Proliferative and cytotoxic immune functions in aging mice. IV. Effects of suppressor cell populations from aged and young mice

The changes with age in three splenic suppressor cell populations were studied in C57BL/6 mice. Allospecific Ts cells and nonspecific non-T suppressor cells were both generated in vitro in allogeneic MLC. The presence of "pre-existing" suppressor cells in fresh spleen cells from normal mice was examined. Suppressor cell activities were assayed for their ability to suppress proliferation in a fresh allogeneic MLC after treatment to prevent their own proliferation. The ability to generate both specific and nonspecific suppressor cells decreased with age, whereas pre-existing suppressor cells were detected in spleens from the majority of the aged animals but not in spleens from young animals. The decrease in suppressor cell activity was not due to any requirement for age matching between donors of suppressor and target cells. The specific and nonspecific MLC-generated suppressor cells inhibited both the proliferative response in the assay MLC and the generation of cytotoxic cells. The pre-existing suppressor cells only suppressed the proliferative response and not the generation of cytotoxic cells. The changes seen with age in these suppressor cell populations suggest that the ability to generate suppression (both allospecific and nonspecific) to newly encountered Ag declines with age, whereas a resident splenic suppressor cell population accumulates over the lifetime of the animals.     

The mixed lymphocyte response of senescent mice: sensitivity to alloantigen and cell replication time.

The age-dependent alteration in the proliferative response of C57B1/6J lymph node cells to stimulation by H-2- and M-locus alloantigens was examined in one-way mixed lymphocyte cultures (MLC). Balb/c (H-2^d, Mls^b) and DBA (H-2^d, Mls^a) spleen cells served as stimulating cells differing from C57B1/6J (H-2^b, Mls^b) at the H-2 and H-2 plus Mls loci, respectively. The day of peak response and the ratio of responder to stimulator cells required for optimal stimulation were the same for all the age groups (3 to 29 months) tested, irrespective of the stimulator strain used. Results obtained in MLC under optimal conditions showed a maximal response to both Balb/c and DBA/2 stimulation at the age of 6 months, followed by a gradual decline in the response with age. In order to determine whether the decline with age in mixed lymphocyte reactivity can be attributed to a reduction in the proliferative capacity of the responding lymphocytes of aged mice, cell cycle analyses were performed. Auto-radiographic studies of MLC containing lymphocytes from CS7B1/6J mice aged 6 and 24 months showed no difference in generation time, S, G₂, G₁, and M phases of the cell cycle. In addition, lymphocytes of both age groups underwent two identical mitotic waves within the period of examination. Our results determine that the functional decline with age in proliferative activity in mixed lymphocyte cultures is attributable to a neither decrease in sensitivity to alloantigen nor to a decrease in generation time or the ability to undergo several mitotic divisions, and suggest that such a decline is caused by fewer cells capable of response in old mice.     

Inhibition of murine suppressor cell function by progesterone

The effects of progesterone on murine suppressor cell function generated in allogeneic MLCs were investigated. BALB/c splenic lymphocytes stimulated in vitro with C3H/He cells significantly suppressed the proliferative response of BALB/c lymphocytes in a secondary MLC. This suppression was highly specific for the sensitizing alloantigens since the suppressor cells had no effect on the proliferative response of BALB/c lymphocytes to third-party alloantigens. In addition, BALB/c lymphocytes stimulated with syngeneic cells were observed to nonspecifically suppress the MLC response to a lesser extent. One to 10 micrograms/ml progesterone added at initiation to suppressor cell generating cultures diminished the ability of both alloantigen specific and nonspecific suppressor cell populations to suppress the proliferative response of homologous lymphocytes to alloantigens. Experiments with pyrilamine, an antihistamine, which blocks cytotoxic T lymphocyte (CTL) generation, suggests that progesterone has a direct inhibitory effect on suppressor cell function independent of its ability to block CTL induction. The effects of progesterone on suppressor cells were not due to shifts in peak response time in MLC or induction of radiosensitive cells in progesterone-treated cultures. Estradiol at doses between 5 and 10 micrograms/ml, and cortisol at dose of 1 microgram/ml, also significantly inhibited suppressor cell function. These results suggest that the steroid hormone milieu within the placenta may effect the activity of allogeneic or nonspecific suppressor cell activity.     

Cell-mediated immune response in vitro. II. The mechanism(s) involved in the suppression of the development of cytotoxic lymphocytes.

Spleen cells harvested from mixed leukocyte cultures (MLC) on day 2 or 3 suppress the development of CL from a fresh MLC across a cell-impermeable membrane, but day 4 MLC cells which have the maximum level of CL showed only a limited effect. Inhibition was observed only when suppressor cells were restimulated with the same H-2 type cells used during induction. However, the suppressive effect was not strain specific; that is, CBA-induced C57BL/6 spleen cells effectively inhibited the development of CL from DBA/2-induced C57BL/6 cells. In addition, DBA/2-induced C57BL/6 spleen cells effectively inhibited the development of CL from CBA cells. B10 spleen cells stimulated by B10.D2 cells gave rise to a suppressor cell population, indicating that H-2 differences alone can induce the response. The suppressive effect seemed to be exerted on an early phase of the response since no detectable inhibition was observed when suppressor cells were added 48 hr after culture initiation. The suppressive effect is not exerted on the accessory cell function but seems to inhibit DNA synthesis of the reacting cells in the MLC.     

Activation of specific suppressor cells with heat-treated allogeneic tumor cells

The ability of heat-treated allogeneic cells to induce suppressor cells was examined. The tumor cell lines EL-4 (H-2^b) and P815-X2 (H-2^d), were heated to 56 °C for 10 min and injected intravenously into mice of the DBA/2J (H-2^d) and C57BL/6J (H-2^b) strains, respectively. After 4 days, the splenocytes of the treated mice were mixed with normal spleen cells and cultured for 5 days with allogeneic tumor cells. The cytotoxic T-cell response was reduced in cultures of these cell mixtures. An allogeneic difference was required to induce suppression because the syngeneic combination did not induce suppressor cell activity. Furthermore, the induction of cytotoxic T cells to the C118 cell line (H-2^k) was not suppressed by this procedure, which suggests that the suppression was haplotype specific. These suppressor cells were sensitive to anti-Thy 1.2 and complement, cortisone, and cyclophosphamide, but insensitive to irradiation. These are characteristics similar to suppressor cells activated by intact cells. Heat treatment abrogated the tumor cell's ability to induce a proliferative and a primary, but not a secondary, cytotoxic T-cell response. The heat-treated cells also lost their ability to function as cold target inhibitor cells, but retained the same quantity of serologically detected antigens as the intact cells. These results suggest that the serologically detected antigens are responsible for the activation of the suppressor cells of the cytotoxic T-cell response.     

Allogeneic T-cytotoxic reactivity of senescent mice: affinity for target cells and determination of cell number

Experiments were performed to determine the cause(s) of the reduced T-cytotoxic-cell response observed in senescent mice. The cytotoxic cells studied developed in mixed lymphocyte cultures (MLC) of 6 × 10⁶ C57B1/6J (H-2^b) spleen cells from mice of various ages which were stimulated by doses of irradiated Balb/c (H-2^d) cells giving responder to stimulator ratios of 10:10 and 10:1. The cytotoxic response, as determined in a ⁵¹Cr-release assay against P815 (H-2^d) mastocytoma cells in culture, declines with age. This age-related decline is more pronounced with the lower dose of stimulator cells (10:1). The cytotoxic response developing in 10:10 and 10:1 MLC of spleen cells from young mice is comparable in magnitude, whereas the lower dose (10:1) is much less stimulatory in cultures of spleen cells from mice above 12 months of age. In order to better understand this age-dependent decline in cytotoxic response, the affinity of effector cells to their target and the percentage of cytotoxic cells which develop in the cultures of spleen cells from mice of various ages were determined. The affinity of cytotoxic cells developing in 10:10 MLC does not change with age. The affinity of cytotoxic cells developing in 10:1 MLC from young mice is significantly higher than the affinity of those developing in 10:10 MLC. This dose-dependent increase in affinity is not apparent in 20-month-old mice, which show equal affinity of cytotoxic cells in 10:10 and 10:1 MLC. The percentage of cytotoxic cells in the cultures was found to decrease with age. This decrease was more pronounced after 10:1 stimulation. Thus the decline with age in cytotoxic response can be attributed to a decrease in number of functional cytotoxic cells developing in MLC cultures, regardless of stimulator cell dose and a decrease in affinity for target cells at low stimulator cell doses.     

Simultaneous suppression of allogeneic cytolytic activity and stimulation of lectin-dependent cytolytic activity by con A.

It is well known that when mouse lymphocytes are cultured with irradiated allogeneic stimulator lymphocytes, T cells capable of mediating specific cytolysis are generated. We noted that when the stimulator cells were pretreated with concanavalin A (Con A), the generation of T cell-mediated specific lysis (TSL) is largely abolished, but large amounts of T cell-mediated lectin-dependent lytic (LDL) activity are nevertheless produced. Data are presented indicating that generation of TSL is inhibited by suppressor cells which are generated by the Con A in the culture.One possible source of the LDL would be a specific clonal response to Con A-altered H-2 molecules. Evidence concerning this was equivocal since the amount of lectin required tor expression of the LDL is suffcient to cause non-specific T cell-mediated lysis of any target cell type, making inoperative the conventional tests for H-2 restricted recognition.Use of dead cell fragments for stimulation in MLC has previously been reported to produce LDL without TSL, but Con A-induced premature death of the stimulator cells was ruled out as a source of the LDL in our cultures.Another possible source of LDL would be suppressor-induced blockade of killer cell differentiation at a hypothetical stage expressing LDL but not TSL. However, delayed addition of Con A induced suppressor cells to M LC's never allowed generation of LDL when TSL was suppressed. Therefore, such a blocked differentiation mechanism did not contribute significantly to the LDL produced.The LDL activity results largely from Con A-induced polyclonal activation of cytolytic progenitor cells. It is shown that generation of LDL by Con A is able to be suppressed by Con A-induced suppressor cells added at the initiation of culture. However, in cultures in which Con A is simultaneously generating LDL and suppressor activities, the LDL is generated rapidly (in 2 days) and apparently passes the suppressable stage before the suppressor cells become active.     

Age restriction in antigen-specific immunosuppression

Age-related alterations of antigen-specific T cell-mediated suppression have been examined in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. Inducer suppressor T cells (Tsi) were activated in mice at the age of 3 mo (young) or 18 mo (old) by i.v. injection of NP-conjugated syngeneic spleen cells (SC). Spleen cells from the NP-SC-injected mice were subcultured in vitro with spleen cells from normal young or old mice to generate transducer suppressor T cells (Tst). Four days later subcultured cells were added to responder cell cultures 1 day before the PFC assays to trigger effector suppressor T cells (Tse). Responder cell cultures, containing NP-conjugated horse red blood cells (HRBC) and spleen cells from HRBC-primed young or old mice, were assayed on day 4 for anti-NP and anti-HRBC PFC. Suppression was found to be antigen specific and age restricted. NP-specific suppressor cells are easily induced in subculture if the Tsi and Tst cell populations are both derived from young or old mice. Conversely, if Tsi cells from young or old mice are subcultured with Tst cells from mice of a different age, suppression of the anti-NP PFC response is hardly observed. Age restriction was also found to operate in the interactions between subcultured and responder cell populations, indicating that age-matching is required for effective triggering of Tse cells by Tst cells. These results altogether suggest that aging may affect the recognition repertoire expressed in suppressor T cell subsets. Moreover, the finding that suppression is less efficient when exerted on responder spleen cells from old than from young mice provides an explanation for the increased frequency of autoimmune disorders in aging.     

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.     

Soluble suppressor of T cell proliferation in primary in vitro response to murine minor histocompatibility antigens

Normal mouse lymphocytes are not capable of mounting a primary cytotoxic T cell response to Mls encoded, non H-2, allodeterminants, although a strong lymphoproliferative response is observed in primary MLR between Mls incompatible cells. In this study it is reported that in the supernatant of primary cultures between AKR macrophages and CBA/H lymphocytes (H-2 identical, incompatible for Mls and other minor antigens) a suppressor of T cell proliferation in MLR is detected. By contrast, a suppressor is not detected in supernatants from primary cultures between BALB/C macrophages and CBA/H lymphocytes (H-2 incompatible, Mls identical), B10.BR macrophages and CBA/H macrophages and CBA/H lymphocytes (syngeneic) suggesting that the production of the suppressor factor occurs only when an Mls incompatibility exists. The suppressive activity of the Mls incompatible culture supernatant upon MLR between incompatible macrophages and lymphocytes is neither antigen specific nor Mls or H-2 restricted, nor is it due to an irreversible toxic effect on T lymphocytes or macrophages. The inhibition of T cell proliferation could be explained by inhibition of IL 2 production, by blocking its union to T cells or by a combination of both effects. Our findings could help explain previous observations that lymphocytes from mice preimmunized with Mls incompatible cells have a depressed proliferative response as well as depressed cytotoxicity against alloantigens.     

Aging, oxidative responses, and proliferative capacity in cultured mouse aortic smooth muscle cells

The cellular mechanisms that contribute to the acceleration of atherosclerosis in aging populations are poorly understood, although it is hypothesized that changes in the proliferative capacity of vascular smooth muscle cells is contributory. We addressed the relationship among aging, generation of reactive oxygen species (ROS), and proliferation in primary culture smooth muscle cells (SMC) derived from the aortas of young (4 mo old) and aged (16 mo old) mice to understand the phenotypic modulation of these cells as aging occurs. SMC from aged mice had decreased proliferative capacity in response to alpha-thrombin stimulation, yet generated higher levels of ROS and had constitutively increased mitogen-activated protein kinase activity, in comparison with cells from younger mice. These effects may be explained by dysregulation of cell cycle-associated proteins such as cyclin D1 and p27Kip1 in SMC from aged mice. Increased ROS generation was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Accrual of oxidant-induced damage and decreased proliferative capacity in SMC may explain, in part, the age-associated transition to plaque instability in humans with atherosclerosis.     

Generation of cytotoxic T lymphocytes in vitro. VII. Suppressive effect of irradiated MLC cells on CTL response.

Irradiated cells obtained from MLC at the peak of the CTL response caused profound suppression of generation of CTL when added in small numbers at the initiation of primary MLC prepared with normal spleen cells. The inhibitory activity of the MLC cells was not affected by irradiation (1000 rads) but was abolished by treatment with anti-theta serum and complement. The suppression was immunologically specific. The response of A (H-2a) spleen cells toward C3H (H-2k) alloantigens was suppressed by irradiated MLC cells obtained from MLC prepared with A spleen cells and irradiated C3H-stimulating cells, whereas the response of A spleen cells toward DBA/2 (H-2d) alloantigens was affected relatively little. However, if irradiated C3H X DBA/2 F1 hybrid spleen cells were used to stimulate A spleen cells in MLC, addition of irradiated MLC cells having cytotoxic activity toward C3H antigens abolished the response to both C3H and DBA/2 antigens. The response to DBA/2 antigens was much less affected when a mixture of irradiated C3H and DBA/2 spleen cells was used as stimulating cells. Thus, the presence of MLC cells having cytotoxic activity toward one alloantigen abolished the response to another non-cross reacting antigen only when both antigens were present on the same F1 hybrid-stimulating cells. This suppression of generation of CTL by irradiated MLC cells apparently involves inactivation of alloantigen-bearing stimulating cells as a result of residual cytotoxic activity of the irradiated MLC cells. This mechanism may be active during the decline in CTL activity noted in the normal immune response in vivo and in vitro.     

Immunoregulation by mouse T-cell clones. I. Suppression and amplification of cytotoxic responses by cloned H-Y-specific cytolytic T lymphocytes

H-Y-specific and H-2Db-restricted, Lyt-1-2+ T-cell clones ( CTLL ) with graded specific cytotoxic activities on male C57BL/6 (B6) target cells ( 1E3 , ; 2C5 , ++; 2A5 , +, 3E6 , +/-) were tested for their capacity to inhibit the generation of H-Y-specific cytotoxic T lymphocytes (CTL) in vitro. Addition of irradiated lymphocytes of CTLL 1E3 and CTLL 3E6 but not those of CTLL 2A5 or CTLL 2C5 abolished the generation of CTL from in vivo primed H-Y-specific precursor cells (CTLP) when added to fresh mixed-lymphocyte cultures (MLC). Exogenous sources of T-cell growth factors (TCGF) did not overcome suppression. Rather the presence of TCGF resulted in a further enhancement of suppressive activities in CTLL 1E3 and 3E6 and the induction of similar activities in cells from CTLL 2A5 and 2C5 , which by themselves were not inhibitory. Moreover when added to similar MLC on Day 1 instead of Day 0, only irradiated cells of CTLL 3E6 but not those of the other three CTLL were suppressive. Induction of suppressive activities in H-Y-specific CTLL was independent of the appropriate male stimulator cells since it was also observed in MLC induced by irrelevant antigens (H-2, trinitrophenol). Furthermore at low cell numbers, irradiated lymphocytes from any of the CTLL consistently enhanced CTL activities generated from H-Y-specific CTLP. This augmenting activity, which was not TCGF, could be transferred by soluble mediators present in antigen-sensitized CTLL cultures. Thus, these data indicate (i) that cytotoxic effector cells can function as suppressor cells in the generation of CTL, (ii) that the cytotoxic activity of cloned CTL does not correlate with their capacity to suppress CTL responses, (iii) that the inhibition of CTL responses by CTLL is not due to simple consumption of T-cell growth factors produced in MLC, and (iv) that different CTL clones may interfere with the generation of CTL at different stages of their maturation. Moreover, the experiments suggest an antigen-independent enhancement of suppression by the interaction of CTL with lymphokines. Together with the augmenting activity evoked by cloned CTL the data provide strong evidence for the expression of multiple immunological functions by one particular subset of T cells and suggest that cytotoxic effector cells can differentially regulate the maturation and/or clonal expression of their precursor cells.     

Con A-inducible suppression of MLC: evidence for mediation by the TH2 + T cell subset in man.

Human peripheral lymphoid cells pretreated with Concanavalin A for 48 hr can markedly suppress the proliferative response of untreated autologous lymphoid cells in MLC. Isolation studies with Sephadex G-200 anti-F(ab')2 affinity chromatography, nylon adherence, and E rosetting indicate that the Con A-induced suppressor cell is a T cell. Further fractionation into TH2+ and TH2- cell subsets with an equine-anti TH2 serum show that both subsets can be activated by Con A to an equivalent degree. After activation only the TH2+ subset can suppress autologous responder cells in MLC. The TH2- subset, which comprises 80% of peripheral human T cells, although induced by Con A to proliferate, cannot itself suppress the MLC response. Nevertheless, the TH2- subset can be shown to modulate the generation of suppressor TH2+ cells at 24 hr but not at 48 hr. These studies support the notion that the Con A-induced suppressor cell is confined to a distinct T cell subset in man and that T-T interactions are important in the overall expression of the immune response.     

Properties of reticulum cell sarcomas in SJL/J mice. III. Promotion of tumor growth in irradiated mice by normal lymphoid cells.

Experiments were performed to elucidate the mechanisms by which lymphocytes obtained from an M-antigen-incompatible strain reduce the specific mixed lymphocyte culture (MLC) response of lymphoid cell populations after injection into allogeneic recipients. Mice of strain CBA were injected with spleen cells from hybrids of the H-2-compatible, M-antigen-incompatible strain C3H. Normal C3H × CBA spleen cells increased the MLC reactivity of the host's lymphocytes during the first 1–3 days, and thereafter the response against C3H was drastically reduced. Mitomycin-treated or antibody-coated C3H × CBA cells rather enhanced the MLC responsiveness. Roughly similar results were obtained by injecting untreated H-2-incompatible C3H hybrid lymphocytes. Lymph node or spleen cell populations from CBA mice, injected with C3H × CBA cells up to 2 weeks earlier, were found to depress the MLC reactivity against C3H when transferred to new CBA hosts. The results indicate that injected cells had survived for 2 weeks in the host. On the other hand, H-2-incompatible C3H hybrid cells could not be detected even at day 3 after injection into CBA mice. The results also indicate that C3H hybrid lymphocytes have to be functionally intact and able to survive in the host for a relatively long period of time to be able to reduce the specific MLC response of the host's lymphocytes.     

Regulation of primary cytotoxic T lymphocyte responses generated during mixed leukocyte culture with H-2d identical Qa-1-disparate cells

Cytotoxic lymphocyte (CTL) responses are not usually generated during primary mixed leukocyte culture (MLC) with H-2 identical cells. Thus NZB mice are unusual in that their spleen cells do mount CTL responses during primary MLC with H-2d identical stimulator cells; the predominant target antigen for these NZB responses is Qa-1b. Considering the numerous immunoregulatory defects in NZB mice, we postulated that these NZB anti-Qa-1 primary CTL responses were due to an abnormality in T suppressor cell activity. Cellular interactions capable of suppressing NZB anti-Qa-1 primary CTL responses were investigated by using one-way and two-way MLC with spleen cells from NZB mice and other H-2d strains. Although H-2d identical one-way MLC with the use of NZB responders resulted in substantial CTL responses, only minimal CTL responses were detected from two-way MLC with the use of NZB spleen cells plus nonirradiated spleen cells from other H-2d mice. Thus the presence of non-NZB spleen cells in the two-way H-2d identical MLC prevented the generation of NZB CTL. Noncytotoxic mechanisms were implicated in the suppression of the NZB CTL responses during two-way MLC, because only minimal CTL activity was generated when NZB spleen cells were cultured with semiallogeneic, H-2d identical (e.g., NZB X BALB) F1 spleen cells. The observed suppression could be abrogated with as little as 100 rad gamma-irradiation to the non-NZB spleen cells. The phenotype of these highly radiosensitive spleen cells was Thy-1+, Lyt-1+, Lyt-2-, L3T4+. The functional presence of these cells in the spleens of semiallogeneic, H-2d identical F1 mice indicated that their deficiency in NZB mice was a recessive trait. These data suggest that NZB mice lack an L3T4+ cell present in the spleens of normal mice that is capable of suppressing primary anti-Qa-1 CTL responses. This model system should facilitate additional investigations of the cellular interactions and immunoregulatory mechanisms responsible for controlling primary CTL responses against non-H-2K/D class I alloantigens. The model may also provide insight into the immunoregulatory defects of autoimmune NZB mice.     

Regulation of immune response in allogeneic mixed spleen cell cultures. I. Influence of I-region on the generation of suppressor cells

The immune responses of allogeneic mixed spleen cell cultures (MLC) to the T-dependent antigen, SRBC, and to the T-independent antigen, DNP-PAA, were investigated. The immune response to DNP-PAA in MLC with certain strain combinations was always suppressed as compared with the expected PFC response calculated from the PFC responses of the individual strains. This suppression was eliminated by treating the spleen cells with RAMB antiserum plus complement before the incubation of the MLC with DNP-PAA. It can be concluded that the suppression in the PFC response to the T-independent antigen DNP-PAA in MLC is due to the generation of suppressor T-cells. The PFC response to the T-dependent antigen, SRBC, in MLC showed either suppression, no change, or rarely augmenation, suggesting that the allogeneic mixed spleen cell cultures can generate both suppressor and helper T cells and that the balance between helper and suppressor activity regulates the PFC response to a T-dependent antigen. Suppressor activity was also generated in a one-way MLC, but the degree of suppression depended upon which of the two strains was responding. Similar amounts of thymidine were incorporated in the one-way MLR irrespective of which strains was responding. Thus, the extent of proliferation in one-way MLR is not related to the degree of suppressor activity generated. The results further indicate that a difference between two strains in the I-C, S, and G regions of the major histocompatibility complex is required to generate suppressor activitiy that can depress the response to a T-independent antigen, MLC between strains differing in K, I-A, I-B, I-J, I-E, and D regions generate little or no suppressor activity in this system.     

Regulation of the cytotoxic activity of alloreactive cytotoxic T lymphocytes by helper cells and lymphokines

The cytotoxic activity of alloreactive cytotoxic T lymphocytes (CTL) was maintained and augmented by transferring cells from a 5-day mixed lymphocyte culture MLC into a host culture (HC) containing indomethacin, freshly explanted normal spleen cells, and peritoneal cells which were syngeneic to the MLC cells. The MLC cells used in the transfer experiments were generated by culturing untreated H-2b splenic responders with irradiated H-2d stimulators, or were generated by culturing Lyt-2-depleted H-2b splenic responders with irradiated H-2d stimulators. The allo-CTL were found to be derived from the donor MLC (first culture) when unfractionated MLC cells were transferred into a host (second) culture and incubated for 5 days. In contrast, the allo-CTL were derived from host culture cells when Lyt-2-depleted MLC cells were transferred and the combined cultures incubated for 5 days. In the former case, the augmentation of MLC-derived cytotoxicity did not result from nonspecific expansion of all donor T cells; instead it was mediated by lymphokine(s), distinct from IL-2, produced by helper T cells generated in host culture, which appeared to selectively expand the antigen-specific CTL or to increase the cytotoxic activity of these CTL. The helper T cells were Thy-1+, L3T4+, and Lyt-2-. These findings indicate that antigen-nonspecific help was provided by helper cells or helper factors (lymphokines) generated in the host culture, which maintained and augmented the cytotoxic activity of the fully generated allo-CTL. This helper effect was also seen in the induction of primary allo-CTL responses which could be generated with fewer stimulating cells and with a stronger cytotoxic response at different R/S ratios tested. The generation of allo-CTL in second culture following transfer of Lyt-2-depleted MLC cells to host cultures appears to have involved antigen carryover from the MLC; however, antigen carryover alone was not sufficient. It appears that in the absence of Lyt-2+ suppressor T cells, antigen-specific help might be generated in donor cultures (Lyt-2-depleted MLC) which promoted or recruited the generation of antigen-specific CTL in host culture.     

Cyclosporine A and peripheral tolerance. Inhibition of veto cell-mediated clonal deletion of postthymic precursor cytotoxic T lymphocytes.

Veto cell-mediated suppression of CTL responses has been proposed as one mechanism by which self tolerance is maintained in mature T cell populations. We have reported that murine bone marrow cells cultured in the presence of high-dose IL-2 (activated bone marrow cells) mediate strong veto suppressor function in vitro and in vivo, and that such veto activity is effected through clonal deletion of cytotoxic T cell precursors. In our studies, we have determined that bone marrow cell populations from athymic NCr-nu mice (H-2d) mediate strong veto cell activity without exposure to exogenous IL-2 in vitro. To examine mechanisms by which these naturally occurring veto cell populations in BM suppress precursor CTL (pCTL) responses, we used as a responding cell population in MLC, spleen cells of transgenic mice expressing at high frequency TCR specific for H-2 Ld encoded Ag with stimulation by H-2d-expressing cells in culture. Flow cytometric analysis was performed by staining the responding MLC cell population with the mAb 1B2 specific for the transgene-encoded TCR and determined changes of 1B2+ T cells. Such experiments demonstrated that the anti-H-2d cytotoxic response by these cell populations was specifically suppressed by NCr-nu (H-2d) bone marrow, and that 1B2+ pCTL were in fact specifically deleted from the responding cell population by incubation with such naturally occurring veto cell populations expressing the appropriate target Ag. In addition, to further understand the interactions of pCTL and veto cells and possible contributions by the latter to peripheral tolerance, we evaluated the effect of cyclosporine A (CsA) on veto cell-mediated suppression of pCTL of the transgenic mice. CsA inhibited veto cell-mediated suppression of cytotoxic T cell responses, and this inhibition correlated with a lack of clonal deletion of pCTL by veto cells in the presence of CsA. Furthermore, CsA exerted its effect through pCTL and not through veto cells, indicating that pCTL may play an active role in their own deletion by veto cells.     

Stimulatory cells in the generation of T-cell-mediated cytotoxicity: Potent stimulating activity of a small radioresistant spleen cell population (RSCs)

The combined effects of irradiation followed by cultivation on a total spleen cell population in order to study the evolution of the stimulating potential in the in vitro generation of allogeneic cytotoxic T lymphocytes (CTLs) were tested. Results revealed that, after 3 days and up to at least 7 days of cultivating irradiated (1000 rad) spleen cells, the remaining living cells (radioresistant spleen cells or RSC) have the same potential to generate CTLs as irradiated noncultivated spleen cells. RSC can resist a 5000-rad irradiation and induce a primary cytotoxic response pattern similar to that of total spleen cells; they act in primary as well as in secondary cultures with optimal responder to RSC ratios of about 100, but are still stimulatory at MLC ratios up to 1000 or 5000. They are lysed by specific allogeneic CTLs and readily inhibit the specific lysis of H-2-identical labeled targets by CTLs. RSCs do not express unusual levels of H-2 or Ia antigens and do stimulate purified T cells. Alloantisera anti-H-2 are able to completely block the RSC-induced generation of CTL. This RSC population may prove to be a good model to study non-H-2- or H-2-associated, nonserologically detectable determinants interacting in the generation of T-cell-mediated cytotoxicity.