Suppression of alloimmune cytotoxic T lymphocyte (CTL) generation by depletion of NK cells and restoration by interferon and/or interleukin 2

By using rabbit antiserum to a glycolipid, ganglio-n-tetraosylceramide (ASGM1), the accessory effect of natural killer (NK) cells on the generation of alloimmune CTL in mice was investigated. When normal C3H/He mice were immunized with C57BL/6 or BALB/c spleen cells, they generated alloimmune CTL with a surface marker phenotype of Thy-1+ Lyt-1-2+ ASGM1-, preceded by early augmentation of cytotoxic activity of NK cells with a Thy-1-Lyt-1-2-ASGM1+ phenotype. Administration of anti-ASGM1 (10 microliters) in mice resulted in a complete depletion of NK activity and ASGM1+ cells in the spleen even 1 day after injection, but no changes in the proportions of T (Thy-1+) cells and their Lyt-1 and Lyt-2 subsets as revealed by an immunofluorescence analyzer (FACS) and phagocytic cells. When these anti-ASGM1-treated mice were immunized with allogeneic cells, they showed neither augmented NK activity nor generation of alloimmune CTL, and spleen cells isolated from these anti-ASGM1-treated mice produced no CTL response to alloimmunization in vitro. Normal spleen cells treated with the antiserum and complement in vitro also showed a complete NK depletion without any deterioration of T cells and their Lyt-1 and Lyt-2 subsets, and when stimulated with allogeneic cells they generated no CTL. Spleen NK (ASGM1+) cells were purified by Percoll-gradient centrifugations followed by complement-dependent killing of T cells with the use of anti-Thy-1 monoclonal antibody, and were further purified by panning methods with anti-ASGM1, giving a preparation consisting of greater than 90% ASGM1+, Ly-5+ cells, and less than 0.5% of Thy-1+, Lyt-1+, and Lyt-2+ cells. These purified ASGM1+ Thy-1- cells alone generated no alloimmune CTL in response to alloantigens, suggesting that ASGM1+ NK cells contained no precursors of alloimmune CTL. When added into NK-depleted spleen cells, they restored the normal alloimmune CTL response of the spleen cells, indicating that ASGM1+ fractions contained cells to provide an accessory function for CTL generation. Lyt-1+ cells purified by panning methods did not restore the CTL response of NK-depleted spleen cells. These results indicate that ASGM1+ NK cells, but not Lyt-1+ helper T cells contaminating ASGM1+ fractions at undetectable levels, are responsible for the accessory function. When these purified ASGM1+ Thy-1- cells were stimulated with allogeneic cells, they produced IL 2 and IFN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of the antibody response by the acute phase reactant: mouse serum amyloid P-component (SAP)

Serum amyloid P-component (SAP) is the major acute phase reactant (APR) of mice. Purified mouse SAP at 0.1 to 10.0 micrograms/ml selectively suppressed the secondary in vitro IgG antibody plaque-forming cell (PFC) response to the T-dependent antigen TNP-KLH but not to the T-independent antigens TNP-LPS and DNP-Lys-Ficoll. The suppression was antigen nonspecific. The mechanism of suppression occurred primarily through the activation of Lyt-1+, I-J+ suppressor-inducer cells, which in turn activated a Lyt-2+ suppressor T-cell population. The activity of preexisting, antigen-specific Lyt-2+ suppressor T cells was not influenced by SAP. The antigen-nonspecific suppressor T cells generated by SAP were sensitive to cyclophosphamide. Removal of SAP from the culture fluid with rabbit anti-Mo SAP antibody or agarose beads abrogated the suppression. Pentraxin proteins closely related to mouse SAP, such as human SAP and hamster female protein (FP), also displayed immunoregulatory activity of the antibody response by the same cellular mechanism. The results suggest that SAP regulates antibody responses by the activation of suppressor-inducer T cells and that the regulation of the antibody response during the acute stage of inflammation may occur via SAP.     

Resistance in murine schistosomiasis is contingent on activated IL-2 receptor-bearing L3T4+ lymphocytes, negatively regulated by Lyt-2+ cells, and uninfluenced by the presence of IL-4

These studies assess the roles of subpopulations of T lymphocytes in inducing and modulating resistance to Schistosoma mansoni. C57BL/6 mice were depleted in vivo of L3T4+, Lyt-1+, Lyt-2+, IL-2R+ cells, or IL-4 by administration of appropriate mAb. Resistance and various correlative parameters of the immune response were studied in normal, depleted, and congenitally athymic mice. Depletion of T lymphocytes by anti-L3T4 or anti-IL-2R mAb reduced the development and expression of resistance, IgG2a and IgE antibody formation, and delayed type hypersensitivity reactivity against schistosome Ag. Depletion with anti-IL-4 antibody led to profound suppression of IgE-eosinophil-mediated antibody-dependent cell-mediated cytotoxicity and passive cutaneous anaphylaxis responses against the parasite and no effect on IgG2a antibody, Ag-mediated blast transformation, or resistance. Depletion of Lyt-2+ cells produced augmented development and expression of resistance and an increase in the immunological parameters of anti-schistosome reactivity. These studies suggest that protective immunity to S. mansoni in mice, induced by irradiated cercariae, is dependent on L3T4+, IL-2R+ lymphocytes and negatively regulated by Lyt-2+ cells. IL-4 does not appear to be essential for the development of resistance but is essential for the IgE response to the parasite.     

Origin and differentiation of natural killer cells. II. Functional and morphologic studies of purified NK-1.1+ cells

Cells bearing the NK-specific marker NK-1.1 were purified from mouse spleens by utilizing a monoclonal anti-NK-1.1 antibody and cell sorting. In normal adult mice, all of the splenic NK activity against YAC-1 cells was found in the NK-1.1+ fraction, whereas NK-1.1- cells were depleted of NK activity. The NK activity of sorted NK-1.1+ cells was enriched 15- to 30-fold over unfractionated spleen cells. Light and electron microscopic studies of purified NK-1.1+ cells showed a homogeneous population of cells, each containing one to four cytoplasmic granules. Mice whose bone marrow has been destroyed by chronic exposure to 17-beta-estradiol have very low NK activity. However, spleen cells of estradiol-treated mice contained a normal frequency of NK-1.1+ cells which bound to YAC-1 cells, but failed to lyse them even after purification and subsequent exposure to interferon-alpha/beta in vitro. It appears, therefore, that in the absence of intact bone marrow, NK-1.1+ cells may be arrested in a nonlytic and interferon-unresponsive state. Spleens of neonatal mice which have low NK activity were analyzed to ascertain whether immature NK-1.1+ cells, similar to those found in estradiol-treated mice, could be demonstrated. Spleens of 8- to 9-day-old mice also contained NK-1.1+ cells which had very low NK activity even after purification. Sorted NK-1.1+ cells were examined for cytotoxicity in mice whose NK activity was suppressed by pretreatment with Corynebacterium parvum (-15 days). In contrast to cells from estradiol-treated and neonatal mice, NK-1.1+ from mice treated with C. parvum had normal functional activity. Similarly, although NK activity of unfractionated bone marrow cells is low, sorted NK-1.1+ cells were greatly enriched for lytic activity. Thus, we conclude that cell sorting with monoclonal anti-NK-1.1 antibody provides a powerful tool for examining the mechanisms underlying various states of low NK activity, and there exist NK-1.1+, nonlytic, interferon-unresponsive cells which apparently require an intact marrow microenvironment for differentiation into mature, lytic NK cells.     

Regulation of IgM expression by adjuvant activated splenic suppressor T cells

Adjuvant-activated Lyt-2 positive suppressor T cells (Ts) are able to inhibit the expression of IgM plaque-forming cells (PFC) during a primary in vitro response to sheep red blood cells. Under the same experimental conditions these suppressor T cells do not affect a secondary IgM PFC response against SRBC. Activated Ts cells were also found to suppress the spontaneous IgM secretion of cultured B cells as well as the IgM production of B cells stimulated by lipopolysaccharide or by supernatant from a T-helper-cell clone.     

Isotype-specific immunoregulation. Systemic antigen induces splenic T contrasuppressor cells which support IgM and IgG subclass but not IgA responses

In the present study, we have isolated and characterized the Lyt-1+, -2- T contrasuppressor (Tcs) cells from mice systemically primed with SRBC. Adoptive transfer of splenic Tcs cells from these mice abrogates oral tolerance and supports IgM and IgG anti-SRBC plaque-forming cell (PFC) responses; however, unlike the responses seen after transfer of Tcs cells derived from orally primed mice, low IgA responses were seen. Mice systemically primed with lower SRBC doses (0.01 to 1%) exhibited contrasuppression only within the L3T4- T cell subset, whereas mice primed with a high dose of SRBC (10%), harbored Lyt-1+, -2- Tcs cells in both the L3T4+ and L3T4- subsets. Both the L3T4- and L3T4+ Tcs cell subsets supported IgM and IgG responses when adoptively transferred to orally tolerized mice, and when added to tolerized spleen cell cultures. Splenic Tcs cells from systemically primed mice supported mainly IgG1 and IgG2b subclass anti-SRBC PFC responses, a pattern also seen with Tcs cells derived from orally primed mice. Both L3T4+ and L3T4- Tcs cells from systemically primed mice exhibited well established characteristics of contrasuppressor cells including binding to Vicia villosa lectin and expression of I-J. The splenic effector Tcs cells which support IgM, IgG1 and IgG2b anti-SRBC PFC responses are antigen-specific, since both L3T4- and L3T4+ Tcs cells from spleens of mice primed with 10% SRBC reverse tolerance to SRBC, but not to horse erythrocytes (HRBC). Further, both L3T4- and L3T4+ Tcs cells from HRBC-primed mice reverse tolerance to IgM and IgG anti-HRBC, but not to anti-SRBC responses. Isolation of T3-positive Lyt-1+, -2- and L3T4- Tcs cell subsets by flow cytometry followed by adoptive transfer, showed that effector Tcs cells express T3 and presumably contain an Ag-R (TCR-T3 complex). These studies show that systemic priming with heterologous RBC induces splenic Ag specific Tcs cells in a dose-dependent manner, which support IgM and IgG subclass responses, but not IgA responses.     

T cells subsets responsible for clearance of Sendai virus from infected mouse lungs

T cell subsets responsible for clearance of Sendai virus from mouse lungs determined by adoptive transfer of immune spleen cell fractions to infected nude mice. T cells with antiviral activity developed in spleens by 7 days after intranasal infection. Spleen cell fractions depleted of Lyt-2+, Lyt-1+, or L3T4+ cells showed antiviral activity in vivo, although the degree of the activity was lower than that of control whole spleen cells. The antiviral activity of the Lyt-2+ cell-depleted fraction was consistently higher than that of L3T4+ (Lyt-1+)-depleted cells. In vitro cytotoxic activity against Sendai virus-associated, syngeneic lipopolysaccharide-blast cells was detected in stimulated cells from intraperitoneally immunized mice but was lost after depletion of Lyt-2+ cells. Multiple injection of anti-Sendai virus antibody into infected nude mice had no effect on lung virus titer. These results indicate that L3T4+ (Lyt-1+) and Lyt-2+ subsets are cooperatively responsible for efficient clearance of Sendai virus from the mouse lung.     

Isotype restriction during infection of mice with the cestode Mesocestoides corti: role of immune suppression

Mice infected with the parasite Mesocestoides corti produce a vigorous antibody response that is restricted to the IgM and IgG1 heavy chain classes. The isotypic restriction observed is apparently associated with active infection and is not a unique characteristic of responses to M. corti antigens. Thus, animals immunized with intact but nonviable parasites respond with the production of a variety of antibody isotypes in addition to IgM and IgG1. To delineate immunoregulatory mechanisms involved in the isotypic restriction of antibody responses to M. corti, an in vitro lymphocyte suspension culture was established. The data indicate that there are two cell subsets in the spleens of infected mice that contribute to an overall suppression of the in vitro antibody response. Thus, both Lyt-2+ cells and G-10-adherent cells must be removed to maximize antibody production. However, the anti-parasite response obtained in vitro after depletion of Lyt-2+ cells and G-10-adherent cells is restricted to the IgM and IgG1 isotypes as observed in vivo, indicating that suppression is not actively involved in the IgM, IgG1 dominance of the response. The cellular regulation associated with this restriction was then studied by using isolated helper T cells derived from parasite-infected animals to stimulate B cells from uninfected animals. The antibody produced was again restricted to IgM and IgG1, indicating that the helper T cells were regulating the preferential expression of the IgM and IgG1 antibody classes.     

Regulation of IgG1 and IgG2 subclass expression by adjuvant-activated splenic suppressor T cells

Spleen cells from mice immunized with different adjuvants are able to suppress secondary in vitro IgG plaque-forming cell (PFC) responses. The suppressive effect is mediated by Lyt-2-positive T cells. IgG subclasses are affected differentially depending on the number of T suppressor (Ts) cells added in the assays. At low Ts cell concentration IgG2a and IgG2b PFC responses are selectively inhibited. At higher Ts cell concentration IgG1 responses could also be completely inhibited, but IgA and IgM responses are not affected. Suppressor cells responsible for IgG1, IgG2a, and IgG2b suppression are never found in the draining lymph nodes of adjuvant-immunized animals.     

Cell interactions in alveolar macrophage-mediated suppression of the immune response: an unusual suppressor pathway involving a population of T-cells that express Lyt-1, L3T4, and I-J

Studies from this laboratory have demonstrated that incubation of murine alveolar macrophages (AM) with SRBC-primed spleen cells (SC) results in suppression of the in vitro plaque-forming cell (PFC) response and that suppression is mediated by a soluble factor contained in supernatants obtained from cultures of AM and SC. In the present study, immunological techniques employing monoclonal antibody (MoAb) were used to isolate various T-cell subsets in order to determine the phenotype of the cells which interact with AM to produce suppression. Spleen cell populations depleted of Thy-1+-, Lyt-1+-, L3T4+-, or I-J+-bearing cells failed to generate suppressive supernatants when cultured with AM. Depletion of Lyt-2+ T-cells (the classical suppressor/effector subset) did not alter the ability of the remaining cell population to cooperate with AM for generation of suppressive supernatants. Direct suppression of the PFC response in cultures containing AM was abrogated after treatment of the spleen cells with anti-I-J, but not anti-Lyt-2 MoAbs. Reconstitution of the AM-mediated suppressive response with enriched populations of SC required the presence of T-cells which expressed Lyt-1, L3T4, and I-J. These results suggest the existence of an unusual suppressor pathway involving I-J restriction but which appears to be mediated by the interaction of AM with a population of T-cells that expresses surface markers characteristic of T-helper cells.     

Immunoregulatory activity of human bone marrow. Identification of suppressor cells possessing OKM1, SSEA-1, and HNK-1 antigens

Human bone marrow contains natural regulatory cells capable of suppressing the in vitro primary IgM response of normal tonsillar cells. The suppression is mediated by non-T cells possessing Fc receptors, OKM1, SSEA-1, and HNK-1 antigens on their surface. The suppression was abrogated by treatment of bone marrow cells (BMC) with anti-HNK-1 or anti-SSEA-1 antisera and complement. Furthermore, BMC depleted of HNK-1+ cells could respond in a primary in vitro antibody response when provided with accessory T cells and macrophages from tonsillar cells. Our findings support the idea that HNK-1+ and HNK-1- BMC populations act antagonistically in the regulation of antibody synthesis. Further, the finding of HNK-1+, SSEA-1+, and OKM1+ suppressor cells in human bone marrow may represent a precursor phenotype of mature natural killer cells with potent immunoregulatory activity.     

Antiviral effect of lymphokine-activated killer cells: characterization of effector cells mediating prophylaxis

Lymphokine-activated killer (LAK) cells generated by cultivation of C57BL/6 mouse spleen cells in the presence of recombinant interleukin-2 were transferred into natural killer (NK) cell-deficient suckling mouse recipients. These mice were then challenged with either murine cytomegalovirus (MCMV) or lymphocytic choriomeningitis (LCMV) and sacrificed 3 days later. No interleukin 2 infusions were given. Mice receiving as few as 5 x 10(5) LAK cells had several 100-fold decreases in spleen MCMV titers as compared with untreated mice. This treatment had no effect on spleen LCMV titers. The LAK cell cultures contained 10 to 17% NK 1.1+, 50 to 55% Lyt-2+, and 33 to 50% immunoglobulin D+ cells. Double fluorescence labeling and in vitro cytotoxicity assays with fluorescence-activated cell sorting revealed at least two mutually exclusive killer cell populations. NK 1.1+ LAK cells resembled freshly isolated activated NK cells with regard to target cell range (YAC-1 cell killing greater than L-929, P815, and EL-4 cell killing), large granular lymphocyte (LGL) morphology, and decreased ability to lyse interferon (IFN)-treated target cells. Lyt-2+ LAK cells lysed the targets mentioned above but at lower levels and without the differences in susceptibility mentioned above. These Lyt-2+ LAK cells also had a decreased ability to lyse IFN-treated targets, in contrast to classic cytotoxic T lymphocytes, which lyse IFN-treated targets far more efficiently than untreated targets. Purified populations of LAK cells obtained by fluorescence-activated cell sorting were used in the antiviral protection model. The results showed that protection against MCMV could be mediated by NK 1.1+, NK 1.1-, Lyt-2+, Lyt-2-, and IgD- populations but not by IgD+ cells. The five protective populations all had in common the LGL phenotype and cytotoxic activity in vitro. The IgD+ population did not contain LGLs, lyse target cells in vitro, or mediate an antiviral effect in vivo. These results suggest that LAK cells may be therapeutically useful against certain virus infections (MCMV) but not others (LCMV) and that despite their heterogeneity in antigenic phenotype and cytotoxic activity, their pattern of antiviral activity in vivo resembles that of NK cells, which protect against MCMV but not LCMV.     

Generation of cytotoxic cells from murine bone marrow by human recombinant IL 2

Murine bone marrow (BM) cells were cultured in recombinant IL 2 (rIL 2) and interferon-alpha, -beta, and -gamma, and cytotoxic activity against YAC cells was determined in a 4-hr 51Cr-release assay. rIL 2 at 20 U/ml was the only lymphokine that consistently induced significant cytotoxic activity within 3 days of culture, peaking around 5 to 7 days. The cytotoxic cells generated are heterogeneous, consisting of at least two populations of cells: a) NK-1+, Qa-5+, AsGm-1+ Thy-1+/-, Lyt-2- cells, similar to natural killer (NK) cells, and b) NK-1-, Qa-5+, AsGm-1+ Thy-1+, Lyt-2+ cells, similar to cytotoxic T lymphocytes. The precursor/accessory cells of these BM cytolytic cells maintained in 20 U/ml of rIL 2 were Qa-2+, Qa-5+, Thy-1+/-, AsGM-1+/-, and NK-1+/- but Lyt-2-. They also lysed NK-resistant targets, P815 and BW5147, and the antigenic phenotypes of these cells were similar to those that lysed YAC cells. These studies indicate that IL 2 alone is adequate to generate cytotoxic activity from BM and that these cytotoxic cells were similar to splenic NK cells.     

Characterization of suppressor cells regulating in vitro expression of IgG2A and IgG2B antibody responses

In vitro cooperative responses between hapten-primed anti-Thy-1.2 plus C-treated spleen cells and carrier-primed T cells have different isotypic patterns depending on the source of the T helper cells. T helper cells from primed lymph node induce IgG1, IgG2a, and IgG2b PFC responses, whereas T helper cells from primed spleen induce only an IgG1 type of response. The addition of activated spleen cells to the lymph node cells suppresses their ability to generate IgG2a and IgG2b PFC responses. The suppressor cells involved have been characterized. Functionally, they appear as nonantigen specific and isotype specific, because they never reduce the IgG1 response. They are Lyt-2.2 positive, Lyt-1 negative, and radiosensitive. Their relative resistance to anti-Thy-1.2 plus C treatment indicates that they express low amounts of this antigen or that they are heterogeneous concerning the expression of Thy-1.2.     

The NK-1.1(-) mouse: a model to study differentiation of murine NK cells

The NK-1.1(-) mouse was constructed by weekly injections of monoclonal anti-NK-1.1 antibody from birth through adulthood. Spleen cells from these mice have decreased NK-1.1+ cells and null (Thy-1- and B220-) cells. Their splenic NK activity to YAC targets was low and was not enhanced by IFN-alpha or IFN-beta. Bone marrow (BM) of these NK-1.1(-) mice have normal precursors to NK cells: 1) NK activity could be generated from NK-1.1(-) BM cells cultured in rIL 2 for 5 to 6 days. These cultured BM cells expressed Qa-5, Thy-1, AsGm-1, and NK-1.1 antigens. The precursor cells of these BM cytotoxic cells are NK-1.1-; 2) transfer of BM cells from the NK-1.1(-) mice reconstituted the NK activity of irradiated, NK-depleted recipients. Lymphokine-activated killer cells could also be generated from spleens of these NK-1.1(-) mice. Therefore, the NK-1.1(-) mice were specifically depleted of mature cytotoxic NK cells, but not the NK-1.1- precursors of NK cells. This mouse model is valuable to study ontogeny and physiologic relevance of NK cells.     

T cell subsets regulating antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in virgin and immunized nonresponder mice

T cell subsets from virgin and immunized mice, which are Ir gene controlled nonresponders to GAT, which regulate antibody responses to GAT have been characterized. Virgin nonresponder B10.Q B cells develop GAT-specific antibody responses to GAT, B10.Q GAT-M phi, and GAT-MBSA when cultured with virgin or GAT-primed Lyt-1+, I-J-, Qa1- B10.Q helper T cells. Virgin T cells are radiosensitive, whereas immune T cells are radioresistant (750 R); qualitatively identical helper activity is obtained with T cells from mice immunized with soluble GAT, B10.Q GAT-M phi, and GAT-MBSA. Responses to GAT and GAT-M phi are not observed when virgin or GAT-primed Lyt-1+, I-J+, Qal+ T cells are added to culture of virgin or GAT-primed Lyt-1+, I-J-, Qa1- helper T cells and virgin B cells; the GAT-specific response to GAT-MBSA is intact. The Lyt-1+, I-J+, Qa1+ T cells from mice primed with GAT, GAT-M phi, and GAT-MBSA were qualitatively identical in mediating this suppression. Virgin Lyt-2+ T cells have no suppressive activity alone or with virgin Lyt-1+, I-J+, Qa1+ T cells, whereas responses to GAT, GAT-M phi, and GAT-MBSA are suppressed in cultures of GAT-primed helper T cells containing GAT-primed Lyt-2+ T cells (with or without GAT-primed Lyt-1+, I-J+, Qa1+ T cells). Suppression of responses to GAT-MBSA in cultures of GAT-M phi-primed helper T cells requires both GAT-M phi-primed Lyt-1+, I-J+, Qa1+ T cells and Lyt-2+ T cells; the Lyt-1+, I-J+, Qa1+ T cells appear to function as inducer cells in this case. In cultures containing GAT-MBSA-primed helper T cells, either GAT-MBSA-primed Lyt-1+, I-J+, Qa1+ or Lyt-2+ T cells suppress responses to GAT and GAT-M phi; under no circumstances are responses to GAT-MBSA suppressed by GAT-MBSA-primed regulatory T cells. This regulation of antibody responses to GAT by suppressor T cells is discussed in the context of the involvement of suppressor T cells in responses to antigens under Ir control, and of the evidence that nonresponsiveness to GAT is not due to a defect in the T cell repertoire, but rather is due to an imbalance in the activation of suppressor vs helper T cells.     

Asialo-GM1+ natural killer cells directly suppress antibody-producing B cells

Natural killer (NK) cells were tested for their ability to suppress antigen-induced antibody responses in vitro. Asialo-GM1+ (ASGM1+) cells were prepared from nylon-wool-nonadherent spleen cells obtained from normal mice. After depletion of Ig+, L3T4+ and Lyt-2+ cells, the ASGM1+-enriched cell population had high NK activity which was abrogated by treatment with anti-ASGM1 and C'. This NK-enriched ASGM1+ cell fraction significantly suppressed the generation of antibody-producing cells when added to in vitro immunization cultures of primed spleen cells. Treatment of the NK-enriched cell population with anti-ASGM1 and C' abrogated the ability of these cells to suppress antibody responses. In vitro antibody production by purified B cells was also suppressed in the presence of the NK-enriched cell population, although the kinetics of the suppression differed from that observed with unfractionated spleen cells. In addition, the NK-enriched cell population suppressed the proliferation of the B cell line WEHI-279.1. Suppression of WEHI-279.1 cells was abrogated when the NK-enriched cell population was treated with anti-ASGM1 and C'. These results suggest that normal NK cells suppress the generation of antibody-producing B cells and that this occurs, at least in part, through a direct regulation of the B cell.     

Feedback suppression of the immune response in vivo. III. Lyt-1+ B cells are suppressor-inducer cells

We previously demonstrated that injection of a high dose (4 X 10(9] of sheep erythrocytes (SRBC) into C57BL/6 mice results in the generation of splenic B cells (plastic nonadherent, Thy-1- and Ig+) which, when transferred to normal syngeneic recipients, subsequently induce antigen-specific suppressor T cells to suppress the recipient's plaque-forming cell (PFC) responses to SRBC. In the present study we characterized the suppressor-inducer B cells phenotypically. Cytotoxic treatment of the donor's immune spleen cells with anti-Lyt-1 antibody plus complement (C'), but not with anti-Lyt-2 antibody plus C', relieved the suppression of PFC responses in recipients. The FcRr+ population separated by EA-rosette formation showed enriched suppressor-inducing activity, whereas the FcRr- population showed no activity. Our findings, taken together with the previous ones, suggest that suppressor-inducer cells are Thy-1-, Lyt-1+, Lyt-2-, FcRr+, and Ig+.     

Natural cytotoxicity against mouse hepatitis virus-infected cells. II. A cytotoxic effector cell with a B lymphocyte phenotype

The effector cell in mouse spleen which mediates natural cytotoxicity against mouse hepatitis virus (MHV)-infected target cells was characterized. The target cells were MHV-infected BALB/c 3T3, and the assay time was 3 hr. The effector cell, designated virus killer (VK) cell for the purpose of discussion, had the following phenotype: lymphocyte morphology, plastic-nonadherent, nylon wool-adherent, nonphagocytic, cyclophosphamide-sensitive; by antibody plus complement (C) depletion studies, it was asialo GM1-, NK 1.2 alloantigen-negative, Thy-1.2-, Lyt-5-, and macrophage antigen-negative; by rosetting techniques, it was Fc receptor-positive and surface Fab+; by flow cytometry (FACS) analysis, it was Lyt-2-, MAC-1-, Ia+, IgG (gamma)+, IgM (mu)+, IgD (delta)+, and B cell lineage antibody B-220+. NK cells, measured for cytotoxicity on YAC-1 cells, were similarly tested and were found to differ from the VK cell in the following properties: nylon wool-nonadherent, asialo GM1+, NK alloantigen-positive, Lyt-5+, surface Fab-, MAC-1+, Ia-, IgG-, IgM-, IgD-, and B-220-. The VK effector cell had a phenotype highly distinguishable from NK cells, effectors most commonly associated with antiviral natural cytotoxicity. The VK cell had a phenotype identical to that of a B lymphocyte and was identified as such. Although the effector cells displayed cell surface antibody, the antibody did not appear to be involved in lysis, because lysis could not be blocked by F(ab)'2 directed against Fab, mu, or delta. Cytotoxicity was more likely associated with recognition of the B lymphocyte surface by the MHV glycoprotein E2, as shown in the accompanying companion paper. This is the first demonstration that natural cytotoxicity can be mediated by B lymphocytes.