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J A Kapp

Publications and source records attributed to J A Kapp.

At least 37 records · Page 2Linked to original sources
PubMed1995

Antigen recognition by CTL is dependent upon ectoATPase activity.

Alloantigen-specific and OVA-specific CD8+ CTL were shown here to express an ectoATPase. These CTL also express an ectoADPase, but do not express detectable levels of an ectoAMPase. CD8+ CTL transported adenosine into their cytoplasm at a rate of 2.3 x 10(-11) mmol/min/10(5) cells. In contrast, adenosine uptake was 34-fold lower when ATP was used as the source of the nucleoside. This was consistent with the lack of ectoAMPase and suggests that the role of ectoATPase is not in the salvage of extracellular nucleotides. 5'-p-(fluorosulfonyl)benzoyl adenosine (5'-FSBA) is an ATP analogue affinity label that irreversibly inhibits CTL ectoATPase. Cells made ectoATPase activity deficient by modification with 5'-FSBA were not susceptible to potential lytic effects of extracellular ATP with less than 20% specific lysis at 20 mM of exogenous ATP. However, cells modified by 5'-FSBA were unable to kill their respective target cells. Complete inhibition of cell-mediated killing was observed with 1 mM 5'-FSBA. CTL modified by 5'-FSBA also failed to secrete TNF-alpha and IFN-gamma after activation by the appropriate Ag. Killing was also inhibited by 5'-adenylylimidodiphosphate (a nonhydrolyzable ATP analogue), but not by ATP, ADP, alpha, beta-methylene ADP (a nonhydrolyzable ADP analogue), AMP, or adenosine. Blockage of CTL activity by 5'-FSBA was not reversed by addition of ADP, suggesting that hydrolysis of ATP is an essential ectoATPase-mediated signal for CTL activation. These results suggest that ectoATPase is essential for Ag recognition and/or effector activities of CTL.

Adenosine↗
PubMed1995

Ovalbumin injected with complete Freund's adjuvant stimulates cytolytic responses.

CD8+ cytotoxic T lymphocytes (CTL) recognize antigens (Ag) associated with class I major histocompatibility complex (MHC) molecules. Endogenously synthesized protein Ag are processed into peptides in the cytoplasm and transported to the endoplasmic reticulum where they are bound by class I proteins. Exogenous Ag do not enter the class I processing pathway of most cells and thus do not activate CD8+ CTL. Nevertheless, several investigators have reported that immunization with exogenous Ag can activate CD8+ T cells that have immunoregulatory activity. To determine how exogenous Ag entered the class I pathway in vivo and whether immunosuppressive CD8+ T cells were cytolytic, we have shown in this report that injection with OVA emulsified in the complete Freund's adjuvant (CFA) primed CD8+, class I MHC-restricted, OVA-specific CTL in mice. These CTL recognize the OVA257-264 epitope, produce tumor necrosis factor-alpha and interferon-gamma upon activation. Both oil and mycobacteria components in CFA were required for inducing CTL responses. Priming was not attributed to direct sensitization of class I-bearing cells by contaminating peptides. Rather, phagocytic cells, but not CD4+ helper T cells, were required for priming CD8+ CTL by OVA-CFA. Thus, OVA in CFA is taken up by antigen-presenting cells and processed into the class I pathway by phagocytic cells in vivo. In addition, CTL induced by OVA-CFA suppressed the antibody response to OVA in adoptive recipients. These results suggest that CD8+ CTL specific for exogenous proteins might be routinely stimulated by injecting proteins in conventional adjuvants and that such cells have the potential to regulate immune responses in vivo.

Animals↗
PubMed1995

Characterization of the epitope recognized by a mAb that reacts differentially with murine suppressor T cells.

Although reliable antibodies are available that distinguish human suppressor T (Ts) cells from CTL and other T cells, few are available for murine Ts cells. We have developed a mAb (984D4.6.5) that, in the presence of complement, depletes alloantigen-specific Ts cells but not CTL. This antibody recognizes activated Ts cells but not their precursors. In these studies, flow cytometric analysis demonstrates that 984D4.6.5 reacts with several Ts cell hybridomas, cloned Ts cell lines and WEHI-3 (a myelomonocytic tumor cell line). Reactivity was not detected with BW5147, Th cell hybridomas, cloned Th cells, CTL lines and hybridomas, B cell lines, thymocytes, splenocytes, bone marrow cells nor a variety of tumor cells. Among 984D4.6.5 positive lines, expression is heterogeneous and the number of cells expressing high levels of the epitope is increased when the hybridomas are maintained at a relatively high cell density. Neuriminidase and pronase deplete the epitope recognized by mAb 984D4.6.5. Protein synthesis and glycosylation inhibitors also reduce expression of this epitope. These observations suggest that the epitope recognized by 984D4.6.5 is a carbohydrate linked to a polypeptide. This antibody was tested by ELISA for binding to a large panel of carbohydrates and glycolipids coupled to BSA. The only one that bound 984D4.6.5 was LS tetrasaccharide c (NeuNAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), an O-linked carbohydrate. Comparative analysis shows that both the sequence and the linkage of these sugars are essential to the reactivity with the 984D4.6.5 antibody. This epitope is expressed by a glycoprotein of approximately 200 kDa, as shown by Western blots. The identity of this glycoprotein remains to be determined, but indirect evidence suggests that it is not CD45.

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PubMed1995

Human lactoferrin induces phenotypic and functional changes in murine splenic B cells.

The immunotropic activities of human lactoferrin were studied with respect to phenotypic and functional changes in murine splenic B cells. Phenotypic changes were induced by human lactoferrin in splenic B-cell fractions separated by buoyant density. B cells from 7-8-day-old BALB/c mice isolated from a 50/60% Percoll gradient, gained characteristic features of more mature B cells manifested by an increase of surface IgD and complement receptor expression. Incubation of the analogous B-cell fraction from adult mice with human lactoferrin resulted in minor changes in relation to IgM and IgD expression. Besides induction of phenotypic changes on immature B cells, human lactoferrin enabled B cells from normal newborn and adult immunodeficient CBA/N mice to present antigen to an antigen-specific T-helper type 2 (Th2) cell line. We conclude that human lactoferrin acts as a maturation factor for B cells with regard to their phenotype and function.

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PubMed1995

Presentation of antigen by B cell subsets. V. Effect of interleukin 7 (IL-7) and interleukin 10 (IL-10) on phenotype and antigen presenting function of immature B cells.

In this communication we demonstrate that B cells from CBA/N mice with x chromosome-linked immunodeficiency and B cells from newborn mice, that have very limited ability to present antigen to antigen-specific T cells lines, acquire this function following preincubation with IL-7 or IL-10. These interleukins do not affect the antigen presenting function of B cells from normal, adult CBA mice. Incubation of B cells from xid and newborn mice with IL-7 or IL-10 but not with IL-2 induces expression of Lyb-5 marker on these cells. The study shows that the property of IL-7 and IL-10 to induce antigen presenting cell (APC) activity in immature B cells is associated with appearance of Lyb-5 antigen on these cells.

Animals↗
PubMed1995

CD4+ T cells from mice primed for humoral immunity to sheep erythrocytes (SRBC) specifically inhibit cell-mediated immunity and vice versa.

The aim of this study was to demonstrate whether development of the humoral or cellular immunity to sheep erythrocytes (SRBC) can be inhibited by a transfer of antigen-specific CD4+ T cells isolated from mice exhibiting cellular or humoral response. For generation of the humoral immune response and suppressor T cells for the cellular immunity, mice were immunized with a high dose of SRBC intraperitoneally (i.p.). On the other hand, for development of the cellular response and generation of T cells with inhibitory activity in relation to the humoral response, mice were immunized subcutaneously (s.c.) with SRBC emulsified with complete Freund's adjuvant or intravenously (i.v.) with low dose of SRBC (10(5)). We showed that depletion of CD4+ but not CD8+ T cells in the populations of transferred T cells abolished their inhibitory activity. The phenomenon of this suppression was dependent on a dose of T cells transferred and was antigen-specific, since T cells specific for chicken erythrocytes did not inhibit anti SRBC-response. The data indicate the importance of antigen specific CD4+ T cells in the mutual regulation of the two major type of the immune response.

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PubMed1994

Delivery of exogenous antigen into the major histocompatibility complex class I and class II pathways by electroporation.

Exogenous, nonreplicating protein antigens (Ags) are usually taken up by antigen-presenting cells (APCs) via endocytosis or pinocytosis and enter the major histocompatibility complex (MHC) class II processing and presentation pathway. Although exogenous Ags are not processed and presented in the class I pathway by most cells, soluble proteins can enter the class I processing and presentation pathway if they are introduced directly into the cytoplasm of APCs. The purpose of these studies was to determine whether exogenous proteins could be processed and presented to T cells if they were delivered into cells by electroporation. The conditions for electroporation were optimized so that the viability of the electroporated cells was high, and the majority of electroporated cells had protein incorporated. Electroporated B cells not only presented exogenous ovalbumin to CD8+, class I MHC-restricted T cells but also stimulated CD4+, class II MHC-restricted T cells. Electroporated cells also primed Ag-specific cytotoxic T lymphocytes (CTLs) in vivo, stimulated CTL precursors in vitro, and served as target cells for lysis by Ag-specific CTLs, indistinguishable from transfected cells. Thus, electropermeabilized cells were structurally intact, and the introduced exogenous protein was processed and presented in association with both class I and class II MHC molecules. This approach is as efficient and reproducible as other techniques of delivering exogenous proteins into the intracellular processing pathways. These studies suggest that electroporation could be employed for the study of cell-mediated immunity to various exogenous proteins.

Animals↗
PubMed1994

Thymocyte-macrophage interactions. Interleukin 6 produced by activated thymocytes induces interleukin 1 production by macrophages.

Coculture of paraformaldehyde-fixed macrophages with syngeneic thymocytes resulted in synthesis of a lymphokine which triggered IL-1 production in untreated, peritoneal macrophages. The release of that lymphokine was MHC restricted, however, the stimulation of IL-1 production was not MHC-controlled event. Preliminary studies showed that the lymphokine had molecular weight (MW) of around 10-30 kDa, was heat stable (56 degrees C), resistant to reduction to DTT and exhibited a weak pyrogenic activity. Fusion of activated thymocytes with the thymoma BW5147 resulted in obtaining several hybridoma clones producing the lymphokine. Preparation of culture supernatants by means of Amicon membranes and Sephadex G-100 filtration allowed to determine the MW of the lymphokine as 25 kDa. Lastly, the lymphokine was identified as IL-6 by means of anti-IL-6 antibodies which blocked its function and by the use of IL-6-sensitive cell line 7TD1.

Animals↗
PubMed1994

Presentation of antigen by B cells subsets. I. Lyb-5+ and Lyb-5- B cells differ in ability to stimulate antigen specific T cells.

We have examined the antigen presenting cell (APC) function of different B cells. Resident, peritoneal B cells from normal mice were more efficient than splenic B cells in presenting antigen to CD4+ T cell lines. Peritoneal B cells from X-linked immunodeficient (Xid) mice, by contrast, stimulated no detectable responses. Xid splenic B cells were much less efficient APC than normal splenic B cells. B cells from neonatal mice also were very poor APC until the mice were 3 to 4 weeks old. Xid B cells presented antigen to T cell hybridomas as well as normal B cells showing that they process antigen normally. Thus, the defect is most likely in providing secondary signals. The ability of B cells to present antigen efficiently correlates with the percentage of B cells reported to express the Lyb-5 antigen. Anti-Lyb-5 serum and complement abrogated the APC activity of B cells suggesting that Lyb-5+, but not Lyb-5- cells are efficient APC. We also found that activated and resting normal splenic B cells, separated by buoyant density, presented antigen equally. Both populations also contained Lyb-5+ B cells although they were a larger fraction of the activated cells. Lyb-5 is now thought to be an activation antigen rather than a differentiation antigen. If this idea is correct, then our data indicate that anti-Lyb-5 more cleanly separates activated and resting B cells than buoyant density techniques.

Animals↗
PubMed1994

Presentation of antigen by B cell subsets. II. The role of CD5 B cells in the presentation of antigen to antigen-specific T cells.

We demonstrate that peritoneal B cells have a much higher ability to present antigen to antigen-specific T cell lines than splenic B cells. Presentation of antigen by B cells is abrogated or drastically reduced after removal of Lyb-5+ cells from the population of splenic or peritoneal B cells. Peritoneal B cells, precultured for 7 days prior to the antigen presentation assay, retain their antigen presenting cell (APC) function. Enrichment for CD5+ cells in the peritoneal B cell population results in a more effective antigen presentation. Lastly, stimulation of B cells via CD5 antigen, by treatment of cells with anti-CD5 antibodies or cross-linking of CD5 receptors, enhances APC function of these cells. The results indicate, both indirectly and directly, that CD5+ B cells play a predominant role in the presentation of conventional antigens to antigen-specific T cells.

Animals↗
PubMed1994

Presentation of antigen by B cell subsets. III. Effects of interleukins on antigen presenting function and phenotype of immature B cells.

In this report we demonstrate that B cells from CBA/N or (CBA/N x BALB/c)F1 male mice with x-linked immunodeficiency, that have very limited ability to present antigen to antigen-specific T cells, acquire this function following preincubation with IL-1, IL-4 and to a lesser degree with IL-6 and IL-5. Preincubation of normal B cells with these B-tropic interleukins does not lead to enhancement of their APC function. Incubation of B cells from the peritoneal cavity and spleen of xid mice with B cell tropic interleukins (IL-1, 4, 5 and 6), but not with IL-2 or IL-3, induces appearance of Lyb-5 antigen on these cells. The study demonstrates that the property of inducing APC activity in immature B cells is correlated with the acquirement of Lyb-5 antigen.

Animals↗
PubMed1994

Presentation of antigen by B cell subsets. IV. Defective T-B cell signalling causes inability to present antigen by B cells from immunodeficient mice.

The purpose of this study was to investigate differences in T-B cell signalling between B cells from normal and immunodeficient mice. B cell blasts from normal and immunodeficient mice expressed comparable levels of membrane-associated IL-1. B cells from normal, but not immunodeficient mice, prefixed with glutaraldehyde and cultured with thymocytes or a T cell line BK33, induce in T cells production of a factor which causes release of IL-1 by macrophages. This factor, preincubated with B cells from immunodeficient mice significantly enhances their APC function. Furthermore, this cytokine induces expression of Lyb-5 alloantigen on B cells from immunodeficient mice. This effect could be blocked by neutralizing antibodies to IL-6 but not to IL-2, IL-4 or GM-CSF. We conclude that immature B cells from immunodeficient (CBA/N x BALB/c)F1 mice are unable to stimulate interacting T cells to produce IL-6 and therefore are inefficient antigen presenting cells.

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PubMed1992

Comparison of the T cell receptors on insulin-specific hybridomas from insulin transgenic and nontransgenic mice. Loss of a subpopulation of self-reactive clones.

Transgenic mice expressing the human insulin gene do not produce insulin-specific antibody after injection of human insulin. Nevertheless, they have some peripheral T cells that proliferate to human insulin in vitro. To investigate the nature of these T cells, human insulin-specific T cell hybridomas were produced from transgenic and nontransgenic mice. Transgenic hybridomas required more insulin to achieve maximum responses and they produced lower levels of lymphokines than nontransgenic hybridomas. The majority of nontransgenic hybridomas recognized only human and pork insulin whereas transgenic hybridomas recognized beef, sheep, and/or horse insulin in addition to human and pork insulin. The TCR expressed by transgenic and nontransgenic hybridomas were determined by Northern analysis. Both types of hybridomas used several different V alpha and V beta gene families and no favored association between V alpha and V beta gene usage was detected in either type. V beta 1 was used by 7 of 16 nontransgenic hybridomas but only by 1 of 16 transgenic hybridomas. V beta 6 receptors were predominantly expressed by the transgenic hybridomas and all V beta 6-bearing hybridomas recognized beef as well as human insulin. The differences in Ag reactivity and TCR gene usage suggest that V beta 1-bearing human insulin-reactive T cells were clonally deleted or inactivated in the transgenic animal. Other clones, representing a minor subpopulation in nontransgenic mice, were recovered from transgenic mice.

Animals↗
PubMed1992

Immune processing of proteolipid protein by subsets of antigen-presenting spleen cells.

Myelin proteolipid protein (PLP) contains one established antigenic epitope within the 139-151 amino acid sequence, with encephalitogenic activity for SJL mice (PLP139-151). In the current study, the processing and presentation of PLP by subsets of splenic antigen-presenting cells (APC) were examined by comparing their capacity to stimulate PLP-responsive T-cell clones, two of which are specific for PLP139-151, and one which is not specific for this peptide. In order to study whether PLP requires processing before its presentation by APC, PLP-pulsed and fixed APC were shown to stimulate PLP-specific T cells. However, the addition of PLP to unpulsed, fixed APC resulted in the absence of T-cell stimulation, while the ability of these fixed APC to bind antigenic peptide and efficiently present it to T cells, was demonstrated by their ability to use a synthetic peptide for the stimulation of the T cells. In order to study potentially different processing efficiencies among APC subsets, spleen cells were fractionated by adherence to plastic, and their respective APC activities were studied separately. The non-adherent (NAd) APC were unable to stimulate PLP139-151 specific T-cell clones with PLP as antigen. In contrast, a T-cell clone specific for a separate, but unidentified epitope on PLP was stimulated by NAd APC efficiently. In addition, stimulation of PLP139-151-specific T-cell clones by NAd APC did occur when the synthetic peptide instead of intact PLP was used as antigen, indicating a defect in PLP processing by the NAd APC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Involvement of two distinct regulatory T cell populations in the antigen-specific suppression of cytolytic T cell generation.

Alloantigen-specific, radiation-resistant T cells generated in mixed-lymphocyte cultures inhibited the generation of allospecific CTL responses in vitro. This regulatory T cell population was studied using mAb generated to Ag-specific suppressor factors that regulate the response to the synthetic terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). Both monoclonal 984 D4.6.5 and a pool of four mAb 2441, when added in the presence of complement, eliminated alloantigen-specific inhibition of the CTL response. When separate cell cultures treated with mAb 984 or 2441 plus complement were recombined, inhibition was reestablished, suggesting that two or more populations of cells are required for active inhibition. Furthermore, neither the mAb 984 nor the mAb 2441 plus complement had any effect on any stage of CTL development. This suggests that the inhibition of the CTL response was not the result of cytolytic activity via the regulatory T cells. Experiments in which these antibodies were added without complement treatment showed that the mAb 2441 neutralized the inhibitory activity, whereas mAb 984 augmented inhibition. It is concluded from these studies that regulatory T cells originally identified in humoral immune responses also regulate cell-mediated immune responses. Suppressor epitopes are displayed on the surface of these cells that allow them to be distinguished from other T cells. These data also show the utility of the mAb 984 and 2441 raised against specific suppressor T cell products in different experimental models of immunity. These studies suggest that phenotypically distinct Ts cell populations can play a normal regulatory role in both cell-mediated and humoral immunity.

Animals↗
PubMed1991

Control of interleukin 1 (IL-1) activity. I. Inhibition of IL-1 activity by soluble immune response suppressor (SIRS) in vitro.

Soluble immune response suppressor (SIRS), a nonspecific inhibitor of cellular and humoral immune responses and cellular proliferation, reversed IL-1-induced inhibition of autologous rosette formation by thymocytes. In addition, SIRS prevented the IL-1-induced increase in resistance of thymocytes to the lytic action of hydrocortisone. Kinetic experiments showed that the action of SIRS on thymocytes was rapid (less than 15 minutes), although a longer time was required to exert protective effects on thymocytes. SIRS also inhibited the stimulation of thymocyte proliferation induced by Con A and IL-1 a costimulatory assay of IL-1 activity. Moreover, SIRS inhibited the IL-1-stimulated expression of complement receptors on neonatal B cells. The inhibitory effects of SIRS were selectively directed towards IL-1, since SIRS did not interfere with induction of LAK cells by IL-2, and did not reverse inhibition of autologous rosette formation induced by factors other than IL-1, such as IL-4, a proline rich polypeptide and lactoferrin. The results presented in this report demonstrate that SIRS may be a selective inhibitor of IL-1 activity with respect to T and B cells, rendering them unresponsive to IL-1 activation and/or maturation signals.

Animals↗
PubMed1990

A peripheral mechanism preserves self-tolerance to a secreted protein in transgenic mice.

We have examined mechanisms of tolerance to circulating self-proteins in mice that are transgenic for human insulin. Normal, nontransgenic mice develop serum antibody responses when injected with human insulin in CFA; syngeneic transgenic mice do not. B cell responsiveness was assessed by immunizing with human insulin coupled to a T-independent Ag, Brucella abortus. No differences were found in the numbers of insulin-specific splenic plaque-forming cells between transgenic and nontransgenic mice suggesting that insulin-specific B cells are not tolerant in transgenic mice. Similarly, APC from transgenic and nontransgenic mice display no differences in their ability to process and present human insulin to human insulin-specific T cells in vitro. However, marked differences were detected between transgenic and nontransgenic T cells. Lymph node T cells from transgenic mice primed with human insulin provided no detectable helper activity for secondary antibody responses to human insulin whereas, lymph node T cells from nontransgenic mice did. Nevertheless, lymph node T cells from transgenic mice developed significant proliferative responses to human insulin. Lymph node T cells obtained from transgenic and nontransgenic mice were fused to BW5147 and human insulin-specific T cell hybridomas were generated. The fact that human insulin-specific T cell hybridomas were obtained from the transgenic mice suggests that these T cells were not clonally deleted. In addition, APC from transgenic mice did not stimulate human insulin-specific hybridomas from normal mice in the absence of exogenous insulin. We suggest that T cells specific for human insulin are not deleted in the thymus of transgenic mice because APC in the thymus do not bear the requisite levels of endogenous human insulin/Ia complexes. Therefore, we conclude that tolerance in the transgenic mice is preserved by peripheral mechanisms.

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