The T-cell response to haptenated insulins. I. The proliferative response

Mice were primed with TNP-derivatized insulin, or TNP-Mycobacteria, and lymph node cells were challenged in vitro with haptenated and unhaptenated antigens. Using either priming antigen, T-cell proliferative responses could be obtained to TNP-insulin. In B10 (H-2b), mice, which are responders to beef insulin (BI), but not to pork insulin (PI), TNP-BI or TNP-PI primed a response to TNP beef and TNP pork insulins, and to beef but not pork insulin, suggesting that a proportion of the response was directed to the modified portion of the molecule. However, priming with BI resulted in responsiveness to TNP-PI, but not to PI. Also, TNP-BI stimulated an augmented proliferative response in BI-primed mice. These results suggest that TNP modification can alter the antigenicity of the carrier molecule, perhaps by enhancing weak interactions with MHC molecules on presenting cells. Finally, there was no evidence that the TNP-dependent response to TNP-pork insulin was down-regulated by suppressor cells directed at the carrier molecule.     

In vitro translation of 42 S virus-specific RNA from cells infected with the flavivirus West Nile virus.

Virus-specific proteins synthesized in BHK cells infected with the flavivirus West Nile (WN) virus and in vitro using the virus-specific infectious 42 S plus-strand RNA as messenger RNA, have been studied. Mapping of the tryptic peptides indicates that the viral core protein V2 and the viral glycoprotein V3 do not share common sequences. No tryptic peptides identifiable by mapping were obtained from the viral membrane-associated protein V1. Seven apparently virus-specific intracellular proteins were detected by comparative SDS-PAGE of mock-infected and virus-infected [³⁵S]methionine-labeled cell lysates: p_i 15, p_i 20, p_i 27, p_i 37, p_i 49, p_i 71, and p_i 100 (the index i indicates the intracellular origin of the proteins, the number gives the apparent molecular weight in 10³ daltons). The proteins p_i 15 and p_i 49 represent the intracellular forms of the viral structural proteins V2 and V3, respectively. p_i 15 and V2 are not identical but differ slightly from each other. V1 has not been detected in infected cells. Mapping has shown that the other intracellular proteins (with the possible exception of p_i 37, which has not been analyzed) are unrelated to either V2 or V3 and do not share common sequences. They represent nonstructural proteins. The total molecular weight of the apparently unrelated nonstructural and structural proteins is about 290,000 daltons. Data obtained in a number of laboratories have shown that a virus-specific 42 S RNA molecule, which is structurally indistinguishable from the viral genome, probably functions as mRNA for all virus-specific proteins in vivo. This RNA has been isolated from WN virus-infected cells and translated in vitro in the wheat germ and the rabbit reticulocyte lysate system. Digestion of the total [³⁵S]methionine-labeled proteins synthesized in vitro in either of both systems with trypsin followed by peptide mapping has shown that the great majority of the resulting peptides are present in the structural proteins V2 and V3 and vice versa. No evidence was obtained for the in vitro synthesis of nonstructural proteins. Proteins synthesized in the reticulocyte lysate were fractionated by SDS-PAGE and isolated polypeptides studied by peptide mapping. Polypeptides of molecular weights between 11,500 and 90,000 daltons were obtained. Their peptide maps indicate that all polypeptides are translated from a single initiation sequence. The map of the smallest in vitro synthesized polypeptide P_retic 11.5, having a molecular weight of 11,500 daltons, was almost identical to that of V2. The map of the largest protein synthesized in significant amounts in vitro, the protein P_retic 90, was very similar to the map of a mixture of the viral proteins V2 and V3. The analyses suggest the following gene order on the 42 S RNA: 5′-terminus-V2-V3-(V1, p_i 20, p_i 27, p_i 37, p_i 71, p_i 100)-3′-terminus. The order of genes indicated in brackets remains to be determined. Some implications of these results concerning the possible mode of translation of flavivirus-specific 42 S RNA are discussed.     

T-cell proliferative response to antigens secreted by Mycobacterium tuberculosis.

An infection model of human tuberculosis was established with C57BL/6J mice. The lymphocyte proliferative responses to antigens from Mycobacterium tuberculosis were investigated during the course of infection and compared with results obtained with a group of mice immunized with large amounts of killed bacteria. The two groups responded similarly to a number of mycobacterial antigens, but marked differences in responses against secreted antigens were found; only infected mice responded vigorously to these. The responding lymphocyte subpopulation was made up of L3T4+ T lymphocytes under restriction of the Ia molecule.     

Human T-cell hybrids with HLA-restricted proliferative response to Epstein-Barr virus-infected B cells.

Human T-cell hybrids were constructed from an HGPRT-negative mutant of the acute lymphoblastoid leukaemia cell-line CEM and an uncloned population of T cells from donor SW (SW-T; partner cell) known to have a strong specificity for the autologous Epstein-Barr virus (EBV)-transformed B cell, SWEBV. The resulting hybrids, 1A9, 1D12 and 2C8, were shown not to be cytotoxic to SWEBV, nor did they have natural killer-like (NK) activity. However, when presented with the target SWEBV in a mixed lymphocyte reaction (MLR), all of the hybrids rapidly increased their rate of proliferation by up to a factor of seven. Hybrid 1D12 also produced interleukin-2-like material (IL2) under these conditions. The hybrids did not react with the autologous PHA-blasts (SWPHA), nor with various unrelated targets. When tested against a bank of EBV-transformed B-cell targets, it was observed that the human T-cell hybrids 1A9 and 2C8 responded only to those targets bearing the antigen HLA Bw35. This response could be blocked by treating the target with the monoclonal antibody W6/32, specific for a shared determinant of the HLA-A, -B and -C antigens. Similarly, the human T-cell hybrid 1D12 reacted only against those targets bearing the antigen HLA DrW2, and this activity could be blocked by the monoclonal antibody DA6.231, specific for a common region of the HLA-DR and SB antigens. Thus, human T-cell hybrids can be produced which exhibit HLA-restricted responses to antigenic stimulation.     

An infectious clone of the West Nile flavivirus

West Nile (WN) virus is the most widespread among flaviviruses, but until recently it was not known on the American continent. We describe here design of a subgenomic replicon, as well as a full-length infectious clone of the lineage II WN strain, which appeared surprisingly stable compared to other flavivirus infectious clones. This infectious clone was used to investigate effects of 5'- and 3'-nonrelated sequences on virus replication and infectivity of synthetic RNA. While a long nonrelated sequence at the 3'-end delayed but did not prevent establishment of the productive infectious cycle, a much shorter extra sequence at the 5'-end completely abrogated virus replication. Replacement of the conserved 5'-adenosine residue substantially delayed, but did not prevent, establishment of virus infection. In all cases, the recovered virus had restored its authentic 5'- and 3'-end genome sequences. However, the presence of extensive nonrelated sequences at both 5'- and 3'-ends could not be repaired.     

In vitro proliferative response to M. leprae and PPD of isolated T cell subsets from leprosy patients

In vitro proliferative response to Mycobacterium leprae and PPD to T cell subsets, isolated by selective depletion procedure from peripheral blood using OKT4 or OKT8 monoclonal antibodies plus complement, was investigated in leprosy patients. Whole peripheral blood mononuclear cells (PBMC) developed a strong proliferative response to both M. leprae and PPD in most tuberculoid patients. This proliferation was confined to T cells, and concerned predominantly OKT4+ cells. Both antigens, however, induced a smaller, but significant proliferation oF OKT8+ cells. In lepromatous patients, proliferative response of whole PBMC incubated with M. leprae was in most cases unsignificant, at variance with PPD-induced proliferation, which was not significantly lower than that of PBMC from tuberculoid patients. In a majority of M. leprae non-responders, neither OKT4+ nor OKT8+ enriched PBMC developed a proliferative response to M. leprae. Unexpectedly in four M. leprae unreactive patients, control treatment of PBMC with complement alone restored a strong proliferative response to M. leprae. Taken together, these results suggest that in vitro unresponsiveness to M. leprae results at least in some patients, from an active suppressor mechanism but that the effector phase of such suppression does not directly involve OKT8+ T cells.     

T-cell proliferative response to human papillomavirus type 16 peptides: relationship to cervical intraepithelial neoplasia.

The incidence of human papillomavirus (HPV)-related cervical intraepithelial neoplasia (CIN) and cervical cancer is increased with immunodeficiency, but the role of immune response, including cell-mediated immunity, in disease prevention is not well understood. In this study, T-cell proliferative responses to six synthetic peptides with predicted immunogenic determinants from the HPV-16 E4, E6, E7, and L1 open reading frames were analyzed in 22 sexually active women with new-onset CIN and 65 sexually active women without cervical disease, characterized by cytology, colposcopy, and HPV testing. T-cell proliferative responses were demonstrated to all six HPV-16 peptides. Although not statistically significant, rates of reactivity to E6 (24-45) were higher among sexually active women without disease (26%) than among women with current CIN (7%), as was the overall number of peptides stimulating a response. Women with CIN may not respond to selected HPV antigens as well as women without disease do.     

Heat-killed Bacillus subtilis inhibits T-cell proliferative response to mitogens and recall antigens

Heat-killed vegetative forms of Bacillus subtilis were found to impair considerably the capacity of human T-lymphocytes to secrete interleukin-2 (IL-2) and to proliferate (in terms of [³H]thymidine incorporation) after phytohaemagglutinin (PHA) stimulation. B. subtilis was also found to interfere with T-cell proliferation induced by concanavalin A (Con A) and the recall antigen tetanus toxoid (TT). The suppressive activity was dependent on bacterial concentration, and was not ascribed to mitogen, medium-nutrient absorption or cell killing. Moreover, B. subtilis did not interfere with mitogen-induced IL-2 receptor expression on the T-cell surface. On the other hand, B. subtilis did not interfere with T-cell proliferation induced by phorbol myristate acetate (PMA) and ionomycin stimulation. All data obtained suggest the binding of B. subtilis subcomponents to — or very close to — the T-cell receptor (TCR). Identification and purification of the basic structure(s) or component(s) of B. subtilis with TCR antagonist activity in vitro will help to exploit different aspects of T-cell activity and development, and possibly, will provide a means of specific control or modification of the immune response.     

Primary proliferative and cytotoxic T-cell responses to HIV induced in vitro by human dendritic cells.

In earlier studies, primary proliferative and cytotoxic T-cell (CTL) responses to influenza virus were produced in vitro by using mouse dendritic cells (DC) pulsed with virus or viral peptide as the stimulus for syngeneic T cells in 20-microliters hanging-drop cultures. We have now adapted this system for producing primary responses with cells from non-immune donors to produce primary proliferative and CTL responses to human immunodeficiency virus I (HIV) and to HIV peptides in vitro using cells from normal human peripheral blood. All donors in this study were laboratory personnel with no history of HIV infection. DC enriched from peripheral blood were exposed to HIV in vitro and small numbers were added to T lymphocytes in 20-microliters hanging drops. Proliferative responses to virus-infected DC were obtained after 3 days in culture. After 6 days, CTL were obtained that killed virus-infected autologous--but not allogeneic--phytohaemagglutinin (PHA)-stimulated blast cells. Proliferative and CTL responses were obtained using cells from 14 random donors expressing a spectrum of major histocompatibility complex (MHC) types but the CTL, once produced, showed killing restricted by the MHC class I type. Treatment of cultures with monoclonal antibody (mAb) to CD4-positive cells at the beginning of culture blocked the development of both proliferative and CTL responses, but treatment after 5 days had no effect on the CTL activity. Treatment with MCA to CD8-positive cells at the beginning of culture did not block proliferation significantly, but treatment either before or after the 5-day culture period blocked CTL responses. Collaboration between proliferating CD4-positive cells and CD8-positive cells may thus be required to produce CTL of the CD8 phenotype. DC exposed to HIV also produced CTL that killed autologous blast cells pulsed with gp120 envelope glycoprotein. However, DC infected with whole virus did not produce CTL that lysed target cells pulsed with a synthetic peptide, which included a known T-cell epitope of gp120 (representing amino acids 111-126). DC pulsed with gp120 were a poor stimulus for the development of CTL. In contrast, DC pulsed with the peptide (111-126) stimulated both proliferative and CTL responses. The latter killed not only target cells pulsed with the peptide itself or with gp120 but also killed virus-infected autologous blast cells. CTL were again obtained reproducibly with this peptide using donors expressing a spectrum of MHC types.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro human lymphocyte proliferative responses to a glycoprotein of the yeast Saccharomyces cerevisiae.

Following reports of enhanced humoral immunity to Saccharomyces cerevisiae in patients with Crohn's disease, and identification of an immunodominant, high molecular weight glycoprotein (gp200), we have investigated the cellular immune response to this yeast in normal individuals. Following exposure to a crude saline extract (Sacc), peripheral blood mononuclear cells (PBMC) from these subjects demonstrated dose-dependent increases in tritiated thymidine incorporation, the time-course of which resembled that of the response to the known recall antigens PPD and TT. This was accompanied by increased cytotoxicity of the cultured cells for natural killer (NK)-sensitive and NK-resistant target cell lines. Furthermore, using a purified, high molecular weight, glycoprotein fraction of Sacc in culture, a dose-dependent lymphoproliferative response was again observed. Stimulation indices (SI) for thymidine incorporation by umbilical cord blood lymphocytes exposed to Sacc were low compared with those of normal adults. These results provide evidence for possible antigen-specific, cellular, immune sensitization of normal individuals to a ubiquitous dietary component.     

T-cell recognition of lysozyme. I. Localization of regions stimulating T-cell proliferative response by synthetic overlapping peptides encompassing the entire molecule

The systematic localization of the full antigenic profile for T-cell recognition of a complex multivalent protein antigen has not been accomplished. Recently, this laboratory has developed a comprehensive strategy for the localization of all the continuous antigenic (as well as other binding) sites of a protein. The strategy depends on the synthesis of consecutive overlapping peptides that together systematically account for the entire polypeptide chain of the protein. This strategy was applied here for the localization of the 'continuous' T-cell recognition sites of hen egg lysozyme. Eight overlapping peptides encompassing the entire primary structure of lysozyme were synthesized and examined for their ability to stimulate in vitro proliferation of T cells from two high responder mouse strains (H-2k and H-2q) that had been primed with lysozyme. This approach enabled the identification of a full profile of in vitro active lysozyme peptides and the localization of four major T-cell recognition sites.     

In vitro proliferative response of BALB/c mouse spleen cells stimulated with trinitrophenylated syngeneic spleen cells.

Spleen cells from normal BALB/c mice showed in vitro proliferative response against hapten-conjugated syngeneic spleen cells. Trinitrophenylated (TNP) spleen cells were prepared by treating normal spleen cells with sodium 2,4,6-trinitrobenzenesulphonate (TNBS). Four-day cultures of TNP-labelled spleen cells incorporated 2.5-7.4 times more [3H]thymidine than similar cultures of untreated spleen cells. An obviously positive mixed lymphocyte reaction (MLR) by normal spleen cells against mitomycin C (MC) treated TNP-labeled syngeneic spleen cells was observed after 4 days of culture. The MLR to TNP-labelled syngeneic cells was inhibited in the presence of epsilon-TNP-L-lysine by 23-37%. The spleen cells from the mice injected intraperitoneally with TNP-labelled syngeneic spleen cells showed a higher MLR against TNP-labelled spleen cells than normal spleen cells. The sensitized spleen cells also showed an increased response to MC-treated spleen cells. These results suggest that normal spleen cells include cells which can recognize the hapten and new antigenic determinants introduced into syngeneic spleen by chemical modification.     

Murine epidermal antigen-presenting cells in primary and secondary T-cell proliferative responses to herpes simplex virus in vitro.

The role of epidermal Langerhans' cells in infection with herpes simplex virus (HSV) was investigated using a culture system that supports antigen-specific primary and secondary T-cell proliferative responses. Epidermal cell suspensions were capable of restimulating the response of in vivo primed T cells to UV-inactivated HSV. This capability was also present in cell suspensions enriched for Langerhans' cells, but was abrogated by the depletion of I-A-bearing cells. The magnitude, kinetics and phenotype of the responding cells were similar to those elicited when HSV was presented to primed T cells by antigen-presenting cells from the spleen. In marked contrast, whereas splenic antigen-presenting cells induced strong antigen-specific proliferation of unprimed T cells (primarily of the helper phenotype), Langerhans' cells failed to invoke any detectable reaction of such cells.     

In vitro response of human fibroblasts to commercially pure titanium.

The generation of metal particles through surface wear of prosthetic joints has been associated with biological reactions that may lead to prosthetic component loosening. The role of the macrophage in these reactions has been studied extensively, but that of the fibroblast has not. The few fibroblast studies that there have been have shown that particles of several metals, with sizes over a wide range, can promote cytokine release and may cause cell necrosis. The intent of this study was to determine if there are metal particle exposure threshold levels that result in morphological changes and cell necrosis of fibroblasts in peri-articular tissues. Retrieved human fibroblasts (superior medial plica) were cultured in standard fashion and then were exposed to various particle dosages of commercially pure Titanium (cpTi). Cell morphological changes and necrosis were observed to occur when the total mass of the particle dosage exceeded a threshold level. These data imply that these cell responses occur at threshold levels of wear particle exposure.     

Human gamma/delta T-cell response to Listeria monocytogenes protein components in vitro.

Listeria monocytogenes is a facultative intracellular pathogen that replicates inside mononuclear phagocytes and induces specific cellular immunity. Listeriosis encompasses many clinical syndromes and meningitis is the most frequent clinical manifestation. Human alpha/beta and gamma/delta T cells have been shown to respond to L. monocytogenes antigens and to play an important role in resistance against listerial infection. We investigated the nature of listerial ligands and the influence of the major virulence factor, listeriolysin (hly), on the stimulation of human gamma/delta T cells from healthy individuals. We found that a listerial somatic protein ligand, which is sensitive to proteinase treatment, stimulated gamma/delta T cells in vitro; the majority of Listeria-responsive gamma/delta T cells expressed V gamma 9V delta 2 T-cell receptor chains and human leucocyte antigen-DR molecules; gamma/delta T-cell responses to hly+ and hly- Listeria strains were comparable; L. monocytogenes strains of different virulence stimulated gamma/delta T cells equally. Thus, protein components of L. monocytogenes unrelated to virulence activate human gamma/delta T cells in vitro.     

Lymphatic mast cells in response to in vitro stimulation by non-specific T-cell mitogens.

Activation of lymph nodes by the T-cell mitogens PHA and Con A is correlated with a depletion of lymphatic mast cells. This result, and our earlier reports on the depletion of mast cells in lymph nodes stimulated by allogeneic and tumour cells, as well as on the elevation of mast cell number in thymus-less 'nude' mice, lead us to the conclusion that antigen- or mitogen-stimulated T lymphocytes produce lymphokine(s) which degranulates mast cells and/or is responsible for their negative chemotaxis.     

In vitro T-cell responses to beta-lactam drugs in immediate and nonimmediate allergic reactions.

BACKGROUND: beta-Lactam drugs may induce both cellular and humoral allergic reactions, and there is evidence that T cells play an important role in the pathogenesis of these reactions. The aim of this work was to assess the sensitivity and specificity of the lymphocyte transformation test (LTT) as an in vitro diagnostic tool, in patients with either an immediate or a nonimmediate reaction to penicillin G and/or amoxicillin. METHODS: Fifty patients with a well-documented history of allergic reactions to beta-lactams (31 immediate and 19 nonimmediate) were studied by means of skin tests (prick and intradermal), radioallergosorbent test (RAST), and, when necessary, controlled administration of the drug. Twenty-eight healthy subjects with good tolerance to penicillins served as controls. LTT was performed in all subjects. RESULTS: Skin tests were positive in 77.4% of the patients with immediate reactions and in 36.8% of those with nonimmediate reactions. The overall sensitivity of LTT in the allergic patients was 62%, but, when analyzed separately, sensitivity was 64.5% for the immediate group and 57.9% for the nonimmediate group. The LTT specificity was 92.8%. CONCLUSION: The LTT should be considered a useful in vitro diagnostic tool to identify subjects allergic to penicillins, especially patients with nonimmediate reactions where the LTT has a better diagnostic value than skin tests. Interestingly, positive T-cell proliferative responses can be observed 10 or more years after the occurrence of the reaction without further exposure to the drug.     

Adrenergic regulation of lymphocyte proliferative response in cultures with T-cell mitogens

The effects of epinephrine, β-adrenergic agonist terbutaline sulfate, and cAMP phosphodiesterase inhibitor theophylline on proliferative response of peripheral blood lymphocytes from healthy subjects were studied in cultures with phytohemagglutinin and concanavalin A. Both adrenergic agonists inhibited lymphocyte blastogenesis, but the effect of epinephrine was more pronounced. Translated fromByulleten' Eksperimental' noi Biologii i Meditsiny, Vol. 128, No. 8, pp. 207–209, August, 1999     

Murine epidermal antigen-presenting cells in primary and secondary T-cell proliferative responses to a soluble protein antigen in vitro.

The capacity of epidermal cells (EC) to present antigen to primed and non-immune T cells was investigated using a culture system that supports antigen-specific primary and secondary proliferative responses. Although both naive and bovine serum albumin (BSA)-immune T cells reacted against BSA in the presence of either splenic or epidermal antigen-presenting cells (APC), important differences were noted in the kinetics and the magnitudes of the various responses. Most conspicuous was the relatively poor primary response supported by EC which evidently elicited very few BSA-immune T-helper cells. Despite this, the primed antigen-specific T cells recovered were phenotypically similar to those resulting from the stronger primary responses induced by spleen cells. In contrast to this disparity in the ability to prime, EC and spleen cells stimulated secondary reactions of comparable magnitude. We therefore consider that, in comparison with splenic APC, EC may require some additional stimulus to acquire the capacity to prime.