Cultured Lyt-2- L3T4- T lymphocytes from normal thymus or lpr mice express a broad spectrum of cytolytic activity

T lymphocytes expressing the surface phenotype Lyt-2- L3T4- represent a minor population of immature thymocytes that appear to be the precursors of mature T cells. Cells with the same apparent surface phenotype also accumulate in vast numbers in the lymphoid tissues of the autoimmune lpr mouse. Lyt-2- L3T4- T lymphocytes from lpr lymph node (LN) or normal thymus express low to undetectable levels, respectively, of surface antigen receptor. In addition, they produce reduced amounts of lymphokines compared with normal T cells and lack precursors of alloantigen-specific cytolytic T lymphocytes. We previously showed that after culture with phorbol esters and interleukin 2, lpr Lyt-2- L3T4- T lymphocytes proliferate and differentiate, acquiring increased levels of surface antigen receptor by most cells, as well as Lyt-2 by a portion. We now show that cultured Lyt-2- L3T4- T cells from lpr LN or normal thymus are very efficiently cytolytic toward not only allogeneic tumor targets, but also natural killer (NK)-susceptible targets and syngeneic targets. Such killing was not inhibited by antibodies to H-2 or Lyt-2. In contrast, cultured mature Lyt-2+ L3T4- T cells from normal LN, thymus, or lpr LN were cytolytic only toward allogeneic targets and were dependent on Lyt-2 expression and H-2 recognition. The similarities of cultured Lyt-2- L3T4- T cells to NK and lymphokine-activated killer cells are discussed.     

Human eosinophils express CR1 and CR3 complement receptors for cleavage fragments of C3

The functional and antigenic characteristics of C3 receptors expressed on human eosinophils were investigated using rosette assays with sheep erythrocytes coated with C3 fragments and flow cytometric analysis of cells stained with anti-receptor antibodies. Purified peripheral blood eosinophils from 13 patients with hypereosinophilia expressed CR1 antigens. In 8 patients, a mean of 14 + 9.5% eosinophils formed C3b-dependent rosettes that were inhibited by F(ab')2 anti-CR1 antibodies. This number increased to 33% following stimulation with leukotriene B4 (LTB4) (10(-7) M). Similar numbers of C3b rosettes were formed by hypodense and normodense eosinophils. Eosinophils from 2 patients from this group expressed 20,000 125I-labeled monoclonal anti-CR1 antibody binding sites/cell. In another group of patients, 55 +/- 9% eosinophils spontaneously formed C3b-dependent rosettes that could not be enhanced by LTB4. In all patients, a mean of 16 +/- 9% eosinophils formed cation-dependent rosettes with C3bi-bearing intermediates that were inhibited by anti-CR3 antibody OKM1. All eosinophils stained with monoclonal antibodies against the alpha chain of CR3. There was no C3d-dependent rosette formation with eosinophils and no eosinophils stained with monoclonal anti-CR2 antibody. Thus, human eosinophils express CR1 and CR3. Since CR3 is required for the adhesion of granulocytes to surfaces and antibody-dependent cellular cytotoxicity of neutrophils, the interaction of C3 fragments with CR3 and CR1 on eosinophils may be of importance in eosinophil-mediated damage of opsonized targets.     

The role of tumor-specific Lyt-1+2- T cells in eradicating tumor cells in vivo. I. Lyt-1+2- T cells do not necessarily require recruitment of host's cytotoxic T cell precursors for implementation of in vivo immunity

The present study determines the Ly phenotype of T cells mediating tumor cell rejection in vivo and investigates some of cellular mechanisms involved in the in vivo protective immunity. C3H/HeN mice were immunized to syngeneic X5563 plasmacytoma by intradermal (i.d.) inoculation of viable X5563 tumor cells, followed by the surgical resection of the tumor. Spleen cells from these immune mice were fractionated by treatment with anti-Lyt antibodies plus complement, and each Lyt subpopulation was tested for the reconstituting potential of in vivo protective immunity in syngeneic T cell-depleted mice (B cell mice). When C3H/HeN B cell mice were adoptively transferred with Lyt-1-2+ T cells from the above tumor-immunized mice, these B cell mice exhibited an appreciable cytotoxic T lymphocyte (CTL) response to the X5563 tumor, whereas they failed to resist the i.d. challenge of X5563 tumor cells. In contrast, the adoptive transfer of Lyt-1+2- anti-X5563 immune T cells into B cell mice produced complete protection against the subsequent tumor cell challenge. Although no CTL or antibody response against X5563 tumors was detected in the above tumor-resistant B cell mice, these mice were able to retain Lyt-1+2- T cell-mediated delayed-type hypersensitivity (DTH) responses to the X5563 tumor. These results indicate that Lyt-1+2- T cells depleted of the Lyt-2+ T cell subpopulation containing CTL or CTL precursors are effective in in vivo protective immunity, and that these Lyt-1+2- T cells implement their in vivo anti-tumor activity without inducing CTL or antibody responses. The mechanism(s) by which Lyt-1+2- T cells function in vivo for the implementation of tumor-specific immunity is discussed in the context of DTH responses to the tumor-associated antigens and its related Lyt-1+2- T cell-mediated lymphokine production.     

Characterization of the interaction of human eosinophils and neutrophils with opsonized particles

The interaction of human eosinophils with opsonized particles was compared with that of human neutrophils. When eosinophils are stimulated with serum-opsonized zymosan particles, the lag time in H2O2 production is twice as long as found with neutrophils. Moreover, the concentration of these IgG + C3-coated particles required for optimal stimulation is about four times as high for eosinophils as for neutrophils. Under these conditions, the two cell types generate similar amounts of H2O2. However, eosinophils produce twice as much H2O2 as do neutrophils when stimulated with the soluble agent phorbol myristate acetate. Thus, although the oxidase capacity of eosinophils is larger than that of neutrophils, opsonized zymosan is a weak trigger for this activity in eosinophils. This phenomenon may be due to differences between the two cell types in the plasma membrane receptors or in the receptor oxidase transducing signal. The following are indications for the first possibility. i) IgG interacts poorly with the Fc gamma receptors on the eosinophil surface compared with those on neutrophils. This was shown by the inability of IgG-coated zymosan or IgG-coated latex to trigger any substantial H2O2 production by eosinophils unless brought into close contact with these cells by centrifugation. In contrast, neutrophils are stimulated by these particles both in suspension and in a pellet. The dissimilarity of the Fc gamma receptors on eosinophils and neutrophils was also shown with respect to antigenicity, determined by the monoclonal antibodies 3G8 and CLB-FcR-1. ii) Eosinophils contain about half as many receptors for C3b and C3bi on their surface as do neutrophils, also detected with monoclonal antibodies. The interaction of IgG subclasses with functional Fc gamma receptors on eosinophils and neutrophils showed that eosinophils release twice as much H2O2 as do neutrophils upon interaction with IgG1-, IgG2-, or IgG3-coated Sepharose beads, but this difference becomes fivefold with IgG4-coated Sepharose. This might be of relevance to the situation of chronic antigenic stimulation, e.g., in chronic schistosomiasis, in which eosinophil numbers and IgG4 antibody levels are elevated.     

Lyt phenotype of cytotoxic T cells: shift from Lyt-1+2+3+ to a mixed population of Lyt-1+2+3+ and Lyt-1-2+3+ during in vitro culture

The Lyt phenotype of cytotoxic T cells generated in the primary H-2 response was investigated kinetically. The cytotoxicity generated in the early stage of culture was abolished by treatment with alpha Lyt-1,2,3, and complement (C), whereas that generated in the late stage was only partially eliminated by alpha Lyt-1, but was abolished by alpha Lyt-2, 3, and C. This suggested late expansion of the Lyt-1-2+3+ population. Lack of Lyt-1 antigen was confirmed with cells that were depleted of Lyt-1+ from primary culture and then stimulated in the secondary response by elimination of cytotoxicity and by direct Lyt typing. Results indicated that the response of proliferative and cytotoxic T cells of the Lyt-1+2+3+ phenotype in the early stage of culture was followed by activation of Lyt-1-2+3+ T cells. Cytotoxic T cells in the late stage were shown to be a mixture of Lyt-1+2+3+ and Lyt-1-2+3+ cells. This was confirmed with cytotoxic T cells from secondary culture and uncloned long-term T cell lines.     

Phenotypic variation in clonal Abelson virus lymphoma cells

Two clonal A-MuLV lymphoma cell lines have the capacity to generate phenotypic variants when grown in vivo as ascites tumors. Variant lines differed from parental lymphoma cells in their expression of enzymatic or cell surface differentiation markers. Parental lines expressed the B220 and Lyb-2 glycoproteins characteristic of pre-B cells and bound B220-specific monoclonal antibodies such as 14.8. The parental cells expressed low levels of TdT activity but did not synthesize detectable mu-heavy chain, a cellular phenotype that may correspond to lymphoid progenitor cells. Three classes of phenotypic variants were recovered from the Thy-1- parental lines: 1) 14.8+, Lyt-1+, Thy-1- cells; 2) 14.8 +/-, Lyt-1+, Thy-1+ cells, and 3) 14.8-, Lyt-1+, Thy-1+ cells. Cell cloning experiments indicated that Thy-1+ variant cells can be recovered within 14 days of in vivo inoculation as a minor proportion (1/10(6] of the tumor cell population and subsequently become the predominant tumor cell population. These clonal tumor lines provide a model for the study of cellular and molecular alterations that occur during neoplastic differentiation and progression in the lymphoid system.     

Subsets of Lyt-2+ cells defined by differential expression of the 9F3 antigen: alterations in mice of the lpr/lpr genotype

Recently, we found that a novel murine cell surface antigen recognized by the 9F3 monoclonal antibody demonstrates T cell heterogeneity in normal mice and in congenic strains homozygous for the lymphoproliferation- and autoimmunity-inducing lpr gene. The objective of the present work was to further characterize this heterogeneity by studying the distribution of the 9F3 marker among Lyt-2+ T cells. By using dual parameter flow cytofluorometry analysis, at least two subsets of peripheral Lyt-2+ cells differing in 9F3 expression could be distinguished. In MRL/Mp-++, C57BL/6, and C3H/HeJ mice, 9F3- and 9F3+ cells accounted, respectively, for 65 to 80% and 20 to 35% of the whole Lyt-2+ population. Interestingly, in lpr/lpr-bearing congenic strains, a three- to fivefold selective increase in the frequency and absolute number of 9F3+ Lyt-2+ cells was found to take place during aging. Functional analysis of Lyt-2+ cells isolated by panning revealed that in +/+ mice, these cells respond better to phytohemagglutinin and concanavalin A than in lpr/lpr mice, indicating that phenotypic changes of Lyt-2+ cells correlate with functional changes. Further evidence for the functional relevance of 9F3-defined Lyt-2+ cell heterogeneity was provided by the study of sorted cells from +/+ mice, showing that 9F3- cells exhibit higher mitogenic reactivities than 9F3+ cells. This finding suggests that 9F3+ Lyt-2+ cells in +/+ and lpr/lpr mice are functionally homologous. Together, these data indicate that, in addition to the known expansion of a population of abnormal Lyt-2-T cells, mice of the lpr/lpr genotype have an alteration of their Lyt-2+ cell population, characterized by an imbalance of 9F3-defined subsets and decreased mitogenic reactivities.     

Expression of a bone marrow-associated Ly-6 determinant on the T cell population expanding in the lymph nodes of the autoimmune mouse strain MRL/Mp-lpr/lpr

In this report we describe the reactivity patterns of two monoclonal anti-Ly-6.2 antibodies toward lymph node (LN) cells of the autoimmune MRL/Mp-lpr/lpr (lpr) and the normal congenic MRL/Mp-+/+ (+/+) mouse strains. The monoclonal antibody 34-11-3, which has previously been found to detect a LN-associated Ly-6.2 antigen, reacted with both lpr and +/+ LN cells. Another monoclonal antibody, 34-10-7, which has been demonstrated to detect a bone marrow- (BM) associated determinant, was found to react with a small proportion of +/+ LN cells and unexpectedly with a high percentage of lpr LN cells. The expression of this BM determinant found in the LN of lpr mice was age dependent, and its presence on Thy-1.2+ cells increased with the expansion of a subset of Lyt-1+2- cells. In contrast, dual labeling experiments showed that in +/+ and young lpr mice a small subset of Lyt-1+2+ cells express this BM-associated Ly6.2 determinant. Therefore these findings demonstrate that the lpr gene-dependent T cell expansion in the LN results in both aberrant expression and association of lymphocyte cell surface markers.     

Distinction of virgin and memory T lymphocytes. Stable acquisition of the Pgp-1 glycoprotein concomitant with antigenic stimulation

The Pgp-1 glycoprotein was identified on a minor (27%) subset of peripheral Lyt-2+ or L3T4+ T cells. In contrast, mature medullary-type thymocytes (Lyt-2+ L3T4-, Lyt-2- L3T4+) were nearly devoid of cells expressing detectable surface Pgp-1. The appearance of peripheral Pgp-1- T cells was found to be thymus dependent, as demonstrated by the diminished proportion of Pgp-1- T cells after thymectomy and their virtual absence in athymic nude mice. The subsequent acquisition of surface Pgp-1 was found to be a stable differentiation event occurring concomitantly with primary antigenic stimulation; selected Pgp-1- mature T cells from thymus or periphery acquired constitutive expression of Pgp-1 after stimulation in vitro with alloantigen or mitogens. These observations were extended by studies in vivo showing that immunization with various antigens augmented the percentage of Pgp-1+ spleen cells within the Lyt-2+ subset. Furthermore, the frequencies of antigen-specific CTLp, after immunization by any of three different antigens tested, were greatly enriched in the Pgp-1+ compared with the Pgp-1- subpopulations. Peritoneal exudate Lyt-2+ cells, after a localized allograft rejection, demonstrated a particularly prominent Pgp-1+ subpopulation (78%) that contained virtually all the allospecific cytolytic activity. A model consistent with all of these data proposes that mature thymocytes lacking surface Pgp-1 upon emigration to the periphery acquire its expression at the time of primary antigenic stimulation. Hence, expression of Pgp-1 among peripheral T cells is an important differentiation marker for identifying antigen-stimulated memory T cells.     

Phenotypic heterogeneity of antisyngeneic tumor killer cells (ASTK) generated in allogeneic mixed lymphocyte reactions

By using monoclonal antibodies to Thy-1, Lyt-2, and Qa-5 differentiation antigens, we demonstrated a heterogeneity of cytotoxic cells developed in allogeneic mixed lymphocyte responses that lyse tumor cells syngeneic with the responder cells. There are minimally two Thy-1+ populations, one of which is Lyt-2+ and the other Lyt-2-. There is probably also a Thy-1- population. Most of the Lyt-2- tumor killer cells are Qa-5+, and most of the Lyt-2+ tumor killer cells are Qa-5-.     

The influence of dexamethasone treatment on the lymphoid and stromal composition of the mouse thymus: a flowcytometric and immunohistological analysis

The effect of injection of a range of doses of dexamethasone on the distribution of T-cell subpopulations and stromal cells in the thymus of BALB/c mice was investigated with flowcytometry and immunohistology. To this purpose we used monoclonal antibodies directed to the T-cell differentiation antigens Thy-1, T200, Lyt-1, Lyt-2, T4, MEL-14, and monoclonal antibodies directed to various classes of stromal cells. Injection of dexamethasone in increasing doses of 5-130 mg/kg body weight gradually leads to a depletion of the cortical thymocyte population, i.e., bright Thy-1 + ve, dull T-200 + ve, bright Lyt-2 + ve, and bright T4 + ve cells. These cortical cells are very dull MEL-14 + and express variable numbers of Lyt-1 molecules. Also the medulla is affected by dexamethasone although to a lesser extent. Dexamethasone injection at 130 mg/kg selects for a dull Thy-1 + ve, bright T-200 + ve, and bright Lyt-1 + ve medullary population. These cells are either T4 + ve Lyt-2-ve or T4-ve Lyt-2 + ve. Under these conditions, MEL-14 + ve cells were no longer present in the cortex but accumulated in medullary perivascular spaces. Staining of sequential sections showed that this particular subpopulation has a typical "helper" phenotype. This observation provides strong evidence that perivascular compartments are an exit pathway for emigrating T cells. The medullary population contains a phenotypically distinct, dexamethasone-sensitive subpopulation. This conclusion is based on two findings: 130 mg/kg dexamethasone depletes the thymus of all but 4% of the thymocytes, which form a much smaller subpopulation than the population of dull Thy-1 + ve cells (amounting to 15% of the total thymocytes). The medulla contains a subpopulation of dull Lyt-2 + ve cells, which are resistant to 20 mg/kg dexamethasone, but depleted by 130 mg/kg. Dexamethasone also has a severe effect on thymic nonlymphoid cells. Even at low doses, dexamethasone induces TR4 + ve cortical epithelial-reticular cells to become spherical ("nurse cell-like") structures, depleted of lymphoid cells. These stromal cells no longer express MHC antigens in a membrane-bound fashion. In contrast, the medullary epithelial cells appear morphologically unaffected even at a dexamethasone dose of 130 mg/kg.     

Immunohistology of lymphoid and non-lymphoid cells in the thymus in relation to T lymphocyte differentiation

The present paper reports the distribution of lymphoid and non-lymphoid cell types in the thymus of mice. To this purpose, we employed scanning electron microscopy and immunohistology. For immunohistology we used the immunoperoxidase method and incubated frozen sections of the thymus with 1) monoclonal antibodies detecting cell-surface-differentiation antigens on lymphoid cells, such as Thy-1, T-200, Lyt-1, Lyt-2, and MEL-14; 2) monoclonal antibodies detecting the major histocompatibility (MHC) antigens, H-2K, I-A, I-E, and H-2D; and 3) monoclonal antibodies directed against cell-surface antigens associated with cells of the mononuclear phagocyte system, such as Mac-1, Mac-2, and Mac-3. The results of this study indicate that subsets of T lymphocytes are not randomly distributed throughout the thymic parenchyma; rather they are localized in discrete domains. Two major and four minor subpopulations of thymocytes can be detected in frozen sections of the thymus: 1) the majority of cortical thymocytes are strongly Thy-1+ (positive), strongly T-200+, variable in Lyt-1 expression, and strongly Lyt-2+; 2) the majority of medullary thymocytes are weakly Thy-1+, strongly T-200+, strongly Lyt-1+, and Lyt-2- (negative); 3) a minority of medullary cells are weakly Thy-1+, T-200+, strongly Lyt-1+, and strongly Lyt-2+; 4) a small subpopulation of subcapsular lymphoblasts is Thy-1+, T-200+, and negative for the expression of Lyt-1 and Lyt-2 antigens; 5) a small subpopulation of subcapsular lymphoblasts is only Thy-1+ but T-200- and Lyt-; and 6) a small subpopulation of subcapsular lymphoblasts is negative for all antisera tested. Surprisingly, a few individual cells in the thymic cortex, but not in the medulla, react with antibodies directed to MEL-14, a receptor involved in the homing of lymphocytes in peripheral lymphoid organs. MHC antigens (I-A, I-E, H-2K) are mainly expressed on stromal cells in the thymus, as well as on medullary thymocytes. H-2D is also expressed at a low density on cortical thymocytes. In general, anti-MHC antibodies reveal epithelial-reticular cells in the thymic cortex, in a fine dendritic staining pattern. In the medulla, the labeling pattern is more confluent and most probably associated with bone-marrow-derived interdigitating reticular cells and medullary thymocytes. We discuss the distribution of the various lymphoid and non-lymphoid subpopulations within the thymic parenchyma in relation to recently published data on the differentiation of T lymphocytes.     

Ly1+ PRO-B lymphocyte clones. Phenotype, growth requirements and differentiation in vitro and in vivo.

Several clones obtained from the bone marrow of a BALB/c mouse were found to contain the heavy and light chain Ig genes in the germline configuration, to express Ly1 and to carry the B cell lineage markers B-220, Lyb8 and BP-1; these clones are Pgp-1+, LFA-1+, J11d+, Mac-1+ and Thy1-, Lyt2-, L3T4-, GM1.2- and Ia-. Three clones analyzed in detail (Lyd9, LyH7 and Lyb9) have receptors for interleukin (IL) 2 and IL3 as assessed with the 7D4 and CC11 monoclonal antibodies respectively. They grow in rIL3 but not in rIL2 or rIL1; both rIL4 and rIL5 also promote their proliferation, albeit to a much lesser extent than rIL3. None of the interleukins tested alone or in various combinations promoted the clones to differentiate in vitro along the B cell pathway. Treatment with 5-Azacytidine (5-Aza) induced cell surface Ia expression but not rearrangement or expression of Ig genes. However, 5-Aza-treated Lyd9, LyH7 and Lyb9 cells co-cultured with X-ray irradiated accessory cells and LPS gave rise to Ly1+, IgM+ B lymphocytes (range 14-51%) including mu + kappa + (78-93%), and mu + lambda + (9-25%) B lymphocytes. In vivo, the Lyd9, LyH7 and Lyb9 clones gave rise to IgM+ B lymphocytes (8.5-17%) including mu + kappa +, and mu + lambda +, but not to Lyt2+ or L3T4+ T lymphocytes after 4-6 weeks of transfer into Scid mice. Our results indicate that Ly1+ IgM+ cells comprise a subpopulation of B lymphocytes that is derived from IL3-responsive Ly1+ PRO-B lymphocytes.     

Qualitative and quantitative heterogeneity in Pgp-1 expression among murine thymocytes

A proportion of Pgp-1+ cells in the thymus have been shown to have progenitor activity. In adult AKR/Cum mice the total Pgp-1+ population in the thymus differs from that of the bulk of thymocytes and is antigenically heterogeneous when examined by flow cytometry. Pgp-1+ thymocytes are enriched for several minor cell populations compared to total thymocytes: B2A2-, interleukin-2-receptor+ (IL-2R+), and Lyt-2-, L3T4-. However, these subsets are still a minor proportion of the Pgp-1+ cells, the majority being Lyt-2+ and/or L3T4+ and B2A2+. Pgp-1+ thymocytes also differ from the bulk of thymocytes in having lower amounts of Thy-1 and in showing a higher proportion of single positive (Lyt-2+, L3T4- or Lyt-2-, L3T4+) cells. Populations of adult thymocytes that are enriched in progenitor cells can be isolated by cytotoxic depletion using either anti-Thy-1 antibody (Thy-1 depletion) or anti-Lyt-2 and anti-L3T4 antibody (Lyt-2, L3T4 depletion). Pgp-1+ cells in progenitor cell-enriched populations are also phenotypically heterogeneous. Pgp-1+ cells in both populations may be IL-2R+ or IL-2R- and B2A2+ or B2A2-. The population of Pgp-1+ cells in progenitor cell-enriched populations in the adult differs from that of the fetus at 14 days of gestation in that in the 14-day fetus, most Pgp-1+ cells are IL-2R+. By Day 15 of gestation, distinct populations of Pgp-1+, IL-2R-; Pgp-1+, IL-2R+; and Pgp-1-, IL-2R+ cells are observed. In the 15-day fetus, as in the adult, many Pgp-1+ thymocytes express low to moderate levels of Thy-1. The total percentage of Pgp-1+ cells in the thymus varies among different mouse strains, ranging from 4 to 35% in the thymus of young adult mice. Pgp 1.1 strains contain more detectably Pgp-1+ thymocytes than Pgp 1.2 strains; however, there is variability in the proportion of Pgp-1+ cells, even among Pgp 1.2 strains. In contrast to AKR/Cum mice, the Pgp-1+ thymocyte population in BALB/c mice, which contain a high proportion of Pgp-1+ thymocytes, closely resembles the total thymocyte population.     

Lyt-2+ cells. Requirements for concanavalin A-induced proliferation and interleukin 2 production

The requirements for inducing Lyt-2+ T cell proliferation in response to concanavalin A (Con A) were examined. Purified Lyt-2+ or L3T4+ spleen cells of C57BL/6 origin were stimulated with Con A and syngeneic macrophages (MO) in the presence of monoclonal antibodies to T cell markers or to polymorphic determinants on major histocompatibility complex molecules, and assessed for the ability to proliferate and to produce interleukin (IL) 2. alpha I-Ab failed to inhibit the Con A response of Lyt-2+ cells at dilutions that significantly inhibited the response of L3T4+ cells. In contrast, alphaKb/Db or alpha Lyt-2.2 specifically inhibited the response of Lyt-2+ cells, but not L3T4+ cells. The ability of alpha Kb/Db and of alpha Lyt-2.2 to inhibit the response of Lyt-2+ cells was dependent upon the concentration of Con A. These data demonstrate that optimal triggering of T cell subsets to proliferate and to produce IL-2 in response to Con A requires interactions with the appropriate restricting major histocompatibility complex molecule. The role of accessory cells in Lyt-2+ Con A-induced proliferation and IL-2 production was also investigated. Purified Lyt-2+ cells and purified L3T4+ cells failed to respond to Con A in the absence of MO. IL-1 reconstituted the response when MO were limiting, but failed to restore the response of either Lyt-2+ or L3T4+ cells when T cells were rigorously purified to remove all MO. These results demonstrate that triggering Lyt-2+ T cells, like L3T4+ T cells, requires accessory cells, and that this does not merely reflect a requirement for IL-1 production. Thus, Con A-induced proliferation and IL-2 production by Lyt-2+ T cells requires intimate contact with accessory cells and interactions dependent upon the class I-restricting element.     

Isolation of T cell line capable of protecting mice against collagen-induced arthritis

A T cell line specific to human type II collagen (CII) was selected and propagated from DBA/1J mice immunized with human CII. The line cells were not reactive to type I or type III collagen of human origin, but they were cross-reactive to bovine, rat, and rabbit CII and they recognized both native and heat-denatured human CII. The cells were reactive to an N-terminal three-quarters fragment of human CII, produced by tadpole collagenase digestion of human CII, but not to a C-terminal one-quarter fragment of human CII. The cells showed Thy-1+, Lyt-1+, Lyt-2-, and L3T4+ phenotypes characteristic of T helper cells or delayed-type hypersensitive cells, determined by the immunofluorescence method. To clarify the role of T cells in the pathogenesis of collagen-induced arthritis, we inoculated this cell line into DBA/1J mice and found that they developed clinical arthritis, albeit at a low incidence. The cells attenuated by x-ray were capable of inducing resistance to the subsequent induction of collagen-induced arthritis of DBA/1J mice. The sera from mice protected by inoculation of the cell line exhibited anti-idiotypic antibody response against conventional and monoclonal anti-CII antibodies. Anti-T cell receptor response may be involved in the mechanism for the protective effect of the cell line against autoimmune murine arthritis.     

Construction and properties of new Lyt-congenic strains and anti-Lyt-2.2 and anti-Lyt-3.1 monoclonal antibodies

Hybridomas producing mouse monoclonal IgM antibodies specific for Lyt-2.2 and Lyt-3.1 T-cell surface alloantigens have been constructed. Cytotoxic titers of ascites fluids were found to be 10(-6) or greater and no lysis of thymocytes of congenic strains bearing the alternative allele was observed at the lowest dilutions tested (1:2). The anti-Lyt-2.2 monoclonal antibody (HO-2.2) specifically precipiated from extracts of Lyt-2.2-positive thymocytes molecular species indistinguishable from those precipitated by conventional anti-Lyt-2.2 sera. However, by immunoprecipitation criteria (though not by cytotoxicity), the anti-Lyt-3.1 antibody (HO-3.1) demonstrated some cross-reactivity with similar molecular species from Lyt-3.1-negative thymocytes. In addition, three new strains of mice differing from existing strains in the region of the Lyt-2 and Lyt-3 loci have been constructed. They are: C.C58-Lyt-2a, Lyt-3a and C.AKR-Lyt-2a, Lyt-3a, congenic with Balb/cAn and bearing Lyt-2a and Lyt-3a alleles of C58/J and AKR/J, respectively; and AKR.C-Lyt-2b, Lyt-3b congenic with AKR/J and bearing the Lyt-2b and Lyt-3b alleles of Balb/cJ.     

Changing patterns of lymphocyte proliferation, IL-2 production and utilization, and IL-2 receptor expression in mice infected with Schistosoma mansoni

In murine schistosomiasis mansoni the soluble egg Ag (SEA)-induced L3T4+ T cell-mediated circumoval granulomatous response peaks at the acute stage (8w) and undergoes Lyt-1+, Lyt-2+ suppressor cell mediated down-modulation at the chronic stage (20w) of the infection. In the present study lymphoproliferation, IL-2 production, utilization, and IL-2R display were examined in splenic lymphocytes of infected mice. The L3T4+ subset was the major SEA-specific proliferative population at both stages of the infection. Chronic infection spleen cells or the L3T4+ subset proliferated less compared with their acute infection counterparts. Elimination of the Lyt-2+ subset did not improve diminished proliferation. Chronic infection cells and the Lyt-2+ subset stimulated with SEA and exogenous rIL-2 regained their proliferative ability to a level comparable with acute infection cells. Ag-stimulated acute infection T cells produced 40 to 50 chronic infection cells less than 5 U/ml IL-2. Elimination of L3T4+ T cells diminished, and the Lyt-2+ T cells left unchanged IL-2 production in the acute infection cells. Elimination of Lyt-2+ subset from the chronic infection population did not enhance IL-2 production. Exogenously added rIL-2 was equally utilized by acute or chronic infection T cells. Scatchard plots of [125I]IL-2 binding showed similar numbers of high affinity receptors on acute (189) and chronic infection cells (118), but the number of low affinity receptors was higher on the acute (2229) vs the chronic infection lymphocytes (578). Analysis of IL-2R expression by two-color fluorescence and flow cytometry revealed that 30 to 40% of the acute, chronic infection L3T4+ cells displayed receptor. Receptor expression increased by added SEA, or SEA + rIL-2. R display among Lyt-2+ cells was only 12 to 18%. SEA stimulation enhanced receptor display more among the acute, SEA + rIL-2 stimuli raised receptor expression only among chronic infection lymphocytes. These data do not show inherent defect in IL-2R display, or utilization in chronic infection T cells. Diminished IL-2 production appears to be the cause of decreased T cell responsiveness.     

Surface markers of T cells causing lethal graft-vs-host disease to class I vs class II H-2 differences

Information was sought on the phenotype of lymphoid cells causing lethal graft-vs-host disease (GVHD) in irradiated mice expressing whole or partial H-2 differences. In all strain combinations tested, pretreating donor lymph node (LN) cells with anti-Thy-1 monoclonal antibodies (MAb) plus complement (C) abolished mortality. With GVHD directed to class I H-2 differences, pretreating LN cells with anti-Lyt-2 MAb prevented mortality, whereas MAb specific for Ly-1 or L3T4 cell surface determinants caused severe mortality. These data imply that lethal GVHD directed to class I H-2 differences is mediated by L3T4-, Lyt-2+ cells; this subset of T cells was shown previously to control GVHD directed to multiple minor histocompatibility antigens, i.e., antigens seen in the context of self-class I molecules. With whole H-2 differences, GVHD appeared to be controlled largely but not exclusively by L3T4+, Lyt-2-T cells. This T cell subset was also the predominant cause of GVHD directed to class II differences. With class II incompatibilities, depleting donor cells of L3T4+ T cells, either by pretreatment with anti-L3T4 MAb + C or by fluorescence activated cell sorter selection, greatly reduced but did not completely abolish GVHD. These data might imply that L3T4-, Lyt-2+ cells have some capacity to elicit anti-class II GVHD. A more likely possibility, however, is that the residual GVHD to class II differences observed with Lyt-2+-enriched cells reflected minor contamination with L3T4+ cells.     

Interaction of reovirus with cell surface receptors. IV. The reovirus type 3 receptor is expressed predominantly on murine Lyt-2,3+ and human T8+ cells

Reovirus type 3 binds to approximately 20% of murine and human T cells via the viral hemagglutinin, a small outer capsid polypeptide. By using purified viral particles as a ligand in a standard plate separation technique, we have been able to enrich human peripheral blood and murine splenic T cells for reovirus receptor-positive cells (reovirus 3+) to levels of 88 to 92%. Analysis of reovirus 3+ T cells with monoclonal antibodies that identify inducer and suppressor/cytotoxic cells demonstrated that in the mouse, 68% of reovirus 3+ cells were Lyt-2+, and in the human, 60% were T8+. In reciprocal experiments, when subpopulations of murine and human T cells were prepared with the use of monoclonal anti-T cell reagents, 16% of Lyt-1+ and 81% of Lyt-2+ cells bound reovirus, whereas 30% of T4+ and 65% of T8+ cells bound reovirus. To determine whether reovirus type 3 identified a functional as well as a phenotypic category of cells, an antigen-specific cytotoxic T cell assay was employed. There was complete loss of cytotoxic activity in the reovirus 3+ cell population and slight enhancement of cytotoxic activity in the cell population from which reovirus 3+ cells were removed. This suggested that reovirus was binding to functionally active suppressor cells. Furthermore, adoptive transfer of antigen-specific T cells that were enriched for reovirus 3+ cells demonstrated suppression of cytoxic T cell activity. These results suggest that reovirus type 3 may identify a structure common to a subclass of murine and human T cells and that by using the virus as a natural biologic probe for cell surface receptors, one may be able to functionally segregate murine cytotoxic from suppressor T cells.