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Biomedical subjects

R C Budd

Publications and source records attributed to R C Budd.

53 records · Page 3Linked to original sources
PubMed1990

Shared molecular markers of genetic predisposition to seropositive rheumatoid arthritis.

Rheumatoid arthritis is associated with the human class II major histocompatibility complex antigens known as HLA-DR4. HLA-DR4 can be subdivided by cellular typing into five subtypes: Dw4, Dw10, Dw13, Dw14, and Dw15. By traditional serologic methods, 60-80% of rheumatoid arthritis patients type HLA-DR4 compared to approximately 20% of the general population. It has been demonstrated, using a panel of four alloreactive T-cell clones, each of which recognized HLA-DR4, Dw14 homozygous typing cells, that cells from all of a group of 23 rheumatoid arthritis patients could be recognized by one or more of these clones regardless of the patients' serologic typing. As the expressed polymorphism of the DR molecule is accounted for by the beta 1 gene, this gene was amplified, using the polymerase chain reaction, and sequenced. Seven patients whose cells were recognized by one of the DR4, DW14-specific T-cell clones, T431, were analyzed. All of these patients shared a common sequence in the third hypervariable region of the DR beta 1 chain gene. The sequence identified is the one normally associated with DR4, Dw14 and DR1. Patients and DR4-positive controls whose cells did not stimulate this clone did not share this sequence. These results suggest that this hypervariable region might be an important contribution to a restriction site for the putative causative agent(s) in rheumatoid arthritis.

Amino Acid Sequence↗
PubMed1989

A subset of memory CD4+ helper T lymphocytes identified by expression of Pgp-1.

The Pgp-1 glycoprotein (Ly-24 antigen) is acquired by mature murine T lymphocytes at the time of primary antigen stimulation Pgp-1 was previously shown to be a useful cell surface marker for distinguishing antigen-specific memory CD8+ T lymphocytes after immunization. Here we demonstrate that this observation extends to CD4+ T lymphocytes. Antigen-specific CD4+ T cells in mice immunized with sperm whale myoglobin or keyhole limpet hemocyanin were contained nearly exclusively in the minor Pgp-1+ subset.

Animals↗
PubMed1988

A CD3- subset of CD4-8+ thymocytes: a rapidly cycling intermediate in the generation of CD4+8+ cells.

T lymphocytes with the surface phenotype CD4+8- and CD4-8+ are considered to be representative of functionally mature cells. We show here that adult murine thymus contains a subpopulation of CD4-8+ cells that differ from CD4-8+ cells found in the periphery in that they do not express the T cell receptor-associated CD3 molecular complex. Such CD3-4-8+ thymocytes are cortisone sensitive and rapidly cycling in situ. Furthermore, in contrast to mature T cells, most CD3-4-8+ thymocytes express low levels of CD5 and high levels of the B2A2 antigen. CD3-4-8+ thymocytes fail to respond to a variety of mitogenic stimuli in vitro but do give rise upon short-term culture to CD4+8+ cells. It is suggested that CD3-4-8+ thymocytes represent a transitional stage of thymus differentiation between the CD4-8- and CD4+8+ compartments.

Animals↗
PubMed1987

T cell lineages in the thymus of lpr/lpr mice. Evidence for parallel pathways of normal and abnormal T cell development.

Autoimmune mice homozygous for the lpr/lpr (lpr) gene develop a profound lymphadenopathy resulting from the accumulation of immature Thy-1+ Lyt-2- L3T4- cells in peripheral lymphoid tissues. The source of these cells is not known although the presence of a thymus is necessary to manifest both the lymph node enlargement and the autoimmunity. For this reason and the fact that the abnormal lpr cell phenotypically resembles immature thymocytes, we studied the thymus in lpr mice. Adult lpr thymuses were found to contain an immature population phenotypically identical to the peripherally accumulating cells, including the expression of B220 and Pgp-1 antigens as well as the presence of surface T cell receptor molecules as defined by the antibody KJ16-133. Evidence is presented that some of these lpr precursor T cells are capable of intrathymic differentiation, whereas the vast majority are exported unchanged to the lymph nodes where a portion differentiate further into mature T cells. This lpr-specific lineage could be distinguished from a normal component of the lpr thymus by surface phenotype and immunohistology. The results suggest that the massive accumulation of cells in lpr lymph nodes is not so much the result of abnormal proliferation of T cells as abnormal intrathymic differentiation. In addition, a minor subpopulation of normal Lyt-2- L3T4- thymocytes was identified that resembles the phenotype of the lpr cell and similarly expresses surface T cell receptor molecules. The presence of two parallel lineages in the lpr thymus thus also provides insight into normal T cell development.

Animals↗
PubMed1987

Developmentally regulated expression of T cell receptor beta chain variable domains in immature thymocytes.

A minor subset of immature (CD4-,8-) thymocytes that lack expression of the B2A2 antigen was found to express low levels of surface TCR protein as detected by mAbs F23.1 and KJ16 (reacting with protein products of the V beta 8 gene family). Interestingly, F23.1/KJ16 determinants were expressed on a two- to three-fold higher proportion of B2A2- thymocytes than mature lymph node T cells in four independent haplotypes. When expanded in short-term culture with PMA and calcium ionophore, B2A2- thymocytes retained their overexpression of F23.1/KJ16 determinants and showed a fivefold elevated level (relative to lymph node) of V beta 8-specific mRNA. Taken together, these findings suggest that expression of TCR V beta genes, like Ig genes, is developmentally regulated.

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

Abnormal expression of T cell receptor genes in Lyt-2- L3T4- lymphocytes of lpr mice: comparison with normal immature thymocytes.

The autoimmunity and lymphoproliferation characteristic of lpr mice are age and thymus dependent. The accumulating Lyt-2- L3T4- T cells express only minimal cell-surface antigen receptor. After stimulation with phorbol myristic acetate (PMA) and interleukin 2, essentially normal levels of surface antigen receptor were expressed by the lpr Lyt-2- L3T4- subpopulation but remained undetectable in the corresponding normal immature thymocyte population. We have found near normal T cell receptor (TCR)-alpha and -beta mRNA expression by these lpr cells. The phenotypically comparable Lyt-2- L3T4- subset of normal thymocytes expressed approximately 10-fold less TCR-alpha mRNA than normal lymph node cells and somewhat higher TCR-beta mRNA. After cultivation of this immature thymocyte subpopulation with PMA and interleukin 2, TCR-alpha mRNA levels remained low, and TCR-beta transcripts were found to be subnormal. By using the same culture conditions, the corresponding mRNA levels of the lpr subset tended to resemble those of Lyt-2- L3T4- thymocytes. Fresh and cultured Lyt-2- L3T4- normal thymocytes showed comparably high levels of full-length TCR-gamma transcripts. In contrast, the fresh lpr subset had barely detectable levels of TCR-gamma mRNA. Upon cultivation, however, these levels increased over 200-fold to within the range observed in the Lyt-2- L3T4- thymocyte subset. No induction of the gamma gene was observed in similarly cultured normal lymph node T cells. The lpr cells may therefore correspond to an as yet undefined stage of normal T cell differentiation.

Animals↗
PubMed1987

Phenotypic identification of memory cytolytic T lymphocytes in a subset of Lyt-2+ cells.

Murine peripheral Lyt-2+ T cells could be subdivided according to surface expression of the Pgp-1 glycoprotein into major (71%) Pgp-1- and minor (29%) Pgp-1+ subsets. A striking correlation was observed between Pgp-1 expression and enrichment for antigen-specific memory cytolytic T lymphocyte precursors (CTLp). After immunization with the male minor transplantation antigen H-Y, virtually all the H-Y-specific CTLp were found in the minor Pgp-1+ subset of Lyt-2+ cells. In addition, after alloimmunization the frequency of allospecific CTLp resistant to inhibition by anti-Lyt-2 antibody was markedly enriched within the Pgp-1+ cells, suggesting an enrichment for CTLp bearing high avidity antigen receptors. Taken together, these data suggest that surface Pgp-1 expression is stably acquired at the time of primary antigenic stimulation by virgin T cells. As such, Pgp-1 represents an important marker for identifying a subset of Lyt-2+ T cells with the quantitative and qualitative properties of memory CTLp.

Animals↗
PubMed1986

Interleukin-2 monoclonal antibody affinity adsorption. The critical role of binding kinetics for optimal immunoadsorption.

With the ready availability of monoclonal antibodies reactive with an extensive spectrum of antigens, immunoaffinity adsorption has become more widely applicable for protein purification. However, given several monoclonal antibodies reactive with the same antigen, most investigators have found that only a few antibodies are useful for solid-phase immunoaffinity antigen purification. Accordingly, in order to determine the parameters of monoclonal antibody-antigen binding most important for effective immunoaffinity adsorption, equilibrium and kinetic binding experiments were performed using radiolabeled interleukin-2 (IL-2) as antigen and four different IL-2-reactive monoclonal antibodies. The antibodies were found to differ primarily with respect to their kinetic binding characteristics; at 37 degrees C IL-2 bound to two of these antibodies very rapidly, while it bound to the other two more slowly. When binding was performed at 4 degrees C, the equilibrium dissociation constants for all of the antibodies decreased due to a more marked prolongation of the dissociation rate than the association rate. However, at 4 degrees C the association rates of the two slow-reactive antibodies became retarded so markedly that efficient affinity adsorption did not occur. By comparison, for both of these antibodies, efficient removal of IL-2 could be obtained if adsorption was performed at 37 degrees C, provided the column flow rate was adjusted according to the IL-2 association rate. Kinetic considerations also dictated IL-2 adsorption to mixtures of two or more monoclonal antibodies: IL-2 immunoadsorption correlated with the association rates of the individual antibodies, rather than the equilibrium binding constants. These results indicate that the most important parameter for efficient affinity adsorption is the association rate constant. In addition, the results obtained indicate that monoclonal antibodies may differ markedly as regards their kinetic binding characteristics, and that all antibodies can serve as effective immunoadsorbents, provided their antigen binding characteristics are known.

Antibodies, Monoclonal↗
PubMed1986

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.

Animals↗
PubMed1985

Functional analysis of T cell subsets from mice bearing the lpr gene.

The autosomal recessive lpr (lymphoproliferation) gene is responsible for a thymus-dependent massive lymphoproliferation associated with the development of lupus-like autoimmune disease. Phenotypic analysis of adult lpr/lpr lymph nodes has demonstrated accumulation of a dull Lyt-1+, Thy-1+ population that expresses neither Lyt-2 nor L3T4 antigens. With the use of a depletion method based on complement-mediated lysis with an anti-Lyt-2 monoclonal antibody (31 M) and a new anti-L3T4 monoclonal antibody (RL 172.4), we have purified the Lyt-2- L3T4- subset from lymph nodes or spleens of C57BL/6-lpr/lpr mice and determined whether they are immunologically functional in vitro. Production of neither interleukin 2 nor interferon-gamma was detected by the double-negative subset after stimulation with concanavalin A and/or phorbol myristate acetate. The frequencies of allospecific cytotoxic T lymphocyte (CTL) precursors and lectin-induced antigen-nonspecific CTL precursors were diminished to almost undetectable levels, whereas the Lyt-2+ population from lpr/lpr mice had CTL-precursor frequencies comparable with that of +/+ mice. These results show that the major cell subset of adult lpr/lpr lymph nodes or spleens is composed of lymphocytes with markedly limited potential for lymphokine production or antigenic stimulation.

Animals↗
PubMed1985

Growth and differentiation in vitro of the accumulating Lyt-2-/L3T4- subset in lpr mice.

Mice bearing the recessive gene lpr develop an autoimmune syndrome associated with a massive lymphadenopathy, both of which are age and thymus dependent. The predominant accumulating cells in lymphoid tissue of lpr/lpr mice are Thy-1+ but express neither of the mature T cell markers, Lyt-2 or L3T4. We have purified this Lyt-2-/L3T4- subset and examined its phenotype. These cells are not actively cycling, do not express interleukin-2 (IL 2) receptors nor significant levels of antigen receptor, but do express the B cell marker B220. In vitro growth conditions were examined for the lpr Lyt-2-/L3T4- subset. By using a combination of phorbol ester and IL 2, these cells acquired transient expression of IL 2 receptors and grew in an IL 2-dependent manner. Furthermore, these proliferating cells underwent differentiation to a more mature T cell phenotype, with loss of cell surface B220 and acquisition, by a portion, of antigen receptor and Lyt-2. The possible parallels with normal T cell maturation are discussed.

Animals↗