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D A Carlow

Publications and source records attributed to D A Carlow.

At least 19 recordsLinked to original sources
PubMed2001

IL-2, -4, and -15 differentially regulate O-glycan branching and P-selectin ligand formation in activated CD8 T cells.

The glycosyltransferase core 2 beta1-6 N-acetylglucosaminyl transferase (C2GnT1 or C2GlcNAcT1) is responsible for formation of branched structures on O-glycans present on cell surface glycoproteins. The O-glycan branch created by C2GnT1 is physiologically important insofar as only this structure can be extended and modified to yield P-selectin ligands that promote initial interactions between extravasating lymphocytes and endothelia. In mature T cells, C2GnT1 activity is thought to be induced as an intrinsic consequence of T cell activation. Through analysis of C2GnT1-dependent epitopes on CD43 and CD45RB we have found that in activated CD8(+) T cells expression of C2GnT1 was dependent upon exposure to specific cytokines rather than being induced as a direct consequence of activation. Activated CD8(+) cells became receptive to strong induction of C2GnT1 expression and P-selectin ligand expression in response to IL-2, moderate induction by IL-15, and minimal induction in response to IL-4. Our observations clarify the relationship between T cell activation and C2GnT1 expression, demonstrate the differential impact of distinct cytokines on expression of C2GnT1 activity and P-selectin ligand, and reinforce the concept that the cytokine milieu subsequent to activation can influence adhesion systems that dictate lymphocyte homing properties.

Animals↗
PubMed2001

Absence of CD43 fails to alter T cell development and responsiveness.

Genetic elimination of CD43 has been associated with increased T cell adhesiveness and T cell hyperresponsiveness to mitogens and alloantigens. Therefore, we investigated whether T cell development was perturbed in CD43-deficient mice by breeding CD43(null) mice with male Ag (Hy)-specific TCR-transgenic mice. Neither positive nor negative thymic selection of male Ag-specific T cells were affected by CD43 status. Furthermore, we did not observe a substantial or consistent hyperresponsive pattern in HY-CD43(null) lymph node cells compared with littermate HY-CD43(+/-) lymph node cells upon analysis of in vitro T cell stimulation with male Ag or mitogen. These observations challenged original conclusions associating absence of CD43 with T cell hyperresponsiveness and led us to re-examine this association. Reported phenotypes of CD43(null) mice have been based on mice with a mixed 129xC57BL/6 genetic background. To exclude a possible influence of genetic background differences among individual mice we analyzed CD43(null) littermates that had been back-bred onto the C57BL/6 background for seven to eight generations. We found that CD43(+) and CD43(null) littermates with the C57BL/6 background exhibited no differences in response to mitogen or alloantigen, thereby establishing that T cell hyperresponsiveness is not a general correlate of CD43 absence.

Animals↗
PubMed1999

A novel CD8 T cell-restricted CD45RB epitope shared by CD43 is differentially affected by glycosylation.

The mAb 1B11 has been characterized as recognizing the activation-associated glycoform of murine CD43, a heavily O-glycosylated protein implicated in leukocyte homing. When hemopoietic cells from CD43-/- mice were stained with 1B11, CD43-independent binding of 1B11 was observed on peripheral CD8 T cells and at low levels on thymocytes, while no binding was detected on CD4 T cells, B cells, or bone marrow cells. Levels of 1B11 staining were comparable in lymph node CD8+ T cells from both CD43-/- mice and CD43+/+ mice. We sought to identify the CD43-independent target of 1B11 expressed on CD8 T cells. Previous work had demonstrated that neuraminidase treatment of lymph node cells (LNC) enhanced 1B11 binding on CD43+/+ LNC; this enhancement was also observed in CD43-/- LNC. We show that neuraminidase-enhanced 1B11 binding in CD43-/- LNC and EL4 thymoma cells is CD43 independent and that 1B11 detects a novel target of apparent mass of approximately 200 kDa identified as a hyposialylated form of CD45RB preferentially expressed on peripheral CD8, but not CD4, T cells. Our data also show that the recognition of CD43 and CD45RB by 1B11 is differentially affected by O-linked glycosylation and sialic acid. Whereas 1B11 recognition of CD43 on activated T cells required both core 2 O-glycan branching and sialic acid, 1B11 recognition of CD45 only occurred in the absence of both core 2 glycosylation and sialic acid.

Animals↗
PubMed1998

Specific antiviral activity demonstrated by TGTP, a member of a new family of interferon-induced GTPases.

The GTPase superfamily includes a diversity of molecules whose functions are regulated through the binding and hydrolysis of GTP. This superfamily can be segregated into families of functionally related molecules that typically share amino acid sequence similarity within and around the nucleotide-binding domains. A new family of putative GTPases, including IRG-47, LRG-47, IGTP, and TGTP/Mg21, has recently emerged that share significant sequence identity (25-40%). Expression of these molecules has been shown to be selectively induced by IFN-gamma and in some cases by IFN-alpha beta or bacterial LPS. This induction pattern implicates these putative GTPases as part of the innate defense of cells to infection, but their role in such defense has not yet been defined. We have previously described the cloning of TGTP and now confirm its intrinsic activity as a GTPase. We found that TGTP is strongly induced by endogenous IFN-alpha beta produced in response to standard lipofection of plasmid DNA or polyinosinic polycytidylic acid. The ability of endogenously produced IFN-alpha beta to efficiently induce expression of TGTP under these conditions suggested that TGTP might participate in defense against viral infection. This proposal was borne out when TGTP-transfected L cells displayed relative resistance to plaque formation by vesicular stomatitis virus but not herpes simplex virus. This observation places TGTP among a small family of innate antiviral agents and has implications for the functions of other members of this family of GTPases.

Animals↗
PubMed1996

Differential activation of phospholipase C-gamma 1 and mitogen-activated protein kinase in naive and antigen-primed CD4 T cells by the peptide/MHC ligand.

In this study, we determined the functional and biochemical differences in naive and primed CD4 T cells that expressed a TCR specific for the pigeon cytochrome c (pcc) peptide presented by I-Ek MHC class II molecules. Naive CD4 T cells expressing the transgenic TCR were isolated from the peripheral lymphoid organs of transgenic mice and stimulated with pcc peptide and IL-2 for 10 to 14 days. After this culture period, the Ag-primed cells were quiescent, as judged by the lack of expression of the early activation marker CD69, low expression of CD25 (IL-2R), and failure to incorporate thymidine. The primed cells required 10-fold less peptide than naive cells to achieve the same degree of proliferation and for the induction of CD69. Primed cells also mobilized calcium more efficiently with regard to Ag dose and magnitude of the response. The biochemical signal-transduction events in naive and primed T cells were compared by stimulating them with different concentrations of pcc peptide presented by adherent Ek-transfected fibroblasts. It was found that tyrosine phosphorylation and activation of mitogen-activated protein kinase (MAPK) in primed cells required 10-fold less Ag and occurred more rapidly and intensively. Interestingly, peptide stimulation induced tyrosine phosphorylation of phospholipase C (PLC)-gamma 1 exclusively in primed cells. RasGAP was also more efficiently tyrosine phosphorylated in primed cells. By contrast, Shc was tyrosine phosphorylated to the same extent in naive and primed cells. PI3Kp85 was not tyrosine-phosphorylated in naive and primed cells either before or after peptide stimulation. We propose that the higher sensitivity of the primed cells to Ag stimulation is most likely dependent, at last in part, on the more efficient activation of PLC-gamma 1, MAPK, and calcium-dependent pathways.

Animals↗
PubMed1995

Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif.

In this study, we describe a novel full length cDNA clone designated Tgtp that encodes a predicted 415-amino acid a T cell-specific guanine nucleotide triphosphate-binding protein (TGTP) bearing the characteristic motifs of a guanine nucleotide triphosphate (GTP) binding protein. Tgtp is expressed preferentially, if not exclusively, in T cells, and is up-regulated in both unfractionated and in purified CD4+8+ thymocytes upon TCR cross-linking. In contrast, expression of Tgtp is peripheral T cells is maintained at relatively high levels and is not grossly affected by TCR cross-linking. Antiserum generated against synthetic peptides from the predicted TGTP amino acid sequence recognized a single protein with a molecular mass of approximately 50 kDa, corresponding well with the computed molecular mass of 47 kDa. The only known relative of Tgtp is MUSGTP, which is reportedly expressed in B cells and bears a GTP binding motif. Thus, the discovery of Tgtp resolves a subfamily of molecules with GTP binding motifs and apparent lymphoid lineage-restricted expression. Given the restricted expression pattern in T cells, the up-regulated expression observed in response to TCR signaling in immature thymocytes, and the presence of the motifs characteristic of GTP binding proteins, we suggest that TGTP may have an important function in T cell development and/or T cell activation.

Amino Acid Sequence↗
PubMed1992

Altered thymocyte development resulting from expressing a deleting ligand on selecting thymic epithelium.

The maturation of CD4+8- and CD4-8+ thymocytes from CD4+8+ thymocytes is dependent on the mandatory interaction of their alpha beta TCR with selecting ligands expressed on thymic epithelial cells (TE). This is referred to as positive selection. The deletion of CD4+8+ thymocytes that express autospecific TCR (negative selection) is mediated primarily by bone marrow-derived cells. Previous studies have shown that TE is relatively ineffective in mediating the deletion of CD4+8- thymocytes expressing autospecific TCR but TE can render them anergic, i.e., nonresponsive, to the self Ag. The mechanism by which anergy is induced in these cells is unknown. In this study, we used thymocytes expressing a transgenic TCR specific for the male Ag presented by H-2Db class I MHC molecules to examine how expression of the deleting ligand by TE affects thymocyte development and phenotype. The development of female TCR-transgenic thymocytes was examined in irradiated male hosts or in female hosts that had received male fetal thymic epithelial implants. It was observed that the development of transgenic-TCR+ thymocytes was affected in mice with male TE. CD4+8+ thymocytes with reduced CD8 expression and markedly enhanced transgenic TCR expression accumulated in mice with male TE. Development of CD4-8+ thymocytes was also affected in these mice in that fewer were present and they expressed an intermediate CD8 coreceptor level. These CD4-8+ thymocytes expressed a high level of the transgenic TCR, retained the ability to respond to anti-TCR antibodies, but were nonresponsive to male APC. However, the maturation of CD4+8- thymocytes, which are also derived from CD4+8+ precursor cells, was relatively unaffected. In an in vitro assay for assessing negative selection, male TE failed to delete CD4+8+ thymocytes expressing the transgenic TCR under conditions where they were efficiently deleted by male dendritic cells. Collectively these results support the conclusion that male TE was inefficient in mediating deletion. Furthermore, expression of the deleting ligand on thymic epithelium interferes with the maturation of functional male-specific T cells and results in the accumulation of CD4+8+ and CD4-8+ thymocytes expressing a lower level of the CD8 coreceptor but a high level of the transgenic TCR.

Animals↗
PubMed1992

Deletion of antigen-specific immature thymocytes by dendritic cells requires LFA-1/ICAM interactions.

An in vitro assay was used for assessing the participation of various cell surface molecules and the efficacy of various cell types in the deletion of Ag-specific immature thymocytes. Thymocytes from mice expressing a transgenic TCR specific for the male Ag presented by the H-2Db class I MHC molecule were used as a target for deletion. In H-2d transgenic mice, cells bearing the transgenic TCR are not subjected to thymic selection as a consequence of the absence of the restricting H-2Db molecule but, nevertheless, express this TCR on the vast majority of immature CD4+8+ thymocytes. In this report we show that CD4+8+ thymocytes from H-2d TCR-transgenic mice are preferentially killed upon in vitro culture with male APC; DC were particularly effective in mediating in vitro deletion when compared with either B cells or T cells. Deletion of CD4+8+ thymocytes by DC was H-2b restricted and could be inhibited by mAb to either LFA-1 alpha or CD8. Partial inhibition was observed with mAb to ICAM-1, whereas mAb to CD4 and LFA-1 beta were without effect. These results are the first direct evidence of LFA-1 involvement in negative selection and provide further direct support for the participation of CD8/class I MHC interactions in this process. Like the requirements for deletion, activation of mature male-specific CD4-8+ T cells from female H-2b TCR-transgenic mice was also largely dependent on Ag presentation by DC and required both LFA-1/ICAM and CD8/class I MHC interactions; these results support the view that activation and deletion may represent maturation stage-dependent consequences of T cells encountering the same APC. Finally, our results also support the hypothesis that negative selection (deletion) does not require previous positive selection because deletion was observed under conditions where positive selection had not occurred.

Animals↗
PubMed1992

Disruption of CD8-dependent negative and positive selection of thymocytes is correlated with a decreased association between CD8 and the protein tyrosine kinase, p56lck.

The CD4 and CD8 coreceptor molecules on immature thymocytes participate in T cell repertoire selection. To examine more definitively the role of CD4 and CD8 in the negative and positive selection of immature thymocytes, we generated transgenic mice with elevated surface CD4 expression and mated them with mice expressing a transgenic T cell receptor. Augmented CD4 expression was found to markedly alter CD8-dependent negative and positive selection of T cells specific for the male (H-Y) antigen presented by H-2Db major histocompatibility complex class I molecules. Moreover, the cytoplasmic tail of CD4 was essential for effecting these alterations, since the overexpression of tailless CD4 molecules failed to influence the outcome of CD8-dependent selection. The inhibition of positive and negative selection in double-transgenic mice expressing the full-length CD4 molecule was associated with a decreased interaction between the protein tyrosine kinase p56lck and CD8. These results strongly implicate p56lck in T cell repertoire selection.

Animals↗
PubMed1992

Peripheral tolerance through clonal deletion of mature CD4-CD8+ T cells.

Transgenic mice bearing the alpha beta transgenes encoding a defined T cell receptor specific for the male (H-Y) antigen presented by the H-2Db class I MHC molecule were used to study mechanisms of peripheral tolerance. Female transgenic mice produce large numbers of functionally homogeneous CD8+ male antigen-reactive T cells in the thymus that subsequently accumulate in the peripheral lymphoid organs. We have used three experimental approaches to show that male reactive CD8+ T cells can be eliminated from peripheral lymphoid organs after exposure to male antigen. (i) In female transgenic mice that were neonatally tolerized with male spleen cells, male reactive CD8+ T cells continued to be produced in large numbers in the thymus but were virtually absent in the lymph nodes. (ii) Injection of thymocytes from female transgenic mice into female mice neonatally tolerized with the male antigen, or into normal male mice, led to the specific elimination of male-reactive CD8+ T cells in the lymph nodes. (iii) Four days after male lymphoid cells were injected intravenously into female transgenic mice, male antigen-reactive CD8+ T cells recovered from the lymph nodes of recipient mice were highly apoptotic when compared to CD4+ (non-male reactive) T cells. These data indicate that tolerance to extrathymic antigen can be achieved through elimination of mature T cells in the peripheral lymphoid organs.

Animals↗
PubMed1991

Participation of CD4 coreceptor molecules in T-cell repertoire selection.

During thymocyte development, progenitor cells bearing both CD4 and CD8 coreceptor molecules mature into functional T lymphocytes that express these proteins in a mutually exclusive way. Although T-cell specificity is determined primarily by the structure of the T-cell antigen receptor (TCR) heterodimer, a developmentally regulated process acts to ensure that cells bearing class II-restricted TCRs are CD4+ and those bearing class I-restricted TCRs express only CD8. To investigate this maturation process, we have engineered transgenic mice in which CD4 is expressed in all thymocyte subsets and in all peripheral T cells. Peripheral CD4+8+ T lymphocytes from these mice react with both class I and class II alloantigens. Moreover, expression of the CD4 transgene disrupts the positive selection of doubly transgenic thymocytes bearing a class I-restricted TCR specific for the male (H-Y) antigen. Hence the CD4 coreceptor participates directly in T-cell repertoire selection.

Animals↗
PubMed1991

Capacity of CD8+ T cells to reject immunogenic variants of a spontaneous murine carcinoma: lack of non-specific (NK1.1+) effector mechanisms.

Class I MHC-expressing (Ia-) immunogenic (imm +) variants, which elicit a strong syngeneic host immune rejection response, can be isolated following 5-azacytidine treatment from the MHC-negative non-immunogenic (imm-) murine carcinoma cell line SP1 (10.1 subclone). In the present study, we have shown that CD4-depleted CD8+ T cells are both necessary and sufficient for the rejection process. Treatment of semi-syngeneic B6 X CBA F1 mice with anti-NK1.1 antibodies had no effect on the rejection of immunogenic variants, although the splenic NK (natural killer) activity of recipients was fully abrogated. Thus NK1.1+ effectors, which include most NK and LAK (lymphokine activated killer) cells, are most likely not involved in the rejection process. This finding was supported by a complete lack of NK susceptibility of SP1 cells in vitro, although variable killing by LAK and poly-I: C-induced killer cells was observed. To assess the role of NK1.1-LAK and other non-T killers (e.g. cytolytic macrophages) in vivo, we determined the specificity of the rejection process. We examined the ability of immune animals to reject a mixture of non-immunogenic parent tumour cells (or cells of an unrelated syngeneic tumour) and of the variant tumour cells used for the initial immunization. Growth of the parent tumour cells was unaffected while the same animals rejected the immunogenic tumour cells. Our findings support a primary role of tumour-specific CD8+ T cells in the rejection of imm+ variants with no detectable involvement of non-specific effector cells in the tumour destruction process.

Adenocarcinoma↗
PubMed1990

Class I (H-2Kb) gene transfection reduces susceptibility of YAC-1 lymphoma targets to natural killer cells.

A "hybrid gene" (MTKb) comprised of the human metallothionein IIA promoter ligated to the genomic sequence of the major histocompatibility complex class I (H-2Kb) gene was subcloned into the expression vector pSV2neo and transfected into the natural killer (NK) cell-sensitive YAC-1 lymphoma. The Kb gene product was readily detectable on the cell surface of G418-resistant transfectants using both Kb-specific monoclonal antibodies and H-2b-specific cytolytic T cells. Unlike control pSV2neo transfectants, MTKb-pSV2neo transfectants were relatively resistant to lysis by NK cells from H-2a, H-2b, H-2k or H-2 (a x b)F1 haplotype mice. These data strongly suggest that the effects of MHC expression on susceptibility to NK cells can be mediated by a single and well-defined class I molecule, Kb.

Animals↗
PubMed1989

Failure of expression of class I major histocompatibility antigens to alter tumor immunogenicity of a spontaneous murine carcinoma.

We have shown previously that clonal immunogenic variants of murine mammary adenocarcinoma 10.1 can be isolated after treatment in vitro with the DNA-hypomethylating agent 5-azacytidine (5-aza). Such immunogenic variants frequently express elevated class I major histocompatibility complex antigens relative to the level of expression in the parent tumor and are rejected in syngeneic mice by a T-cell-dependent process. To ascertain whether elevated immunogenicity is a function of increased class I antigen expression, we isolated high class I antigen expressors from 5-aza-treated 10.1 cells by using the fluorescence-activated cell sorter. Clonal variants displaying any increase in class I antigen expression were more efficient stimulators of allo-class I antigen-specific cytolytic T-cell precursors. However, these variants displayed unaltered tumorigenicity in immunocompetent syngeneic mice. Thus, phenotypic changes other than, or in addition to, elevated class I antigen expression cause the reduced tumorigenicity of immunogenic clones of 10.1 cells isolated after 5-aza treatment.

Adenocarcinoma↗
PubMed1987

In situ augmentation of class I major histocompatibility antigen expression on immunogenic variants of a spontaneous murine mammary carcinoma.

The relationship between expression of cell surface glycoproteins encoded by the major histocompatibility complex (MHC) and immunogenicity of a recently obtained spontaneous murine mammary adenocarcinoma (designated CBA.SP1) was examined. Immunogenic and nonimmunogenic variant clones were isolated from a subclone of the parent tumor after treatment with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or the DNA hypomethylating agent and "gene activator," 5-aza-2'-deoxycytidine (5-aza-dCyd). All clones from the untreated tumor population were tumorigenic in normal syngeneic recipients. In contrast, immunogenic variant clones, isolated at high frequencies after drug treatment [ranging from 5% (5-aza-dCyd treated) to greater than 90% (MNNG treated)], were rejected in normal syngeneic mice but grew progressively in T-cell deficient nude mice. Consistent with our previous report (J. Natl. Cancer Inst., 75: 291, 1985), all 5-aza-dCyd induced immunogenic clones expressed elevated levels of class I (particularly Dk) MHC antigens. However some (three out of nine) nonimmunogenic clones also showed enhanced class I MHC expression, implying that not all high MHC expressors were immunogenic. In contrast to 5-aza-dCyd induced variants, only 50% of MNNG induced immunogenic variants showed elevated levels of Dk or Dk and Kk antigens in vitro. Strong augmentation of class I MHC antigens in situ was observed on all immunogenic, but not nonimmunogenic, clones following transplant into syngeneic mice; no increase in MHC expression on variants during progressive growth in athymic nude mice occurred. Although no class II (Ak or Ek) antigens were detected on the parent line or any of the immunogenic variants, a strong infiltration of host I-A bearing cells occurred during immune rejection of SP1 variants. These results are consistent with the hypothesis that induction of class I MHC antigen expression on certain low MHC expressing tumors, although not the sole requirement for immunogenicity, can facilitate immune rejection of the SP1 tumor and, conversely, that the reduced level of MHC observed in certain clinical cancers may significantly affect the immunological aspects of the tumor-host relationship.

Adenocarcinoma↗