T cell-mediated cytotoxic immune responsiveness of chimeric mice bearing a thymus graft fully allogeneic to the graft of lymphoid stem cells

Fully allogeneic, chimeric mice were established by adult thymectomy of (A × B) F1animals, grafting parental A-type thymus under the kidney capsula, followed by lethal (900 rd) irradiation and reconstitution with B parental-type bone marrow cells treated with xenogeneic anti-T cell antiserum plus complement. Following in vivo sensitization with inactivated Sendai virus (SV) suspensions, no virus-specific T cells could be detected within the spleen cells of the mice. Upon stimulation with third-party allogeneic cells in a primary mixed lymphocyte culture, spleen cells of all animals generated alloreactive cytotoxic T lymphocytes (CTL). More interestingly, upon secondary in vitro stimulation …

Depressed Prostaglandin Release from Peritoneal Cells Induced by a T Cell Adjuvant, Lentinan

Abstract PGE and PGF release from peritoneal exudate cells was studied in mice after injection with two s (1–3) glucans, the antitumor active lentinan and the inactive pachyman. 4 days after injection of both polysaccharides, the spontaneous and phagocytosis-induced PGE and PGF release was markedly suppressed. However, only the immunopotentiator lentinan induced peritoneal exudate cells which exhibited a longer lasting diminished PG release. The data suggest that the T cell adjuvant lentinan may potentiate cellular immune responses by reducing synthesis of immune suppressive prostaglandins from peritoneal exudate cells.

Impact of thymus on the generation of immunocompetence and diversity of antigen-specific MHC-restricted cytotoxic T-lymphocyte precursors.

The role of Interleukin-2 during the activation of cytotoxic T-lymphocytes

Der vorliegende Artikel stellt eine Ubersicht dar Uber die derzeitigen Vorstellungen des Zusammenwirkens zellularer und humoraler Faktoren, die zu T-Zell-vermittelten zytotoxischen Immunreaktionen fuhren. Da im Ablauf derartiger Immunreaktionen hormonahnliche Wachstumsfaktoren (interleukine) eine entscheidende Rolle spielen, liegt der Schwerpunkt der Diskussion auf der Beschreibung dieser Mediatoren; insbesondere wird die Bedeutung von Interleukin-2 (Il-2) diskutiert. Il-2 ist ein losliches, nicht antigenspezifisches Glykoprotein mit einem Molekulargewicht von 15 000 Dalton (humanes Il-2) bzw. 30 000 Dalton (murines Il-2). Es wird in vitro von T-Helfer-Lymphozyten sezerniert, die in der Mau…

T-cell-derived helper factor allows in vivo induction of cytotoxic T cells in nu/nu mice

T-cell immunocompetence and diversity are thought to be generated in the thymus1,2. This view is based on the findings that (1) T-cell ontogeny is thymus dependent3,4, (2) the major histocompatibility restrictions of T-cell interactions are phenotypically related to the H–2 type of the thymus5–9, and (3) the phenotypic manifestation of H–2-linked immune responsiveness parallels the restriction elements selected in thymus10–12. However, it is unclear whether pre-thymic cells programmed to develop into T cells do already express a receptor diversity, also whether pre-thymic cells have the potential to react against self-antigens, and whether the mechanism of self-tolerance is initiated in the…

H-2-linked murine cytotoxic T cell responses specific for sendai virus-infected cells

CBA (H-2k) mouse-derived lymphochoriomeningitis virus and herpes simplex virus-specific cytotoxic T lymphocytes lyse virus-infected target cells compatible on either the H-2k or H-2D region. In contrast, CBA, C3H and AKR (H-2k) mouse-derived sendai virus-specific cytotoxic T lymphocytes (CTL) fail to lyse H-2D-compatible virus-infected cells. A similar lack of H-2D region-associated lytic activity was found with C57BL/6 and C57BL/10 (H-2b) mice as well as with the recombinants B10.A (2R) [Kb-Db] and B10.A (4R) [Kk-Db]. On the other hand, BALB/c (H-2d) mice and A/J (H-2a) mice do generate H-2Dd-associated sendai virus-specific CTL. These results are in contrast to those obtained with (CBA X …

Generation of Cytotoxic T Lymphocytes Against Ly Alloantigen

Cytotoxic T lymphocytes specific for immune alloantigens controlled by alleles of the Ly system have been induced in vivo. These results were obtained either in a secondary type of response or by treating mice before immunization with a single dose of cyclophosphamide (80 mg/kg).

Interleukin 2 Induction in Lyt 1 + 23 − T Cells from Listeria monocytogenes -Immune Mice

Peritoneal exudate T lymphocytes from mice experimentally infected with the intracellular bacterium Listeria monocytogenes secreted high interleukin 2 activities after interaction with syngeneic normal macrophage presenting listerial antigen in vitro. L. monocytogenes -immune cells secreting IL 2 were radioresistant and bore the phenotype Thy 1 + Lyt 1 + 23 − .

Shared determinants between virus-infected and trinitrophenyl-conjugated H-2-identical target cells detected in cell-mediated lympholysis

Infection of H-2-identical mice with either lymphocytic choriomeningitis (LCM) virus, vaccinia virus, or paramyxo (Sendai) virus resulted in the generation of specifically sensitized cytotoxic T lymphocytes (CTL). CTL generated in vitro against 2,4,6-trinitrophenyl (TNP)-conjugated syngeneic stimulator cells were specifically cytotoxic for TNP-conjugated H-2K (D) region identical targets. Both LCM and vaccinia-induced CTL, however, were found to be strongly cytotoxic towards TNP-conjugated, H-2K(D) region-identical target cells. In contrast, Sendai virus-induced CTL did not lyse TNP-conjugated, syngeneic target cells. Inhibition experiments using cold targets suggested that shared antigenic…

Fine specificity and cytolytic activity of continuously growing alloreactive cytotoxic T lymphocyte clones.

The role of Lyt 1+ T-cell-derived secondard CTL inducing factor allowed the cloning of alloreactive cytotoxic T lymphocytes (CTL) by the limiting dilution approach. Several monoclonal cell lines were established in vitro. The lytic activity of some of the cell lines exceeded that of CTL from bulk cultures; that is, 50% of the target cells were lysed at an effector to target cell ratio of 0.04:1. The fine specificity of individual CTL clones is discussed.

Growth, interleukin-2 production, and responsiveness to IL-2 in T4- positive T Lymphocyte populations from malignant cutaneous T cell lymphoma (Sezary's syndrome): the effect of cyclosporin A

Abstract Functional analysis and surface phenotyping using monoclonal antibodies have revealed that malignant T lymphocyte populations in the peripheral blood of patients with Sezary's syndrome resemble the T helper cell populations from normal individuals. In this article we have studied the effects of the immunosuppressive drug cyclosporine A (CsA) on growth, interleukin-2 (IL-2) production, and the induction of IL-2 responsiveness of peripheral blood monocytes (PBMs) from five patients with Sezary's syndrome in vitro, using the lectin phytohemagglutinin (PHA) and the phorbol ester phorbol myristate acetate (PMA) as stimuli. The following results were obtained: PHA-induced cell proliferat…

Frequency-Analysis of Precursors of Cytotoxic T Lymphocytes in Radiation Chimeras: Enumeration of Antigenspecific CTL-P Restricted to Thymic MHC- and Bone Marrow-MHC-Determinants

The mechanisms controlling the acquisition of T cell restriction specificity and immunocompetence are, despite of numerous investigations, not well understood. From studies of the CTL-immune responsiveness in thymus- and bone marrow-grafted chimeric mice, it became apparent, that it is the thymus which is crucial not only for the maturation or T cells, but also for the specificity repertoire of the T cells (1,2). From these data it was suggested, that during intra-thymic maturation both mutational events and positive selection mechanisms influence the repertoire such that only T cells restricted to thymic epithelial cell MHC determinants mature and will be exported to the peripheral lymphoi…

Virion Antigens Introduced Exogeneously into the Cell Membrane Render Syngeneic Target Cells Susceptible for T Cell-Mediated Cytolysis

Non-infectious sendai virus renders H-2 matched target cells susceptible to the lytic effect of sendai virus immune cytotoxic T lymphocytes. This observation suggests that exogeneous insertion of virion antigen in the membrane of the target cell is sufficient for T cell cytotoxicity. The finding is incompatible with the concept that H-2K or H-2D gene products of the target cells must be altered in their primary structure (pretranslational effect of the virus infection) for T cell-mediated cytolysis to occur.

Influenza virus-specific T cell-mediated cytotoxicity: integration of the virus antigen into the target cell membrane is essential for target cell formation.

This study deals with the requirements for target cell recognition by influenza A virus-specific cytotoxic T lymphocytes (CTL). H-2-identical cells were incubated with infectious or UV light-inactivated influenza A virus expressing either cleaved or uncleaved hemagglutinin (HA). Thereafter, the treated cells were tested in a 4-h 51Cr assay for susceptibility to CTL-mediated cytolysis. Regardless whether the influenza virus was infectious, virions expressing cleaved HA were efficient in target cell formation. In contrast, cells incubated with either active or UV-inactivated virions expressing uncleaved HA were not lysed by virus-specific CTL. Yet, after mere trypsin-mediated cleavage of the …

T cell-mediated cytotoxicity: discrimination between antigen recognition, lethal hit and cytolysis phase.

Using a 51Cr release cytotoxicity assay, the cytotoxic effector phase of in vitro activated mouse T lymphocytes (killer cells) against 51Cr-labeled target cells has been investigated. It is shown that within 5–10 minutes of contact between killer cells and target cells, the target cells are already committed to lysis, therefore, antigen recognition and “lethal hit” must have taken place within this period of time. In contrast, target cell lysis (cytolysis phase) requires up to 3–4 h in order to be completed; it occurs independently of killer cells and it is highly temperature dependent. The killer cell-dependent phase (antigen-recognition and “lethal hit”) is dissociated into two consecutiv…

The in Vivo Effects of Interleukin 2 (TCGF)

This brief review of our experiments concerning the in vivo activity of crude Il-2 led us to the following conclusion: The first is the existence, in vivo, of a cyclophosphamide-sensitive T-cell controlling the activity of a serum born Il-2 inhibitor in thymus-bearing normal mice. Under in vivo conditions which are characterized by high Il-2 inhibitor activities, locally applied Il-2 administered along with antigen amplified in vivo CTL-responsiveness, yet the effect observed was poor. Crude Il-2 proved to be a potent immuno-enhancing agent in the athymic (nu/nu) mouse, which lacks Il-2 inhibitor activity. It was found that together with antigen administration of Il-2 to nude mice results i…

T cell proliferation in the mixed lymphocyte culture does not necessarily result in the generation of cytotoxic T effector cells.

It was tested whether T lymphocytes, when stimulated in vitro by M locus-coded lymphocyte activating determinants (LAD), are able to mediate cytotoxic effector functions. The assay for cytotoxicity included both the use of purified appropriate target cells (i.e. purified lipopolysaccharide blasts) as well as the use of phytohemagglutinin dependent cytolysis as a model for detecting cytotoxic T lymphocytes (CTL). Although strong proliferative responses were obtained in the mixed lymphocyte culture, the T cell blast generated did not display any detectable cytotoxic effector function. Thus, it is concluded that LAD, at least in the M locus-dependenet system, do have the capacity to induce T c…

Anti H-2Dd alloreactivity mediated by herpes-simplex-virus specific cytotoxic H-2k T lymphocytes is associated with H-2Dk.

Herpes-simplex-virus (HSV) specific, H-2k-restricted, immune cytotoxic T lymphocytes also lyse noninfected H-2d target cells. Genetic mapping studies revealed that HSV-specific Dk-restricted CTL cross-react with allogeneic targets expressing Dd alloantigens. Cold target inhibition experiments indicate that only a minority of HSV-specific CTL mediate cross-reactive cytolysis. The data give an example of where the phenomenon of H-2-restricted versus nonrestricted responsiveness is not due to distinct subsets of T cells but solely depends on the antigenic determinants recognized.

Cyclosporin A mediates immunosuppression of primary cytotoxic T cell responses by impairing the release of interleukin 1 and interleukin 2

The site of action of the immunosuppressive drug cyclosporin A in in vitro cytotoxic allograft responses has been localized. General cytotoxic effects of the drug on proliferating T cells became apparent at concentrations of 500-1000 ng/ml, while selective effects were observed at concentrations of 10-100 ng/ml. The selective effects included a blockade of interleukin 2 release from activated T helper cells on the one hand and inhibition of interleukin 1 release from splenic adherent cells on the other. While cyclosporin A did not interfere with the intracellular events required for the activation and subsequent clonal expansion of alloreactive T cells, the lack of interleukin 1 and interle…

T-T cell collaboration during in vivo responses to antigens coded by the peripheral and central region of the MHC.

MIXED lymphocyte culture (MLC)1 has been used extensively as an in vitro model to analyse the reactivity of T cells to antigens coded by the major histocompatibility complex (MHC). When murine T responder cells are exposed in vitro to allogeneic lymphoid cells (stimulator cells) they proliferate and cytotoxic T lymphocytes (CTL) are generated2,3. Antigens coded by the central I region of the MHC are chiefly responsible for triggering proliferation4,5, whereas the target antigen of the CTL generated is either a H–2K or H–2D region or a I–A subregion gene product5–8. This dichotomy in the antigenic requirement of a MLC seems to be reflected at the level of the responding T lymphocytes. Two di…

T-T cell interactions during in vitro cytotoxic T lymphocyte responses. III. Antigen-specific T helper cells release nonspecific mediator(s) able to help induction of H-2-restricted cytotoxic T lymphocyte responses across cell-impermeable membranes.

T helper cell induction and the specificity of T cell-mediated help as generated during alloreactive and H-2-restricted, virus- or hapten-specific cytotoxic T lymphocyte (CTL) responses have been compared. With the use of a double-chamber culture system, it was possible to dissect and separately analyze the induction phase of T helper cells from the T helper cell effector function. The data obtained revealed that during alloreactive as well as H-2-restricted T cell responses, antigen-specific T helper cells are induced. Upon specific restimulation of T helper cells, helper cell function is mediated across a cell-impermeable membrane via soluble products in an apparently nonspecific and nonr…

T-CELL-MEDIATED CYTOLYSIS : FROM THE LYSIS OF H-2 NEGATIVE TARGET CELLS TO THE INDUCTIVE EFFECT OF XENOGENEIC SERUM

Publisher Summary This chapter describes the results of a series of experiments. In the first series, evidence was found in favor of some specificity of cytolysis against embryonic antigens at the surface of the F9 cells. In a second series of experiments, it was found that P815 and EL4 cells were lysed. From a systematic investigation of the parameters of this system, two facts emerged. F9 cells were not necessary during the in vitro boost incubation. There was some preferential lysis of targets having the same H-2 as the mice receiving the H-2-less F9. For instance, if F9 cells were injected into B1O mice, and after 2–3 weeks, the spleen cells were incubated for 5 days in conventional tis…

Quantitative representation of all T cells committed to develop into cytotoxic effector cells and/or interleukin 2 activity-producing helper cells within murine T lymphocyte subsets

A limiting dilution culture system based on stimulation with concanavalin A (Con A) has been used to study the quantitative distribution of helper and of cytotoxic precursor cells in Lyt-2-defined subpopulations of murine T cells. Virtually all of the selected Lyt-2+ and Lyt-2-T cells grow and expand to large clonal colonies within an 8-9-day culture period. Our data show that upon stimulation with Con A, 90% of the Lyt-2-T cells were capable to produce interleukin 2 (IL 2) activity. In addition, IL 2 activity is produced by 8-10% of Lyt-2+ T cells. However, at the clonal level, the average of the IL2 activity produced by Lyt-2+ T cells is about 8-fold less as compared to Lyt-2-T cells. Pre…

Deficiency in the Transcription Factor Interferon Regulatory Factor (Irf)-2 Leads to Severely Compromised Development of Natural Killer and T Helper Type 1 Cells

Interferon (IFN) regulatory factor (IRF)-2 was originally described as an antagonist of IRF-1–mediated transcriptional regulation of IFN-inducible genes. IRF-1−/− mice exhibit defective T helper type 1 (Th1) cell differentiation. We have used experimental leishmaniasis to show that, like IRF-1−/− mice, IRF-2−/− mice are susceptible to Leishmania major infection due to a defect in Th1 differentiation. Natural killer (NK) cell development is compromised in both IRF-1−/− and IRF-2−/− mice, but the underlying mechanism differs. NK (but not NK+ T) cell numbers are decreased in IRF-2−/− mice, and the NK cells that are present are immature in phenotype. Therefore, like IRF-1, IRF-2 is required for…

Randomized clinical study on intratumoral BCG-cell wall preparation (CWP) therapy in patients with squamous cell carcinoma in the head and neck region

Based on animal experiments a clinical study with BCG cell wall preparation (CWP) was developed. Patients with head and neck carcinomas stage T1/2N0–2M0 were randomized. One group received surgical treatment only and a second group received preoperative intralesional BCG-CWP. So far 12 patients have been included in each group. After 3 years the CCR (complete cancer remission) in the surgery only group was 39% and that in the preoperative BCG-CWP group, 69% (P=11%). The cumulative proportion of surviving patients was 50% in the surgery only and 73% in the BCG-CWP group (P=21%). BCG-CWP injection was followed by an increase in body temperature and a decrease in peripheral blood lymphocytes. …

Different response of TH1 cells for stimulation with anti-CD3 antibodies.

In this report, evidence is provided for a further subdivision of CD4+ T helper cell lines. The earlier definition of the TH1 and TH2 subtypes was confirmed by their differential response to interleukin (IL) 1. An additional subdivision of the TH1 subset was revealed when TH1 cell lines were costimulated with anti-CD3 antibodies and IL2. The IL2-induced proliferation of three of the resulting TH1 lines was blocked by anti-CD3 antibodies. By contrast, no such block was observed in a fourth TH1 cell line. In all four lines anti-CD3 triggering caused production of IL2. The block of proliferation was reversed neither by antigen-presenting cells nor by phorbol 12-myristate 13-acetate, a protein …

T-T cell interactions during cytotoxic T cell responses. IV. Murine lymphoid dendritic cells are powerful stimulators for helper T lymphocytes.

Enriched populations of Ia+ Fc receptor-negative dendritic cells were compared to other cell types for their stimulatory activity in primary mixed lymphocyte reactions to alloantigens and 2,4,6,-trinitrophenylated syngeneic cells. Dendritic cells were 20-100 times more effective than unfractionated splenocytes. A second cell type exhibiting strong stimulatory activity was an Ia+ Fc receptor-positive transiently adherent cell. Both types of stimulatory cells were only effective when able to produce the monokine interleukin 1. Thus glutaraldehyde-fixed cells were not stimulatory unless extraneous interleukin 1 was added. Stimulation of helper cells by either dendritic cells or Ia+ Fc receptor…

Alloreactive and H-2-restricted Lyt 23 cytotoxic T lymphocytes derive from a common pool of antecedent Lyt 123 precursors.

If the collaborative requirement of Lyt 1 T helper cells is bypassed by the Lyt 1 T cell-derived mediator of T help, termed Il-2, upon antigenic stimulation, PNA+ Lyt 123 thymocytes differentiate into either alloreactive or H-2-restricted PNA- Lyt 23 cytotoxic effector cells. Along the differentiation pathway from Lyt 123 leads to 23 effector cells, cytolytic activity is carried out by T cells that still express the Lyt 123 phenotype. The data establish that Lyt 23 CTL are produced by differentiation from antecedent Lyt 123 cells.

T-cell-derived helper factor allows Lyt 123 thymocytes to differentiate into cytotoxic T lymphocytes.

IT is generally accepted that the diversity of T-cell responsiveness is generated in the thymus1. It is also known that except for a few Lyt 1 cells all thymocytes express the Lyt 123 phenotype2,3. Surprisingly, thymocytes are poorly responsive in vitro4, and only the medullary thymocytes, comprising 5–10% of the total thymic cell population, show an in vitro responsiveness comparable with that of peripheral T cells5. Cortical thymocytes, comprising 90–95% of all thymocytes, have previously been considered to be immature and immunologically incompetent4. The result reported here show that thymocytes are able to generate alloantigen-, virus- and hapten-specific cytotoxic T lymphocytes (CTL),…

Secondary cytotoxic allograft responsein vitro. I. Antigenic requirements

The antigenic requirementsfor in vitro induction of secondary murine cytotoxic allograft responses were tested. The proliferative responses were assayed by the [3H]thymidine uptake technique; the generation of cytotoxic T lymphocytes (CTL) was tested in a 51Cr-cytotoxicity assay. Spleen cells from normal or alloantigen preimmunized CBA mice (H-2k) were used as responder cells. Allogeneic x-irradiated splenic lymphocytes (normal stimulator cells) were UV light treated, heat treated or glutaradehyde fixed and subsequently tested for their capacity to induce CTL in a primary or secondary mixed lymphocyte culture (MLC). In addition allogeneic fibroblasts were tested as stimulator cells. The res…