CTL Induction Research Articles

Limiting Dilution Analysis of Alloreactive T Helper Cells: Precursor Frequencies Similar to that of Alloreactive Cytotoxic T Cells

Precursor frequencies for alloreactive T helper cells involved in the generation of primary cytotoxic responses from thymocytes were determined in splenic T cells and selected Lyt-1 lymphocytes by limiting dilution analysis. T helper precursors at frequencies ranging from 1/5000 to 1/13,500 were found in individual experiments in unsensitized selected Lyt-1 populations reacting to H-2 alloantigens. After preactivation of Lyt-1 lymphocytes with antigen in limiting dilution, the frequencies of T helper cells were increased 2–3-fold when cultured in the absence and 10–50-fold when cultured in the presence of T cell growth factor. The frenquencies for T helper precursors found in Lyt-1 cells were comparable to those of unselected T cells, indicating that a significant portion of T helper cells resides in the Lyt-123 population. Activation of T helper precursors with H-2 antigens or with H-2 and non-MHC (plus MLs) antigens resulted in similar frequencies, suggesting that the same T cell can respond to H-2 and non-MHC determinants. The data suggest that alloreactive T helper precursors exist at frequencies similar to that of CTL precursors. In addition, the results indicate that the induction of CTL by T helper cells is subject to regulation presumably by suppressive cells and that Lyt-1 inducer cells may be involved in the development of suppression for CTL responses.

Blocking of the induction and expression of immunologically functional T lymphocytes by rat antiactivated T-cell serum

The preparation of a xenogeneic rat anti-mouse CTL serum, RAT ∗, which potently inhibits CTL function in the absence of complement, has recently been described. In the present study, this serum is further characterized. The membrane antigens recognized by RAT ∗ were shown by FACS analysis to be detected most intensely on CTL, but are also present in lesser amounts on splenic T cells, thymocytes, and B cells. The T-cell proliferative response induced by Con A, PHA, TCGF, and modified self were all inhibited by RAT ∗, but no effect was seen on LPS-induced B-cell proliferation. In addition, the in vitro induction of secondary CTL was not observed in the RAT ∗-treated cultures. Finally, the effect of RAT ∗ pretreatment is shown to be irreversible. It is proposed that RAT ∗ serum reacts with receptors on T cells which are intimately involved in the “activation” step during the induction or expression phases of the response.

Regulatory T cells in pregnancy: VI. Evidence for T-cell-mediated suppression of CTL generation toward paternal alloantigens

The reactivity of spleen cells from allogeneically pregnant mice was assayed versus paternal strain target cells by a direct 51Cr-release assay. Despite multiple allogeneic parities, the lytic indexes of spleen cells were equivalent to those observed with nonpregnant controls. In view of previously obtained in vivo and in vitro results, spleen cells of allogeneically pregnant mice were added at the onset of MLC-CMLs 3 3 Abbreviation used: CML, cell-mediated lympholysis; CTL, cytolytic T lymphocyte; FCS, fetal calf serum; MLC, mixed lymphocyte culture; MLR, mixed lymphocyte reaction; NMS, normal mouse serum. of maternal strain responder cells versus paternal strain stimulator and target cells and studied for regulatory capacities. They did exert a suppressive effect, assessed by 51Cr release per culture. For the most, this effect was on the CTL induction, not the effector phase. The suppression of CTL generation was specific and mediated by a Thy 1 +, Ly 2 + cell.

Chromosome 1 locus required for induction of CTL to H-2-compatible cells in NZB mice

Chromosome 1 locus required for induction of CTL to H-2-compatible cells in NZB mice

Absorption of normal lymphocytes on allogeneic spleen monolayers: depletion of alloreactivity is due to inhibition of CTL induction by released monolayer cells.

A technique for the haplotype-specific depletion of precursors of alloreactive cytolytic T lymphocytes (CTL-P) by absorption of unprimed spleen cells on allogeneic lymphocyte monolayers has been examined. When H-2d spleen cells were incubated on monolayers of H-2b spleen cells and the nonadherent cells were cultured in mixed leukocyte culture (MLC), CTL activity generated against the monolayer haplotype was 30- to 1000-fold lower than the equivalent cultures of unabsorbed H-2d cells. Cytolytic reactvity to a 3rd-party haplotype, H-2k, was reduced no more than 6-fold. However, culture of the same cells at limiting dilution indicated that the frequency of CTL-P against the monolayer haplotype in monolayer-nonadherent H-2d populations was not significantly different from the frequency in normal H-2d spleen. It was noted that MLC of monolayer-nonadherent H-2d populations were highly cytolytic for H-2d target cells. This reaction was reduced and the ability to generate anti-H-2b CTL was restored when the nonadherent cells were treated with anti-H-2b antibody and complement to eliminate contaminating monolayer cells released during absorption, and when monolayers were prepared from spleen cells depleted of Lyt 2-bearing cells (and hence CTL-P). These data suggested that the reduction in anti-H-2b reactivity of monolayer-nonadherent cells in MLC was due to inhibition by the competing anti-H-2d reaction of contaminating, monolayer-derived CTL-P. Although fluorescent labeling of monolayer cells indicated that these cells represented only 10% of the nonadherent population, flow cytometry and further limiting dilution analysis demonstrated that cells that detached from H-2b monolayers during absorption were enriched 2-fold in T lymphocytes and CTL-P over normal spleen. The strength of the anti-H-2d reaction by a minority of monolayer cells can thus be attributed both to the higher frequency of H-2b anti-H-2d than H-2d anti-H-2b CTL-P in spleen populations and to the preferential detachment of monolayer T lymphocytes during absorption.

Noncodominant expression of target antigens recognized by human cytotoxic T lymphocytes

Human CTL that recognize MHC-controlled target determinants distinct from HLA-A, B, C, and D/DR antigens were tested in a family with nine siblings. Segregation analysis of positive CML reactions showed strong lysis of target cells of three HLA-identical siblings, inheriting the b and c MHC haplotypes ( blc ), but not of the parents or siblings inheriting only one of these haplotypes. Some cytotoxicity was seen against parental target cells, although it seemed to be qualitatively distinct from that directed against the blc targets. Studies using the competitive inhibition technique showed that cells inheriting only the b or c haplotypes were not effective in decreasing the specific cytotoxicity. Furthermore, simultaneous inclusion of inhibitor cells of both the b and c haplotypes did not hinder the specific cytotoxicity. Induction of CTL recognizing this new determinant did not occur when either antigens of the b or c haplotypes were used as MLC stimulating cells, or when antigens of both haplotypes were presented simultaneously, but on separate stimulating cells, in a three-cell MLC. These results suggest that the target determinant recognized by these unusual CTL is complex; it may be formed through interactions of two surface molecules or by genetic complementation yielding a hybrid antigen.

Specific induction of syngeneic cytotoxic T lymphocytes by solubilized tumor antigen: fractionation of the specific R-MuLV-induced leukemia antigen.

The parameters of the secondary in vitro generation of syngeneic CTL by solubilized tumor antigens have been investigated. With PM and SPM, stimulation was achieved over a 10-fold antigen concentration, 5 microgram/ml to 0.5 microgram/ml, and with whole cells the optimum R/S cell ratio was 80:1 to 100:1. Assuming a 50-fold enrichment of plasma membrane antigens over cells by purifying the PM, this observation suggests that cells are at least 200 times more efficient than PM. A specific tumor antigen that induces CTL in vitro was solubilized with sodium deoxycholate and fractionated on Ultrogel AcA34 followed by affinity chromatography on Lens culinaris lentil lectin-Sepharose 4B column. The purification scheme was shown to have enriched for the specific CTL-inducing activity. The specificity of the induction and effector phases of the reaction was assessed and the data clearly indicate that the reactions were specific. Furthermore, we ruled out the possibility that the induction of CTL by soluble tumor antigen in a secondary reaction could be due to the nonspecific induction of helper activity, which then caused a polyclonal expansion of memory cells.

The induction of virus-specific cytotoxic T lymphocytes with solubilized viral and membrane proteins.

Reconstituted membranes were prepared from detergent solubilized P815 (H-2d) tumor cell membranes and solubilized Sendai virus protein. These reconstituted membranes stimulated a virus-specific H-2-restricted secondary CTL response. Stimulating activity was dependent upon the presence of both viral and P815 protein in the same membrane and was restricted to the H-2 specificity present in the reconstituted membrane. Liposomes prepared from solubilized Sendai virus proteins and partially purified H-2 alloantigen also had activity for CTL induction. The results demonstrate the feasibility of using detergent solubilized membrane proteins to study antigen recognition by virus-specific, H-2 restricted cytolytic T lymphocytes.

Cytotoxic T Lymphocytes to Murine Plasmacytoma Cells in Allogeneic and Syngeneic Mice: H-2 Antigens Are Essential in the Induction and Effector Stages of Ctl

Abstract T cell-mediated cytotoxicity and transplantation immunity to immunoglobulin-producing and nonproducing mouse plasmacytoma cells have been investigated in allogeneic and syngeneic mice. By using the 51Cr-release assay it was found that cytotoxic activity was developed in vitro by culturing C57BL/6 spleen cells with immunoglobulin-producing plasmacytoma MOPC 11 (producer) cells that were susceptible to lysis by C57BL/6 anti-BALB/c cytotoxic T lymphocytes (CTL). In contrast, CTL were not induced when C57BL/6 spleen cells were cultured in vitro with immunoglobulin-nonproducing variant cells of MOPC 11 (nonproducer) that contained fewer H-2 antigens as revealed by the cytotoxic responses and cold inhibition tests of C57BL/6 anti-BALB/c CTL. MOPC 11 producer cells, when injected i.p. into C57BL/6 mice, were completely rejected, but the tumor was developed in C57BL/6 mice that had been previously inoculated with MOPC 11 nonproducer cells. When spleen cells from BALB/c mice that had been sensitized in vivo with MOPC 11 producer cells were restimulated in vitro with MOPC 11 producer cells, CTL could be generated to the tumor-associated antigens (TAA) of MOPC 11 producer cells. No syngeneic CTL were induced against MOPC 11 nonproducer cells. Transplantation analyses showed that, although the anti-tumor immunity against MOPC 11 producer cells was observed when BALB/c mice were primed with MOPC 11 producer cells, MOPC 11 nonproducer cells failed to prime BALB/c mice either to MOPC 11 producer or nonproducer cells. The cytotoxic activity of syngeneic CTL generated against MOPC 11 producer cells was significantly inhibited by anti-H-2d serum, suggesting the involvement of H-2 antigens on the membrane of MOPC 11 producer cells in lysis by anti-TAA CTL. These findings, therefore, indicate: 1) H-2 antigens and TAA present on plasmacytoma cells are essential in the induction and effector stages of CTL and 2) H-2 restriction of CTL exists also in plasmacytoma systems.

Inhibition of the Induction of Cytolytic T Lymphocytes with Alloantisera Directed against H-2K and H-2D Gene Products

Alloantisera directed at gene products of the H-2Kd or H-2Dd loci on the stimulator cell were shown to inhibit specifically the generation of cytolytic T lymphocytes to those antigens. Thus, masking the antigens of one H-2 locus on the stimulator cell inhibits the induction of CTL to products of that locus but does not inhibit the induction of CTL to antigens of another H-2 locus. Alloantisera inhibition of the induction of cytolytic T lymphocytes occurred with both normal and primed responder cells and also occurred when the stimulating antigens were on whole cells or purified plasma membrane. Absorption on the appropriate spleen cells removed the inhibitory capacity of these alloantisera.

Kinetics of CTL Induction by Concanavalin A

Induction of maximal CTL activity was achieved within 12 hr of exposure to Con A in vitro in various mouse lymphoid cell populations. These included spleen cells from normal unsensitized mice, spleen cells from mice previously immunized with alloantigen, and mouse spleen cells exposed to alloantigen in long-term mixed leukocyte culture (LTMLC). Although induction of maximal incorporation of tritiated thymidine was accomplished within this same period in the cells obtained from LTMLC, a much longer period of Con A exposure (greater than 24 hr) was required for freshly prepared spleen cells from normal or previously immunized mice. These findings indicate that the increased tritiated thymidine uptake induced in freshly prepared spleen cells on continued exposure to Con A beyond 12 hr is not associated with the development of cytolytic activity, and that it probably represents stimulation of subpopulations no longer present in the LTMLC population where positive selection for cells responsive to cellular alloantigens has taken place.

Macrophages Suppress CTL Generation in Rat Mixed Leukocyte Cultures

The generation of CTL in rat MLC was actively suppressed by a cell population present in spleen cell preparations from normal rats. These suppressor cells were characterized by using a variety of cell fractionation techniques. Suppressor cells were removed by passage of spleen cells through nylon wool columns or by treatment with carbonyl iron. Suppressive activity was present in the mononuclear cell fraction of spleen cells obtained by Ficoll-Hypaque density gradient centrifugation. After velocity sedimentation at unit gravity, enrichment of suppressive activity was demonstrated in the fractions containing large cells as compared to the fractions containing small cells. Populations rich in macrophages were shown to have similar suppressive activity upon CTL induction in MLC. These studies suggest that macrophages present in normal rat spleen cell preparations account for the difficulty in generating CTL in MLC prepared with rat cells.