Expression of a monoclonal antibody-defined, B-lineage transformation antigen specifically identifies Abelson-diseased animals. Genetically determined resistance to Abelson murine leukemia virus acts before induction of gp160(6C3).

Mice genetically susceptible or genetically resistant to the leukemogenic effects of A-MuLV(Mo) were tested for their expression of the B-lineage neoplastic transformation-associated antigen, 6C3Ag. Genetically resistant inbred strains and recombinant inbred lines developed neither cells expressing high levels of 6C3Ag (6C3Aghi) in their hematolymphoid tissues nor Abelson leukemias. Genetically susceptible inbred strains and recombinant inbred lines developed high percentages of 6C3Aghi hematolymphoid cells concomitant with development of Abelson leukemias and lymphomas. Thus the genetically-determined resistance to A-MuLV(Mo) leukemogenesis appears to act at some step(s) after virus infection but before the stage of malignant progression, which is marked by 6C3Ag expression.

Transformed lymphocytes from Abelson-diseased mice express levels of a B lineage transformation-associated antigen elevated from that found on normal lymphocytes.

Animals injected with Abelson murine leukemia virus (A-MuLV) rapidly develop fatal bone marrow-derived lymphosarcomas. In all such diseased animals tested, a subpopulation of bone marrow cells expressed a monoclonal antibody-defined, B lineage transformation-associated antigen (6C3 Ag) at levels increased from that detected on normal lymphocytes. Cells bearing a high level of this antigen were found to be transformed as measured by clonal growth in agar, and they expressed surface antigen markers characteristic of early pre-B cells. High-level antigen-expressing cells were found in the bone marrow, lymph nodes, and spleen, but never in the thymus of diseased animals. This distribution agrees with the published pathology of Abelson disease.

The specificity of cellular immune responses II. The structure of antigenic determinants leading to T-lymphocyte stimulation.

T cells from guinea pigs immunized with the hapten 2,4-dinitrophenyl (DNP)-coupled directly to mycobacteria are of interest since they recognize and respond to DNP conjugated to many but not all carriers. The experiments reported here further analyze the structure of the complex, chemically defined antigenic determinants recognized by such T cells. These antigenic determinants can have DNP coupled either to the xi-amino group of lysyl residues or to the hydroxyl group of tyrosyl residues. Furthermore, essential contributions to the determinant recognized by such T cells are made by amino acid residues to which the hapten is not attached. Such residues are thought to be close to the hapten group itself, since introducing a small spacer between hapten and carrier prevents recognition. The hapten itself is also recognized and discriminated from other haptens with great precision by these T lymphocytes. The strain of guinea pig immunized affects the precise specificity characteristics of the responding T cells, in a way that may reflect the activity of histocompatibility-linked immune response genes. Finally, the characteristics of the immunogen have been studied. It is thought that the lipid content of the mycobacteria may be critical in inducing the hapten-reactive T cells, and this is supported by finding similar responses in T cells from guinea pigs immunized with DNP protein to which lipid has been covalently attached. Thus, the T-cell population being studied, while recognizing haptens with great precision, appears to require a larger determinant for activation than do hapten-specific B lymphocytes.

Expression of the 6C3 antigen on murine hematopoietic neoplasms. Association with expression of abl, ras, fes, src, erbB, and Cas NS-1 oncogenes but not with myc.

The monoclonal antibody 6C3 was used to test a wide variety of murine hematopoietic neoplasms for cell surface expression of a 160 kD glycoprotein (gp160(6C3)) previously shown to be expressed by neoplastic pre-B and some B lymphocytes transformed by Abelson murine leukemia virus (A-MuLV). This antigen was expressed on many pre-B and B cell lymphomas, but not on A-MuLV-transformed fibroblasts, T cell lymphomas, or myelomonocytic leukemias, gp160(6C3) was expressed by most early B-lineage spontaneous tumors, and early B tumors induced by replication-defective MuLV-containing oncogenes the products of which are associated with the cytoplasmic aspect of the plasma membrane, i.e., fes, abl, H-ras, bas, src, erbB, and Cas NS-1. By comparison, none of the early B lineage lymphomas induced by the "nuclear" oncogene avian v-myc MuLV, or arising in mice transgenic for a murine c-myc gene, or later B cell lineage stages bearing translocations of the c-myc locus expressed this antigen.

Expression of T lymphocyte adhesion molecules: regulation during antigen-induced T cell activation and differentiation.

The pattern of lymphocyte traffic and migration in vivo is a composite of constitutive recirculation and transient changes induced by interaction with antigen. Naive T lymphocytes in their basal, unstimulated state continuously recirculate throughout the entire host, poised to react to specific antigens that they are programmed to recognize. After interaction with antigen, T cell traffic changes, first with the trapping of reactive cells in antigen-containing lymphoid tissue. Subsequently, the effector cells responding to antigen, accompanied by nonspecific T cells and monocytes, traffic in large numbers to sites of antigen localization, resulting in the localized inflammatory response. Then, as the immune response wanes, memory T cells develop, many of which exhibit still different routes of recirculation. The traffic and tissue localization of leukocytes is regulated by a series of cell surface adhesion molecules that recognize specific ligands on endothelial cells and in the extracellular matrix. Modulation of the expression of these adhesion molecules results in the changes in T cell traffic that are characteristic of each stage of T cell differentiation. Thus, during T cell activation and differentiation, the down-regulation of adhesion receptors specific for lymphoid tissue endothelium and up-regulation of integrins facilitate the targeting of effector cells to sites of inflammation. Subsequent changes in adhesion receptors regulate the traffic of the antigen-specific memory cells. T cell adhesion molecule expression is therefore regulated as a function of the stage of activation and differentiation and, in addition, is influenced by cytokines and the local lymphoid microenvironment.

A novel and inexpensive source of allophycocyanin for multicolor flow cytometry.

Allophycocyanin (APC) belongs to a family of phycobiliproteins that are well suited as fluorescent reagents for flow cytometric analysis, since they have a broad excitation spectrum, a large Stoke's shift and they fluoresce with a high quantum yield. The widespread use of APC has been limited by the availability of raw material and high cost of the purified phycobiliprotein. We have assessed the suitability of dry, powdered Spirulina platensis, available at health food stores, as an inexpensive source of APC. APC was extracted from Spirulina platensis by overnight treatment with lysozyme, followed by ammonium sulfate precipitation. APC was then separated from phycocyanin (the only other major phycobiliprotein in Spirulina) by elution of bound material from an hydroxylapatite column using an increasing continuous phosphate gradient. APC isolated in this manner retained its normal trimeric structure. The absorbance and fluorescence excitation and emission spectra of the purified phycobiliproteins were identical to those previously shown for C-PC and APC. APC can be stored concentrated at 4 degrees C, frozen at -70 degrees C, or as a saturated ammonium sulfate precipitate, with no subunit dissociation or change in spectral properties. Moreover, APC has been conjugated to monoclonal and polyclonal antibodies for use in multicolor FACS analysis, with the conjugated antibody activity remaining stable for at least 2 years. Thus, this procedure is a simple, cost-effective method for preparing reagents for multicolor immunofluorescence and flow cytometry.

Splenic injury caused by therapeutic irradiation.

Splenic irradiation in the course of therapy for lymphoma can result in functional deficit, sometimes as severe as that caused by splenectomy, placing the patient at risk for fatal infection. We examined 33 spleens obtained at necropsy from patients irradiated for lymphomas (mainly Hodgkin's disease) and compared them with 18 nonirradiated spleens from similar patients. One to 8 years after a mean radiation dose of 3899 rads, fractionated over 5-6 weeks, most irradiated spleens were small (average weight 75 g) and had thick, wrinkled capsules, often with focal hemorrhage. There was collapse of the parenchyma, with close apposition of trabeculae and mild to severe diffuse fibrosis of the red pulp. Lymphocyte depletion was obvious in more than 50% of the specimens. The most consistent alteration was myointimal proliferation of arteries. Significant intimal thickening was seen only in the irradiated specimens. Similar myointimal changes were found in the veins of three cases. While none of these changes is specific, their combination appears to be characteristic of delayed radiation injury to the spleen.

Pharmacological characterization of histamine H2 receptors on clonal cytolytic T lymphocytes. Evidence for histamine-induced desensitization.

Cultured cytolytic T lymphocytes of clonal origin were screened for histamine-stimulated cyclic AMP production. Histamine caused a 2- to 8-fold elevation of cyclic AMP levels in five independent clones. The EC50 for histamine of 1.7 X 10(-5) M and the rank order of potencies of H1 and H2 agonists [impromidine greater than histamine greater than dimaprit greater than 4-methylhistamine greater than 2-methylhistamine greater than 2-(2-aminoethyl)-thiazole] were characteristic of the conventional histamine H2 receptor. H1 and H2 antagonists inhibited histamine-stimulated cyclic AMP elevation with inhibition constants typical for those found on other H2 receptor systems. Prior incubation of cells with histamine resulted in a marked loss in responsiveness to subsequent histamine challenge. We demonstrate that this desensitization is dose and time dependent and results in a change in the efficacy and not the potency of histamine. Although cyclic AMP increases could also be elicited with isoproterenol, prostaglandin E1 or forskolin, desensitization of histamine had no effect on the ability of these agents to stimulate cyclic AMP production. In contrast to the rapid rate of histamine-induced desensitization, recovery of histamine responsiveness could not be detected for several hours.

Expression of the Leu-8 antigen by B-cell lymphomas.

The Leu-8 antigen is found on the surface of many hematologic cells, including many T- and B-lymphocytes. With the use of a frozen-section immunoperoxidase technic, 152 B-cell non-Hodgkin's lymphomas were examined for Leu-8 expression. Of these lymphomas, 53% expressed Leu-8. Subclassification of the lymphomas with the use of the International Working Formulation showed that most small lymphocytic, intermediate lymphocytic, and diffuse large cell lymphomas and about half of diffuse small cleaved, diffuse mixed, and follicular lymphomas expressed Leu-8. In contrast, all 17 cases of small noncleaved cell (Burkitt's) lymphoma and 9 of 10 cases of multiple myeloma/plasmacytoma were Leu-8 negative. These results indicate that Leu-8 is expressed on a wide variety of B-cell lymphomas and that differences in Leu-8 expression may be useful in the diagnostic separation of small lymphocytic lymphoma with plasmacytoid features from multiple myeloma/plasmacytoma, and diffuse large cell lymphoma from Burkitt's lymphoma.

Regulation of adhesion molecule expression by CD8 T cells in vivo. I. Differential regulation of gp90MEL-14 (LECAM-1), Pgp-1, LFA-1, and VLA-4 alpha during the differentiation of cytotoxic T lymphocytes induced by allografts.

A variety of adhesion molecules regulate the traffic and tissue localization of lymphocytes in vivo by mediating their binding to vascular endothelial cells. The homing receptor gp90MEL-14 (gp90), also known as LECAM-1 or L-selectin, mediates the adhesion of lymphocytes to specialized high endothelial venules in lymph nodes (LN) and is the primary molecule regulating lymphocyte recirculation and homing to LN, whereas other adhesion molecules have a major role in the localization of lymphocytes in inflammatory sites. We used four-color flow cytometric analysis to examine the regulation of adhesion receptor expression on LN CD8 T cells responding to skin allografts in vivo. In normal mice, greater than 95% of LN CD8 T cells are gp90+, being either gp90+Pgp1- (Population (Pop.) 1 or gp90+Pgp-1+ (Pop.2). Allografting induces the down-regulation of gp90 and up-regulation of Pgp-1 on a subset of cells, resulting in the appearance of CD8+gp90-Pgp-1hi (Pop. 3) cells. Pop. 3 cells also express high levels of LFA-1, ICAM-1, and ICAM-2, and a subset of them are VLA-4 alpha-positive. Purified Pop. 3 cells have potent cytolytic activity directed against donor alloantigen, whereas no such activity is present in Pop. 1 or 2 cells. Correlating with this is the high granzyme activity in Pop. 3 cells. In addition, Pop. 3 lymphocytes, but not Pop. 1 or 2, secrete a large amount of IFN-gamma in response to Ag. Finally, the CD8 T cells that infiltrate sponge matrix allografts are markedly enriched for the Pop. 3 subset. These results show that, during the immune response to alloantigen in vivo, a small subset of CD8 T cells down-regulates the LN homing receptor while increasing the expression of other adhesion molecules, as they differentiate into highly active cytolytic T lymphocytes. Thus, the differential regulation of LN homing receptors and receptors for peripheral vascular endothelium provides a mechanism that would redirect the traffic of activated effector cells away from lymphoid tissue and to sites of Ag deposition, where they would participate in the inflammatory response.

Expression of adhesion structures during B cell development in man.

We have used three-color flow cytometry to study the expression of adhesion structures during B cell development in man. The results indicate that the cell-surface molecule(s) recognized by 515, a mAb which defines a broadly expressed family of cell-surface glycoproteins that includes lymphocyte homing receptors, exhibit a clear bimodal distribution (515lo and 515hi); 515hi cells were found exclusively on more mature B cells which already expressed high levels of CD20. Earlier, less mature B cells, identified by their expression of CD10, were uniformly 515lo. In contrast, the CD11a/LFA-1 Ag was acquired gradually over the course of B cell development. B cells which expressed high levels of CD20 expressed three to six times as much CD11a/LFA-1 as cells which expressed CD10. Interestingly, expression of the 515hi phenotype was tightly correlated with that of Leu-8, a marker previously shown to define maturational and functions subsets of B cells. These data document the coordinated regulation of multiple cell surface structures during B cell ontogeny, and demonstrate that adhesion structures necessary for proper B cell function are precisely up-regulated during B cell differentiation in man.

Induction of cell-mediated immunity to chemically modified antigens in guinea pigs. I. Characterization of the immune response to lipid-conjugated protein antigens.

Bovine serum albumin (BSA) conjugated with a lipid, dodecanoic acid, is capable of inducing strong delayed-type hypersensitivity (DTH) in guinea pigs. This paper reports experiments on the nature and specificity of this hypersensitivity. The response to lipid-conjugated BSA (L-BSA) was found to be classical DTH, as evidenced by its ability to be transferred passively by immune cells, but not by serum. In addition, special histologic examination of skin test sites demonstrated the characteristics of DTH rather than cutaneous basophil hypersensitivity. Similar results were obtained when lipid-conjugated purified protein derivative of tubercle bacilli (L-PPD) was used. The increased immunogenicity of L-BSA was not caused by the presence of protein aggregates, but seemed to be related to the hydrophobic nature of the conjugated side chains. A series of cross-reacting serum albumins was used for a study of the specificity of the antibody and DTH responses to BSA. It was found that the degree of enhancement of immunogenicity for DTH caused by lipid conjugation varied for different antigenic determinants on BSA.

Traffic of L-selectin-negative T cells to sites of inflammation.

T cells responding to antigen in vivo down-regulate L-selectin, the lymph node homing receptor, as they develop into activated effector cells. The concomitant up-regulation of the proinflammatory adhesion molecules LFA-1, CD44, and VLA-4 suggests that, after their release into the circulation, they traffic to sites of antigen deposition and inflammation. Previous evidence, however, has suggested a role for L-selectin in the recruitment of both neutrophils and lymphocytes into sites of inflammation, which would indicate that these L-selectin(-) effector cells could not be the precursors of inflammatory cells. We therefore directly tested whether L-selectin(-) T cells activated in vivo are capable of homing to model inflammatory sites. L-selectin(-) cells isolated from mice primed with alloantigen or with a contact sensitizer migrated to inflammation markedly better than L-selectin(+) cells from the same animals. Furthermore, the analogous population of CD44(hi)integrin(hi) cells from intravenously primed L-selectin knockout mice traffic efficiently to inflammatory sites and reject allogeneic skin grafts with normal kinetics. These data demonstrate that the previously described L-selectin(-) population of T cells that differentiate into effectors in spleen and lymph nodes subsequently traffic to inflammatory sites, due in part to their increased expression of other proinflammatory adhesion molecules.

Rapid modulation of homing receptors (gp90MEL-14) induced by activators of protein kinase C. Receptor shedding due to accelerated proteolytic cleavage at the cell surface.

Lymphocyte entry into lymph nodes and Peyer's patches is initiated by the adhesion of the lymphocytes to specialized postcapillary high endothelial venules (HEV). The binding of lymphocytes to lymph node HEV is mediated by the cell surface receptor gp90MEL-14 (gp90). Previous work has shown that gp90 is down-regulated over a period of days after mitogenic or mixed lymphocyte reaction stimulation of T lymphocytes. In our study, it is shown that stimulation of lymphocytes with activators of protein kinase C (PKC), such as PMA or 1-oleoyl 2-acetyl-glycerol, results in the nearly complete loss of surface expression of gp90 within 1 h. Pretreatment of the cells with H-7 or staurosporine, PKC inhibitors, but not HA1004, a general protein kinase inhibitor, prevents the loss of gp90MEL-14. Within 15 min of stimulation of PKC, a novel form of gp90 can be immunoprecipitated from the supernatant of stimulated cells. Upon deglycosylation, this soluble gp90 polypeptide is shown to be 12 kDa smaller than the cell surface protein. Peptide mapping showed identical patterns for surface and soluble receptor, confirming that the soluble Ag is related to the cell membrane protein. Together, these experiments suggest that activation of PKC results in the proteolytic cleavage of gp90MEL-14, resulting in receptor shedding and the inability of the lymphocytes to adhere to HEV endothelium. Furthermore, because supernatant from unstimulated, normal lymphocytes also contains a small amount of the low Mr form of gp90, cell surface proteolysis may be part of the normal turnover of this receptor glycoprotein. These experiments suggest that PKC may play a role in the regulation of lymphocyte traffic to lymphoid tissues.

Mechanisms of L-selectin regulation by activated T cells.

The activation of T cells through the TCR results in the differential regulation of a set of adhesion molecules that dramatically alters lymphocyte migration and tissue localization properties in vivo. L-selectin, the lymph node homing receptor, is central to the control of lymphocyte recirculation. We examined the regulation of L-selectin as a function of time after activation in vitro. Within an hour of stimulation, T cells down regulate L-selectin, with a 90% loss by 4 h, due to accelerated proteolytic cleavage. Over the course of the following 48 h, surface receptor expression increases markedly. This is due to an increase in L-selectin mRNA, which, in turn, results from increased message stability. During the next several days after activation, L-selectin levels decrease, resulting in L-selectin-negative T cells by 5 to 7 days after stimulation. This decrease occurs faster in CD8 than in CD4 T cells. During this phase of regulation, L-selectin message remains stable even as the level of specific mRNA continuously decreases. This indicates that the L-selectin-negative phenotype of T cells late after activation is due to the down-regulation of gene transcription. These results demonstrate that after stimulation through the TCR, the expression of L-selectin changes in a triphasic pattern, with an initial marked decrease, followed by a transient phase of superinduction and then a loss of expression. These changes are regulated through the complex interactions between several mechanisms at the transcriptional, post-transcriptional, and protein turnover levels.

Regulation of adhesion molecule expression by CD8 T cells in vivo. II. Expression of L-selectin (CD62L) by memory cytolytic T cells responding to minor histocompatibility antigens.

Activation of murine T cells by Ag or mitogens results in changes in the expression of several cell-surface adhesion molecules that alter the way in which these cells migrate and localize in tissues in vivo. As naive CD8 precursor cells in lymph nodes (LN) differentiate into effector CTL in response to a skin allograft, they up-regulate their expression of Pgp-1 (CD44), VLA-4, and LFA-1 (CD11a/18), while down-regulating L-selectin (CD62L). All cytolytic activity is therefore present in this minor population of L-selectin- Pgp-1high LN T cells. We now report that, late after rejection of minor histocompatibility Ag-disparate skin grafts, the majority of memory CD8 T cells express both L-selectin and Pgp-1 and thus would be expected to migrate via the classical route of recirculation through LN. Furthermore, restimulation of these memory cells by Ag causes them to down-regulate L-selectin, so that memory-effector cells have the same L-selectin- Pgp-1high phenotype as do primary effector cells. These results are in contrast to recent reports that murine and ovine CD4 memory cells do not express L-selectin or recirculate through LN high endothelial venules. In addition, although virgin and naive CD4 cells may be divided based upon their expression of CD45RA or CD45RB, memory CD8 cells cannot be differentiated by their expression of CD45 isoforms.