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H R MacDonald

Publications and source records attributed to H R MacDonald.

At least 127 records · Page 7Linked to original sources
PubMed1993

Mouse intestinal epithelial cells express the self superantigen Mls1a.

In previous studies, we demonstrated that intestinal epithelial cells of the mouse small intestine could present exogenous antigen to specific CD4+ T cell hybridomas. We now report on the ability of normal enterocytes to present the self superantigen Mls1a. Enterocytes from Mls1a but not from Mls1b strains stimulated interleukin-2 production through a V beta 6+ T cell hybridoma specific for Mls1a determinants. Antibody inhibition experiments showed that enterocytes presented Mls determinants via a major histocompatibility complex class II-dependent mechanism. Furthermore, the ability of enterocytes to activate V beta 6+ Mls1a-specific T cells was inhibited by monoclonal antibodies against the Orf protein encoded by an Mtv-7 provirus which is associated with Mls1a expression. These findings provide evidence for the first time that Mls determinants are expressed on normal enterocytes and support the theory of a possible role of these cells in extrathymic selection of T cell receptor V beta repertoire of intraepithelial T lymphocytes.

Animals↗
PubMed1993

Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.

Animals↗
PubMed1993

The CD8 T cell response to staphylococcal enterotoxins.

Staphylococcal enterotoxins (SE) are superantigens that bind to MHC class II molecules and activate T lymphocytes via the T cell receptor (TCR) V beta domain. By analogy with T cell responses to conventional peptide antigens, it might be predicted that CD4 (MHC class II restricted) cells would respond better to SE than CD8 (MHC class I restricted) cells. Here we summarize evidence that in fact CD8 cells respond as well as CD4 cells to SE both in vitro and in vivo. These findings emphasize the unique character of TCR-superantigen interactions.

Animals↗
PubMed1993

Subversion of host immune responses by viral superantigens.

Recent experiments with mouse mammary tumor virus indicate that expression of a virally encoded superantigen by B cells and its subsequent recognition by T cells are essential steps for amplification of infection and virus transmission. Preliminary results suggest that superantigens may also be expressed during retroviral infection in humans.

Animals↗
PubMed1993

Skewed T cell receptor V alpha repertoire among superantigen reactive murine T cells.

Reactivity of murine T cells with viral or bacterial superantigens is clearly correlated with the expression of TCR V beta domains. Thus, T cells responding to the minor lymphocyte stimulatory locus (Mls-1a) or staphylococcal enterotoxin B (SEB) express predominantly TCR V beta 6 or V beta 8.2 respectively. We have investigated the involvement of the other major variable element of the TCR, the V alpha domain, in these superantigen responses. Using a panel of anti-TCR V alpha mAbs, it is demonstrated that the TCR V alpha repertoire among superantigen stimulated V beta 6+ or V beta 8.2+ blasts (responding to Mls-1a or SEB respectively in vitro) is altered in comparison with anti-CD3 stimulated cells expressing the same V beta domains. Furthermore, the TCR V alpha repertoire is strongly skewed in TCR V beta 8.2 transgenic mice that have undergone extensive peripheral clonal deletion after SEB injection. These data imply that the V alpha domain influences superantigen recognition by the TCR.

Animals↗
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

Enigma variations.

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Journal Article↗
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↗