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H R MacDonald

Publications and source records attributed to H R MacDonald.

At least 19 recordsLinked to original sources
PubMed1992

Inhibition of mouse mammary tumor virus-induced T cell responses in vivo by antibodies to an open reading frame protein.

Minor lymphocyte stimulating (Mls) antigens specifically stimulate T cell responses that are restricted to particular T cell receptor (TCR) beta chain variable domains. The Mls phenotype is genetically controlled by an open reading frame (orf) located in the 3' long terminal repeat of mouse mammary tumor virus (MMTV); however, the mechanism of action of the orf gene product is unknown. Whereas predicted orf amino acid sequences show strong overall homology, the 20-30 COOH-terminal residues are strikingly polymorphic. This polymorphic region correlates with TCR V beta specificity. We have generated monoclonal antibodies to a synthetic peptide encompassing the 19 COOH-terminal amino acid residues of Mtv-7 orf, which encodes the Mls-1a determinant. We show here that these antibodies block Mls responses in vitro and can interfere specifically with thymic clonal deletion of Mls-1a reactive V beta 6+ T cells in neonatal mice. Furthermore, the antibodies can inhibit V beta 6+ T cell responses in vivo to an infectious MMTV that shares orf sequence homology and TCR specificity with Mtv-7. These results confirm the predicted extracellular localization of the orf COOH terminus and imply that the orf proteins of both endogenous and exogenous MMTV interact directly with TCR V beta.

Amino Acid Sequence↗
PubMed1992

CD4+8- thymocytes bearing major histocompatibility complex class I-restricted T cell receptors: evidence for homeostatic control of early stages of CD4/CD8 lineage development.

During thymus development CD4+ CD8+ precursor cells differentiate into mature CD4+ and CD8+ T cells expressing T cell receptors (TCR) that recognize foreign antigens in association with major histocompatibility complex (MHC) class II or I molecules, respectively. Studies with TCR transgenic mice have shown that the accumulation of mature CD4+ and CD8+ thymocytes is strongly skewed by the MHC restriction specificity of the TCR, thus suggesting that commitment of CD4+ CD8+ precursors to the CD4 or CD8 lineage is a direct consequence of TCR/MHC interactions. However, we show here that CD4+ cells expressing an inappropriate (MHC class I-specific) TCR appear transiently in the neonatal thymus of TCR transgenic mice and can also be found in the periphery of adult TCR transgenic recombination-deficient SCID mice. These data argue that the early stages of CD4 and CD8 lineage development in the thymus are (at least in part) controlled by homeostatic mechanisms independent of appropriate TCR/MHC interactions.

Animals↗
PubMed1992

H-2-restricted cytolytic T lymphocytes specific for HLA display T cell receptors of limited diversity.

We previously showed that H-2Kd-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasmodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human class I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2Kd. We first sequenced the TCR alpha and beta genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR alpha and beta cDNA junctional regions of 23 independent H-2Kd-restricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of V alpha, J alpha, V beta, and J beta segments, and in terms of length and sequence of the CDR3 alpha and beta loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2Kd selects CTLs that bear TCRs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.

Amino Acid Sequence↗
PubMed1992

An exogenous mouse mammary tumor virus with properties of Mls-1a (Mtv-7).

The classical minor lymphocyte stimulating (Mls) antigens, which induce a strong primary T cell response in vitro, are closely linked to endogenous copies of mouse mammary tumor viruses (MMTV). Expression of Mls genes leads to clonal deletion of T cell subsets expressing specific T cell receptor (TCR) V beta chains. We describe the isolation and characterization of a new exogenous (infectious) MMTV with biological properties similar to the Mls antigen Mls-1a. In vivo administration of either Mls-1a-expressing B cells or the infectious MMTV (SW) led to an increase of T cells expressing V beta 6 followed by their deletion. Surprisingly, different kinetics of deletion were observed with the exogenous virus depending upon the route of infection. Infection through the mucosa led to a slow deletion of V beta 6+ T cells, whereas deletion was rapid after subcutaneous infection. Sequence analysis of the open reading frames in the 3' long terminal repeat of both this exogenous MMTV (SW) and of Mtv-7 (which is closely linked to Mls-1a) revealed striking similarities, particularly in the COOH terminus, which has been implicated in TCR V beta recognition. The identification of an infectious MMTV with the properties of a strong Mls antigen provides a new, powerful tool to study immunity and tolerance in vivo.

Amino Acid Sequence↗
PubMed1992

Propidium iodide staining correlates with the extent of DNA degradation in isolated nuclei.

Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37 degrees C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluorescence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogenous endonuclease: Zn2+ and EGTA. However, the presence of Zn2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations.

Animals↗
PubMed1992

Minimal growth requirements of mature T lymphocytes: interleukin (IL)-1 and IL-6 increase growth rate but not plating efficiency of CD4 cells stimulated with anti-CD3 and IL-2.

We show that interleukin (IL)-2 is necessary and sufficient for the proliferation of both CD4 and CD8 subsets of peripheral murine T cells activated by plastic-bound anti-CD3 monoclonal antibodies (mAb). The frequency of proliferating cells (f) was 0.32 for CD4 cells and 0.63 for CD8 cells. These frequencies were not increased by the addition of IL-1 or IL-6, alone or in combination. These cytokines were unable to induce responsiveness to IL-2 in T cells confirming that they cannot substitute for the signal delivered via the TcR/CD3 complex. On the other hand, IL-1 and IL-6 increase the growth rate of CD4 cells. The addition of IL-6 significantly lowered the mean doubling time (dt) of CD4 cells (dt: 26 h vs. 38 h in the presence of IL-2 alone, p less than 0.01), while the addition of IL-1, ineffective by itself, combined with IL-6 further increased the growth rate of CD4 cells (dt: 23 h, p less than 0.001). The growth rate of CD8 cells stimulated with anti-CD3 and IL-2, was markedly faster than that of CD4 cells (dt: 18 h vs. 38 h, p less than 0.001) and was not significantly influenced by addition of IL-1 and/or IL-6.

Animals↗
PubMed1992

In vivo responses of CD4+ and CD8+ cells to bacterial superantigens.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds to major histocompatibility complex (MHC) class II molecules and specifically activates T cells bearing V beta 8 T cell receptor domains. We have compared several aspects of the response of CD4+ and CD8+ T cell subsets to SEB in vivo. V beta 8+ cells in both subsets proliferated to a similar extent upon SEB injection. Furthermore, mRNA for interferon-gamma was induced in both subsets with similar kinetics and SEB dose-response. Finally CD8+ (but not CD4+) T cells from SEB-injected mice exhibited SEB-specific lysis of MHC class II-bearing target cells. Collectively, these data indicate that the CD4: MHC class II interaction confers no detectable selective advantage to CD4+ cells in the in vivo response to SEB. The observed effector functions of both subsets may contribute to SEB-induced immunopathology.

Animals↗
PubMed1992

The viral superantigen Mls-1a induces interferon-gamma secretion by specifically primed CD8+ cells but fails to trigger cytotoxicity.

Superantigens can be operationally defined by their ability to stimulate CD4+ and CD8+ T cells via the T cell receptor beta chain variable domain (TcR V beta). We show here that effector functions of CD8+ T cells specific for superantigens differ depending upon the nature of the superantigen involved. Hence, activated CD8+ T cells bearing TcR V beta specific for the superantigen Mls-1a [encoded in the open reading frame of the 3' long terminal repeat of endogenous mouse mammary tumor virus (MMTV)] are unable to lyse Mls-1a-bearing target cells despite the fact that they release interferon-gamma (IFN-gamma) upon Mls-1a stimulation. In contrast CD8+ T cells specific for the exogenous superantigen staphylococcal enterotoxin B (SEB) readily mediate both lysis and IFN-gamma secretion when exposed to SEB-bearing target cells. This dissociation between lysis and IFN-gamma production by Mls-1a-specific CD8+ T cells is independent of the initial stimulus used for activation and appears not to be simply explained by a low Mls-1a determinant density. We suggest that this phenomenon reflects differing TcR affinity thresholds for lymphokine secretion and cytolysis. Such differences may be exploited by retroviruses such as MMTV in order to escape immunosurveillance.

Animals↗
PubMed1992

Distinct effects of phorbol esters and exogenous diacylglycerols in the induction of murine thymocyte proliferation.

Murine thymocytes were stimulated with the protein kinase C activating agents Phorbol-12-myristate-13-acetate (PMA) or a more physiological membrane permeant diacylglycerol (dioctanoyl-sn-glycerol, DiC8) in the presence or absence of exogenous lymphokines (rIL-1 beta, rIL-2). Whereas PMA directly induced reactivity to rIL-2, DiC8 did not but had to synergize with the calcium ionophore Ionomycin. Expression of the p55 chain of the IL-2 receptor behaved similarly. In the absence of exogenous rIL-2, thymocytes proliferated in response to a combination of Ionomycin and PMA; however, replacing PMA by a single addition of DiC8 did not result in proliferation. Stimulation with Ionomycin plus repeated addition of DiC8 induced a low level of thymocyte proliferation and further addition of rIL-1 beta resulted in a significant increase. Purified immature (L3T4-Lyt2-) thymocytes behaved similarly, but showed an increased sensitivity to rIL-1 beta. Taken together, the data support the idea that PMA and the more physiological diacylglycerols do not possess totally equivalent activities in lymphocyte stimulation.

Animals↗
PubMed1992

A cortisone sensitive CD3low subset of CD4+CD8- thymocytes represents an intermediate stage in intrathymic repertoire selection.

Two populations of CD4 single positive (SP) thymocytes were found in transgenic mice bearing class I-restricted Mls-1a reactive (V beta 8.1) TCR genes in the absence of the restriction element. CD3high CD4 SP cells were deleted in the presence of Mls-1a and were cortisone resistant, whereas CD3low CD4 SP cells were not deleted in the presence of Mls-1a and were cortisone sensitive. Intravenous transfer of CD3low CD4 SP cells into nude mice resulted in significant peripheral expansion of these cells with apparent upregulation of CD3. These data indicate that CD3low CD4 SP thymocytes represent an intermediate stage in the transition from CD3low double positive (DP) to CD3high SP thymocytes and raise the possibility that these cells may hve undergone positive but not negative selection events (at least to Mls-1a). Furthermore the fact that CD3high DP thymocytes were also deleted by Mls-1a in these mice suggests strongly that sensitivity to Mls-1a deletion is dependent upon stage of thymic maturation (as revealed by TCR density) rather than CD4/CD8 phenotype.

Animals↗
PubMed1992

Analysis of immature (CD4-CD8-) thymic subsets in T-cell receptor alpha beta transgenic mice.

Introduction of a transgenic alpha beta TCR (V alpha 2, V beta 8.1) specific for lymphocytic choriomeningitis virus (LCMV), in the context of H-2Db into the genome of C57BL/6 mice, has many effects on the development and selection of T cells in both the thymus and the periphery. These mice produce increased numbers of CD4-8+ mature T cells, all of which express the transgenic TCR, and small numbers of CD4+8- cells using endogenous TCRs are also produced. This study follows the intrathymic development of T cells in these TCR alpha beta transgenic mice, in particular the earliest CD4-8- stages. As expected, the transgenic TCR is expressed on the cell surface at an earlier developmental stage than endogenous TCRs in nontransgenic littermate controls. Of the three major subsets expressing the heat-stable antigen (HSA), only the most mature, the CD25-CD44- expresses the transgenic TCR, and the earlier CD25-CD44+ and CD25+CD44- do not. Furthermore, in contrast to other TCR alpha beta transgenic lines, TCR gamma delta lineage cells appear to develop normally.

Animals↗
PubMed1991

A recombinant C-terminal fragment of staphylococcal enterotoxin A binds to human MHC class II products but does not activate T cells.

Binding of staphylococcal enterotoxin A (SEA) to MHC class II encoded proteins is a prerequisite for its subsequent activation of a large fraction of T lymphocytes through interaction with variable segments of the TCR-beta chain. We cloned SEA in Escherichia coli and produced four recombinant fragments covering both the N- and C-terminal regions. These fragments were used to analyze the interaction between SEA and the human MHC class II products. A C-terminal fragment of SEA, representing amino acids 107-233 bound to HLA-DR and HLA-DP but did not activate T cells. The three other fragments (amino acids 1-125, 1-179 and 126-233) neither bound to MHC class II Ag nor activated T cells. SEA apparently bind to HLA-DR and HLA-DP through its C-terminal part, whereas T cell activation is dependent on additional parts of the protein.

Base Sequence↗
PubMed1991

Staphylococcal enterotoxin-dependent lysis of MHC class II negative target cells by cytolytic T lymphocytes.

The enterotoxins of Staphylococcus aureus (SE) are extremely potent activators of human and mouse T lymphocytes. In general, T cell responses to SE are MHC class II dependent (presumably reflecting the ability of SE to bind directly to MHC class II molecules) and restricted to responding cells expressing certain T cell receptor beta-chain variable (TCR V beta) domains. Recently we demonstrated that CD8+ CTL expressing appropriate TCR V beta could recognize SE presented on MHC class II-bearing target cells. We now show that MHC class II expression is not strictly required for T cell recognition of SE. Both human and mouse MHC class II negative target cells could be recognized (i.e., lysed) in a SE-dependent fashion by CD8+ mouse CTL clones and polyclonal populations, provided that the CTL expressed appropriate TCR V beta elements. SE-dependent lysis of MHC class II negative targets by CTL was inhibited by mAb directed against CD3 or LFA-1, suggesting that SE recognition was TCR and cell contact dependent. Furthermore, different SE were recognized preferentially by CTL on MHC class II+ vs MHC class II- targets. Taken together, our data raise the possibility that SE binding structures distinct from MHC class II molecules may exist.

Animals↗
PubMed1991

CD8+ T cell response to Mls-1a determinants involves major histocompatibility complex class II molecules.

Recent studies indicate that both CD4+ and CD8+ T lymphocytes proliferate in vitro in response to Mls-1a-encoded determinants. Using both immunogenetic and antibody blocking approaches we show here that Mls-1a responses of both subsets require expression of major histocompatibility complex (MHC) class II molecules (I-A and/or I-E) by the stimulator cells. Furthermore, CD8+ T cell responses to Mls-1a/class II MHC do not require (and are in fact inhibited by) the presence of functional CD8 molecules. Taken together, our data underscore the dramatic differences between CD8+ T cell responses to conventional peptide antigens as opposed to "superantigens" such as Mls-1a.

Animals↗
PubMed1991

Clonal expansion precedes anergy and death of V beta 8+ peripheral T cells responding to staphylococcal enterotoxin B in vivo.

Staphylococcal enterotoxin B (SEB) selectively stimulates T cells bearing T cell receptor V beta 8 domains and hence provides a useful model to study immunity and tolerance in vivo. We show here that V beta 8+ T cells in both CD4+ and CD8+ subsets expand dramatically (fivefold) in lymphoid tissues of mice 2-4 days following injection with SEB. This initial clonal expansion, which is accompanied by a transient hyper-reactivity to SEB, is followed by a rapid decrease in V beta 8+ cells and a concomitant induction of specific non-responsiveness which persists for at least 30 days. Selective death of V beta 8+ cells occurs during this latter phase. Taken together, our data indicate that clonal expansion, anergy and death can occur as sequential stages of an immune response in vivo.

Animals↗
PubMed1991

Monoclonal antibodies raised against engineered soluble mouse T cell receptors and specific for V alpha 8-, V beta 2- or V beta 10-bearing T cells.

We have characterized a panel of monoclonal antibodies (mAb) produced by immunizing rats with two distinct soluble mouse alpha/beta T cell receptor (TcR). Fifty mAb were found to react with the corresponding surface-bound TcR. Such observations suggest that the soluble TcR molecules used as immunogen are folded in a conformation similar to the native structure. Furthermore, the binding to T cells of four antibodies was found to correlate with the expression of the V alpha 8, V beta 2 or V beta 10 gene segments. Finally, staining of T lymphocytes from various mouse strains suggests that (a) the two anti-V alpha 8 antibodies recognize different epitopes, and each on only a fraction of V alpha 8+ cells; (b) the anti-V beta 10 mAb identifies a V beta 10 polymorphism among mouse strains, and (c) T cells expressing the V beta 2 or V beta 10 gene segments are not subject to major clonal deletion events induced by the major histocompatibility complex class II and Mls products which were tested.

Animals↗
PubMed1991

Does negative selection involve accumulation of self-reactive thymocytes in thymic rosettes?

Thymic rosettes, the natural associations between thymocytes and either macrophages or dendritic cells, were isolated from the thymus by collagenase digestion and unit-gravity elutriation. Rosettes from mouse strains where either the V beta 6-bearing thymocytes are deleted because of reactivity with products of the Mlsa allele of the minor lymphocyte stimulating locus, or where V beta 17a-bearing thymocytes are deleted because of reactivity with IE class II MHC molecules, were compared with rosettes from appropriate control strains to test if a selective association with stromal cells preceded deletion. Rosettes from an Mlsa-bearing strain were able to stimulate an Mlsa-reactive T-hybridoma, but much of this stimulatory activity was attributable to the few B cells associated with the rosette preparations; the stromal components of the rosettes appeared to be poor presenters of Mlsa gene products. There was no enrichment of thymocytes bearing high or low levels of V beta 6 TcR in the rosettes from the Mlsa-bearing strain, which might have reflected the poor presentation by the stromal cells. However, nor was there detectable selective association of thymocytes bearing C beta 17a in the rosettes from an IE-positive mouse strain. This argues against binding and immobilisation on stromal cells as part of the deletion process, but not against the stromal cells delivering a rapid signal during a transient association, leading later to deletion.

Animals↗