Cytotoxic T cell responses to haptenated cells IV. Requirements for in vivo priming

Hapten-specific cytotoxic T cells (CTL) can be generated in cultures containing mouse spleen cells and hapten-coupled syngeneic stimulator cells. A response to sparsely hapten-coupled stimulator cells is only obtained with responder cells from immunized H-2^k mice. Immunization was effective with hapten coupled to syngeneic, allogeneic or xenogeneic nucleated cells or membranes thereof. Hapten-coupled erythrocytes, bacteriophages or soluble proteins did not induce CTL precursors (CTL-P) nor responses of other lymphocytes which would interfere with the response of CTL-P. The results show that antigen presentation to CTL-P is very efficient in vivo. Haptens could be presented to and recognized by CTL-P only if coupled to surface membranes of nucleated cells.     

Cytotoxic T cell responses to haptenated cells. I. Primary, secondary and long-term cultures.

Cytotoxic T lymphocyte (CTL) responses were obtained in vitro to cells coupled with several different haptens. The degree of lysis of target cells was dependent on the amount of hapten coupled to stimulator and target cells. Spleen cells from normal mice responded to high-hapten density cells but not to low-hapten density cells. However, spleen cells from immunized CBA mice could be stimulated in vitro by low-hapten density cells to generate effector cells able to lyse low-hapten density cells. In vitro primed responder cells could be restimulated in the presence of the original hapten-coupled stimulator cells or in the presence of supernatant from concanavalin A-stimulated mouse or rat spleen cells. Large number of hapten-specific and H-2-restricted CTL could be generated by repeated exposure to fresh supernatant.     

Cytotoxic T cell responses to haptenated cells II. On the role of H-2 genes

Cytotoxic T cell (CTL) responses to cells coupled with high or low doses of the hapten 3-(p-sulfophenyldiazo)-4-hydroxyphenyl acetic acid (SP) were studied. CTL responses to densely coupled cells were obtained with all mouse strains tested. Only mice carrying the K^k allele responded to sparsely coupled cells. Mice expressing I-A^kbut not K^k were nonresponders. (Responder × nonresponder) F₁ hybrids responded in vitro to sparsely coupled cells from responder but not from nonresponder mice. However, responder mice could be cross-primed in vivo with SP-coupled cells from nonresponder mice. CTL responding to sparsely coupled cells were restricted to K^k. Cross-reactive lysis of target cells not carrying the K^k allele was dependent on the hapten density and the H-2 haplotype carried by the target cells.     

Fine specificity of a continuously growing killer cell clone specific for H-Y antigen.

H-Y-specific cytotoxic T cells were first cloned in soft agar and grown over a period of 8 months in media conditioned with supernatants from mouse and rat spleen cells stimulated with concanavalin A. The specificity of cloned cells and their cytolytic potential remained essentially unchanged over the entire culture period. In addition to lysing male target cells expressing H-2Db antigens, the cytolytic cells lysed also male as well as female cells expressing H-2Dd alloantigens. Seventeen out of eighteen subclones derived from the original clone revealed the same activity. The cells divide about every 17--20 h can be obtained in large quantities.     

Cytotoxic T-memory cells in virus infection and the specificity of helper T cells.

Infective influenza virus primes mice and increases at least ten-fold the level of splenic cytotoxic T-memory and precursor cells in comparison with normal mice. Intranasal virus infection or intraperitoneal injection of infective virus results in frequencies of 1-2 x 10(-4) cytotoxic T-cell precursors in spleen as determined by limiting dilution assays. With both types of immunization, T-helper cells amplifying the generation of T-killer cells are limiting, and optimal clone frequencies depend on addition of excess T-helper cells. We find that at least part of the T-helper cells amplifying the generation of cytotoxic T cells are cross reactive for the type A influenza viruses and therefore have a similar virus specificity to type A influenza-specific cytotoxic T cells (tc). Help for T-killer cells can be replaced by supernatants derived from Concanavalin A-stimulated rat spleen cells, but presence of antigen is still required to stimulate the Tc precursor or memory cells before they respond to antigen non-specific T cell-growth factor(s) present in the stimulated rat spleen cell medium.     

Cytolytic T cell hybridomas. III. The antigen specificity and the restriction specificity of cytolytic T cells do not phenotypically mix

We have obtained cytolytic T-T hybrids by fusing an H-2Kk restricted clone specific for the hapten 3-(p-sulfophenyldiazo)-4-hydroxyphenyl acetic acid (SP) with an H-2Dd-restricted clone specific for the hapten fluorescein (FL). Several hybrid clones express both parental specificities but fail to lyse SP-coupled H-2Dd and FL-coupled H-2Kk target cells. We also fused the H-2Kk-restricted, SP-specific clone with a clone which recognizes FL in the context of any class I major histocompatibility complex antigen. Again several hybrids show both parental specificities but fail to recognize SP coupled to target cells which are not recognized by the parental SP-specific clone. These findings indicate that the observed cytotoxic T lymphocyte specificities for haptens on the one hand and polymorphic as well as nonpolymorphic class I major histocompatibility complex antigenic determinants on the other hand are not carried by independent proteins.     

Autoreactive T-cell clones which suppress cytotoxic T cell responses.

Autoreactive T-cell clones (Thy 1+, CD4+, CD3+) which suppress generation of cytotoxic T lymphocytes (CTL) were established in long-term in vitro culture by stimulation with GM3-liposomes or soluble melanoma (B16) antigen composed of GM3. The T-cell receptors (TCR) of two representative clones analyzed used the same TCR alpha- and V13+ beta-chains. The clones produce only interferon gamma(IFN-gamma) but not interleukins (IL)2 and 4, despite their CD4+ phenotype, suggesting that they are not a typical TH1 or TH2 type. The clones are effectively stimulated by IFN-gamma treated (I-Ab/GM3+) B16 melanoma or I-Ab-transfected GM3+ L cells, but not by GM3-/I-Ab mutant melanoma, EL 4, or I-Ad/k-transfected L cells. This strongly suggested the involvement of GM3/class II in T-cell recognition. Antigen specificity was required for stimulation of the clones. However, once stimulated, they suppressed CTL generation in an antigen non-specific fashion. As class II+ B16 melanoma cells effectively function as antigen-presenting cells to stimulate the autoreactive suppressor T cell (Ts) clones of this type, this negative circuit between class II+ tumor cells and IFN-gamma-producing Ts would be a possible mechanism whereby tumor cells could escape the immune system.     

Cytotoxic T cell recognition of Epstein-Barr virus-infected B cells. III. Establishment of HLA-restricted cytotoxic T cell lines using interleukin 2

Epstein-Barr virus (EBV)-specific cytotoxic T cell precursors, present in the circulation of previously infected (seropositive) individuals, have been reactivated in vitro by challenging with autologous EBV-transformed cells, and the reactivated populations subsequently expanded as interleukin 2 (IL2)-dependent cell lines. These lines were dominated by T cells possessing the cytotoxic/suppressor cell surface phenotype and, when tested for effector function in chromium-release assays, demonstrated potent EBV-specific, HLA-A and -B antigen-restricted cytotoxicity even when derived from seropositive donors whose initial cytotoxic response to in vitro reactivation was relatively weak. With all the lines tested from 10 seropositive donors, strong killing of autologous EBV-transformed cells was observed in the absence of any significant lysis of autologous mitogen-stimulated lymphoblasts or of a panel of EBV genome-negative cell lines sensitive to natural killing. Furthermore, the availability of IL2-expanded effectors cell populations allowed their being tested upon a wide panel of allogeneic EBV-transformed targets such that the dominant HLA-restricted reactivities within these populations could be identified. Monoclonal antibody blocking experiments confirmed that lysis of the autologous EBV-transformed cell line by IL2-expanded effectors could be specifically inhibited (a) by pretreatment of the target cells with antibodies binding to the HLA/beta 2-microglobulin complex, and (b) by pretreatment of the effector cells with the cytotoxic/suppressor T cell-specific antibody Leu 2a.     

Immunoglobulin-specific T-B cell interaction. III. B cell activation by immunoglobulin-recognizing T cell clones

Immunoglobulin (Ig)-specific T-B cell interactions were studied in the model of T cell recognition of Ig kappa chain Ig kappa-1b allotype in Ig kappa-1-congenic rats. Using Ig kappa-1b-recognizing major histocompatibility complex (MHC)-restricted T helper clones from August rats we have shown that Ig kappa-1b+ B cells from congenic August.1b rats presented Ig kappa-1b epitope of the processed self-synthesized Ig to T clones. This interaction was found to be a bidirectional regulatory event inducing specific MHC-dependent proliferation of both interacting T cell and B cell as well as Ig(Ig kappa-1b) synthesis. Small Ig kappa-1b+ B cells were capable of inducing T clone proliferation and becoming activated in response to the same T clone. Limiting dilution analysis suggested that every tenth cell in Ig kappa-1b+ B cell population is involved in this interaction. The bystander activation of Ig kappa-1a+ B cells by T clones in the presence of irradiated Ig kappa-1b+ spleen cells, if observed, was less than the level of specific Ig kappa-1b+ B cell proliferation. In contrast to a 20-fold increase of Ig(Ig kappa-1b) levels upon stimulation of Ig kappa-1b+/1a+ B cell population from heterozygous (August x August.1b)F1 rats by T clones, a "nonspecific" increase of Ig(Ig kappa-1a) was not observed. This result demonstrates the requirement for direct T-B contact for B cell activation to occur. The data suggest a great functional potency of T-B interactions mediated by T cell recognition of Ig-derived peptide/MHC class II complexes on the B cell surface. The implication of the data for idiotypic regulation enables us to propose the existence of putative idiopeptidic network T-B cell interactions.     

Cytotoxic T lymphocyte and natural killer cell responses to non-typeable Haemophilus influenzae

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells have a key role in host defence against infectious pathogens, but their response to bacteria is not well characterized. Non-typeable Haemophilus influenzae is a major cause of respiratory tract infection including otitis media, sinusitis, tonsillitis and chronic bronchitis (especially in chronic obstructive pulmonary disease and bronchiectasis). This bacterium is also present in the pharynx of most healthy adults. The primary factor that may determine whether clinical disease occurs or not is the nature of the lymphocyte response. Here we examined the CTL cell and NK cell responses to nontypeable H. influenzae in healthy control subjects and in subjects who had bronchiectasis and recurrent bronchial infection with this bacterium. Cells were stimulated with live H. influenzae and intracellular cytokine production and release of cytotoxic granules measured. Control subjects had significantly higher levels of interferon gamma production by both CTL and NK cells, while levels of cytotoxic granule release were similar in both groups. The main lymphocyte subsets that proliferated in response to H. influenzae stimulation were the CTL and NK cells. The results suggest that CTL and NK cell responses may be important in preventing disease from nontypeable H. influenzae infection.     

Isolation of antigen-specific T cell clones from nonresponder mice

The mechanisms responsible for major histocompatibility complex (MHC)-linked unresponsiveness are still poorly understood. Here we examine the cellular events that follow when B10. A mice are immunized with cow insulin, an antigen to which they make no apparent immunologic response. Despite the fact that there is no detectable antibody or T cell proliferative response to cow insulin, we have been able to clone out responding T cells after priming and restimulating in vitro with this "nonimmunogenic" antigen. These cells are L3T4+, and co-recognize specific antigen and class II MHC gene products. The data demonstrate that "nonresponder" mice to cow insulin have both the capacity to present antigen and T cells capable of recognizing that antigen. The diversity within this population was investigated by analyzing various parameters of cellular activation. These include fine specificity of both antigen and MHC recognition, as well as recognition of allogeneic MHC and M1s determinants. In addition, the antigen-presenting cell requirements were studied. The results demonstrate that this population comprise a surprisingly heterogeneous group in terms of its repertoire of receptors.     

T cell clones which share T cell receptor epitopes differ in phenotype, function and specificity

Recently, we described a monoclonal antibody (3D6) that reacts with the T cell receptor (Ti) of the T leukemic cell line HPB-ALL and that cross-reacts with 2-10% of the T cells of normal healthy individuals. In this study we report the establishment of T cell clones that are 3D6+ but that differ in function and phenotype. These clones were established according to two different protocols: T cells of donor HY (10% 3D6+) were stimulated with the Epstein-Barr virus-transformed cell line JY. The proliferating 3D6+ T cells were enriched using a rosetting technique and cloned. T cells of donor HY were stimulated with the 3D6 antibody and subsequently expanded in recombinant interleukin 2-containing medium. This yielded 70% 3D6+ T cells which after activation with either Daudi cells or with TT in the presence of autologous non-T cells, followed by cloning, resulted in antigen-specific 3D6+ T cell clones. The 3D6+ T cell clones were also tested on their reactivity with 4 other monoclonal antibodies (1C1, 1C2, 2D4, 65) specific for the Ti of HPB-ALL. The antibodies 1C1 and 1C2 reacted with all 3D6+ T cell clones and recognize probably the same epitope as 3D6. The antibodies 2D4 and 65 reacted with two mutually exclusive subsets of T cell clones. All the anti-Ti antibodies reacted with functional epitopes, since they were able to block the function of the T cell clones. The specificity of the clones was investigated by blocking studies using monoclonal antibodies specific for different major histocompatibility complex antigens. No correlation was found between the expression of the different Ti epitopes and the specificity, the CD4/CD8 phenotype or function of the clones.     

Dendritic cells need T cell help to prime cytotoxic T cell responses to strong antigens.

Peptide-pulsed mouse dendritic cells (DC) primed peptide-specific CD8+ cytotoxic T cell responses very effectively if they expressed minor histocompatibility antigens, which could stimulate a CD4+ T helper cell response. These DC could also prime most syngeneic mice, although there was no deliberate immunization for help (the DC were prepared in syngeneic mouse serum, to avoid any response to fetal calf serum antigens). In contrast, DC were unable to prime MHC class II-deficient mice for cytotoxic responses to the classical helper-dependent antigens Qa1a and HY. More strikingly, Balb.B DC failed to prime B6 MHC class II-deficient mice for cytotoxic responses to Balb minor antigens, even though these two strains differ at more than 40 minor histocompatibility loci. When peptide-pulsed DC were prepared without enzymes (used to release DC from lymphoid tissues), they failed to prime the majority of normal syngeneic mice, even though they expressed high levels of B7 and ICAM-1 co-stimulatory molecules, suggesting that help was provided by responses to antigens in the enzyme cocktail. The enzyme treatment itself did not provide signals that could substitute for help, since DC prepared with enzymes could not prime MHC class II-deficient mice. The observation that highly immunogenic minor-incompatible DC failed to prime MHC class II-deficient mice suggests that in the absence of inflammatory signals, even strong antigens cannot stimulate CD8+ T cell responses without help.     

Isolation and characterization of human peripheral blood CD4+ T cell clones expressing gamma delta T cell receptors.

Rare T cell clones bearing both CD4 and gamma delta T cell receptors (TcR gamma delta) were obtained from human peripheral blood by cell sorting using anti-CD4 and anti-TCR delta 1 antibodies. All the clones established were reactive with anti-TcR gamma delta 1 antibody, whereas only about 20% of the clones showed reactivity with anti-delta TCS1 antibody. Unlike most CD4+ T cells bearing TcR alpha beta, all the clones tested showed lectin-dependent and anti-CD3 antibody-redirected cytolytic activity. About 60% of the clones exhibited natural killer cell-like activity. Immunoprecipitation analysis of TcR gamma delta showed that each clone expressed either a disulfide-linked or non-disulfide-linked heterodimer consisting of 37-44-kDa TcR gamma and TcR delta chains.     

Secondary IgG responses to type 3 pneumococcal polysaccharide. III. T cell requirement for development of B memory cells.

Mice primed with a thymus-dependent form of Type 3 pneumococcal polysaccharide (S3), i.e. S3 coupled to erythrocytes (S3-RBC) produces S3-specific IgG antibody after secondary challenge with S3-RBC. When mice are depleted of T cells by treatment with anti-lymphocyte serum (ALS) at the time of priming, no IgG antibody is produced after secondary challenge. In order to determine the cellular basis for this phenomenon, various combinations of T and/or B cells from ALS-treated or normal primed mice were transferred to irradiated recipients prior to secondary challenge with S3-RBC. The results indicated that T cells were required at the time of priming with S3-RBC in order to (a) prevent the induction of tolerance in S3-specific B cells in mice primed with high doses of S3-RBC, and (b) induced differentiation of IgG-producing B cell precursors to Bgamma memory cells in mice primed with low doses of antigen.     

Allergen-specific human T cell clones: derivation, specificity, and activation requirements

The interaction between allergen and immune cells plays a pivotal role in the development of human allergy. In an attempt to understand this interaction, we have studied allergen-specific T cells in vitro. These T cells are derived from rodent-allergic individuals and are specific for a major allergen found in mouse urine (MA-1). Antigen-specific, major histocompatibility complex-restricted, human T cell clones have been generated by limiting dilution from lines derived from the peripheral blood T cells of allergic individuals. Antigen (Ag)-presenting cells are necessary for this response, and they can be modulated by appropriate agents. These clones can be propagated in vitro under conditions of restimulation with Ag in the presence of Ag-presenting cells without the continuous use of exogenous interleukin-2. Most clones are CD3+ or CD4+, but one clone is CD3+ CD8+ by fluorescence-activated cell sorter and monoclonal antibody-killing data. Ag stimulation of these clones induces them to produce interleukin-2 and proliferate. These T cell clones can provide a basis for studying the structure of allergenic epitopes and the potential role of altered Ag in the induction of T cell tolerance. If the determinants of T cell allergen recognition and tolerance are solved, it might provide a basis for a new approach to the immunotherapy of allergic disease.     

Epitope specificity and Ia restriction of T cell responses to insulin in a system of complementing Ir genes: analysis with primed lymph node T cells and a long-term cultured T cell line

The antibody response of (H-2b X H-2k)F1 mice to pig insulin (PI) has previously been shown to be under the control of H-2-linked, complementing Ir genes. In addition, this response was reported to depend on the genetic background of the parental strains (Keck, K., Eur. J. Immunol. 1977. 7: 811). Here it is demonstrated that the secondary in vitro response of proliferating T cells shows the same dependence on H-2-linked Ir genes yet an influence of the background genes could not be detected. The complementing genes were mapped to the Kb, I-Ab and Kk, I-Ak regions. For restimulation of F1 T cells by PI, the Ir genes of both parental chromosomes have to be expressed in the same antigen-presenting cell, suggesting complementation at the molecular rather than at a cell interaction level. With a long-term cultured, PI-specific T cell line (ST2) of (B10 X B10.BR)F1 origin the complementation data could be confirmed by mapping the Ia restriction elements to Kb, I-Ab and I-Ak. The reactivity pattern of this line towards species variants of insulin and the isolated A and B polypeptide chains in the presence of syngeneic accessory cells suggests that the glutamic acid residue in position 4 of the A polypeptide chain (Asp in mouse insulin) is essential for recognition in conjunction with an (I-Ab X I-Ak)F1 hybrid Ia complex. I-Ab-encoded molecules carrying specificity Ia. W39 which, according to Rosenwasser, L. J. and Huber, B. T. are essential for the presentation of BI to (CBA/N X C57BL/6)F1 T cells, are not required as components of the F1-unique restriction element recognized by the F1 T cells of the ST2 line in conjunction with PI. This is indicated by the fact that accessory cells of (CBA/N X B10)F1 hybrids, regardless of their sex, could present PI as well as beef, sheep and horse insulin to the F1-restricted ST2 cells.     

Cytotoxic T cell clone-specific monoclonal antibodies used to select clonotypic antigen-specific cytotoxic T cells

Two rat monoclonal antibodies (mAb) have been produced which recognize a clone-specific determinant on the alloreactive cytotoxic T lymphocyte (CTL) clone 3 F9. CTL clone 3F9 of BALB/c origin is specific for H-2Db and can be grown by weekly restimulation with irradiated stimulator spleen cells expressing H-2Db in the presence of interleukin 2. Two mAb against T cell clone 3F9, 44-22-1(IgG2a) and 46-6 B5(IgM), have been proven to be clone specific: they inhibit cytotoxic activity of 3F9 only and bind specifically to 3 F9 when compared in a panel of different CTL clones, or cells from different mixed lymphocyte cultures (MLC), BALB/c thymus and spleen cells. The mAb 44-22-1 has been used to sort cells from a primary MLC BALB/c anti-H-2Db by fluorescence-activated cell sorter (FACS) to select CTL expressing 3 F9 clonotype-specific determinants. The lymphocytes reactive with 44-22-1 represent a minor subpopulation of the CTL of the primary MLC. The specific alloreactive cytotoxicity of unsorted lymphocytes of the bulk primary MLC could not be inhibited by the mAb 44-22-1 and 46-6 B5 whereas the sorted 3 F9 clonotype-positive cultures could be inhibited very effectively. All the CTL clones derived from the FACS-sorted clonotype-positive culture show all the same properties and are identical with clone 3 F9 with respect to antigen-specific cytotoxicity, inhibition of cytotoxicity by the mAb and surface markers.