56BL/6 (B6) mice. However, only two Kir genes are found in the B6 mouse genome sequence data base. To clarify this discrepancy, we cloned Kir cDNAs from multiple strains of mice. Sequencing of the cDNA clones showed that the Kir3dl1 gene is found in C3H/HeJ and CBA/J but not in B6 mice. Analysis of the single nucleotide polymorphism data base suggested that Kir3dl1 is the C3H/HeJ and CBA/J allele of Kirl1. We generated mAb to the recombinant KIRL1 protein to investigate its expression pattern. The anti-KIRL1 mAb bound to NK1.1⁺ T cells but only very weakly or at undetectable levels to other lymphocytes including natural killer (NK) cells and conventional T cells. Among NK1.1⁺ T cells, conventional NK T cells stained with CD1d tetramer did not significantly bind anti-KIRL1 mAb, whereas CD1d-tetramer-negative subset was KIRL1-positive. Furthermore, the expression of KIRL1 is readily detected on NK1.1⁺ T cells from β₂-microglobulin-deficient B6 mice. Thus, KIRL1 is predominantly expressed on CD1d-independent NK1.1⁺ T cells.     

Strain difference in mouse natural killer activity augmented by mouse interferon and poly I.C

The level of natural killer (NK) activity was found to vary considerably among several mouse strains. In vivo and in vitro, interferon (IFN) and IFN inducers have been shown to augment mouse NK activity. C3H/He mice showed high NK activity, DDD/1 and A/J mice low NK activity, and C57BL/6, BALB/c and DBA/2 mice intermediate NK activity after injection with polyinosinic polycytidylic acid (poly I. C.). The same NK activity correlation was observed in nontreated mice, but the NK activities were lower compared with the poly I. C.-injected mice. Moreover, the DDD/1 and A/J mice showed almost no augmentation of NK activity on injection with poly I.C. In vivo, C3H/He, BALB/c and C57BL/6 mice injected with IFN showed augmented NK activity, but DDD/1 mice showed no such reaction. In vitro, C3H/He, BALB/c and C57BL/6 mouse spleen cells treated with IFN also showed augmented NK activity, but DDD/1 mouse spleen cells showed almost none. F1 hybrids between high (C3H/He) and low (DDD/1) NK-activity strains showed high NK activity. Thus, activity is dominant over low activity. The segregation of (DDD/1 X C3H/He) Fl X DDD/1 back-cross mice suggested that the strain differences in NK activity are under polygenic control.     

Mechanisms of killing of measles virus-infected cells by human lymphocytes: interferon associated and unassociated cell-mediated cytotoxicity

The recent interest in natural killer (NK) cells in immunosurveillance and the ability of infection with certain organisms to modulate NK activity led us to examine the influence of Toxoplasma gondii infection on mouse NK cells. Infection of BALB/c mice with 5 × 10³ virulent Toxoplasma intraperitoneally (ip) resulted in significantly enhanced NK activity in peritoneal exudate cells (PC) and in spleen cells (SC). Intravenous (iv) and subcutaneous (sc) challenge of BALB/c mice with Toxoplasma also resulted in enhanced natural killer (NK) activity in PC and SC. In BALB/c mice, as well as in other strains (A/J, C57BL/6, C3H/HeJ, CeH/HeN, [A/J × C3H]F₁), peak augmentation of PC and SC NK activity was observed 3 days following ip Toxoplasma challenge. Administration of silica to mice abolished Toxoplasma-induced NK cytotoxicity. BALB/c mice chronically infected with Toxoplasma had significantly higher endogenous NK activity than did controls in PC but not in SC. Chronically infected BALB/c mice boosted with virulent Toxoplasma ip exhibited significantly enhanced NK activity in PC but not in SC. Thus, acute and chronic infection with Toxoplasma modulates NK activity in addition to macrophage activation and thereby provides a system that should facilitate study of the relative contribution of NK cells and activated macrophages in resistance to tumor growth and spread.     

Mouse CD94 participates in Qa-1-mediated self recognition by NK cells and delivers inhibitory signals independent of Ly-49

Inhibitory receptors expressed on NK cells recognize MHC class I molecules and transduce negative signals to prevent the lysis of healthy autologous cells. The lectin-like CD94/NKG2 heterodimer has been studied extensively as a human inhibitory receptor. In contrast, in mice, another lectin-like receptor, Ly-49, was the only known inhibitory receptor until the recent discovery of CD94/NKG2 homologues in mice. Here we describe the expression and function of mouse CD94 analyzed by a newly established mAb. CD94 was detected on essentially all NK and NK T cells as well as small fractions of T cells in all mouse strains tested. Two distinct populations were identified among NK and NK T cells, CD94(bright) and CD94(dull) cells, independent of Ly-49 expression. The anti-CD94 mAb completely abrogated the inhibition of target killing mediated by NK recognition of Qa-1/Qdm peptide on target cells. Importantly, CD94(bright) but not CD94(dull) cells were found to be functional in the Qa-1/Qdm-mediated inhibition. In the presence of the mAb, activated NK cells showed substantial cytotoxicity against autologous target cells as well as enhanced cytotoxicity against allogeneic and "missing self" target cells. These results suggest that mouse CD94 participates in the protection of self cells from NK cytotoxicity through the Qa-1 recognition, independent of inhibitory receptors for classical MHC class I such as Ly-49.     

Changes in number and density of large granular lymphocytes upon in vivo augmentation of mouse natural killer activity

NK activity of mice as well as humans and rats has been clearly associated with large granular lymphocytes (LGL). To better understand the effects of interferon (IFN) and IFN inducers on natural killer (NK) cells, we have compared the LGL in the spleens of normal and boosted mice. Cells were fractionated by centrifugation on discontinuous Percoll density gradients, and each fraction was tested for NK activity against YAC-1 targets and for the presence of LGL. In vivo treatment with C. parvum (0.7 mg/mouse, i.p., day-3), MVE-2 (25 mg/kg, i.p., day-3), poly I:C (4 mg/kg, i.p., day-3), or IFN (10(5) U/mouse, i.p., day-1) resulted in a marked augmentation and a change of distribution of cytotoxic activity. Most of the NK activity of boosted spleen cells was associated with lower density fractions 1 and 2, whereas active normal spleen cells had somewhat higher density (fractions 2 and 3). In parallel to their increased reactivity, the boosted spleens had a marked increase in the percentage of LGL, particularly in fractions 1 and 2. The augmented activity appeared to be mediated by the LGL, because treatment with anti-asialo GM1 or anti-Thy-1.2 plus complement reduced NK as well as the number of LGL. These results indicate that IFN-mediated boosting of NK activity in the spleen is due to an increase in the lower density LGL, as well as to an increase in the function of preexisting NK cells.     

A "chimeric" C57l-derived Ly49 inhibitory receptor resembling the Ly49D activation receptor.

Ly49D is a natural killer (NK) cell activation receptor that is responsible for differential mouse inbred strain-determined lysis of Chinese hamster ovary (CHO) cells. Whereas C57BL/6 NK cells kill CHO, BALB/c-derived NK cells cannot kill because they lack expression of Ly49D. Furthermore, the expression of Ly49D, as detected by monoclonal antibody 4E4, correlates well with CHO lysis by NK cells from different inbred strains. However, one discordant mouse strain was identified; C57L NK cells express the mAb 4E4 epitope but fail to lyse CHO cells. Herein we describe a Ly49 molecule isolated from C57L mice that is recognized by mAb 4E4 (anti-Ly49D). Interestingly, this molecule shares extensive similarity to Ly49D(B6) in its extracellular domain, but its cytoplasmic and transmembrane domains are identical to the inhibitory receptor Ly49A(B6), including a cytoplasmic ITIM. This molecule bears substantial overall homology to the previously cloned Ly49O molecule from 129 mice the serologic reactivity and function of which were undefined. Cytotoxicity experiments revealed that 4E4(+) LAK cells from C57L mice failed to lyse CHO cells and inhibited NK cell function in redirected inhibition assays. MHC class I tetramer staining revealed that the Ly49O(C57L)-bound H-2D(d) and lysis by 4E4(+) C57L LAK cells is inhibited by target H-2D(d). The structural basis for ligand binding was also examined in the context of the recent crystallization of a Ly49A-H-2D(d) complex. Therefore, this apparently "chimeric" Ly49 molecule serologically resembles an NK cell activation receptor but functions as an inhibitory receptor.     

Suppression of IgE antibody production in SJL mice. II. Expression of Ly-1 antigen on helper and nonspecific suppressor T cells.

Under appropriate conditions of immunization combined with irradiation, SJL/J mice show a high and persistent anti-DNP IgE antibody response. Spleen cells transferred from normal untreated SJL mice suppress this response. Elimination of Ly-1+ cells, but not of Ly-2+ cells, abolished the capacity of spleen cells to suppress the IgE response. Thus of the three T cell Ly subclasses presently identified, Ly-1, Ly-2,3, and Ly-1,2,3, the normal SJL spleen cell which suppresses the IgE response of irradiated-immunized SJL mice belongs to the Ly-1 set. It is not known whether this Ly-1 cell suppresses the IgE response directly or by helping another cell in the recipient. The carrier-specific helper cell activity for IgE and probably IgG1 antibody response belongs to Ly-1 subclass in the SJL strain also.     

Germinal center cells are a major IL-5-responsive B cell population in peripheral lymph nodes engaged in the immune response

Germinal center formation and the development of B cell memory in lymphoid tissue is a T cell-dependent process. The specific B cell-T cell interactions, and/or cytokines, resulting in germinal center cell growth have not yet been identified. Germinal center B cells were separated from other lymph node (LN) B cells by panning on peanut agglutinin (PNA)-coated dishes. Resulting fractions enriched for PNA+ (germinal center) B cells, and the PNA- (other) LN B cells from immune SJL mice were assayed for proliferation in the presence of cytokines. PNA+ and PNA- B cells responded equally to IL-4 in the anti-mu co-stimulator assay. In contrast, PNA+ B cells responded to murine (r)IL-5 or human B cell growth factor in the dextran sulfate (DxS) co-stimulator assay, to a much greater degree than did PNA- B cells. The same results were obtained with PNA+ and PNA- cells from LAF1 mice. Unfractionated LN B cells from nonimmunized SJL or BALB/c mice did not respond to IL-5 with or without DxS. B cell populations from BALB/c mice such as from spleen and peritoneal cavity, which are known to be high in Ly-1+B cells, responded to IL-5 alone, and more dramatically, to IL-5 as a co-stimulator with DxS. Such populations of cells from SJL mice, which are known to contain low numbers of Ly-1+B cells, responded markedly less. These results are consistent with those of others which show that in nonimmunized mice, Ly-1+B cells are a major IL-5 responsive subpopulation. IL-1 enhanced the proliferation of PNA+ cells in response to rIL-5 and had no effect on PNA- cells. IL-4 and IL-5 did not enhance each other's effects as co-stimulators of proliferation. In contrast to PNA+ B cells from immune LN, B cells activated by Escherichia coli endotoxin exhibited no responses to rIL-5. The present results indicate that in immune LN, PNA+, germinal center B cells constitute a prominent IL-5-responsive population.     

Granulated metrial gland cells of pregnant mouse uterus are natural killer-like cells that contain perforin and serine esterases

The mouse uterus during pregnancy contains a large population of lymphoid cells termed granulated metrial gland (GMG) cells. Our observations suggest that these cells are highly activated cytolytic lymphocytes related to NK or lymphokine-activated killer cells. Immunostaining demonstrated asialo GM1 and Thy-1 on GMG cells, both of which are expressed by NK cells. Decidua basalis tissue and isolated GMG cells contained three proteins that are characteristic of activated cytolytic lymphocyte granules: perforin, serine esterase 1, and serine esterase 2. These mediators were demonstrated in GMG cells by Western blot analysis using polyclonal antisera and by Northern blot analysis using specific cDNA probes for their mRNA. The proteins were not detected in normal spleen or liver or in asialo GM1+ cells isolated from those organs, consistent with the absence of these mediators from resting cytolytic cells. The amount of perforin in GMG cells was similar to that present in cloned, IL-2-stimulated, CTL shown previously to contain a large amount of this protein. A large population of NK cells bearing the surface marker LGL-1 was demonstrated at the implantation site by labeling with monoclonal antibody 4D11, but T cells were not detected. Many LGL-1+ cells at the implantation site expressed the GMG cell markers asialo GM1, Thy-1, and perforin. Staining intensities were inversely correlated, with LGL-1-bright cells showing little or no staining of GMG cell markers and LGL-1-faint cells showing more obvious staining of GMG cell markers. This suggests that LGL-1+ NK cells may differentiate in situ to GMG cells, losing LGL-1 and gaining a high concentration of GMG cell markers in the process. Activated cytolytic cells related to NK or lymphokine-activated killer cells may function in the pregnant rodent uterus to intercept and kill aberrant placental or embryonic cells that might otherwise enter the female and proliferate.