Characterization of the immunodeficiency of RIIS/J [corrected] mice. I. Association with the CD5 (LY-1) [corrected] B cell lineage

RIIIS/J mice produce low antibody responses to several polysaccharide Ag of bacterial origin. They have low levels of serum IgM and IgG3 and high levels of serum IgG2a and IgG2b. Low serum IgM and IgG3 have been attributed to a low frequency of CD5 (Ly-1) B cells, which play an important role in the production of natural antibodies. Indeed, RIIIS/J mice have a low frequency of CD5 (Ly-1)+, IgM bright+, Ly-5 (B220)dull+ (i.e., CD5 (Ly-1) B) cells in their peritoneum. RIIIS/J mice treated with LPS produce a low anti-bromelain-treated mouse RBC splenic plaque-forming cell response and a normal anti-mouse transferrin splenic PFC response. Those data are compatible with the fact that CD5 (Ly-1) B cells contain the precursors of B lymphocytes secreting anti-bromelain-treated mouse RBC antibody. However, they have a higher frequency of IgM bright+, Mac-1+ cells in their peritoneum. These cells represent the CD5 (Ly-1) "sister population" of CD5 (Ly-1) B cells described by others. This suggests that characteristics usually associated with the CD5 (Ly-1) lineage are applicable only to the CD5 (Ly-1)+ Mac-1+ IgM+ population, but not the related CD5 (Ly-1)- Mac-1+ IgM+ population. RIIIS/J mice should thus prove a valuable model to study the CD5 (Ly-1) B cell lineage.     

The age-dependent loss of bone marrow B cell precursors in autoimmune NZ mice results from decreased mitotic activity, but not from inherent stromal cell defects

The formation of B lymphocytes is abnormal in autoimmune NZB and (NZB x NZW)F1 mice. With age, the proportion of sIg- Ly-5(220)+ pre-B cells and less mature B cell progenitors in the bone marrow progressively declines, reaching only approximately one-third of normal levels in 20-wk-old NZ mice. To determine the mechanisms responsible for the deficiency of NZ B lineage precursors, the mitotic activity of sIg- Ly-5(220)+ bone marrow cells in vivo was determined in NZ and conventional inbred mice as a function of age. The proportion of sIg- Ly-5(220)+ B cell precursors in (S + G2/M) stages of the cell cycle steadily decreased with age in NZ autoimmune mice. Furthermore, upon metaphase arrest, the rate of entry of sIg- Ly-5(220)+ bone marrow cells into G2/M also decreased with age in NZ mice. Therefore, the mitotic activity of sIg- Ly-5(220)+ B cell precursors is substantially decreased in NZ mice greater than or equal to 20 wk of age. The capacity of the bone marrow stromal microenvironment of NZ mice to support B lineage precursor growth was tested in two ways: 1) the capacity of preformed NZ bone marrow stroma to support B lineage cell growth in long term bone marrow cell culture under lymphopoietic conditions was assessed and 2) the capacity of NZ bone marrow B lineage precursors to expand in vivo after sublethal (200 rad) whole body irradiation was determined. Stroma derived from adult NZ mice supported the growth and development of B lineage lymphocytes in long term bone marrow cell culture to a greater extent than did age-matched conventional murine stroma. Furthermore, sublethal irradiation of older adult NZ mice resulted in some expansion of bone marrow sIg- Ly-5(220)+ B cell precursors in vivo. Therefore, the deficiency of B cell progenitors in the bone marrow of older NZ autoimmune mice is associated with diminished mitotic activity. However, this does not result from defects in the capacity of NZ bone marrow stroma to permit B lineage cell expansion as determined by both in vitro and in vivo experiments. In the absence of a detectable stromal cell defect, it is possible that an active inhibitory process within the bone marrow influences the mitotic activity of B cell precursors in NZ mice.     

Cellular control of abscess formation: role of T cells in the regulation of abscesses formed in response to Bacteroides fragilis

Although abscesses are a major sequela of infection, little is known about which cellular events initiate and which prevent this pathologic response. These studies are the first to indicate a role for T cells in the important pathogenic process of abscess development and also in immunity to abscesses induced by Bacteroides fragilis. We have shown that T cells initiate the formation of abscesses in mice after i.p. challenge with B. fragilis. These T cells bear both Ly-1 and Ly-2 surface markers. Nude mice (which have been shown by others to have T cell or T cell precursors) are also able to form abscesses. Cyclophosphamide-treated mice (with depressed T cell function) were not capable of developing abscesses. Reconstitution with normal or nude mouse spleen cells restored this ability. However, reconstitution with anti-Thy-1.2-treated, anti-Ly-1, or anti-Ly-2-treated spleen cells (or a mixture of the two cell populations) failed to allow abscess formation after bacterial challenge. Immunity to abscesses caused by B. fragilis requires two T cells. The first Ly-1-2+ T cell has an IJ surface marker and has been shown to release a small m.w. soluble factor (ITF) that is antigen specific. Immunity to abscesses, however, depends on the interaction of ITF with a second Ly-1-2+ T cell, demonstrated in reconstitution experiments with nude mice. The data presented document a critical role for T cells in abscess induction and suggest the existence of a suppressor-like T cell circuit in immunity to abscesses.     

Regulation of B lymphocyte responses to IL-4 and IFN-gamma by activation through Ly-6A/E molecules

The Ly-6 family of cell surface molecules has previously been shown to participate in T cell activation. We show that Ly-6A/E proteins also modulated the response of normal B lymphocytes in three separate in vitro assays. First, unfractionated or small resting B cells proliferated when cultured with IFN-gamma, IL-4, and an anti-Ly-6A/E mAb. Second, this anti-Ly-6A/E mAb restored B cell proliferation responses that were inhibited when coculturing the B cells in IFN-gamma, IL-4, and anti-IgM. Third, anti-Ly-6A/E specifically up-regulated the cell surface expression of its own Ag, and this response was dependent upon co-stimulation with IFN-gamma. Mixing of T and B cells in culture suggested that T cells did not contribute substantially to the B cell proliferative response. Moreover, up-regulation of Ly-6A/E was observed for one B cell lymphoma, WEHI-231. Therefore, it appeared that modulation of B cell function by anti-Ly-6A/E was due to a direct effect of the mAb binding to the B cells. Taken together, these data suggest Ly-6A/E proteins are functional on B cells and may play a regulatory role in B cell activation.     

Origin of autoreactive T helper cells. I. Characterization of Thy-1+, Lyt-, L3T4- precursors in the spleen of normal mice

A new population of dull Thy-1+, Ly-1-, Lyt-2-, L3T4- PNA- cells, resistant to a double cytotoxic treatment by monoclonal antibodies to these T cell markers plus complement, has been isolated from the spleen of normal adult BALB/c and DBA/2 mice (Tkr cells). These cells exhibit no spontaneous autoreactivity or alloreactivity but can be activated with concanavalin A (Con A). Once activated, they differentiate into bright Thy-1+, Ly-1+, Lyt-2-, L3T4+ PNA- T lymphocytes. Con A-activated Tkr cells also strongly proliferate in the presence of allogeneic or syngeneic dendritic cells in secondary cultures. Moreover, contrary to other Con A-stimulated T cell populations, they induce B lymphocytes to proliferate and to differentiate into Ig-secreting cells at a very high level. Con A-activated Tkr cells are therefore very potent polyclonal B cell activators. Restimulated of Tkr cells by syngeneic dendritic cells can be inhibited by anti-L3T4 or anti-class II monoclonal antibodies. The results suggest that Tkr cells are the precursors of class II-specific autoreactive T helper cells. Tkr cells are absent in the spleen of B6 animals. This indicates that their expression might be genetically controlled. It also suggests that Tkr cells may not be the unique splenic precursors of autoreactive T cells. Con A activation of Tkr cells in Click's medium is 2-mercaptoethanol dependent and highly sensitive to pCO2, like the response of thymocytes. Tkr cells are also absent in the spleen of nude mice. We conclude that Tkr cells represent splenic precursors of autoreactive T helper cells equivalent to Thy-1+, Ly-2-, L3T4- PNA- cortical thymocytes.     

Multiple cytokine interactions regulate Ly-6E antigen expression: cooperative Ly-6E induction by IFNs, TNF, and IL-1 in a T cell lymphoma and in its induction-deficient variants.

The cell surface Ly-6E antigen, known to play a role in T cell activation, is up-regulated by IFNs. In the present study, we investigated the possible interactions between IFNs and other cytokines in this regulation. As a model system, we used the YAC T cell lymphoma, in which Ly-6E is normally absent but can be highly induced both at the mRNA and surface protein levels by IFN-gamma or IFN-alpha/beta. The combination of the two IFNs was found to result in markedly synergistic Ly-6E induction in this cell line. Moreover, mutants of YAC cells were isolated that did not respond to the Ly-6E-inducing action of IFN-gamma or IFN-alpha/beta alone but did respond to their combination. Such a synergistic interaction is consistent with the notion that the two IFN types utilize different intracellular mechanisms to induce Ly-6E expression. Ly-6E induction mediated by IFN-gamma or IFN-alpha/beta was also enhanced by cotreatment with TNF-alpha or IL-1 alpha, which by themselves had no detectable Ly-6E-inducing effect. These two cytokines similarly synergized with IFNs to trigger a response in several Ly-6E-induction-deficient mutants. However, their action could be dissociated in one mutant (B54) where the response to IFN-alpha/beta was enhanced by TNF-alpha, but not by IL-1 alpha. Altogether, these data indicate that Ly-6E antigen expression is regulated by the interaction of several inflammatory cytokines, which may provide a mechanism for the local modulation of T cell activation. The YAC cell mutants described here should facilitate further analysis of the molecular bases of Ly-6E regulation.     

Transgenic Ly-49A inhibits antigen-driven T cell activation and delays diabetes

Activation of islet-specific T cells plays a significant role in the development of type 1 diabetes. In an effort to control T cell activation, we expressed the inhibitory receptor, Ly-49A, on islet-specific mouse CD4 cells. Ag-mediated activation of Ly-49A T cells was inhibited in vitro when the Ly-49A ligand, H-2D(d), was present on APCs. Ag-driven T cell proliferation, cytokine production, and changes in surface receptor expression were significantly reduced. Inhibition was also evident during secondary antigenic challenge. Addition of exogenous IL-2 did not rescue cells from inhibition, suggesting that Ly-49A engagement does not lead to T cell anergy. Importantly, in an adoptive transfer model, Ly-49A significantly delays the onset of diabetes. Together these results demonstrate that the inhibitory receptor Ly-49A effectively limits Ag-specific CD4 cell responses even in the presence of sustained autoantigen expression in vivo.     

A new lymphocyte alloantigen (Ly-10) controlled by a gene linked to the Lyt-1 locus

Spleen cells from a (BALB/c x C57BL/6)F1 mouse immunized with CBA/J spleen cells were fused with the myeloma cell line NS-1. One of the six established hybrid cell lines continuously secreted antibody that recognized a new antigenic specificity, tentatively called "Ly-10.1". This newly found antigen is expressed on thymocytes, on splenic T and B cells, on bone-marrow cells, and on the cells derived from brain, kidney and liver. It is also expressed on a continuous cell line, 416B, with stem-cell characteristics. The unique tissue distribution and, furthermore, a distinct strain distribution pattern distinguishes Ly-10.1 from any known murine lymphocyte alloantigen. On the basis of reactivity with cells of the C57BL/6-Lyt-1a congenic strain, one gene governing Ly-10 expression is assigned to the Lyt-1 region of chromosome 19.     

Activation induced differential regulation of stem cell antigen-1 (Ly-6A/E) expression in murine B cells

Expression of stem cell antigen-1 (Ly-6A/E) is developmentally regulated in murine B cells. However, little is known about its modulation during B cell activation. We report here the differential regulation of Ly-6A/E expression in response to diverse activation signals in mature B cells. Stimulation of resting B cells through the antigen receptor (BCR) inhibited, Ly-6A/E surface expression in dose dependent manner. Activation induced downregulation of Ly-6A/E is specific to BCR mediated signaling events as stimulation of B cells with anti-CD40, lipopolysaccharide or interferon-γ induced upregulation of Ly-6A/E surface expression. The activation induced differential modulation of Ly-6A/E expression is mediated at the mRNA levels. A role for BCR signaling in inhibition of Ly-6A/E expression was further confirmed using STAT-1^−/− B cells, which expressed constitutive, but not inducible Ly-6A/E. The BCR induced inhibition of Ly-6A/E RNA and surface expression was mimicked by ionomycin, but not phorbol myristate acetate, indicating a role for calcium but not protein kinase C dependent signaling events. Inhibition of calcineurin reversed the BCR or ionomycin inhibited Ly-6A/E expression. Interestingly, in vitro differentiation analysis of Ly-6A/E⁺ and Ly-6A/E⁻ splenic B cells revealed the Ly-6A/E⁺ cells to be the major source of antibody production, suggesting a potential role for Ly-6A/E in B cell differentiation. These studies provide the first evidence for activation induced differential modulation and differentiation of Ly-6A/E⁺ B cells.     

Precursors of both conventional and Ly-1 B cells can escape feedback inhibition of Ig gene rearrangement

Experiments with transgenic mice carrying rearranged Ig transgenes have shown that membrane bound Ig molecules cause feedback inhibition of endogenous Ig gene rearrangement. However, this inhibition is never complete. It has been postulated that escape from feedback may be a property of the Ly-1 B cell subset, whereas rearrangement of endogenous Ig genes may be completely inhibited in conventional B cells. This possibility was investigated in transgenic mice carrying a lambda transgene under the control of the H chain enhancer. It was found that kappa producing B cells in these lambda transgenic mice were for the most part, although not exclusively, of the conventional B cell phenotype. Examination of peritoneal exudate cells revealed that a large proportion of Ly-1 B cells also express kappa. Adoptive transfer of bone marrow from adult lambda transgenic mice, a source of conventional B cell precursors, resulted in the production of relatively high levels of serum kappa 2 to 3 mo after transfer into recipient SCID mice. A high proportion of donor B cells in the spleen produced endogenous kappa protein with or without co-production of lambda. It is concluded that precursors of both conventional and Ly-1 B cells can escape feedback inhibition of L chain gene rearrangement.     

A new lymphocyte alloantigen (Ly-10) controlled by a gene linked to theLyt-1 locus

Spleen cells from a (BALB/cxC57BL/6)F₁ mouse immunized with CBA/J spleen cells were fused with the myeloma cell line NS-1. One of the six established hybrid cell lines continuously secreted antibody that recognized a new antigenic specificity, tentatively called Ly-10.1. This newly found antigen is expressed on thymocytes, on splenic T and B cells, on bone-marrow cells, and on the cells derived from brain, kidney and liver. It is also expressed on a continuous cell line, 416B, with stem-cell characteristics. The unique tissue distribution and, furthermore, a distinct strain distribution pattern distinguishes Ly-10.1 from any known murine lymphocyte alloantigen. On the basis of reactivity with cells of the C57BL/6-Lyt-1^a congenic strain, one gene governing Ly-10 expression is assigned to the Lyt-1 region of chromosome 19.We chose the notation Ly-10 rather than Ly-9 to allow for a future decision that Lgp 100 (Ledbetter et al. 1979) should be renamed Ly-9.     

Role of Ly-1:Qa1- and Ly-1:Qa1+ inducer T cells in activation of Ly-23 effectors of suppression of antibody production in mice

Ly-2+ effectors of T cell-mediated suppression require inducing signals from antigen and a helper cell bearing the Ly-1+:Qa1+ surface phenotype. In this report, we have further examined the helper cell requirements for suppressor cell induction of antibody production in mice. By using the T cell subset education procedure in vitro, we have activated T cells to sheep red blood cells (SRBC) antigens and then purified Ly-2 cells before testing for suppressor activity in assay cultures of defined T and B cell subsets. We have confirmed our previous observations that Ly-1+:Qa1+ cells are required for activation of T suppressors, but have found that under the appropriate conditions, there is not a strict requirement for the Ly-123 subset of T cells. Furthermore, if Ly-23 cells are stimulated in the presence of Ly-1+:Qa1- T cells, effective suppressors can be obtained only if a source of Ly-1:Qa1+ inducers is added to the assay culture. If Ly-23 cells are activated by antigen in the absence of Ly-1 cells, subsequent exposure to the Ly-1+:Qa1+ subset under the conditions tested here is not sufficient to activate suppressors. These results show that effectors of suppression, like B cells and cytotoxic T lymphocytes, may respond to two helper cells.     

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)     

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.     

Maturation-dependent adhesion of human B cell precursors to the bone marrow microenvironment

Murine B cell precursors can be induced to proliferate in culture if allowed to bind to bone marrow derived adherent cells prepared under specific conditions. We studied the binding of human B cell precursor subpopulations to various in vitro microenvironments to determine which conditions may potentially be suitable models for human B precursor differentiation. Using the markers CD10, CD34, and CD20, B lineage populations of increasing maturation were quantitated: CD10+/CD34+, CD10+/CD20-, CD10+/CD20+, and CD10-/CD20+ cells in marrow, and CD10-/CD20+ mature B cells in peripheral blood. The adhesion of subpopulations of blood and marrow-derived light density cells to adherent cell layers or matrix was studied following a 2-h incubation in 24-well plates. The absolute number of bound B lineage cells was determined by cell counts and flow cytometry analysis. The adherence of B lineage cells to passaged human marrow fibroblasts (BM-FB) was highest in the most immature CD10+/CD34+ cells (34.3 +/- 4.2%), decreasing steadily with each stage of maturation to the peripheral blood B cells (11.2 +/- 2.4%). Increased adhesion of CD10+ B cell precursors relative to CD10-/CD20+ marrow B cells was confirmed by adhesion studies using sorted cells. The two most immature B lineage cells (CD10+CD34+ and CD10+/CD20-) showed more adherence to BM-FB than any other cell type tested, except for monocytes. Only B lineage precursor cells, erythroid precursors and CD10-/CD34+ cells showed significantly greater binding to BM-FB than to plastic. B lineage precursors bound equally well to primary and passaged human marrow fibroblasts, but bound significantly less well to passaged human foreskin fibroblasts, primary human marrow stroma, extracellular matrix of marrow fibroblasts, or fibronectin. These results suggest that specific binding to marrow fibroblasts is part of the differentiation program of early B lineage precursors. This binding activity gradually and predictably decreases during B lineage differentiation, in contrast to expression of other binding receptors, such as LFA-1 and CD44, which increase during B lineage maturation.     

Identification of early stages of T lymphocyte development in the thymus cortex and medulla

Thymocyte subpopulations with a phenotype suggesting they are early stages of T cell development in the adult mouse thymus were characterized and isolated by using multiparameter flow cytometry and sorting, in conjunction with selective killing with antibody and complement (C). The intrathymic localization of these subpopulations was assessed by dipping the thymus in fluorescent dyes to selectively label outer-cortical cells. The main phenotypic markers used were sensitivity to C-mediated lysis by the monoclonal antibody B2A2 (which spares most prothymocytes but kills most thymocytes), the expression of the T cell lineage specific markers Ly-2 and L3T4, and the levels of the common T cell antigens Ly-1 and Thy-1. A preliminary selection for cells lacking Ly-2 and L3T4, or resistant to B2A2 and C, produced a population of large cells, only 5% of all thymocytes and distinct from the typical cortical blast cells. This population of putative early thymocytes was itself heterogeneous, consisting of eight subpopulations separable by phenotype and intrathymic localization. One group of two subpopulations (B2A2-, Ly-1++, Thy-1+ and either Ly-2+ L3T4- or Ly-2- L3T4+) appeared to be of medullary location, and their phenotype suggested they could have been early members of the medullary lineages. Another group of two subpopulations (B2A2-, Ly-1++, Thy-1-, Ly-2-, L3T4- and B2A2-, Ly-1++, Thy-1+, Ly-2- L3T4-) did not show a clear localization pattern and may have represented cells in an earlier stage of transition to medullary phenotype and location. A quite different group of three subpopulations (B2A2++, Ly-1-, Thy-1-, Ly-2- L3T4-; B2A2++, Ly-1-, Thy-1+, Ly-2-, L3T4-; and B2A2++, Ly-1+, Thy-1++, Ly-2- L3T4-) was concentrated in the outer cortex and seemed to represent a series of stages of a cortical pathway, before the typical cortical blast cells. Finally, a very minor subset (0.2% of thymocytes), lacking all these markers, was concentrated in the outer cortex; this fifth group had the phenotype expected of the earliest intrathymic precursor cells. The results suggest that the separate developmental streams of cortical and medullary thymocytes may be traced back, via these minor early blast subpopulations, to common precursor cells in the outer cortex.     

Characterization of cytotoxic cells generated from bone marrow culture

Bone marrow (BM) harbors precursors (Pre-NK) to NK cells. Recently, we devised an in vitro culture system that induces differentiation of the presumptive BM Pre-NK cells into cytotoxic cells to YAC in the presence of rat concanavalin A (Con A) conditioned medium. We have now analyzed the antigenic phenotype of the effector cells, precursor cells, and the target specificity of these cytotoxic cells. The cytotoxic cells had antigenic profiles similar to endogenous NK cells with the exception of Lyt-2 antigen. They are strongly positive for Qa-5, Thy-1, and partially positive for NK-1, Ly-5, Ly-6, Ly-10, and AsGm-1 and Lyt-2 antigens. The Pre-NK or accessory cells are positive for Qa-5, Ly-10, and Ly-20 and partially positive for NK-1, Thy-1, and AsGm-1 antigens. These Qa-5+ NK cells do not exhibit cytotoxic activity to WEHI or P815. They could also be generated from BM of nude mice as well as beige mice. We concluded from these studies that rat Con A-conditioned medium contained factors that could differentiate Pre-NK cells to mature NK cells and that these cells are heterogeneous. This in vitro culture system is useful in delineating the ontogeny of NK cells.     

Responses of B cells from autoimmune mice to IL-5

Three strains of mice (NZB/W F1 X NZW (NZB/W), BXSB, and MRL/Mp-lpr/lpr (MRL/lpr] develop an autoimmune disease that is clinically and immunologically similar to human SLE. A characteristic of these mice is polyclonal B cell hyperactivity. To explore whether this may be related to hyper-responsiveness to B cell stimulatory factors, we investigated the proliferative and secretory responses of B cells from these mice to semi-purified natural and rIL-5, a major regulator of B cell development in the mouse. As this lymphokine stimulates growth and differentiation of activated B cells, attention was focused on in vivo-activated B cell populations, obtained from the interface of 50/65% Percoll density gradients, from normal or autoimmune mice. This cell population from NZB/W mice secreted IgM and incorporated [3H]TdR at significantly higher levels in response to IL-5, and was more sensitive to IL-5, than a comparable population from several normal murine strains. NZB/W female and male mice displayed heightened responses to IL-5, indicating that this is characteristic of the strain in general and is not associated with the accelerated severe disease of the females. Small resting B cells from NZB/W and normal mice were insensitive to IL-5 stimulation. In contrast to NZB/W mice, no difference was observed in the magnitude of either proliferative or Ig secretory responses between in vivo-activated B cell populations from autoimmune BXSB and MRL/lpr or normal mice. Thus, B cell hyper-responsiveness to IL-5 is a characteristic of NZB/W mice but not of two other lupus-prone murine strains. As one unique feature of NZB/W mouse B cells compared to normal and other autoimmune B cells is an elevated proportion of Ly-1+ B cells, the possibility of IL-5 hyper-responsiveness being associated with this B cell subpopulation was investigated. Fluorescence-activated cell sorter sorted Ly-1+ and Ly-1- B cells both responded to IL-5, however Ly-1+ B cells consistently showed a higher stimulation index in both proliferative and Ig secretory responses to this lymphokine.     

Alcohol impairs the myeloid proliferative response to bacteremia in mice by inhibiting the stem cell antigen-1/ERK pathway

Enhancement of stem cell Ag-1 (Sca-1) expression by myeloid precursors promotes the granulopoietic response to bacterial infection. However, the underlying mechanisms remain unclear. ERK pathway activation strongly enhances proliferation of hematopoietic progenitor cells. In this study, we investigated the role of Sca-1 in promoting ERK-dependent myeloid lineage proliferation and the effects of alcohol on this process. Thirty minutes after i.p. injection of alcohol, mice received i.v. challenge with 5 × 10(7) Escherichia coli for 8 or 24 h. A subset of mice received i.v. BrdU injection 20 h after challenge. Bacteremia increased Sca-1 expression, ERK activation, and proliferation of myeloid and granulopoietic precursors. Alcohol administration suppressed this response and impaired granulocyte production. Sca-1 expression positively correlated with ERK activation and cell cycling, but negatively correlated with myeloperoxidase content in granulopoietic precursors. Alcohol intoxication suppressed ERK activation in granulopoietic precursors and proliferation of these cells during bacteremia. Granulopoietic precursors in Sca-1(-/-) mice failed to activate ERK signaling and could not increase granulomacrophagic CFU activity following bacteremia. These data indicate that Sca-1 expression promotes ERK-dependent myeloid cell proliferation during bacteremia. Suppression of this response could represent an underlying mechanism for developing myelosuppression in alcohol-abusing hosts with severe bacterial infection.