Stat5b is essential for natural killer cell-mediated proliferation and cytolytic activity.

We have analyzed the immune system in Stat5-deficient mice. Although Stat5a-/- splenocytes have a partial defect in anti-CD3-induced proliferation that can be overcome by high dose interleukin (IL)-2, we now demonstrate that defective proliferation in Stat5b-/- splenocytes cannot be corrected by this treatment. Interestingly, this finding may be at least partially explained by diminished expression of the IL-2 receptor beta chain (IL-2Rbeta), which is a component of the receptors for both IL-2 and IL-15, although other defects may also exist. Similar to the defect in proliferation in activated splenocytes, freshly isolated splenocytes from Stat5b-/- mice exhibited greatly diminished proliferation in response to IL-2 and IL-15. This results from both a decrease in the number and responsiveness of natural killer (NK) cells. Corresponding to the diminished proliferation, basal as well as IL-2- and IL-15-mediated boosting of NK cytolytic activity was also greatly diminished. These data indicate an essential nonredundant role for Stat5b for potent NK cell-mediated proliferation and cytolytic activity.

Disease and non-disease-related cell-mediated cytotoxicity in humans.

The human bladder cancer/T24 system was used to investigate disease and non-disease-related cell-mediated cytotoxicity (CMC). CMC was determined in a modification of the microcytotoxicity assay of Takasugi and Klein. Analysis of data of groups of patients confirmed previous findings that effector cells (EC) from bladder cancer patients were more cytotoxic against T24 than were EC from normal individuals or from patients with other genitourinary cancers. Differences between patients with bladder cancer and other patients were not observed for other target cells. During the course of these experiments, non-disease-associated CMC by EC from individual normal donors and patients was observed. This phenomenon was investigated to determine its reproducibility and its relationship to different methods of preparing EC. Reproducibility of non-disease-related CMC was ascertained using EC prepared from heparinized blood by centrifugation over Ficoll-Hypaque (FH). A total of 126 experiments were performed in which 18 normal donors were tested 2 to 7 times each against 4 target cell lines. Of the resulting 46 combinations or groups of repeated assays, only 7 showed significant variability. Each normal donor had consistent CMC with differences from others being reproducible. CMC was therefore not due to crowding or physical effects. CMC mediated by EC prepared in this manner was then compared to that mediated by EC prepared by other methods in simultaneous tests.

Xenotransplantation--federal regulatory considerations.

Xenotransplantation may offer benefits to individual patients, but may also pose significant risks, to the recipient and to the public at large. The United States Food and Drug Administration endeavors to meet the challenge of regulating xenotransplantation to allow its development while safeguarding the public health. The approach includes using the existing FDA regulatory framework, publishing xenotransplantation-specific guidance documents, obtaining public input, and collaborating with other public health agencies and international bodies in order to develop and maintain a safe and rational program for the regulation of xenotransplantation.

Xenotransplantation: regulatory challenges.

During 1999-2000, the US government published three xenotransplantation policy/guidance documents, one by the Public Health Service and two by the Food and Drug Administration (FDA). The FDA also held two public meetings of the xenotransplantation subcommittee of the Biological Response Modifiers Advisory Committee to discuss particular issues in xenotransplantation.

Life events, depressive symptoms, and immune function.

Because both bereavement and depression have been associated with impaired immune responses, the authors studied two indicators of immune function, natural killer (NK) cell activity and measures of T cell subpopulations, in 37 women who differed in the magnitude of recent life events. Women who had experienced major life changes had lower NK cell activity than women who had few changes. Severity of depressive symptoms in these women was associated with an impairment of NK cell activity, an absolute loss of suppressor/cytotoxic cells, and an increase in the ratio of T helper to T suppressor/cytotoxic cells.

Lymphokine-activated killer cells and aging in mice: significance for defining the precursor cell.

The present study was undertaken to define the cell populations which mediate lymphokine-activated killer (LAK) cell activity in mice. Because old mice exhibit markedly decreased to nondetectable natural killer (NK) cell activity, this age-associated change provided an advantageous system to examine the contribution of NK and T cells to LAK activity. Spleen cells from either young (6-9 weeks) or old (20-26 months) mice were cultured with 1000 units/ml of recombinant interleukin 2 (rIL 2) for 3-5 days. The cells were then tested in a 51 Cr-release assay for their cytotoxicity against NK-resistant fresh tumor cells (MCA-102). The LAK activity exhibited by spleen cells from old mice following 5 days of culture was equivalent to that developed by spleen cells of young mice. This result was contrary to what would be anticipated if mature NK cells comprise the primary precursors of LAK activity, and required further elucidation. The Thy-1 and asialo GM1 (ASGM1) phenotypes of LAK precursor and effector cells were therefore examined by depletion techniques using the appropriate antibodies plus complement. The results using spleen cells harvested after 5 days of culture with rIL 2 showed that LAK effector cells which developed from spleen cells of both young and old mice were predominantly Thy-1+ (85.3% young; 91.8% old) and some coexpressed ASGM1. Spleen cells were treated prior to culture to study the precursor cells. Development of LAK activity by spleen cells from both young and old mice was greatly reduced by pretreatment with anti-ASGM1 plus complement. However, since spleen cells of old mice exhibit very low mature NK activity, these data suggest that the LAK precursors, at least in old mice, may be ASGM1+ NK precursor cells rather than mature ASGM1+ NK effector cells. In addition, treatment with anti-Thy-1 plus complement inhibited generation of a significant proportion of LAK activity only in the spleens of old mice, suggesting a qualitative difference in LAK precursor cells with age and supporting the heterogeneity of the cells which are capable of developing LAK activity.

Suppression of murine natural killer cell activity by adherent cells from aging mice.

Natural killer (NK) cell activity declines with age in mice. The purpose of this study was to investigate the effect of peritoneal and splenic adherent cells from young and old mice on NK activity to determine whether adherent cell suppressor function might contribute to this decline. Peritoneal adherent cells from old mice suppressed NK activity of young splenic non-adherent indicator cells more than peritoneal cells from young mice. Splenic adherent cells from old but not from young mice also suppressed this activity. That (1) the suppressive activity of the adherent cell populations was not affected by treatment with anti-Thy-1 plus complement, and that (2) the adherent cell population contained 77-92% cells positive for alpha-naphthyl acetate esterase activity, suggests that the active adherent suppressor cell may be a macrophage. Therefore, the age-related decline in NK activity in mice can be explained, in part, by an increase in adherent cell suppressor function.

Differential effects of interleukin-12 treatment on gene expression by allostimulated T cells from young and aged mice.

Alloantigen stimulation was used to examine the effect of interleukin (IL-12) treatment of stimulated cells from young and aged mice on the expression of mRNAs for perforin and granzyme B, two proteins known to be intimately involved in an important lytic pathway used by CTL, and mRNA for interferon (IFN)-gamma, production of which is highly stimulated by IL-12 As reported previously, IL-12 augmented the lytic activity by cells from both young and aged mice, although the relative increase was greater for the latter. The mRNAs encoding perforin and granzyme B were both marginally enhanced at early time points (for cells from young mice) or throughout the stimulation (for cells from aged mice) following allo-stimulation in the presence of IL-12. The levels of augmentation of these mRNAs was consistent with the augmentation of lytic activity. In contrast, mRNA encoding IFN-gamma was markedly enhanced throughout stimulation in cells from animals of both age groups, corresponding to the more substantial increase in interferon protein in response to IL-12.

Quantification of natural cytotoxicity by human lymphocyte subpopulations isolated by density: heterogeneity of the effector cells.

Natural cell mediated cytotoxicity has been expressed as percent cytotoxicity, as the slope of the titration curve obtained by testing different effector: target cell ratios, and as lytic units. Objections can be raised to each method as used. The present report involves the study of cytotoxicity by subpopulations of lymphocytes obtained by Percoll density gradient centrifugation. The subpopulations vary greatly in cytotoxic activity, making accurate comparisons by traditional means difficult. A method was therefore developed for making objective comparisons between activities of subpopulations of lymphocytes. Cytotoxicity titration curves, which were sigmoidal on linear-linear plots, were found to best fit the sigmoidal curves described by the Von Krogh equation. A best fitting scale family of curves having the identical maximum cytotoxicity and shape parameter was fitted simultaneously to cytotoxicity measurements obtained by titrating all subpopulations of effector cells obtained from each patient. Values expressing relative cytotoxicities were obtained from the ratios of the scale parameters of the different curves or by obtaining lytic units from the fitted curves. In addition to the enriched natural cytotoxic activity found among the lighter cells obtained by Percoll density gradient centrifugation, activity was also increased among cells sedimenting at the very bottom of the gradient. This was found in subpopulations of cells obtained from 16/21 gradients in tests against K562 and in subpopulations obtained from both of 2 gradients in tests against Daudi cells. These findings are consistent with the existence of at least 2 subpopulations of lymphocytes which can mediate natural cytotoxicity, and which are separable by density.

Does the age-related change in CD44-defined T-cell subsets have functional significance for cytotoxic T lymphocyte generation?

CD44 or Pgp-1 is a transmembrane leukocyte adhesion-related glycoprotein which is often expressed in greater density on the membranes of memory T lymphocytes (CD44hi) compared to naive T cells (CD44lo). The proportion of Pgphi or CD44hi cells among T cells is increased with advancing age. We examined the relevance of this alteration for the age-related decrease in the generation of allospecific CTL activity. The findings confirm the age-related increase in the frequency of CD44hi cells in spleens of aged mice of several strains, but also show interstrain variability in the magnitude of the increase (bm1 > C57BL/6 > BALB/c). In contrast, we found that after allo-stimulation, the proportion of cells bearing the memory phenotype is decreased in cells from aged mice, particularly within the CD8+ T cell subset. To determine if these observations reflected an alteration in the frequency or responsiveness of naive T cells, enriched populations of spleen cells depleted of CD44hi cells were prepared from spleen cells of young and aged mice, and stimulated in mixed lymphocyte culture. Enrichment for cells expressing the naive phenotype did not restore the ability of T cells from aged mice to generate allospecific CTL. Together, these findings suggest that (1) the age-related increase in frequency of splenic T cells expressing memory phenotype and concordant decrease in phenotypically naive cells, does not explain the age-related decrease in the ability to generate primary allo-CTL, and (2) naive cells from aged mice exhibit intrinsically compromised ability to generate CTL in response to primary alloantigenic stimulation.

Concentration-dependent augmentation of anti-K562 activity following short-term culture of null cell-enriched human blood lymphocyte populations.

The increase in natural cell-mediated cytotoxicity against K562 cells following short-term culture (e.g. 12 hours) of null cells was dependent upon the concentration of cells during incubation. The increased lytic activity by cells cultured at higher concentrations was associated with increased numbers of target-binding cells. It occurred with a variety of serum supplements in the medium. The augmentation of natural killer activity required close cellular proximity or contact and was not associated with detectable increased release of interferon or interleukin 2, although the participation of these molecules cannot be excluded. The lower recovery of lytic activity from cells incubated at lower concentrations was not associated with increased PGE synthesis.

Immunological responses of aging Japanese A-bomb survivors.

Immune response parameters were studied on 1341 A-bomb survivors residing in Hiroshima, Japan. Mononuclear cells were isolated from venous blood and tested for interleukin-2 production; lymphocytes were purified and tested for natural killer (NK) cell activity and interferon (IFN) production; and serum was tested for IFN and circulating immune complex (CIC) levels. Statistical analyses were performed for each type of assay using a linear models procedure including sex, age at the time of the bomb, radiation exposure, all the interaction variables, and the categorical variable day-of-assay in the model. The findings showed that (1) none of the immunologic variables were significantly affected by radiation exposure; (2) NK activity and CIC levels were positively associated with age; and (3) NK activity was on average higher for males than females. The data exemplify the difficulty in reaching firm conclusions concerning associations with radiation exposure when the dependent variable exhibits a large degree of interindividual and day-of-assay variability.

Immune function in aging atomic bomb survivors residing in the United States.

Immunologic parameters were studied among survivors of the 1945 atomic bombs who now reside in the United States. Of all known survivors living in the U.S., about 40% (n = 189) participated in this study. Of those survivors on whom radiation exposure information was available (n = 168), 96% were exposed to less than 50 rad at the time of the bomb (ATB). Survivors were divided into two groups; those exposed to varying low doses of radiation (S+ group, exposed at less than or equal to 2500 m from the hypocenter) were compared with those exposed to "O rad" (S0 group, exposed at greater than 2500 m from the hypocenter). Of the former group, 92% were exposed to less than 100 rad and 89% to less than 50 rad ATB. Cellular immune responses, including natural cell-mediated cytotoxicity (NCMC), interferon production, and the mitogenic response to PHA, tended to be higher among S+ individuals, although only the difference for NCMC was statistically significant. This was suggestive of a trend which was consistent with the higher serum interferon levels and lower frequencies of detectable immune complexes and antimitochondrial antibodies among the S+ group, although these differences were not statistically significant. Other immunologic parameters which showed no trend included frequency of antinuclear antibodies, rheumatoid factor, levels of serum immunoglobulins, levels of isoantibodies and heteroantibodies, and the magnitude of the mixed lymphocyte reaction.

Immunogenicity and efficacy of a DNA vaccine in aged mice.

The immunogenicity and protective efficacy of a DNA vaccine encoding the circumsporozoite protein of the Plasmodium yoelii malaria parasite was evaluated in young (2 months) versus aged (>26 months) BALB/c mice. The primary and secondary humoral immune response of aged mice was 19- and 7-fold lower, respectively, than that of similarly treated young animals (p < .01). Cytotoxic T lymphocyte activity in aged mice was also lower than in younger animals. The vaccine response of aged animals was characterized by a 6-fold increase in interleukin-4 and a 3-fold increase in interferon-gamma (IFN-gamma) secreting cells, whereas in young animals immunization only stimulated the production of the type 1 cytokine IFN-gamma. Overall, 80% of young vaccinated mice were protected from subsequent challenge with live malaria sporozoites whereas only 40% of aged mice were protected. These results are the first to demonstrate that DNA vaccination induces less effective immunity in aged than young animals.

Target cell-induced apoptosis in IL-2-activated human natural killer cells.

We demonstrated that tumor cells induce cell death in lymphokine-activated NK (LAK) cells, but not in non-activated NK cells. Cell death in LAK cells involves nuclear condensation and DNA cleavage, all of which are characteristic features of apoptosis. The mechanism involves signaling through integrins and requires src family tyrosine kinases and protease activities. Engagement of an apoptotic signal molecule, Fas, may also trigger LAK cell death by apoptosis. It appears that LAK cells rapidly die by apoptosis after attacking tumor cells. This phenomenon may provide a means for potential tumor target cells to escape from natural immunosurveillance during therapeutic interventions such as those using IL-2 or LAK cells.

Delayed effects of low-dose radiation on cellular immunity in atomic bomb survivors residing in the United States.

Several parameters of cellular immune function were assessed among persons who survived the 1945 atomic bombs in Hiroshima and Nagasaki but who now reside in the United States. The subjects in this study were exposed to various low doses (T65D) of radiation at the time of the bomb. More than half received an estimated 0 Gy (S0 group). Of those exposed to more radiation (S+ group), nearly 90% received less than 0.50 Gy (50 rad). Lymphocytes were isolated from the peripheral blood of these individuals and were assessed for the following parameters of cellular immunity: mitogenic response to phytohemagglutinin, mitogenic response to allogeneic lymphocytes, natural cell-mediated cytotoxicity (NCMC), and interferon production. In every case, the response of the S+ group was greater than that of the S0 group, although only the difference for NCMC was statistically significant. Results of studies presently being performed on A-bomb survivors residing in Hiroshima do not confirm this difference. Therefore, it is difficult to say whether the increase in natural cytotoxicity observed among the American and not the Japanese A-bomb survivors exposed to very low doses of radiation is a hormetic effect which was modulated by post-radiation environmental conditions or a result of selective migration.

Xenotransplantation: the potential and the challenges.

Clinical use of xenotransplants is a potential way to provide care for a population of seriously ill patients and alleviate the demand for human organs. However, xenotransplantation also presents a spectrum of concerns, not only for individual patients but also for the public health, that must be discussed and dealt with in a science-based and public manner. Such discussions should take place on a national level and should include scientists, physicians, and policy makers from all countries in which the clinical use of xenografts is being considered.