Limiting dilution analysis of human, tetanus-reactive helper T lymphocytes. A rapid method for the enumeration of helper T lymphocytes with specificity for soluble antigens.

The number of helper T lymphocytes (HTL) in human peripheral blood with specificity for the soluble protein, tetanus toxoid, was estimated by limiting dilution analysis (LDA). HTL were detected via antigen-induced interleukin-2 (IL-2) production, as measured by incorporation of tritiated thymidine by an IL-2-dependent indicator cell line, CTLL-20. Culture conditions optimizing assay sensitivity are described, and the ability to detect antigen-specific HTL using this LDA technique are demonstrated. Observed HTL frequencies in healthy human donors tested for tetanus-reactive helper T cells ranged from less than 1 HTL/268,749 peripheral blood mononuclear cells (PBMC) (undetectable) to 1 HTL/1486 PBMC. The LDA technique was also used to detect frequency shifts in human peripheral blood HTL following challenge with antigen. This assay offers distinct advantages over proliferative LDA techniques in that it is rapid (requiring only 2 days), and defines an antigen-reactive T cell subset with defined function (IL-2 secretion). Furthermore, the LDA technique can be adapted for the detection of other soluble protein antigens, such as PPD and Candida albicans. In general, this LDA technique provides a reliable, quantitative index of human HTL reactivity to any of a variety of soluble protein antigens, and has clinical as well as experimental applicability.     

Human anti-tetanus antibody response in vitro: autologous and allogeneic T cells provide help by different routes.

Human B cells will make anti-tetanus antibody in vitro in the presence of antigen and T cells. These T cells may be autologous, but allogeneic T lymphocytes function equally well provided they are first irradiated. The allogeneic cells provide help by a different route. Co-culture of allogeneic cells produces a degree of polyclonal activation of B cells and a much higher level of IgM anti-tetanus antibody than autologous cultures. Depletion of tetanus toxoid or alloantigen-reactive T cells by 3H-thymidine suicide indicates that in autologous cultures help for anti-tetanus toxoid antibody production is provided by antigen-reactive T cells while in allogeneic cultures antibody production is dependent on the presence of alloreactive T cells. The implications for assessing human T and B cell function are discussed.     

Limiting dilution analysis of the human T cell response to mycobacterial antigens from BCG vaccinated individuals and leprosy patients.

The number of peripheral blood T lymphocytes responding to soluble mycobacterial antigens from Mycobacterium tuberculosis purified protein derivative (PPD) and M. leprae (MLS) was estimated by limiting dilution analysis. Antigen-induced lymphocyte activation was measured by means of [3H]TdR incorporation on day 10 of culture in the presence of suboptimal concentrations of interleukin 2 (IL-2). In the peripheral blood of BCG-vaccinated individuals from the UK, the frequency of T lymphocytes responding to PPD was 1.5 to 4 times greater than to MLS. Frequencies between 1/1970 and 1/13, 982 were observed in response to PPD and between 1/4097 and 1/24, 717 in response to MLS. A proportion of cells in the peripheral blood were also observed to respond to IL-2 only. The frequency of cells observed in limiting dilution analysis for PPD and MLS reflected the relative amounts of proliferation to these two antigens in bulk culture lymphocyte transformation tests. Use of PPD-specific T cell lines suggested that the responsiveness observed to M. leprae antigens in BCG-vaccinated individuals was due to cross-reactivity with antigens shared with M. bovis BCG. In tuberculoid leprosy, the frequency of peripheral blood T lymphocytes responding to M. leprae antigens was either greater than or similar to the frequency of T cells responding to PPD. In contrast, limiting dilution analysis of T lymphocytes from the peripheral blood of lepromatous leprosy patients revealed the complex regulatory heterogeneity of this group. In some patients M. leprae responsive T cells were detected in the presence of exogenous IL-2.     

Quantitative studies of alloantigen-reactive human lymphocytes in primary and secondary MLC

The number of alloantigen-reactive cells in human peripheral blood was estimated by a limiting dilution analysis. In MLC combinations between allogeneic unrelated donors, the frequency of alloantigen-reactive cells ranged between 1:241 to 1:486. The frequency of alloantigen-reactive cells to the specific donor was increased six- to eight-fold after priming in MLC. The results demonstrate that specific "memory" cells are enriched in long-term MLC. In limiting dilution experiments between HLA-identical siblings, the frequency of alloantigen-reactive cells ranged from 1:1160 to 1:1740. The data point to the existence of a lymphocyte-defined antigen system controlled by a genetic region that is not linked to HLA. The results suggest that the lymphocyte clones that are able to react to non-HLA antigens probably consist of a small number of lymphocytes. Finally, the response of these clones of cells to non-HLA antigens was observed only under conditions where responder cells were limiting.     

Cooperative effects in mitogen- and antigen-induced responses of human peripheral blood lymphocyte subpopulations.

Human peripheral blood lymphocytes were separated into T cell-enriched and T cell-depleted fractions by E rosette sedimentation. These two fractions, as well as the unseparated lymphocyte suspension, were tested for their responsiveness to the mitogens phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) and to the antigens PPD (purified protein derivative of tuberculin) and tetanus toxoid. The response to PHA, ConA and the antigens was found to be confined to the purified T cell fraction; PWM could stimulate both purified T and non-T cells. However, the T cell response to ConA, PPD and tetanus toxoid was always decreased by 50-70%, when compared to the unseparated lymphocytes. Addition of monocytes could restore the T cell response. In the response to PHA and tetanus toxoid, the (primarily unresponsive) non-T cell fraction could be recruited into proliferation by gamma-irradiated T cells. Moreover, in the response to tetanus toxoid, lymphocytes (T as well as non-T) from a nonimmune individual could be recruited into proliferation by gamma-irradiated immune T cells.     

Radiosensitivity of T and B lymphocytes. II. Effect of irradiation on response of T cells to alloantigens

The radiosensitivity of T cells was investigated by studying the effect of irradiation in vitro in suppressing the capacity of parental strain thoracic duct lymphocytes (a) to induce splenomegaly in newborn F₁ mice, and (b) to proliferate in adult irradiated F₁ mice as measured by incorporation of tritiated thymidine ([³H]dThd) 4 days after transfer. By these parameters, small T lymphocytes were found to be highly radiosensitive. It was calculated that, of cells with the reactivity to the alloantigens studied, 0.3 % were capable of a proliferative response after exposure to 500 r. Radiosensitivity was considered to be a reflection of lymphocyte death in interphase. The radiosensitivity of H-2-activated T cells (T. TDL) differed from that of small T cells. Thus, [³H]dThd incorporation by T. TDL measured 1 day after transfer to irradiated F₁ hosts was not abolished, although lowered, by exposure to doses as high as 5000 r; [³H]dThd incorporation measured at 2 days, however, was greatly reduced by much smaller doses of irradiation. In view of evidence obtained elsewhere that the response of T. TDL to alloantigens involves DNA synthesis but not cell division, the present studies were interpreted in terms of irradiation causing death of T. TDL in interphase before entry into DNA synthesis. It was concluded that T. TDL were far more resistant to irradiation-induced interphase death than were small T cells. The small numbers of lymphocytes obtained from thoracic duct lymph of mice exposed to whole body irradiation 4 days before consisted almost entirely of T cells; these cells, although viable, were found incapable of mounting a proliferate response when exposed to alloantigens on transfer.     

Human leucocyte responses in vitro. I. Transformation of purified T lymphocytes with and without addition of partially purified monocytes

Highly purified Fc receptor-negative T lymphocytes were obtained by filtration of human blood mononuclear cells through Ig–anti-Ig columns. Monocyte-enriched cells were isolated by density centrifugation in bovine serum albumin solutions. The proliferative in vitro response of the purified T lymphocytes was investigated with and without the addition of monocyte-enriched cells, after stimulation by allogeneic cells, phytohaemagglutinin (PHA), pokeweed mitogen (PWM), purified protein derivative (PPD), Candida albicans, Staphylococcus aureus, E. coli and tetanus toxoid.

The results were as follows:

(1) Fc receptor-negative T lymphocytes respond autonomously, and are the main source of proliferative cells found after stimulation by allogeneic cells and optimal doses of PHA, PWM and PPD. Addition of monocyte-enriched cells increases the responses, presumably by a non-specific feeder-effect in the cultures.

(2) Stimulation of Fc receptor-negative T lymphocytes by C. albicans, S. aureus, E. coli and tetanus toxoid requires the presence of autologous monocytes in the cultures, whereas allogeneic monocytes do not support the responses.

(3) Fc receptor-negative T lymphocytes are the main proliferating cells found after stimulation by C. albicans, whereas other cells (B-lymphocytes and/or Fc receptor-positive T lymphocytes) may be responsible for a substantial part of the proliferation elicited by S. aureus, E. coli and tetanus toxoid.

(4) Fc receptor-negative T lymphocytes can stimulate allogeneic cells in mixed lymphocyte culture, but monocyte-enriched cells and unfractionated mononuclear cells are better in this respect.

     

Utility of flow cytometric detection of CD69 expression as a rapid method for determining poly- and oligoclonal lymphocyte activation.

CD69 is a lymphoid activation antigen whose rapid expression (< or = 2 h postactivation) makes it amenable for the early detection of T-cell activation and for subset activation analyses. In the present study we evaluated the utility of flow cytometric detection of CD69 expression by T cells activated with polyclonal stimuli (anti-CD3 and staphylococcal enterotoxin B [SEB]) and oligoclonal stimuli (tetanus toxoid and allogeneic cells) using flow cytometry. Following activation of T cells with anti-CD3 or SEB, CD69 is detectable at < or = 4 h following activation, with anti-CD3 peaks at 18 to 48 h. Dose titration experiments indicated that CD69 expression largely paralleled that in [3H]thymidine incorporation assays, although the former offered a more sensitive measure of T-cell activation at limiting doses of activator than [3H]thymidine incorporation when cells were activated with either anti-CD3 or SEB. However, activation of T cells with either tetanus toxoid or allogeneic stimulator cells failed to induce detectable CD69 expression at up to 7 days of culture. Subset analyses of anti-CD3- and SEB-activated T cells indicated that populations other than T cells can express CD69 following stimulation with T-cell-specific stimuli, indicating that CD69 can be induced indirectly in non-T cells present in the population. These findings indicate that CD69 is a useful marker for quantifying T-cell and T-cell subset activation in mixed populations but that its utility might be restricted to potent stimuli that are characterized by their ability to activate large numbers of cells with rapid kinetics.     

HLA class II restriction repertoire of antigen-specific T cells. I. The main restriction determinants for antigen presentation are associated with HLA-D/DR and not with DP and DQ

In order to study the HLA class II restriction repertoire in antigen presentation to T cells, T lymphoblasts (T-LB) of ten different HLA class II donors were generated by a simple and rapid technique; peripheral blood lymphocytes (PBL) were restimulated in vitro with purified protein derivative (PPD) or tetanus toxoid (TET), and then propagated in interleukin-2 containing conditioned medium (IL2-CM). These T-LB appeared to be antigen specific and devoid of alloreactivity. Antigen was presented to these T-LB by allogeneic irradiated PBL as antigen-presenting cells (APC) in 179 combinations. T-LB proliferative responses were restricted mainly by determinants associated with HLA-DR and not with -DP or -DQ; in 102 fully DR mismatched T-LB/APC combinations matching for DP or DQ determinants had no significant influence on T-LB responses. For PPD, preferential DR1 restriction was observed, and the results suggest a preferential DRw11 vs. DRw12 restriction for TET. Moreover, DRw13 may be associated with low anti-PPD T-LB responsiveness.     

Stimulation of DNA synthesis in mouse lymphoid cells by polyanions in vitro. I. Target cells and possible mode of action

The effect of dextran sulfate on [³H]dThd incorporation in lymphoid cells was investigated. The polyanion activated DNA synthesis in spleen and bone marrow cells of normal mice. The highest rate of activation was detected in spleen cells of athymic (nude) mice; the ratio of [³H]dThd incorporation was higher in TxBM spleen cells of thymectomized, lethally irradiated and bone marrow protected mice than in spleen cells of normal donors. Thymus cells of normal mice could not be stimulated, but a slight response was obtained in cortisone-resistant thymus cells. A synergetic effect was found in normal thymus cells using a combination of dextran sulfate and phytohemagglutinin (PHA). In contrast, lipopolysaccharide and PHA showed no synergetic effect. The possible mode of action of polyanions as de-repressors of DNA synthesis is discussed.     

Initiation of the blastogenic response of lymphocytes by hyperoptimal concentrations of concanavalin A.

The blastogenic response of human lymphocytes in vitro to hyperoptimal concentrations of concanavalin A (Con A) has been studied by means of volume spectroscopy (measuring cellular and nuclear volume), flow cytofluorometry (measuring cellular DNA content) and incorporation of [3H]thymidine ([3H]dThd). The optimal Con A dose with respect to [3H]dThd incorporation was about 30 micrograms/ml. In cultures given hyperoptimal doses, e.g. 100 micrograms/ml, [3H]dThd incorporation was strongly inhibited, whereas the number of cells entering S-phase and significantly increasing their cellular and nuclear volume was considerably larger than with 30 micrograms/ml. With 200 micrograms/ml Con A, which induced negligible [3H]dThd incorporation, the percentage of responding cells was even larger. Hence, doses of Con A, which were hyperoptimal with regard to [3H]dThd incorporation, induced blastogenic response, including DNA synthesis, in a larger percentage of the cells than did the optimal dose. However, in cultures with hyperoptimal Con A doses, the progression of the cell cycle stagnated mainly during S- and G2-phase and few cells completed mitosis. Thus, the blocking effect of hyperoptimal doses was not confined to any particular point of the cell cycle. The reduced [3H]dTd incorporation, seen with hyperoptimal doses, is attributed partly to a failure of this assay under such conditions.     

Changes in the proliferative response of human lymphocytes in vitro under the influence of subcellular components ofBordetella pertussis

Different subcellular components ofBordetella pertussis were found to have a similar inhibitory action on incorporation of thymidine-³H by lymphocytes stimulated by phytohemagglutinin. Lymphocytes were obtained from donors immunized with tetanus toxoid. However, the same components ofB. pertussis had a differential action on lymphocyte proliferation in the presence of tetanus toxoid: murein-containing membranes increased incorporation of thymidine-³H, the RNA-containing fraction inhibited it, and the water-soluble components of the homogenate had no effect on lymphocyte proliferation.Moscow Research Institute of Epidemiology and Microbiology, Ministry of Health of the RSFSR. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 4, pp. 332–334, April, 1979.     

The effects of cyclosporin on lymphocyte activation in a systemic graft-vs.-host reaction

We have investigated the effects of cyclosporin (CsA) on each of three stages of lymphocyte activation in vivo viz. sequestration of alloantigen-reactive lymphocytes from the circulation into the spleen and lymph nodes, blast transformation and induction of DNA synthesis in the activated cells and release of these cells and their progeny into the circulation. Parental strain lymphocytes injected i.v. into semi-allogeneic rats and recovered from the thoracic duct within 36 h are profoundly unresponsive in a local graft-vs.-host assay to the alloantigens of the F1 hybrid but have normal activity against unrelated alloantigens (negative selection). CsA treatment of the F1 hybrid recipients did not prevent this selective sequestration of antigen-reactive cells. In the untreated F1 hybrid, from 36 h after injection, large numbers of dividing blast cells were released into the lymph. These cells did not appear in the lymph of recipients treated with CsA. However, CsA did not prevent the activation of cells sequestered in the spleen or lymph nodes as assessed by [3H] thymidine incorporation and autoradiography. This unexpected finding suggests that CsA inhibits lymphocyte responses to alloantigens in vivo after DNA synthesis which is a later stage than the in vitro studies have shown.     

Stimulation of lymphocytes by antigen in microplate cultures; absence of an effect of transfer factor in vitro.

The best conditions for the optimum stimulation of human leucocytes by antigens, including protein purified derivative (PPD), streptokinase-streptodornase varidase (SKSD) and tetanus toxoid have been studied in microplate cultures. The leucocytes of each donor have their own characteristic response to antigen which depends on the culturing conditions such as antigen concentrations, cell concentrations and the time of measuring the rate of DNA synthesis. Thus, no conditions provide a universal optimum for antigen stimulation in vitro. Leucocyte dialysates, i.e. potential transfer factors, have been prepared from donors, who between them are strongly positive to the antigens PPD, SKSD, tetanus toxoid, diphtheria toxoid and Keyhole limpet haemocyanin. In contrast to some previous reports these leucocyte dialysates had no effect on the thymidine incorporation by leucocytes grown in the presence of these antigens. It is suggested that the selection of optimal conditions for the response to antigen may have obscured the effect of any non-specific enhancement of reactivity b lyeucocyte dialysates.     

Pregnancy-associated growth factor. I. A proliferative agent which expands adult and cord T4 cells in unfractionated cultures

Experiments were designed to examine the effects of pregnancy-associated growth factor (PAGF), a substance found in commercial preparations of crude human chorionic gonadotropin (hCG), on unfractionated human cord blood cells (CBC) and adult peripheral blood lymphocytes (PBL) cultured in 5% fetal calf serum (FCS). Comparisons of PAGF-induced [3H]TdR incorporation in nine pairs of simultaneously cultured CBC and PBL with phytohemagglutinin (PHA) and tetanus toxoid (TT) showed that all CBC and PBL responded to PAGF and PHA whereas all PBL and one CBC responded to TT. The rank order of potency for CBC and PBL was PHA greater than PAGF greater than TT. To examine phenotypic changes induced by PAGF, flow cytometry was performed on precultured cells, control cultures, and PAGF-stimulated cultures at 2, 5, 7, and 9 days. The monoclonal antibodies (mAbs) included T3, T4, and T8 (T cells), T9 (transferrin receptor), Tac (IL-2 receptor), 12 (Ia or DR-framework antigen), and T10 (putative activation and/or maturation antigen). PAGF-stimulated cultures had statistically significant increased percentages of T3, T4, T9, T10, and Tac but not T8 when compared to precultured cells and control cultures. PAGF also increased PBL but not CBC Ia. In PAGF-stimulated cultures, CBC had more T3 and T4 cells with increased fluorescence intensity than PBL. Maximal expression of phenotypes usually occurred at Days 7 and 9, 2 days after maximal [3H]TdR incorporation. In comparison to PAGF, PHA-stimulated PBL had earlier expression of these phenotypes but included T8. These data indicate PAGF induces proliferation, activation antigens, and T3 expansion predominantly confined to the T4 subset in both CBC and PBL.     

Follicular cells of tonsils metabolise more deoxycytidine than other cell populations

Nine subpopulations of tonsillar lymphocytes and the unseparated cells were compared in their utilization of exogenous deoxycytidine ([5-3H]CdR) and thymidine ([3H]TdR). Uptake phosphorylation and incorporation of labeled precursors were determined in B and T lymphocytes, in low density (LD; enriched in S phase cells) and in high density (HD; enriched in G0/G1 phase cells) cell fractions as well as in LD and HD subfractions of B and T lymphocytes, and in cells isolated from follicles of tonsils. As expected, LD cells and B lymphocytes were more active in TdR incorporation than HD cells and T lymphocytes. However, the ratio of [5-3H]CdR to [3H]TdR in their total phosphorylation and incorporation into DNA was much lower than the expected value of 1: about 0.5 for total phosphorylation and about 0.3 for incorporation in all subpopulations, except for the follicular cells, where these ratios were 1.0 and 0.7, respectively. These results show that the relative utilization of the two pyrimidine deoxyribonucleoside precursors varies among different lymphocyte subpopulations. However, this variation is not due to the different rate of DNA synthesis; rather, it depends on the differentiation stage of lymphocytes occurring in the germinal center of the follicles.     

Suppression of human T-cell mitogenesis and E-rosette formation by the monoclonal antibody OKT11A.

OKT11A, a monoclonal anti-human T-cell antibody was studied for its in vitro effects on lymphocyte functions. At a concentration as low as 10 ng/ml, OKT11A significantly suppressed T-cell proliferation induced by OKT3, purified protein derivative (PPD), tetanus toxoid and allogeneic non-T cells. Total inhibition of proliferation was noticed at concentrations of 1-10 microgram OKT11A/ml. The antibody was only fully effective when added to stimulated cell cultures within the first 2 hr of the culturing period. OKT11A also blocked total and active sheep erythrocyte (E)-rosette formation by T lymphocytes: this activity closely paralleled the suppression of proliferative response. Quantitative studies on the binding of 125I-labelled IKT11A indicated that an average of 2 x 10(4) antibody molecules were bound per T cell. Taken together, these findings show that OKT11A recognizes a sparsely represented T-cell surface determinant that is associated with the inhibition of mitogenic responsiveness and E-rosette formation. Furthermore, our data imply that the E-rosette receptor of T cells is involved in the regulation of immune functions.     

The effect of colchicine and colcemid on the mitogeninduced blastogenesis of lymphocytes

The effect of colchicine and colcemid (1 × 10^?6 M) on the blastogenic response of human lymphocytes to concanavalin A was studied in vitro by three different methods. (a) Measurement of [³H]thymidine ([³H]dThd) incorporation which was strongly suppressed by the drugs. (b) Volume spectroscopy showing that the growth of cellular and nuclear volume was only moderately affected by the drugs during the first two days of culture: in drug-treated cultures, 25% of the cells responded by measureable growth of their nuclear volume as compared to 30% in untreated cultures. After two days, however, growth stagnated in drug-treated cultures, and cell division never occurred. (c) Flow cytofluorometry, showing that in drug-treated cultures the number of cells measurably increasing their DNA content, i.e. entering S-phase, was about 60% of that in untreated cultures. However, the drugs caused a majority of the responding cells to stop DNA synthesis before completing S-phase. This effect could not fully account for the strong suppression of the [³H]dThd incorporation indicating that colchicine and colcemid caused a malfunction of the [³H]dThd assay. It is concluded that colchicine and colcemid do not significantly inhibit initiation of a blastogenic response indicating that microtubuli, which are known to be affected by these drugs, are not essential for the triggering of blastogenesis.     

T90/44 (9.3 antigen). A cell surface molecule with a function in human T cell activation

T90/44 is a cell surface antigen which is present on human T cells of the helper and cytotoxic subsets and which binds the 9.3 monoclonal antibody (9.3 mAb). It is expressed in the form of 90-kDa disulfide-bonded dimers of a 44-kDa polypeptide and of free 44-kDa subunits. The function of T90/44 was investigated in a series of T cell function assays. 9.3 mAb was found to inhibit the activation of class II-restricted cloned T helper cells derived from leprosy patients and reactive with M. leprae antigens. The inhibition was first found at 1-10 ng/ml 9.3 mAb and regularly increased with the antibody concentration. The extent of the inhibition varied among different T cell clones in proportion to the respective different levels of T90/44 expression at their cell surface. The proliferative responses of peripheral blood lymphocytes (PBL) to purified protein derivative of M. tuberculosis (PPD) and tetanus toxoid were enhanced by the 9.3 mAb resulting in up to 20-30-fold increase of [3H]-thymidine incorporation. After phytohemagglutinin-induced activation of PBL, the number of T90/44 molecules per cell expressed at the cell surface rose from day 0 to day 7 by a factor of about 10. High concentrations of 9.3 mAb (5-10 micrograms/ml) at low cell densities and in the presence of monocytes in culture media supplemented by fetal calf serum were directly mitogenic for resting lymphocytes. The cytolytic effector functions of class I-restricted cytotoxic T lymphocytes (CTL) were not modulated by 9.3 mAb. The mixed lymphocyte reactions of three class I-restricted CTL to their specific target cells were found not to be significantly influenced by 9.3 mAb. In conclusion it is proposed that an antigen-independent T cell activation pathway can be entered at T90/44.     

DNA-damaging potential of diethylstilbestrol evaluated in the germ cell unscheduled dna synthesis assay

Despite the fact that the nonsteroidal estrogen diethylstilbestrol (DES) exerts its toxic effects primarily on the reproductive system, little is known about the possible interference of this compound with germ cell DNA. The measurement of unscheduled DNA synthesis (UDS) in spermatocytes and early spermatids of mice germ-cells is a valid indicator for the DNA-damaging potential of a compound. UDS occurrence was thus determined after IP administration of 10, 30, 60 or 180 mg/kg DES to male mice. Tritiated thymidine ([³H]dThd) was then injected into the testes, the spermatozoa were serially collected, the sperm heads isolated, and UDS determined by the amount of [³H]dThd incorporation. [³H]dThd measurements in germ cells of mice which were treated with 10 mg/kg DES were comparable to those of the controls. Higher incorporation of [³H]dThd, indicating UDS, was measured in sperm cells which had been spermatocytes at the time of treatment with 30 and 60 mg/kg DES; this increase was statistically significant at 60 mg/kg. Administration of 180 mg/kg DES caused [³H]dThd incorporation which was comparable to that of the controls, suggesting that DES interfered with repair mechanisms or delayed spermatogenic cycles at high dose levels. General toxicity was manifested in a dose-dependent decrease of the sperm cell numbers in the spermatogenic stages investigated. This study provides evidence that DES, or its metabolite(s), reached the germ cells of adult mice in sufficient amounts to produce DNA damage. The levels of radioactivity measured were comparable to those measured after cyclophosphamide treatment, but [³H]dThd incorporation was about 10 times less than in methylmethane sulfonate-treated animals.