Progressive changes in cytotoxic potential during mixed lymphocyte culture.

In a previous study it was shown that at least one round of DNA synthesis is required for initial expression of cytotoxic function in mouse lymphocytes responding to alloantigen in vitro. In the experiments reported here we ask whether subsequent rounds of cell division are required simply for clonal expansion of this initial level of cytotoxic function within the population, or whether the amount of cytotoxicity per cytotoxic cell is altered during subsequent rounds of cell division. The amount of cytotoxicity per unit number of cells at various stages of culture was compared with the frequency of cytotoxic cells as estimated principally by effector-target cell conjugates. Our results strongly suggest that the amount of cytotoxicity per cell (cytotoxic potential) is not a static property of cytotoxic cells, but can be modulated up or down during the course of a reaction.     

Helper cells in the autologous mixed lymphocyte reaction. I. Generation of cytotoxic T cells recognizing modified self H-2

The autologous mixed lymphocyte reaction (AMLR) in mice measures the proliferative response of T cells to determinants on syngeneic non-T spleen cells. Normally, cytotoxic T lymphocytes (CTL) are not generated in this reaction. However, the addition of trinitrophenyl-modified mitomycin C-treated syngeneic T cells (TNP-Tm) to the AMLR results in the generation of TNP-specific CTL but does not alter the proliferative response. Significant cytotoxic activity is not detectable against TNP in association with Ia unless TNP is present on cells bearing those determinants. Thus, if unselected spleen cells are TNP-modified and used as stimulators in the AMLR, the proliferative response is enhanced and CTL are generated that recognize TNP in association with K, D, and I region-encoded determinants. The CTL generated in the AMLR are H-2 restricted and dependent on the presence of adherent cells in the sensitization cultures. The experiments presented here suggest that the AMLR can provide the help necessary for generating cytotoxic T cells in vitro.     

Activation of suppressor T cells in human autologous mixed lymphocyte culture.

Co-culture of autologous T and mitomycin-C treated B cells results in increased DNA synthesis in the responding T cells. T cells thus activated in AMLC exerted suppressive effects on both the proliferative and cytotoxic responses of fresh unstimulated T cells to allogeneic cells in MLC. The suppressor cells generated are sensitive to treatment with mitomycin-C. AMLC-activated cells treated with mitomycin-C failed to suppress both cytotoxicity and proliferation in fresh primary MLC. It appears that the AMLC reaction reflects an immunologic homeostatic mechanism. Since this reaction is defective in patients with CLL and SLE and the homologous mouse syngeneic MLC is defective in NZB mice, the failure of this T-B interaction may be related to the pathogenesis of certain lymphoproliferative and autoimmune disorders.     

Cytotoxic T cells generated in the autologous mixed lymphocyte reaction. I. Primary autologous mixed lymphocyte reaction

In the course of the culture of an autologous mixed lymphocyte reaction (AMLR), T cells proliferated in response to autologous non-T cells, and differentiated to cytotoxic T cells (AMLR killers). DNA synthesis was necessary to generate AMLR killers, as the elimination of autoreactive proliferating cells with BUdR and UV light completely abrogated AMLR killer cytolysis. Amlr killers lysed various lymphoid cell lines, including autologous B cell lines, autologous or allogeneic mitogen blasts stimulated by Con A, PHA, or pokeweed mitogen, variious nonlymphoid cell lines derived from human, mouse, or rat, and weakly normal autologous or allogeneic non-T cells. KMT-17, methylcholanthrene-induced rat fibrosarcoma, was the only resistant cell line to have been tested. AMLR killers had characteristics similar to NK cells, Major histocompatibility antigens were not the target antigens for AMLR killers. AMLR killers distinguished the blasts stimulated by alloantigens as self from the blasts stimulated by mitogens as non-self.     

Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture

Summary Lymphocytes from cancer patients were stimulated in mixed culture with autologous tumour (MLTC) or pooled allogeneic lymphocytes (MLC). Both protocols induced increased uptake of ³H-thymidine at 5 days and the appearance of lymphoblasts. Blasts were isolated on discontinuous Percoll gradients and either expanded as bulk cultures or cloned directly under limiting dilution conditions in the presence of conditioned medium containing IL-2. Results with MLTC-blast-CTC have been reported elsewhere. MLC-activated cultures lysed autologous tumour but not autologous lymphoblasts. Lysis of some allogeneic tumours, lymphoblasts from members of the inducing pool, and K562 was also apparent. MLC activated cultures did not undergo restimulation in response to autologous tumour or lymphocytes but were restimulated by leukocytes from pool members.MLTC clones showed autologous tumour-specific cytotoxic activity or cross-reactive proliferative responses with tumours of the same site and histology. The majority of MLC clones cytotoxic for autologous tumour were also specific and did not lyse allogeneic tumour, K562, or lymphoblasts from the inducing pool. Two clones lysed autologous tumour and pool members. None of the clones tested proliferated in response to autologous tumour following MLC activation but some were responsive to pool members and one clone was restimulated by autologous monocytes. No association was found between clone phenotype and function. The implication of these data is that the effector cells with activity against autologous tumour induced in MLC arose largely by transstimulation of in vivo-activated tumour reactive lymphocytes by IL-2 release rather than expansion of NK-like effectors or sharing of antigenic specificities between tumour and allogeneic lymphocytes. Since MLC activation of cancer patients lymphocytes does not induce proliferative responses to autologous tumour it is unlikely to be a useful procedure in preparing cells for immunotherapy protocols. Abbreviations used in this paper: PBL, peripheral blood lymphocytes; TIL, tumour infiltrating lymphocytes; MLTC, mixed lymphocyte tumour culture; IL-2, interleukin-2; MLC, mixed lymphocyte culture; LSM, lymphocyte separation medium; BSS, balanced salt solution; HuSe, human serum; PBS, phosphate-buffered saline; CTC, cultured T cells; PHA, phytohaemagglutinin; CM, cultured medium; NK, natural killer; FcR, receptor for the Fc portion of IgG     

A close relationship between cytotoxic T lymphocytes generated in the mixed lymphocyte culture and insulin receptor-bearing lymphocytes: enrichment by density gradient centrifugation.

In contrast to liver, fat, muscle, the fibroblast, and the monocyte, the lymphocyte does not bear insulin receptors unless it is activated by antigen or mitogen. Antigen stimulation by skin graft or in the mixed lymphocyte culture (MLC) generates a population of T lymphocytes, the effector function of which can be augmented by insulin. In the same pool of cells are found T lymphocytes with newly emergent insulin receptors capable of supporting this augmentation. This study demonstrates the close relationship between the augmentable effector T cell and the insulin receptor-bearing cell and strongly suggests that these cells are identical. Splenic lymphocytes from unidirectional murine MLCs were separated into light and heavy fractions by discrete density gradient eentrifugation daily and assayed for cellular-mediated cytotoxicity and for insulin receptors. Receptor-bearing and cytotoxic lymphocytes waxed and waned together primarily in the light fraction. Receptor-positive cells preceded effectors by 24 hr and the two characteristics were highly correlated over time (r ≥ 0.95). T-Cell depletion by specific antisera or by immunoabsorbent column chromatography demonstrated that most, but not all, receptor-bearing cells were T cells and that virtually all effectors were also receptor positive. When the insulin receptor was functionally removed from the lymphocyte membrane by trypsin proteolysis, effector function ceased. The return of cytotoxicity was accompanied by return of the lymphocyte insulin receptor. Receptor-bearing cells were predominantly of the Ly 2⁺3⁺ pedigree but Ly 1⁺ cells were also induced to bear the insulin receptor along with a few non-T cells. These data show that the emergence of a lymphocyte insulin receptor is not just a fortuitous marker event of cellular activation but provides a structure capable of supporting lymphocyte effector function. The appearance of Ly 1⁺ receptor-bearing cells suggests the alloactivation of T helpers and their participation in a T-T cooperative event.     

Generation of cytotoxic lymphocytes in mixed lymphocyte reactions II. Importance of private and publicH-2 alloantigens on the expression of cytotoxicity

MLC were established to test for the generation of specific cytotoxic effector cells in CML. The target cell used to assay for CML in the five combinations tested was of a differentH-2 haplotype from the stimulating cell population. Cytotoxicity was observed against this target only when it shared private alloantigens (antigens that are specific for theH-2D andH-2K region of differentH-2 haplotypes) with the stimulating cell population. Very weak or no Cytotoxicity was found when such alloantigens were not shared, although cross-reactive publicH-2 specificities were. These findings indicate that T cells display a cytotoxic potential against privateH-2 antigens in a primary response in vitro and are not capable of responding to publicH-2 specificities to the same level.BSS balanced salt solution - CML cell-mediated lympholysis - GPC guinea pig complement - ¹²⁵IUdR ¹²⁵I-iodo-deoxyuridine - MLC mixed lymphocyte culture - SE standard error     

Prostaglandin-mediated regulation of the mixed lymphocyte culture and generation of cytotoxic cells

We examined the role of prostaglandins or prostaglandin-producing cells in the regulation of proliferation and generation of specific cytotoxicity in one-way mixed lymphocyte cultures of mouse spleen cells. Cultures treated with indomethacin or other prostaglandin synthesis inhibitors resulted in enhanced proliferation and cytotoxicity. The level of prostaglandins produced in vitro, as measured by RIA, was 10^?8M and was found to be completely blocked by indomethacin. Adding back 10^?8M PG restored baseline (control) proliferative responses. Kinetics of the enhanced MLC response were unchanged from controls as were the specificities of the cytotoxic cells. Cells from indomethacin-treated cultures were more efficient at killing targets than those from control cultures. Prostaglandins appear to have a preferential effect on the induction of cytotoxic cells.     

Allograft immunity in vitro: V. Generation of cytotoxic effector cells in mixed lymphocyte culture and the specificity of target cell lysis

Cells from one-way mixed lymphocyte cultures lyse allogeneic target cells in vitro. Target cell lysis is effected by lymphoid cells; macrophages do not contribute to cell death to any measurable degree. The cytotolytic effect is detected at the earliest on the 4th culture day, and reaches a maximum on Day 10–12, i.e., considerably later than the peak of cell proliferation. On Day 6 the killer cell is a large cell (blast); later it is a small (lymphocyte). No cytotoxic effect is detected if DNA synthesis and cell division are blocked. Killer cells are not damaged during the lytic reaction. Direct cell-to-cell contacts rather than diffusable factors of long range, such as lymphotoxins, are prerequisites for target cell damage. Target cell lysis is specific. Labeled “third party” target cells, not sharing antigens with the stimulating cell, are not damaged.     

Splenocytes incorporated with exogenous gangliosides induce a mixed lymphocyte reaction in autologous lymphocytes

SWR splenocytes incorporated with mixed gangliosides triggered an efficient mixed lymphocyte reaction in autologous thymus lymphocytes. The potency of the ganglioside-incorporated splenocytes as stimulators in the autologous system was eight to ninefold greater than background effect of untreated splenocytes. The induced mixed lymphocyte reaction was not due to free gangliosides in culture medium and occurred only if the gangliosides were incorporated into the triggering splenocytes, but not into the responding thymocytes. The mixed lymphocyte reaction generated by gangliosides incorporation in the autologous system is similar to that reaction in the allogeneic system with regard to the time course of [³H]thymidine incorporation.     

Lack of generation of killer cells in the mixed lymphocyte reaction between mitogen-stimulated and unstimulated autologous lymphocytes.

The present study has demonstrated that the Con A-activated cell-mediated autologous mixed lymphocyte reaction (MLR) is not associated with the generation of cytotoxic effector cells that kill autologous targets. Thus, the suppression of antibody production of PWM stimulated lymphocytes by autologous Con A-activated suppressor cells cannot be explained by detectable cytotoxicity. We have further demonstrated that the stimulator cell in this system is a nonadherent non-T cell.     

Induction of suppressor cells in autologous mixed lymphocyte culture (AMLC) in humans

T cells stimulated for 6-7 days in autologous mixed lymphocyte culture (AMLC) showed suppressive effects when added to fresh mixed cultures where autologous lymphocytes (A) were stimulated by Mitomycin C-treated allogeneic lymphocytes (Xm), in a ratio of A:Xm:AMLC-activated cells of 1:1:0.5. Both cytotoxic and proliferative activities in second cultures, as assayed after 6 days of incubation, were significantly inhibited (percentage suppression of cytotoxic activity observed in 17 experiments was 75.3 +/- 22.4; percentage suppression of proliferation was 60.6 +/- 18.2). Suppressor cells (SC) generated in AMLC were Mitomycin C sensitive and nonspecific in their action; not only A/Xm but also X/Am and X/Ym cultures were suppressed to the same extent. AMLC-Activated cells showed a considerable degree of proliferation in response to alloantigens but failed to express any cytotoxic activity against autologous or allogeneic phytohemagglutinin blasts. Thus, the inhibitory effect observed in this system is not due to cytotoxic elimination of responding or stimulating cells in the second culture but rather reflects a true regulatory (suppressive) mechanism.     

Further characterization of the autologous mixed lymphocyte reaction: induction of double negative gamma delta T lymphocytes.

To investigate the specific nature of the autologous mixed lymphocyte reaction (AMLR), we applied a method in which mixtures of NY-nonadherent responder cells and NY-adherent stimulator cells were treated with neuraminidase before culture and then cultured to assay the AMLR. This method produced a marked enhancement of DNA replication in the responder cells and the results were reproducible, regardless of the individuals tested. Using this method, we were able to make the following observations regarding the specific nature of the AMLR. (i) The AMLR is an IL-2-independent reaction, as revealed by bioassay to detect the presence of IL-2 by a blocking test using anti-IL-2R sera and as shown by the absence of mRNA for IL-2 in Northern hybridization. (ii) It is also HLA-DR dependent as proven by the fact that anti-DR sera almost completely inhibited the reaction. (iii) The AMLR was also found to induce the generation of activated CD4+ helper T cells in direct response to stimulation by NY-adherent cells, in which HLA-DR antigens were involved. (iv) Also, it induced the generation of CD4-CD8- double-negative (DN) lymphocytes, including gamma delta T cells with a cytotoxic activity against NK-resistant target cells and with a variety of lymphocyte activation markers (CD56, HLA-DR, CD25, transferrin receptors, CD38, and LFA-1). However, the AMLR did not induce the generation of NK cell markers CD16 and CD57. (v) The DN lymphocytes and gamma delta T cells appeared to be generated from the precursors of CD4-CD8- DN cells, in direct response to the stimulator cells. These results strongly suggest that the AMLR may be a phenomenon which induces the proliferative response of gamma delta T cells and their precursors, in addition to that of alpha beta T cells, particularly of CD4+ helper T cells.     

Human cytotoxic lymphocytes. V. Frequency and specificity of gamma delta+ cytotoxic lymphocyte precursors activated by allogeneic or autologous stimulator cells

We have investigated the frequency and specificity of gamma delta+ cytotoxic lymphocyte precursors (CLP) under limiting dilution culture conditions. E rosette separated total T cells and CD3+CD4-CD8-TCR alpha beta- double-negative (DN) T cells were cocultured with allogeneic or autologous PBMC stimulator cells, and frequencies of alloreactive and autoreactive CLP were determined after 12 to 14 days against Con A blast target cells. Freshly isolated DN cells consisting of 82.3 +/- 8.2% gamma delta+ T cells did not exert cytolytic activity against K562 or anti-TCR gamma delta mAb-producing hybridoma cells. In striking contrast to E+ cells, the vast majority of alloantigen-stimulated clonally developing DN CLP did not show specificity for stimulator-derived target cells. Thus, frequencies of alloreactive and autoreactive CLP after alloantigenic stimulation were in the range of 1/100 to 1/4800 and 1/450 to 1/5000, respectively. After coculture with autologous stimulator cells, frequencies of autoreactive and alloreactive DN CLP were 1/700 to 1/2700 and 1/1360 to 1/4500, respectively. Split culture analysis revealed that most proliferating DN colonies selected for high probability of clonality simultaneously killed both autologous and HLA-mismatched allogeneic targets. The majority of the DN cells expressed the CD3+/TCR gamma delta+ phenotype after culture, and thus were not CD2+CD3- NK cells. Taken together, our results show that 1) freshly isolated peripheral blood gamma delta+ T cells lack cytotoxic activity, and 2) most cytotoxic gamma delta+ T cells activated by autologous or allogeneic stimulator cells under limiting dilution conditions do not discriminate between autologous and allogeneic targets.     

Generation of cytotoxic lymphocytes in vitro: response of immune rat spleen cells to a syngeneic gross virus-induced lymphoma in mixed lymphocyte-tumor culture.

Spleen cells from W/Fu rats 4 to 6 weeks after immunization with syngeneic Gross virus-induced lymphoma (C58NT)D cells usually lack detectable activity in a short-term 51Cr release assay. The results presented here demonstrate that these spleen cells retain the capacity to generate significant proliferative and cytotoxic activity upon re-exposure to mitomycin C-treated (C58NT)D cells in vitro. Optimal conditions were defined in W/Fu rats for this secondary immune response in vitro to the (C58NT)D cells. The cytotoxic response was observed to be quantitative, reproducible, and specific. Optimal generation occurred 5 days after initiation of cultures with a 30:1 responding cell:stimulating cell ratio. In vitro generated cytotoxic cells inhibit tumor growth in vivo when administered as a mixture with tumor cells.     

Simple sugars inhibit proliferation of human T lymphocytes in autologous and allogeneic mixed lymphocyte reactions

Several oligo- and monosaccharides were studied for their capacity to modulate lymphocyte proliferation in human allogeneic and autologous mixed lymphocyte reactions (MLR). A defined subset of sugars showed a marked inhibitory effect on lymphocyte proliferative response in the majority of the allogeneic MLR combinations studied. The inhibitory effect disappeared when sugars were added to allogeneic MLR 96 hr after the beginning of culture. These sugars also showed a significant inhibitory power on autologous MLR, performed by using T- and non-T-enriched lymphocytes from the same donor. The reported data suggest that carbohydrate determinants are involved in the proliferative response of human lymphocytes in both autologous and allogeneic MLR.     

Cell-mediated immune response in vitro. I. The development of suppressor cells and cytotoxic lymphocytes in mixed lymphocyte cultures.

During the in vitro development of cytotoxic lymphocytes (CL), suppressor cells also develop. Spleen cells or lymph node cells harvested from mixed lymphocyte cultures (MLC) on day 2 (day-2 MLC) and added to a fresh MLC suppressed the development of CL. This suppressive effect was sensitive to treatment with anti-theta and C. The suppressive effect of day-2 MLC was not due to cytotoxic effects nor to altered kinetics of the development of both suppressor cells and CL. Although CL develop from hydrocortisone-treated spleen cells, day-2 MLC of hydrocortisone-treated spleen cells did not suppress the development of CL. These studies suggest that suppressor cells and CL are derived from different T cell subpopulations.