Interleukin-2-induced activation of natural killer activity in spleen cells from old and young mice.

Generation of natural killer (NK) activity in response to a partially purified preparation of rat interleukin-2 (IL-2) was compared in spleen cells derived from young (8-10 weeks old) and old (greater than 2 years old) female C57BL/6 mice. Significant NK activation was observed in both young and old mouse spleen cells incubated with 100 U IL-2/ml for 1-4 days, but the levels of cytotoxic activity generated in old mouse spleen cells was always lower than those of similarly treated young mouse spleen cells. Differences in IL-2-induced NK activation in old and young mouse spleen cells was obtained irrespective of the concentration of IL-2 used (25-400 U/ml). Quantitative comparisons indicated that old spleen cells activated by 3 day incubation with IL-2 acquired about two-fold higher NK activity than fresh young mouse spleen cells but still had only one-fourth of the levels of NK activity attained by IL-2-activated young mouse spleen cells. Cytotoxic activity of IL-2-activated young or old mouse spleen cells were totally abrogated by anti-asialo GM-1 antiserum + C but not by anti-Ly-2 + C treatment, indicating that the activated cytotoxic cells fell in the NK cell category. An analysis of NK precursor (NK-p) frequency by limiting dilution assay indicated that the NK-p frequency was about 4-fold higher in young as compared to old mouse spleen cells. The level of cytotoxic activity attained per NK-p cell was not significantly different for NK-p cells of old or young mice.     

Heterogeneity of soluble T cell products. I. Precursor frequency and correlation analysis of cytotoxic and immune interferon (IFN-gamma)-producing spleen cells in the mouse

In this study, the precursor frequency, relationship and activity of murine splenic cytotoxic and immune interferon (IFN-gamma)-producing cells has been determined. C57BL/6 spleen cells were activated by concanavalin A (Con A) and subsequently grown in microcultures under limiting dilution conditions. The progeny of cells plated in microcultures was divided and tested for (a) total cytotoxic activity on EL4 (H-2b) tumor target cells in the presence of Con A to nonspecifically attach the killer to the target cells, and (b) the quantity of IFN-gamma released by Con A stimulation. Cytotoxic T lymphocytes and IFN-gamma-producing cells were present in Con A-activated C57BL/6 spleen cells at a high frequency of approximately 1 out of 3 and 1 out of 2 cells, respectively. IFN-gamma could be released from both noncytotoxic and a large fraction of cytotoxic T cells and the cytotoxic activity was not necessarily associated with IFN-gamma release. In a few selected cultures with a progeny of cells plated at a low cell number (up to 10/well) very high IFN-gamma titers (greater than 10 000 U/ml) could be found. These results provide the first frequency estimate of IFN-producing cells and are discussed with respect to the physiological role of IFN-gamma release from T cells.     

Immunomodulatory effects of neurotropin through the recovery of interleukin-2 production in autoimmune-prone (NZB/NZW) F1 mice

The immunomodulatory effects of Neurotropin, a substance extracted from inflammatory skin of rabbits inoculated with vaccinia virus, were assessed in autoimmune-prone (NZB/NZW) F1 (B/W F1) mice. The concanavalin A (Con A)-induced proliferative response of spleen cells was markedly decreased in aged B/W F1 mice as compared with young B/W F1 mice. Neurotropin, when administered i.p. to aged B/W F1 mice, significantly increased the Con A-induced proliferative response. In aged B/W F1 mice, interleukin-2 (IL-2) production by Con A-stimulated spleen cells was severely impaired and IL-2 responsiveness of Con A-activated spleen cells was partially decreased in comparison with young B/W F1 mice. Neurotropin, administered to the aged B/W F1 mice, restored IL-2 production by Con A-stimulated spleen cells to the level of young B/W F1 mice. Furthermore, Neurotropin completely restored the IL-2 responsiveness of Con A-activated spleen cells from aged B/W F1 mice. To test whether Neurotropin exerts its immunoregulatory activities in B/W F1 mice by restoring IL-2 production, we directly examined the effect of recombinant IL-2 on the immune functions of spleen cells in vitro. Recombinant IL-2 markedly enhanced Con A-induced proliferative response of aged B/W F1 mice. Furthermore, the suppressive activity of spleen cells which had been activated by Con A in the presence of rIL-2 was significantly increased. These results indicate that some immunoregulatory functions of aged B/W F1 mice can be corrected by IL-2 and suggest that Neurotropin restores immunoregulatory activity in B/W F1 mice by the recovery of IL-2 production.     

Independent and synergistic effects of interleukin-18 and interleukin-12 in augmenting cytotoxic T lymphocyte responses and IFN-gamma production in aging.

Aged mice exhibit diminished CD8(+) cytotoxic T lymphocyte (CTL) response to influenza virus. Previously, interleukin-12 (IL-12) was shown to partially restore in vitro influenza virus-specific CD8(+) CTL activity and interferon-gamma (IFN-gamma) production in aged mice. The present study investigated IL-18 production and its ability to collaborate with IL-12 to enhance these responses to the levels of young mice. IL-18 protein production and mRNA expression in influenza virus-specific CTL from aged mice were higher than from young mice. In contrast, IL-18 receptor (IL-18R) mRNA expression was significantly reduced in CD8(+) CTL from aged mice. Generation of CTL in the presence of IL-12 alone caused a significant increase in IFN-gamma production in both old and young mice. IL-18 treatment alone significantly increased IFN-gamma in CTL from young but not old mice. However, a combination of IL-18 and IL-12 significantly increased IFN-gamma in both old and young mice. IL-18 and IL-12, either alone or in combination, stimulated significant influenza virus-specific cytotoxicity in both old and young mice, but no significant synergistic effect was observed. These results represent an initial demonstration of downregulated IL-18R expression in aging mice and are consistent with age-related cytotoxic T lymphocyte 1 (Tc1) deficiency. Potentially, IL-18 and IL-12 can augment IFN-gamma production and reverse CD8(+) CTL deficiency in aging, independently or synergistically.     

T-cell cytotoxicity and aging: differing causes of reduced response in individual mice

Specific T-cell cytotoxic responses to allogeneic and hapten-modified syngeneic cells decrease with age. In order to determine the causes of these reduced T-cell cytotoxic responses, spleen cells from individual young and senescent C57BL/6J female mice were mixed in various proportions in culture with either X-irradiated BALB/c spleen cells or trinitrophenyl-modified syngeneic cells and the resultant cytotoxic responses determined in comparison to those of spleen cells from young and old mice stimulated alone. In both allogeneic and hapten-modified syngeneic cytotoxicity, it was found that a low percentage of the aged mice suffered from decreased helper-cell activity or from increase of suppressor activity, while the majority of mice showed no synergy, positive or negative, with the cells from the young donor. Studies of interleukin-2 (IL-2) activity were performed on conditioned medium from spleen cells from mice of various ages cultured for 24 h with concanavalin A. Those preparations from senescent mice that showed reduced IL-2 activity did not contain activity suppressive or competitive to IL-2 produced by spleen cells from young mice. Limiting dilution of spleen cells from mice of various ages in the presence of semi-allogeneic stimulatory cells and subsequent assay of the resultant allogeneic cytotoxicity provided a measure of the frequency of cytotoxic units. Parallel experiments in which crude IL-2 was added to the limit dilution cultures provided a measure of the frequency of cytotoxic cell precursors. Once again in these experiments, individual senescent mice demonstrated different defects. Three different types of age-related defects were observed. Certain aged mice were devoid of detectable cytotoxic units and cytotoxic T-lymphocyte precursor at the cell dilutions used. Other senescent mice demonstrated a very low frequency of cytotoxic units (approximately 1/40 000) as compared with young mice (approximately 1/5 000), and the addition of crude IL-2 to cultures from these mice did not improve reactivity. A third group of old mice, those with a moderate age-related decrease in the frequency of cytotoxic units (approximately 1/12 000), demonstrated a cytotoxic T-lymphocyte precursor frequency in the presence of crude IL-2 which was comparable to that of young mice (approximately 1/1000).     

A suppressor B lymphocyte inhibiting IL-2 consumption in spleen cell cultures from Mycobacterium bovis BCG-infected mice.

In concanavalin A (Con A)-activated spleen cell (SC) cultures from normal C57BL/6 mice, the production of IL-2 peaked at 18-20 hr after initiation of cultures and declined rapidly during the next 24 hr, the decline of IL-2 activity being due, at least in part, to its utilization by the Con A-induced IL-2 receptor cells. In Con A-activated SC from BCG-infected mice, significant levels of IL-2 activity persisted in the 48-hr and 72-hr culture supernatants, a situation which seemed to be related to the depressed capacity of infected splenocytes to acquire IL-2 receptors. In cell mixing experiments, SC from infected mice actively depressed the utilization of IL-2 by Con A-activated normal SC, thus indicating that suppressor cells can down-regulate IL-2 responsiveness. These suppressor cells may belong to the B-cell lineage since they possessed the Thy-1-, sIg+ and FcR+ phenotype.     

Intra-epithelial lymphocytes: interferon-gamma production and suppressor/cytotoxic activities.

Human intraepithelial lymphocytes (IEL) proliferate minimally in response to phytohaemagglutinin (PHA), but produce as much interleukin-2 (IL-2) as do peripheral blood lymphocytes (PBL). The addition of sheep erythrocytes during activation of IEL with PHA markedly augments both T cell functions. This study evaluates the ability of IEL to produce interferon-gamma (IFN-gamma) and to develop suppressor and cytotoxic activities when stimulated with mitogens in the presence or absence of sheep erythrocytes. PHA-activated IEL produced as much IFN-gamma as did PHA-activated peripheral blood CD8+ T lymphocytes. IEL activated by concanavalin A (Con A) demonstrated less suppressor activity directed against T cell proliferation than did Con A-activated peripheral blood CD8+ T lymphocytes. IEL generated less mitogen-induced cellular cytotoxicity and lymphokine-activated killer cell activity than did peripheral blood CD8+ T lymphocytes. The addition of sheep erythrocyte lysates during mitogen stimulation of IEL markedly enhanced their proliferation and lymphokine production but did not affect their suppressor or cytotoxic activities.     

Defects in the regulation of anti-DNA antibody production in aged lupus-prone (NZB x NZW)F1 mice: analysis of T-cell lymphokine synthesis.

(NZB x NZW)F1 (B/W) mice spontaneously develop a lupus-like syndrome characterized by an increased level of autoantibodies in old mice. We analysed the role of T cells in the regulation of anti-DNA antibody production by B cells in vitro as a function of age. In cultures of old mouse T and B cells, IgG and IgM anti-DNA antibodies were synthesized at high levels, in contrast to consistently lower amounts, particularly of IgG, measured in cultures of young mouse cells. Addition of young mouse T cells to old B cells inhibited IgG, but not IgM, anti-DNA production, whereas T cells from old mice stimulated IgG synthesis by young mouse B cells. Addition of supernatants harvested from concanavalin A (Con A)-stimulated T cells to B-cell cultures induced similar effects. Therefore, we evaluated possible modifications of lymphokine synthesis compared to that of the healthy NZW parent. T cells from old mice were able to secrete normal levels of interferon-gamma (IFN-gamma) and interleukin (IL)-10; however, secretion of IL-2 and IL-4 was dramatically decreased. Semi-quantitative polymerase chain reaction analysis of constitutive RNA messengers showed increased IFN-gamma levels in young and old B/W mice, and normal IL-10 mRNA levels in young and higher levels in old mice. Constitutive IL-2 and IL-4 mRNA were detected only after Con A stimulation and their levels decreased in old compared to young B/W mice; in particular IL-2 mRNA was considerably lower in old B/W than in control NZW mice. Taken together, these results suggest that, despite constitutive T-cell abnormalities, young B/W mice are able partially to control their lymphokine production, whereas aged mice exhibit a deficient synthesis, associated with an increased capacity to produce IFN-gamma.     

Effect of feeding a diet with half of the recommended levels of all vitamins on the natural and inducible levels of cytotoxic activity in mouse spleen cells.

Groups of 6-week-old female C57Bl/6 mice were fed a normal diet with recommended levels of all vitamins or a vitamin-deficient (VD) diet containing half of the recommended level of each vitamin. At different time periods (1-11 weeks) after the initiation of diets, basal natural killer (NK) activity, interleukin-2 (IL-2) and concanavalin A (Con A)-induced cytotoxic activity, Con A-induced IL-2 production and levels of allospecific cytotoxic T cell activity generated in a mixed lymphocyte culture (MLC), were studied in spleen cells derived from control and VD mice. Results indicated that: (i) spleen NK activity remained normal until 2 weeks after the initiation of VD diet, fell steeply to low levels at the 4 and 5 week time points and remained depressed thereafter; (ii) IL-2- and Con A-induced levels of cytotoxic activity in spleen cells derived from VD mice declined at 4 weeks after the institution of VD diet, and then remained low throughout the study; (iii) the capacity of spleen cells from VD mice to generate IL-2 in response to Con A and cytotoxic T cells in response to allogeneic spleen cells, was normal at 1 and 4 weeks after initiation of the VD diet and was markedly depressed at the 6 and 9 week time points. These results suggest that partial combined deficiencies of dietary vitamins strongly influence assays of immune function.     

Immunopharmacological actions of an extract isolated from inflamed skin of rabbits inoculated with Vaccinia virus (Neurotropin)--II. Restorative effect on immune responses through the recovery of IL-2 production in aging mice

To examine the immunopharmacological actions of an extract isolated from inflamed skin of rabbits inoculated with Vaccinia virus (Neurotropin), its effect on the immune responses in aging BALB/c mice was examined. Neurotropin clearly restored the decreasing T-cell-dependent immune responses such as delayed-type hypersensitivity (DTH) response and plaque-forming cells (PFC) response to sheep red blood cells (SRBC) when administered i.p. from 13 months old (mo) to 16 mo. However, Neurotropin administration from 2 to 5 mo had no effect on the immune responses of young animals. Neurotropin administration from 13 to 16 mo restored not only the T-cell proliferation of spleen cells induced by concanavalin A (Con A) and phytohemagglutinin (PHA), but also the interleukin-2 (IL-2) production by spleen cells activated with Con A. However, Neurotropin did not affect the responsiveness of Con A-activated spleen cells to exogenous recombinant IL-2. An absence of suppressor cells capable of inhibiting the IL-2 production in the spleens was confirmed in the 16 mo mice. Neurotropin administration also restored IL-1 production by peritoneal macrophages stimulated with lipopolysaccharide (LPS). These results suggest that long-term administration of Neurotropin restores the decreasing T-cell-dependent immune responses through the recovery of IL-2 and in part IL-1 production, but not the responsiveness to IL-2 in aging BALB/c mice.     

Human colostrum contains an activity that inhibits the production of IL-2.

The effect of human colostrum on T cell immune function was investigated. Colostrum inhibited the proliferation of human T cells activated by allogeneic, concanavalin A (Con A) or phytohaemagglutinin (PHA) stimulation. Colostrum also inhibited the production of IL-2 by Con A-activated human peripheral blood T cells and by Con A-activated Jurkat cells, a human T lymphoma line. Similarly, human colostrum inhibited the production of IL-2 by EL4 cells, a murine thymoma line, when stimulated with phorbol myristate acetate. The inhibitory activity was not cytotoxic and could not be neutralized by antibody to transforming human growth factor beta.     

Gestational immunosuppression is mediated by specific Lyt 2+ T cells.

Female BALB/c mice were tested during the first week of pregnancy for their lymphocyte-mediated cytotoxic response to paternal alloantigens. Spleen or uterine regional lymph node cells were not spontaneously cytotoxic against concanavalin A-activated paternal target lymphocytes. Female mice immunized i.p. with paternal H-2-matched or third-party allogeneic cells on the fifth day and tested on the 12th day of pregnancy demonstrated total suppression of cell-mediated cytotoxicity to paternal alloantigens and partial suppression to third-party alloantigens. A generalized non-specific immunosuppression to alloantigens seems to be associated with pregnancy, which may indicate that soluble factors were involved in mediating the suppressive effect. Cocultures of spleen cells from virgin mice and the whole population of spleen or regional lymph node cells from allogeneic pregnant female mice demonstrated specifically suppressed responses to alloantigens. Similar cocultures with Thy 1.2- and Lyt 2.2-depleted populations restored the cytotoxicity levels of activated spleen cells. We conclude that antigen-specific Lyt 2+ T cells were activated during pregnancy to regulate the female T-cell response to paternal alloantigens.     

Immunotoxicity of alcohol in young and old mice. II. Impaired T cell proliferation and T cell-dependent antibody responses of young and old mice fed ethanol-containing liquid diet

The effect of extended ethanol consumption of young and old BALB/c mice on the proliferative response to Concanavalin A (Con A) and T cell-dependent antibody response of their spleen cells to sheep red blood cell (RBC) stimulation was determined. Splenic cells of young (3 months) and old (25 months) BALB/c mice, fed with one of three different diets (ethanol, maltose-substitute and standard mouse chow), were first cultured with Con A to assess T cell proliferation and production of interleukin 2 (IL2). Then, Con A-activated T blast cells from young and old mice were assessed for their proliferative responding capacity to exogenous human recombinant IL2 and crude rat IL2 supernatant. Finally, splenic cells of young and old mice were assessed for their ability to generate plaque-forming cells in response to sheep RBC. The results revealed that both T cell mitogenesis and IL2-dependent proliferation of T blast cells from young and old ethanol diet-fed mice were remarkably diminished as compared to that of young and old maltose-substituted diet (isocaloric control) fed mice, respectively. The ability of T cells from both young and old ethanol diet-fed mice to produce IL2, however, was not affected. Finally, the ability of young and old ethanol diet-fed mice to mount a primary antibody response to SRBC was also significantly reduced. These results taken together demonstrate for the first time that both T cell proliferative activity and T cell-dependent antibody response of young and old ethanol diet-fed mice are impaired; however, with respect to age, a differential effect of immunosuppression of ethanol was not noted.     

Production of gamma interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor alpha.

Previous studies of mice have implicated natural killer (NK) cells as mediators of protective activity against Toxoplasma gondii through their production of gamma interferon (IFN-gamma). In the present study, we have compared NK-cell activity in infected and uninfected SCID mice. Our data reveal that infection results in increased levels of IFN-gamma in serum and elevated NK-cell activity but that these NK cells were not cytotoxic for T. gondii-infected P815 cells. Treatment with anti-IFN-gamma antibody abrogated the increase in NK-cell activity and resulted in earlier mortality of infected mice. In vivo treatment with anti-asialo GM1 antiserum reduced NK cell activity and levels of IFN-gamma in serum but did not alter time to death. Spleen cells from infected mice produced higher levels of IFN-gamma than those from uninfected mice when stimulated in vitro with live T. gondii or parasite antigen preparations. Further analysis revealed that interleukin 10 (IL-10) inhibited, whereas tumor necrosis factor alpha (TNF-alpha) and IL-12 enhanced, IFN-gamma production by spleen cells from infected or uninfected mice. The combination of IL-12 and TNF-alpha induced higher levels of IFN-gamma from whole spleen cells of infected mice than from those of uninfected mice. Depletion of the adherent cell population from the spleen cells of infected mice led to a significant reduction in the levels of IFN-gamma produced after stimulation with IL-12 plus TNF-alpha. Similar results did not occur with cells from uninfected mice. These data indicate that other cytokines produced by the adherent cell population from infected mice may be involved in maximal production of IFN-gamma by NK cells stimulated with IL-12 and TNF-alpha. To assess the importance of endogenous IL-12, a polyclonal anti-IL-12 was administered to infected SCID mice. This treatment led to earlier mortality, indicating that endogenous IL-12 mediates resistance to T. gondii.     

Role of mRNA stability in the different patterns of cytokine production by CD4+ cells from young and old mice.

CD4+ cells from young (3 months) and old (19 months) mice were stimulated by plate-bound anti-CD3 monoclonal antibody (mAb) alone or also by soluble anti-CD28 mAb. Supernatants were analysed by enzyme-linked immunosorbent assay (ELISA) to determine cytokine concentrations. Total RNA was extracted from cells, reverse transcribed and the cDNA amplified by polymerase chain reaction (PCR) to evaluate the amount of specific mRNA. The results indicate that anti-CD3 alone is not sufficient to induce interleukin-2 (IL-2) production in CD4+ cells from both young and old mice. However, anti-CD28, together with anti-CD3 mAb, induces a much higher production of IL-2 in CD4+ cells from young as compared with old mice. Conversely, interferon-gamma (IFN-gamma) production is also induced by anti-CD3 alone and is higher in CD4+ cells from old as compared with young mice. Upon addition of anti-CD28 mAb, IFN-gamma production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL-4 and IL-10 is induced by anti-CD3 mAb but it is increased by the addition of anti-CD28 mAb. CD4+ cells from old mice produce more IL-4 and IL-10 as compared with cells from young mice. The amounts of cytokine specific mRNA in CD4+ cells from young and old mice parallel the cytokine levels in culture supernatants. Results on the mRNA turnover indicate that when CD4+ cells are stimulated by anti-CD3 or costimulated also by anti-CD28 mAb, the IFN-gamma, IL-4 and IL-10 specific mRNAs are more stable in old than in young mice, suggesting that mRNA stability has a relevant role in the different patterns of cytokine production.     

Induction of Lyt-2+ cytotoxic T lymphocytes following primary and secondary Salmonella infection.

Investigations of the cytotoxic activity of T cells induced following one or two intraperitoneal doses of live Salmonella revealed that cytotoxicity was restricted to the Lyt-2+ T-cell subset and was enhanced following secondary infection with Salmonella. Initial studies using the lectin-dependent cellular cytotoxicity (LDCC) assay detected Lyt-2+ cytotoxic T cells in peritoneal cell suspensions of S. enteritidis 11RX (11RX)-infected mice, with the peak of activity occurring 5 days after infection. This did not correlate with the proliferative activity of these cells, which peaked 10-12 days after infection. Secondary challenge with 11RX or S. typhimurium C5 (C5) induced a rapid increase in the cytotoxic activity of Lyt-2+ peritoneal T cells and was detected even 21 days later. The antigen specificity of some of these cells was confirmed in cytotoxicity assays using P815 tumour cells infected with 11RX organisms as targets. No cytotoxic activity was detected in the spleen cell suspensions of infected (and normal) mice unless the cells were first activated by in vitro culture with concanavalin A (Con A). Both types of activated spleen cells showed LDCC but Salmonella-specific cytotoxic Lyt-2+ T cells were detected only in spleen cell (SC) cultures prepared from mice challenged with a second dose of Salmonella.     

Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice: natural killer cell activity in cultured spleen leukocytes concomitant with T-cell-dependent immune interferon production

The characteristics and specificities of spleen and peritoneal cytotoxic cells generated during lymphocytic choriomeningitis virus (LCMV) infection of C3H/St mice were examined. Activated natural killer (NK) cell activity was identified in fresh leukocyte populations from the 2nd to 8th days postinfection, whereas virus-specific cytotoxic T-cell activity was detected from the 6th to 14th days. When leukocytes were cultured overnight at 37 degrees C before assay, T-cell activity was still observed, but nonspecific activated NK cell-like cytotoxicity was only detected on the 6th and to a lesser degree the 8th day postinfection. Overnight culture of leukocytes taken earlier in the infection eliminated their NK cell activity. Similar activities were seen with spleen cell, plastic-adherent peritoneal cell, and nonadherent peritoneal cell populations. The virus-specific cytotoxicity observed with adherent peritoneal cells was due to contamination with cytotoxic T cells, as shown by H-2-restricted cytotoxicity and sensitivity to anti-theta antibody and complement. The nonspecific cultured day 6 effector cell from either the spleen or peritoneum displayed killing specificities and other physical properties identical to those of activated NK cells, but had sensitivities to anti-theta antibody and complement intermediate between activated day 3 NK cells and cytotoxic T cells. Culture stable NK-like cells were not found in athymic nude mice, suggesting a T-cell-dependent mechanism. Whereas LCMV spleen homogenates contained 10-fold-higher levels of interferon at day 2 than at day 6 postinfection, substantially more (nearly 20-fold) interferon was made in cultures of day 6 cells than day 2 cells. Spleen interferon was predominantly type I, whereas the culture interferon was predominantly type II, as shown by acid lability studies. Significant levels of interferon were produced by nylon-wool-passed day 6 spleen cells, and virtually all interferon production was eliminated by treatment of either day 2 or day 6 cells with antibody to theta antigen and complement, suggesting that T cells produced the interferon in vitro. Furthermore, athymic nude mice had no culture-stable NK cells 6 days postinfection, and spleen cells from them failed to produce significant levels of interferon in vitro. Addition of interferon (type I, fibroblast) to cultured C3H spleen cells affect the already elevated levels of cytotoxicity in day 6 cultures, suggesting that the NK cells in the day 6 culture were already activated. Our results suggest that T cells responding to LCMV infection secrete interferon type II which causes the continued activation of NK cells in culture. The resulting population of activated NK cells therefore appears to be relatively stable in culture and to express more theta antigen because of this T-cell dependence. Although one could mistakenly or allospecific cytotoxic T cells or cytotoxic macrophages, more careful examination shows that they are most likely activated NK cells...     

Corrective effects of interleukin-12 on age-related deficiencies in IFN-gamma production and IL-12Rbeta2 expression in virus-specific CD8+ T cells.

Interleukin-12 receptor beta2 (IL-12Rbeta2) has been shown to be selectively expressed on Th1 T cell subsets, and we have previously shown that influenza-specific CD8+ cytotoxic T lymphocyte (CTL) deficiency in old mice was associated with deficient Th1 (interferon-gamma [IFN-gamma]) cytokine production. This study tested whether IL-12Rbeta2 expression was also deficient in CD8+ CTL from old mice and the effect of IL-12 treatment on these responses. Splenic lymphocytes from influenza-primed old and young BALB/c mice were stimulated with influenza virus in vitro with and without IL-12 and then enriched for CD8+ T cells. IFN-gamma was significantly reduced, whereas IL-4 and IL-12p40 (an antagonist of IL-12 function) were evaluated in old when compared with young mice. This was true for secreted protein measured by ELISA and for mRNA levels quantitated by RT-PCR. IL-12Rbeta2 mRNA expression in CD8+ CTL was also significantly reduced in old mice. IL-12 treatment in vitro caused significant upregulation of IFN-gamma and IL-12Rbeta2 and downregulation of IL-4 in CD8+ T cells from old mice and young mice. The present demonstration of an age-related downregulation in IL-12Rbeta2 expression and our previous data showing reduced IFN-gamma and elevated IL-4 production provide strong evidence that CD8+ CTL deficiency in aging results from a Th1/Th2 cytokine production switch. Agents that increase IL-12Rbeta2 expression and redirect Th2 to Thl immune responses are likely to enhance CD8+ CTL-mediated control of viral infections in aging.     

Lymphocyte-mediated cytotoxicity against tumor cells II. Characteristics and tissue distribution of concanavalin A-activated cytotoxic effector cells.

In vivo or in vitro activation of cytotoxic effector lymphocytes by concanavalin A (Con A) has been shown to result in a population of effector cells exhibiting a degree of immunological specificity when Con A was present during the cytotoxic assay (Waterfield, J.D. et al., Cell. Immunol. 1975. 17:392). In this communication we have characterized the Con A-activated effector cell by physical criteria and tried to assign it to a given subpopulation of thymus-derived lymphocytes. It was shown that macrophages played no role in target cell lysis in this system, nor was their presence required for Con A activation of the effector cell. The effector cell proved insensitive to anti-theta serum, but was classified as a T cell by the enrichment of cytotoxicity when a purified population of T cells was activated by Con A in vitro and by the failure to activate the effector cell in nude mice. Precursors of the effector cell were shown to reside primarily in the spleen, to be radioresistant up to 400 R, and to be short-lived after adult thymectomy. Thus, the effector cell can be classified with the T1 population of T cells and has similar characteristics as a cytolytically active cell described by Stobo et al. (J. Immunol. 1973. 110: 362, 652).     

IL-2 and IL-4 can co-modulate the generation of cytotoxic T cells through CD8- CD4- splenic lymphocytes.

Following activation with concanavalin A (Con A), murine T cells are able to suppress the generation of allospecific cytotoxic T lymphocytes (CTL). We have analysed the phenotype, tissue distribution, and mode of action of these cells in an effort to understand further the regulation of CTL-mediated immunity. The precursors of such cells are rare (1 cell per 70,000 spleen cells being able to suppress the generation of a particular allospecific response), but are much more abundant in the spleen than in the thymus. By the use of cytotoxic antibodies, we have been able to demonstrate that the splenic precursors of such cells are Thy-1.2+, CD4-, CD8- but, following activation with Con A, these cells acquire the CD8 marker. Cellular suppression by these lymphocytes is dramatically increased in the presence of the Th2-derived lymphokine, IL-4, whereas IL-2, the Th1-derived lymphokine, significantly augments the generation of CTL in a mixed lymphocyte culture even though relative suppression is still evident in the presence of Con A-activated lymphocytes. Suppression is not due to overcrowding of a cell culture since adding Con A-activated cells to an A anti-B + C culture often resulted in the suppression of the A anti-B response but not the A anti-C response, or vice versa. Suppression appears to require cellular interaction since supernatants from Con A-activated lymphocytes are unable to mediate suppression. Such cells may play an important intermediate role in homeostasis.