Expression of Lyt-1 by a subset of B lymphocytes

Using two-color flow cytometry and multiparameter data analysis, we have shown that the IgM bright, large subset of mouse splenic B lymphocytes express Lyt-1. This is not due to B cell uptake of immune complexes of Lyt-1 and antibody from T cells. The IgM bright cells of autoimmune NZB mice express more Lyt-1 than normal controls. This is because IgM containing plasmablasts, which are greatly increased in NZB spleens, are Lyt-1+. NZB spleen also contains more cells that are Lyt-1+ (but perhaps Lyt-1.2-), Thy-1.2 dull, and smaller in size than cells in normal mice. Thus, Lyt-1 is common to the T and B cell precursor or is induced independently during the ontogeny of T and at least one subset of B cells. We suggest that it be called Lyt-1.     

Rat T lymphocyte antigens comparable with mouse Lyt-1 and Lyt-2,3 antigenic systems: characterization by monoclonal antibodies

Rat T lymphocyte antigens were defined by using two distinct monoclonal antibodies (R1-3B3 and R1-10B5). R1-3B3 antibody, when tested for its reactivity with rat lymphoid cells by immunofluorescence, stained almost all of thymus and T cells but not the majority of B cells and bone marrow cells. The antigen defined by R1-3B3 existed more abundantly on medullary thymocytes and peripheral T cells than on cortical thymocytes. Immunochemical data showed that R1-3B3 antibody recognized a single glycoprotein with a m.w. of 67,000, showing marked electric charge heterogeneity with isoelectric points ranging from 5.4 to 7.3. R1-10B5 antibody, on the other hand, had more restricted reactivity with rat T cells and labeled approximately 85% of thymus cells and 30% of the peripheral T cells but neither B cells nor bone marrow cells. These T cells positive for R1-10B5 appeared to be negative for W3/25 antigen, which has been shown to be the marker for the rat T cell subset associated with helper function. R1-10B5 antibody detected a basic glycoprotein complex consisting of sulfhydryl-linked subunits with 30,000 and 34,000 m.w. Although the antigen defined by R1-3B3 was resistant to trypsin digestion, the one detected by R1-10B5 was much more sensitive to trypsin cleavage. All of these data obtained with either R1-3B3 or R1-10B5 are quite comparable to those reported for mouse Lyt-1 or Lyt-2,3 antigens, and thus suggest that the antigens defined by R1-3B3 and R1-10B5 antibodies represent rat homologues of Lyt-1 and Lyt-2,3 antigens in the murine system, respectively.     

Lyt-2- T cell-independent functions of Lyt-2+ cells stimulated with antigen or concanavalin A

Approximately 30% of cytolytic Lyt-2+ clones from primed mice are able to proliferate autonomously, i.e., independent of IL 2 derived from Lyt-2- cells after antigenic stimulation. H-2K- or -D-restricted induction of Lyt-2+ cells to autonomous proliferation requires Ia+ stimulator cells. A strict correlation was observed between the ability of Lyt-2+ clones to proliferative autonomously and to induce DH. Eventually, the growth of all Lyt-2+ cytolytic clones becomes dependent on exogenous IL 2, and their ability to induce DH is lost. Small Lyt-2+ cells can also be induced in primary cultures by antigen or concanavalin A to proliferate in the absence of exogenous IL 2. The frequency of autonomously proliferating small Lyt-2+ cells is the same as that of small Lyt-2+ cells proliferating in the presence of exogenous IL 2. IL 2 derived from Lyt-2- cells can augment proliferation of Lyt-2+ cells, but is not obligatory.     

Positively selected Lyt-2+ and Lyt-2- mouse T lymphocytes are comparable, after Con A stimulation, in release of IL 2 and of lymphokines acting on B cells, macrophages, and mast cells, but differ in interferon production

Purified mouse T lymphocytes were separated into Lyt-2+ and Lyt-2- populations by the procedure of panning, in which a monoclonal rat anti-Lyt-2 antibody and dishes coated with affinity-purified mouse anti-rat Ig antibodies were used. The populations obtained were 95 to 99% pure as determined by immunofluorescence. Graded doses of these T cells were cultured with optimal mitogenic doses of concanavalin A and the 0 to 24 and 24 to 48-hr culture supernatants were collected. The dose-curve assays of the supernatants of Lyt-2+ and Lyt-2- cells showed comparable activity in interleukin 2 (IL 2) and T cell-replacing factor (TRF), assayed on antigen-stimulated culture of T-depleted spleen cells. Limiting dilution assays of IL 2-secreting precursor cells stimulated by Con A showed a high frequency of precursors in both populations, slightly higher among Lyt-2- cells. The supernatants also contained comparable levels of IPA (inducer of plasminogen activator production by the macrophages), MAF (macrophage-activating factor, assayed by induction of their cytolytic function), and MCGF (mast cells growth factor, assayed on a mast cell line). IPA and MAF were not produced with the same kinetics and in the same T cell concentration conditions as IL 2 and TRF. In contrast, interferon was principally produced by the Lyt-2+ cells.     

Mouse alloantibodies capable of blocking cytotoxic T cell function. V. The majority of I region-specific CTL are Lyt-2+ but are relatively resistant to anti-Lyt-2 blocking

In an attempt to solve the conflict concerning the correlation between the Lyt-2 phenotype of T cells subsets and the type of the MHC antigens involved in the recognition by T cells, class 2 (I region) antigen-specific CTL were studied for their Lyt phenotypes and the sensitivity to the blocking effects of anti-Lyt-2,3 antibodies. To avoid contamination by CTL to class 1 antigens such as Qa antigens, A.TH anti-A. TL attackers and A.TH anti-A attackers were tested on LPS blasts of the A strain and the A.TL stain, respectively. By using these combinations, it was shown that the majority of I region-specific killers were Thy-1+, Lyt-1+23+. Specific target cell lysis by these cells were, however, found to be far less sensitive to the blocking effects of various monoclonal antibodies to the Lyt-2,3 antigens than conventional class 1-specific CTL. This conclusion was drawn by directly comparing the sensitivity of the I region-specific and K region-specific killing by identical numbers of the same attacker cells (A.TH anti-A). No significant difference was seen between the primary and the hyperimmune CTL. Lyt-2-, Thy-1+ killer cells with I region specificity could be induced when Lyt-2-depleted A.TH responder cells were stimulated in vitro. Such Lyt-2- killer cells were not induced to the H-2K alloantigen.     

Analysis of Ly-6.2-bearing murine lymphocyte subpopulations in relation to the T-lymphocyte markers,Thy-1, Lyt-1, and Lyt-2

Using immunofluorescence with a monoclonal anti-Ly-6.2 antibody and FACS analysis we have confirmed that the Ly-6.2 antigen is present on approximately 70% of mature T cells and B cells but on few immature lymphocytes. There is a wide range of antigen density among the Ly-6.2⁺ populations, with the mean density higher on T cells than B cells. Following Con A activation of splenocytes there was a sixfold increase in Ly-6.2 antigen density though approximately 20% of the activated lymphocytes were Ly-6.2^?. The increase in Ly-6.2 density was specific since similar density increases did not occur for the closely linked antigens ThB and H $\text{9}\text{25}$. By panning a predominantly T-cell population for Lyt-2-bearing cells, it was found that Lyt-2⁺ lymphocytes were either negative or dully staining for Ly-6.2. However, activated cells bearing the Lyt-2 antigen were all Ly-6.2 positive. Double-staining experiments showed that T cells which had high Ly-6.2 antigen densities also had high Thy-1 antigen densities. Corticosteroid-resistant thymocytes were highly enriched for Ly-6.2-bearing cells compared to untreated thymocytes and had staining profiles for Ly-6.2 which were similar to peripheral T cells, supporting the idea that steroid treatment selects for a phenotypically mature thymic population.     

Role of L3T4 antigen in the activation of various functions of Lyt-1+2- T cells against vaccinia virus

The present study defines assay systems for vaccinia virus-reactive Lyt-1+2- T cells mediating various functions and investigates the positivity of L3T4 antigen on these Lyt-1+2- T cells as well as the role of L3T4 antigen in the activation of these T cells with respect to their functions. C3H/He mice were immunized against vaccinia virus by inoculating viable virus intraperitoneally (i.p.). Anti-vaccinia virus reactivity in lymphoid cells from these immunized mice was assessed by proliferative response, helper T cell activities involved in cytotoxic T lymphocyte (CTL) and B cell (antibody) responses, delayed type-hypersensitivity (DTH) response, and production of lymphokines such as interleukin 2 (IL2) and macrophage-activating factor (MAF). The results demonstrate that all of the above anti-vaccinia virus responses were mediated by Lyt-1+2- T cells and that these Lyt-1+2- T cells expressed L3T4 antigens on their cell surfaces. Moreover, such anti-vaccinia Lyt-1+2- T cell responses were inhibited in the presence of anti-L3T4 antigen antibody. These results indicate that there is a reciprocal relationship between Lyt-2 and L3T4 markers, and that L3T4 antigen is closely related to the activation of various functions of anti-vaccinia virus Lyt-1+2- T cells.     

A human homolog of the mouse CD8 molecule,Lyt-3: genomic sequence and expression

The CD8 antigen is a marker for those cytotoxic T cells that recognize antigen in the context of class I major histocompatibility antigens (MHC) and has now been identified in many species. In rodents the CD8 antigen is a heterodimer of two distinct chains, Lyt-2 and Lyt-3 in the mouse and OX-8 M _r 32 000 and 37 000 chains in the rat. Human CD8 has consistently been described as a homodimer/homomultimer on mature T cells made up of one chain homologous to the Lyt-2 and OX-8 M _r 32 000 chains. This paper identifies a human equivalent of the second rodent CD8 chain (Lyt-3 and OX-8 M _r 37 000 chains) at the genomic level and shows that this gene is transcribed in human thymocytes and in some acute leukemic T-cell lines. The existence of a human Lyt-3 homolog raises the possibility that human CD8, like mouse CD8, may exist as a heterodimer.     

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.     

The cellular and molecular basis of the Lyt-1+2- T cell-mediated tumor-eradicating mechanism in vivo

This article reviews recent findings that bear on the mechanism(s) of tumor-specific Lyt-1⁺2^? T cell-mediated tumor eradication in vivo A tumor-immune Lyt-1⁺2^? T cell subset has been identified which is distinct from T cells mediating in vitro cytotoxicity (Lyt-1⁺2⁺/1^?2⁺). The Lyt-1⁺2^? cells have a crucial role in rejecting tumor cells when adoptively transferred into T cell-deprived B cell mice. This indicates that Lyt-1⁺2^? T cells do not necessarily require recruitment of the host's cytotoxic T cell precursors for implementation of in vivo immunity. Instead, this T cell subset exerts its anti-tumor effect in collaboration with macrophages as shown with an in vivo tumor cell culture system utilizing a diffusion chamber. A pathway of Lyt-1⁺2^? T cell-macrophage interaction leading to tumor cell killing is discussed in terms of its probable relevance to the eradication of tumor cell masses consisting of tumor cells expressing quantitatively and/or qualitatively different tumor antigens.     

Lyt-1 cells mediate acute murine experimental allergic encephalomyelitis

Adult SJL/J mice were depleted of T lymphocytes and were reconstituted with Lyt-1, Lyt-2, or Lyt-1 + Lyt-2 cells. After immunization with MSCH, severe acute EAE developed in the Lyt-1- and the Lyt-1 + Lyt-2-reconstituted mice. Only 25% of the Lyt-2-reconstituted mice developed signs of EAE, and in those EAE onset was delayed. Thus, Lyt-1 cells mediate EAE in the mouse.     

Frequency analysis of class I MHC-reactive Lyt-2+ and class II MHC-reactive L3T4+ IL 2-secreting T lymphocytes

The reactivity of Lyt-2+ or L3T4+ T cells stimulated with either mutant class I or class II MHC alloantigens was studied. Whereas stimulation with class I MHC antigens induced only Lyt-2+ T cells to proliferate and to secrete IL 2, stimulation with class II MHC alloantigens induced L3T4+ but not Lyt-2+ T cells. When the frequencies of precursors of IL 2-secreting T lymphocytes (IL 2TL-p) were determined by limiting dilution analyses, class I MHC-reactive Lyt-2+ T cells displayed frequencies (f = 1/200) as high in magnitude as those within class II MHC-reactive L3T4+ (f = 1/100). Clonally developing IL 2TL of either T cell subset were antigen-specific, as shown in split-culture experiments. Whereas L3T4+ helper TL could be induced to specific IL 2 secretion over a long time period (days 3 to 9), Lyt-2+ TL showed a marked time optimal on day 4; thereafter, the number of TL colonies inducible to secrete IL 2 decreased steadily. IL 2 production and IL 2TL-p frequencies of unseparated T responder cells were not the numerical superposition of the two individual T cell subsets (Lyt-2+ + L3T4+); the latter finding is likely to reflect regulatory influences of Lyt-2+ T cells on IL 2-secreting L3T4+ T cells.     

Redistribution of Lyt-bearing T cells in acute murine experimental allergic encephalomyelitis: selective migration of Lyt-1 cells to the central nervous system is associated with a transient depletion of Lyt-1 cells in peripheral blood

Experimental allergic encephalomyelitis (EAE) was induced in SJL/J mice by using two injections of spinal cord homogenate in incomplete Freund's adjuvant supplemented with mycobacteria. Analysis of circulating Lyt-bearing subsets by indirect immunofluorescence during the course of acute EAE revealed the following: 1) during the pre-clinical phase of EAE (1 to 2 days before the onset of paralysis), there was a decrease in the percentage of Lyt-1- but not of Lyt-2-bearing cells in peripheral blood, and of both Lyt-1- and Lyt-2-bearing cells in spleen; 2) with the onset of clinically evident EAE, there was a decrease in both Lyt-1 and Lyt-2 cells in peripheral blood and an increase in the percentage of Lyt-1-bearing cells in pooled inguinal and axillary lymph node; and 3) after these early changes, there was a rapid reconstitution of the percentages of total Lyt-bearing cells and of both Lyt-1- and Lyt-2-bearing cells in peripheral blood. Immunohistochemical analysis of the central nervous system infiltrate revealed that the earliest lesions consisted predominantly of Lyt-1 T lymphocytes, with few Lyt-2 cells present. These results demonstrate that the influx of cells of the Lyt-1 inducer subset to the central nervous system in acute EAE is accompanied by a transient decrease in Lyt-1 cells in peripheral blood.     

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.     

Heterologous antisera to Lyt-1+, 2- -derived antigen-binding factor detect a subfactor of an antigen-specific suppressor factor and cell surface proteins on Lyt-1+, 2- and Lyt-1-, 2+ T cells

Rabbits were immunized with TNP-specific Lyt-1+, 2- T cell-derived, antigen-binding proteins (PCI-F) released by T cells sensitized by skin painting with picrylchloride. The resulting antiserum (anti-PCI-F) bound to PCI-F and TNP-specific factors that suppressed delayed hypersensitivity (TSF) known to be comprised of PCI-F and Lyt-2+ -derived polypeptides released by cells sensitized by injection of trinitrobenzenesulfonic acid (TNBSF). Anti-PCI-F bound to T lymphocytes and 68,000 to 72,000 m.w. T cell surface proteins but not B cells on their surface proteins. Anti-PCI-F bound to both Lyt-1+ and Lyt-2+ T cells and surface proteins. A comparison of anti-PCI-F with anti-TSF indicates that anti-TSF contains specificity for Ly-2+ T cell-derived components of TSF and T cells not present in anti-PCI-F. The possibility of multiple isotypes of T cell receptors and antigen-binding molecules is discussed.     

Differential helper and effector responses of Lyt-2+ T cells to H-2Kb mutant (Kbm) determinants and the appearance of thymic influence on anti-Kbm CTL responsiveness

The goal of this study was to assess and compare the allorecognition requirements for eliciting Lyt-2+ helper and effector functions from primary T cell populations. By using interleukin 2 (IL 2) secretion as a measure of T helper (Th) function, and cytolytic T lymphocyte (CTL) generation as a measure of effector function, this study compared the responses of Lyt-2+ T cells from wild-type B6 mice against a series of H-2Kb mutant determinants. Although all Kbm determinants stimulated B6 Lyt-2+ T cells to become cytolytic effector cells, the various Kbm determinants differed dramatically in their ability to stimulate Lyt-2+ T cells to function as IL 2-secreting helper cells. For example, in contrast to Kbm1 determinants that stimulated both helper and effector functions, Kbm6 determinants only stimulated B6 Lyt-2+ T cells to become cytolytic and failed to stimulate them to secrete IL 2. The distinct functional responses of Lyt-2+ T cells to Kbm6 determinants was documented by precursor frequency determinations, and was not due to an inability of the Kbm6 molecule to stimulate Lyt-2+ Th cells to secrete IL 2. Rather, it was the specific recognition and response of Lyt-2+ T cells to novel mutant epitopes on the Kbm6 molecule that was defective, such that anti-Kbm6 Lyt-2+ T cells only functioned as CTL effectors and did not function as IL 2-secreting Th cells. The failure of Lyt-2+ anti-Kbm6 T cells to function as IL 2-secreting Th cells was a characteristic of all Lyt-2+ T cell populations examined in which the response to novel mutant epitopes could be distinguished from the response to other epitopes expressed on the Kbm6 molecule. The absence of significant numbers of anti-Kbm6 Th cells in Lyt-2+ T cell populations was examined for its functional consequences on anti-Kbm6 CTL responsiveness. It was found that primary anti-Kbm6 CTL responses could be readily generated in vitro, but unlike responses to most class I alloantigens that can be mediated by Lyt-2+ Th cells, anti-Kbm6 CTL responses were strictly dependent upon self-Ia-restricted L3T4+ Th cells. Because the restriction specificity of L3T4+ Th cells is determined by the thymus, in which their precursors had differentiated, anti-Kbm6 CTL responsiveness, unlike responsiveness to most class I alloantigens, was significantly influenced by the Ia phenotype of the thymus in which the responder cells had differentiated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intrathymic differentiation and tolerance induction of lymphokine-secreting Lyt-2+ T helper cells

The present study has assessed thymic influence on the differentiation and recognition specificity of developing Lyt-2+ lymphokine-secreting T cells, and compared it with those of developing Lyt-2+ CTL. It was demonstrated that development of Lyt-2+ lymphokine-secreting Th cells requires an intrathymic differentiation step, and that peripheral Lyt-2+ lymphokine-secreting Th cells, unlike peripheral Lyt-2+ CTL, are profoundly tolerant to intrathymically expressed alloantigens. These data are interpreted as demonstrating that functionally distinct Lyt-2+ T cell populations are heterogeneous in their requirements for differentiation and/or activation.     

Intramucosal lymphocytes of the gut: Lyt-2 and thy-1 phenotype of the granulated cells and evidence for the presence of both T cells and mast cell precursors

The gut mucosa contains lymphocyte-like cells, a proportion of which contain a small number of granules that resemble those of mast cells in that they contain histamine and stain metachromatically. It has been suggested that these granulated lymphocytes represent transitional forms in the differentiation of T cells into mast cells. We used monoclonal antibodies and the fluorescence-activated cell sorter to analyze the expression of Thy-1 and Lyt-2 antigens on gut intramucosal lymphocytes with particular emphasis on the granulated cells. A minority of the granulated cells (10 to 20%) expressed Thy-1 antigen at high levels equivalent to those on cortical thymocytes. A much higher proportion of the granulated cells (about 90%) expressed readily detectable levels of Lyt-2 antigen and the most prevalent phenotype of the granulated lymphocytes (60 to 70%) was Lyt-2+, Thy-1-. Two operationally specific preparations of growth factors, one maintaining the proliferation of T cells and containing T cell growth factor, and the other containing a factor stimulating the growth of persisting (P) cells that are probably mast cell progenitors, were tested on lymphocytes from the gut mucosa. By using the appropriate preparation of growth factors, both T and P cells could be grown readily from the preparation of gut intramucosal lymphocytes. Estimates of the frequency of P cell precursors among these cells indicated a minimum of one in 300 could give rise to cells resembling mast cells. Fractions of Lyt-2+ cells that were enriched in granulated cells had few detectable P cell precursors, an observation lessening the likelihood that the granulated cells were progenitors of the P cells. The precise relationship of the granulated lymphocytes (mainly Lyt-2+, Thy-1-) to T cells remains to be established.