Sex differences in stress and immune responses during confinement in Antarctica.

BACKGROUND: Antarctica challenges human explorers by its extreme environment. The effects of these unique conditions on the human physiology need to be understood to best mitigate health problems in Antarctic expedition crews. Moreover, Antarctica is an adequate Earth-bound analogue for long-term space missions. To date, its effects on human physiology have been studied mainly in male cohorts though more female expeditioners and applicants in astronaut training programs are selected. Therefore, the identification of sex differences in stress and immune reactions are becoming an even more essential aim to provide a more individualized risk management. METHODS: Ten female and 16 male subjects participated in three 1-year expeditions to the German Antarctic Research Station Neumayer III. Blood, saliva, and urine samples were taken 1-2 months prior to departure, subsequently every month during their expedition, and 3-4 months after return from Antarctica. Analyses included cortisol, catecholamine and endocannabinoid measurements; psychological evaluation; differential blood count; and recall antigen- and mitogen-stimulated cytokine profiles. RESULTS: Cortisol showed significantly higher concentrations in females than males during winter whereas no enhanced psychological stress was detected in both sexes. Catecholamine excretion was higher in males than females but never showed significant increases compared to baseline. Endocannabinoids and N-acylethanolamides increased significantly in both sexes and stayed consistently elevated during the confinement. Cytokine profiles after in vitro stimulation revealed no sex differences but resulted in significant time-dependent changes. Hemoglobin and hematocrit were significantly higher in males than females, and hemoglobin increased significantly in both sexes compared to baseline. Platelet counts were significantly higher in females than males. Leukocytes and granulocyte concentrations increased during confinement with a dip for both sexes in winter whereas lymphocytes were significantly elevated in both sexes during the confinement. CONCLUSIONS: The extreme environment of Antarctica seems to trigger some distinct stress and immune responses but-with the exception of cortisol and blood cell counts-without any major relevant sex-specific differences. Stated sex differences were shown to be independent of enhanced psychological stress and seem to be related to the environmental conditions. However, sources and consequences of these sex differences have to be further elucidated.

Early immune anergy towards recall antigens and mitogens in patients at onset of septic shock.

The pathology of sepsis is typically characterized by an infection and excessive initial inflammation including a cytokine storm, followed by a state of immune suppression or paralysis. This classical view of a two peak kinetic immune response is currently controversially discussed. This study was a sub-study of the randomized clinical Trial SISPCT registered with www.clinicaltrials.gov (NCT00832039, Registration date: 29/01/2009). Blood samples from 76 patients with severe sepsis and septic shock were incubated for 48 h at 37 °C in vitro with bacterial or fungal recall-antigens or specific mitogen antigens within 24 hours of sepsis onset. Recall-antigen stimulation led to a severe dampening of normal cytokine release. This immunologic anergy was similarly observed after mitogen stimulation. Moreover, patients under hydrocortisone therapy or with lowered arterial oxygen tension had further reductions in cytokine levels upon B- and T-cell mitogen stimulation. This investigation reveals an early onset of immunoparalysis during sepsis. This immune incompetence in mounting an adequate response to further infections includes previously sensitized pathogens, as seen with recall-antigens. Also, the immune-suppressive role of hydrocortisone and low PaO2 is highlighted. Aside from early broad-spectrum antimicrobial therapy, our findings reinforce the need for maximal immunological support and protection against further infections at the onset of sepsis.

Hyperbaric hyperoxia alters innate immune functional properties during NASA Extreme Environment Mission Operation (NEEMO).

BACKGROUND: Spaceflight is associated with immune dysregulation which is considered as risk factor for the performance of exploration-class missions. Among the consequences of confinement and other environmental factors of living in hostile environments, the role of different oxygen concentrations is of importance as either low (e.g. as considered for lunar or Martian habitats) or high (e.g. during extravehicular activities) can trigger immune dysfunction. The aim of this study was to investigate the impact of increased oxygen availability--generated through hyperbaricity--on innate immune functions in the course of a 14 days NEEMO mission. METHODS: 6 male subjects were included into a 14 days undersea deployment at the Aquarius station (Key Largo, FL, USA). The underwater habitat is located at an operating depth of 47 ft. The 2.5 times higher atmospheric pressure in the habitat leads to hyperoxia. The collection of biological samples occurred 6 days before (L-6), at day 7 (MD7) and 11/13 (MD11/13) during the mission, and 90 days thereafter (R). Blood analyses included differential blood cell count, ex vivo innate immune activation status and inhibitory competences of granulocytes. RESULTS: The absolute leukocyte count showed an increase during deployment as well as the granulocyte and monocyte count. Lymphocyte count was decreased on MD7. The assessments of native adhesion molecules on granulocytes (CD11b, CD62L) indicated a highly significant cellular activation (L-6 vs. MD7/MD13) during mission. In contrast, granulocytes were more sensitive towards anti-inflammatory stimuli (adenosine) on MD13. CONCLUSION: Living in the NEEMO habitat for 14 days induced significant immune alterations as seen by an activation of adhesion molecules and vice versa higher sensitivity towards inhibition. This investigation under hyperbaric hyperoxia is important especially for Astronauts' immune competence during extravehicular activities when exposed to similar conditions.

Multiple latent viruses reactivate in astronauts during Space Shuttle missions.

Latent virus reactivation and diurnal salivary cortisol and dehydroepiandrosterone were measured prospectively in 17 astronauts (16 male and 1 female) before, during, and after short-duration (12-16 days) Space Shuttle missions. Blood, urine, and saliva samples were collected during each of these phases. Antiviral antibodies and viral load (DNA) were measured for Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and cytomegalovirus (CMV). Three astronauts did not shed any virus in any of their samples collected before, during, or after flight. EBV was shed in the saliva in all of the remaining 14 astronauts during all 3 phases of flight. Seven of the 14 EBV-shedding subjects also shed VZV during and after the flight in their saliva samples, and 8 of 14 EBV-shedders also shed CMV in their urine samples before, during, and after flight. In 6 of 14 crewmembers, all 3 target viruses were shed during one or more flight phases. Both EBV and VZV DNA copies were elevated during the flight phase relative to preflight or post-flight levels. EBV DNA in peripheral blood was increased preflight relative to post-flight. Eighteen healthy controls were also included in the study. Approximately 2-5% of controls shed EBV while none shed VZV or CMV. Salivary cortisol measured preflight and during flight were elevated relative to post-flight. In contrast DHEA decreased during the flight phase relative to both preflight and post-flight. As a consequence, the molar ratio of the area under the diurnal curve of cortisol to DHEA with respect to ground (AUCg) increased significantly during flight. This ratio was unrelated to viral shedding. In summary, three herpes viruses can reactivate individually or in combination during spaceflight.

Reactivation of latent viruses is associated with increased plasma cytokines in astronauts.

Success of long duration space missions will depend upon robust immunity. Decreased immunity has been observed in astronauts during short duration missions, as evident by the reactivation of latent herpes viruses. Seventeen astronauts were studied for reactivation and shedding of latent herpes viruses before, during, and after 9-14 days of 8 spaceflights. Blood, urine, and saliva samples were collected 10 days before the flight (L-10), during the flight (saliva only), 2-3h after landing (R+0), 3 days after landing (R+3), and 120 days after landing (R+120). Values at R+120 were used as baseline levels. No shedding of viruses occurred before flight, but 9 of the 17 (designated "virus shedders") shed at least one or more viruses during and after flight. The remaining 8 astronauts did not shed any of the 3 target viruses (non-virus shedders). Virus-shedders showed elevations in 10 plasma cytokines (IL-1α, IL-6, IL-8, IFNγ, IL-4, IL-10, IL-12, IL-13, eotaxin, and IP-10) at R+0 over baseline values. Only IL-4 and IP-10 were elevated in plasma of non-virus shedders. In virus shedders, plasma IL-4 (a Th2 cytokine) was elevated 21-fold at R+0, whereas IFNγ (a Th1 cytokine) was elevated only 2-fold indicating a Th2 shift. The inflammatory cytokine IL-6 was elevated 33-fold at R+0. In non-shedding astronauts at R+0, only IL-4 and IP-10 levels were elevated over baseline values. Elevated cytokines began returning to normal by R+3, and by R+120 all except IL-4 had returned to baseline values. These data show an association between elevated plasma cytokines and increased viral reactivation in astronauts.

Routine detection of Epstein-Barr virus specific T-cells in the peripheral blood by flow cytometry.

The ability to detect cytomegalovirus-specific T-cells (CD4(+)) in the peripheral blood by flow cytometry has been recently described by Picker et al. In this method, cells are incubated with viral antigen and responding (cytokine producing) T-cells are then identified by flow cytometry. To date, this technique has not been reliably used to detect Epstein-Barr virus (EBV)-specific T-cells primarily due to the superantigen/mitogenic properties of the virus which non-specifically activate T-cells. By modifying culture conditions under which the antigens are presented, we have overcome this limitation and developed an assay to detect and quantitate EBV-specific T-cells. The detection of cytokine producing T-cells by flow cytometry requires an extremely strong signal (such as culture in the presence of PMA and ionomycin). Our data indicate that in modified culture conditions (early removal of viral antigen) the non-specific activation of T-cells by EBV is reduced, but antigen presentation will continue uninhibited. Using this method, EBV-specific T-cells may be legitimately detected using flow cytometry. No reduction in the numbers of antigen-specific T-cells was observed by the early removal of target antigen when verified using cytomegalovirus antigen (a virus with no non-specific T-cell activation properties). In EBV-seropositive individuals, the phenotype of the EBV-specific cytokine producing T-cells was evaluated using four-color flow cytometry and found to be CD45(+), CD3(+), CD4(+), CD45RA(-), CD69(+), CD25(-). This phenotype indicates the stimulation of circulating previously unactivated memory T-cells. No cytokine production was observed in CD4(+) T-cells from EBV-seronegative individuals, confirming the specificity of this assay. In addition, the use of four color cytometry (CD45, CD3, CD69, IFNgamma/IL-2) allows the total quantitative assessment of EBV-specific T-cells while monitoring the interference of EBV non-specific mitogenic activity. This method may have significant utility for the monitoring of the immune response to latent virus infection/reactivation.

Bing de ling, a Chinese herbal formula, stimulates multifaceted immunologic responses in mice.

Bing de ling is a Chinese herbal formula most commonly used in complementary medical settings against viral disorders. We have found that bing de ling potentiates upregulation of immune activity when administered to mice in dosages proportional to those used clinically. These mice demonstrated significant elevation of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production in splenocytes and enhancement of macrophage, natural killer cell, and lymphokine-activated killer cell cytotoxicity. These data are consistent with bing de ling's clinically observed efficacy against viruses and identify the formula as a promising candidate for clinical trials against diverse diseases that may respond to increased immunologic activity.

Altered cytokine production by specific human peripheral blood cell subsets immediately following space flight.

In this study, flow cytometry was used to positively identify the specific lymphocyte subsets exhibiting space flight-induced alterations in cytokine production. Whole blood samples were collected from 27 astronauts at three points (one preflight, two postflight) surrounding four space shuttle missions. Assays performed included serum/urine stress hormones, white blood cell (WBC) phenotyping, and intracellular cytokine production following mitogenic stimulation. Absolute levels of peripheral granulocytes were significantly elevated following space flight, but the levels of circulating lymphocytes and monocytes were unchanged. Lymphocyte subset analysis demonstrated a decreased percentage of T cells, whereas percentages of B cells and natural killer (NK) cells remained unchanged after flight. Nearly all the astronauts exhibited an increased CD4/CD8 T cell ratio. Assessment of naive (CD45RA+) vs. memory (CD45RO+) CD4+ T cell subsets was ambiguous, and subjects tended to group within specific missions. Although no significant trend was seen in absolute monocyte levels, a significant decrease in the percentage of the CD14+ CD16+ monocytes was seen following space flight in all subjects tested. T cell (CD3+) production of interleukin-2 (IL-2) was significantly decreased after space flight, as was IL-2 production by both CD4+ and CD8+ T cell subsets. Production of interferon-gamma (IFN-gamma) was not altered by space flight for the CD8+ cell subset, but there was a significant decrease in IFN-gamma production for the CD4+ T cell subset. Serum and urine stress hormone analysis indicated significant physiologic stresses in astronauts following space flight. Altered peripheral leukocyte subsets, altered serum and urine stress hormone levels, and altered T cell cytokine secretion profiles were all observed postflight. In addition, there appeared to be differential susceptibility to space flight regarding cytokine secretion by T cell subsets. These alterations may be the result of either microgravity exposure or the physiologic stresses of landing and readaptation to unit gravity. Future studies, including in-flight analysis or sampling, will be necessary to determine the cause of these alterations.

Laboratory outreach: student assessment of flow cytometer fluidics in zero gravity.

Due to the the clinical utility of the flow cytometer, the National Aeronautics and Space Administration (NASA) is interested in the design of a space flight-compatible cytometer for use on long-duration space missions. Because fluid behavior is altered dramatically during space flight, it was deemed necessary to validate the principles of hydrodynamic focusing and laminar flow (cytometer fluidics) in a true microgravity environment. An experiment to validate these properties was conducted by 12 students from Sweetwater High School (Sweetwater, TX) participating in the NASA Reduced Gravity Student Flight Opportunity, Class of 2000. This program allows high school students to gain scientific experience by conducting an experiment on the NASA KC-135 zero gravity laboratory aircraft. The KC-135 creates actual zero-gravity conditions in 30-second intervals by flying a highly inclined parabolic flight path. The experiment was designed by their mentor in the program, the Johnson Space Center's flow cytometrist Brian Crucian, PhD, MT(ASCP). The students performed the experiment, with the mentor, onboard the NASA zero-gravity research aircraft in April 2000.

Development of a whole blood staining device for use during space shuttle flights.

BACKGROUND: Exposure to microgravity during space flight results in profound physiologic changes. Numerous studies have shown changes in circulating populations of peripheral blood immune cells immediately after space flight. It is currently unknown if these changes result from exposure to microgravity or are caused by the stress of reentry and readaptation to gravity. METHODS: We have developed the whole blood staining device (WBSD) as a system for the staining of whole blood collected during space flight for subsequent flow cytometric analysis. This device contains all liquids to address safety issues concerned with space flight and also moves the cells through the staining, lyse/fixation, and dilution steps. RESULTS: Data from flow cytometric analysis of samples stained in the WBSD was found to be comparable to data from samples stained by the conventional methods. Cells stained with the WBSD remain stable in the device for up to 14 days. The necessary manipulations required to use the device were tested on the KC-135 aircraft during the reduced gravity segment of parabolic flight. CONCLUSIONS: With the WBSD immunophenotype analysis can be performed at various time points for the duration of an entire Shuttle flight. In addition, this device has significant terrestrial applications for rapid and easy immunofluorescence labeling of whole blood in remote and isolated locations where immediate access to specialized equipment and skilled laboratory personnel may not be available. The WBSD provides a simple mechanism to design specific immunophenotyping tests for use by nontechnical personnel at bedside or in field locations. Cytometry 37:74-80, 1999. Published 1999 Wiley-Liss, Inc.

The use of a spaceflight-compatible device to perform WBC surface marker staining and whole-blood mitogenic activation for cytokine detection by flow cytometry.

Significant changes have recently been described regarding circulating peripheral immune cells immediately following spaceflight. Existing methods for immunophenotype staining of peripheral blood in terrestrial labs do not meet the constraints for flight on the Space Shuttle. We have recently described the development and use of the Whole Blood Staining Device (WBSD), a simple device for staining flow cytometry specimens during spaceflight. When preparing samples with the WBSD, all liquids are safely contained as the cells are moved through staining, lysis and fixation steps. Here we briefly review the use of the WBSD, and then describe another versatile adaptation, a modification to perform intracellular staining of cytokines for detection by flow cytometry. Alterations in cytokine production have been reported both in ground-based simulated microgravity culture and in astronaut samples returning from spaceflight. Data regarding microgravity effects on cytokine production for specific subpopulations of cells is lacking. Flow cytometric cytokine analysis offers the unique ability to perform simultaneous surface marker analysis and positively identity cytokine producing subsets of cells. The utilization of the WBSD provides the ability to perform rapid and routine mitogenic activation during spaceflight coupled with the ability to perform simultaneous surface marker analysis. The only external requirements for this procedure are an in-flight 37-degree incubator and the capacity for 4-degree storage.

Broadened clinical utility of gene gun-mediated, granulocyte-macrophage colony-stimulating factor cDNA-based tumor cell vaccines as demonstrated with a mouse myeloma model.

Effective immunization against the murine B16 melanoma by a nonviral approach in which a gene gun is used to transfer GM-CSF cDNA into tumor cells has been described. We have extended this nonviral approach by using the poorly immunogenic murine myeloma MPC11 model. Vaccination with the transfected, GM-CSF-expressing MPC11 cells induced a potent antitumor cytotoxic T lymphocyte response associated with tumor rejection in the majority of the test mice. Furthermore, nearly 100% (27 of 28) of the tumor-free mice were able to reject a tumor rechallenge. While this approach is clinically attractive because of minimal tissue manipulation/culturing and the absence of infectious agents, a number of tested human primary tumors, including myeloma cells, have failed to produce high levels of GM-CSF after gene gun transfection. To circumvent the low transfection efficiency in certain human tumor cells, we showed that combining irradiated tumor cells to provide tumor antigens together with gene gun-transfected fibroblasts to provide GM-CSF induced effective tumor rejection. We also report that normal human skin fibroblasts transfected by the gene gun produce high levels of human GM-CSF (250 ng/10(6) cells/24 hr). These results suggest that combining irradiated tumor cells with gene gun-transfected fibroblasts results in antitumor immune responses and may allow for a wider application of this approach to cancer immunotherapy.

Interferon-gamma-inducing factor elicits antitumor immunity in association with interferon-gamma production.

Interferon-gamma-inducing factor (IGIF) is a novel cytokine that stimulates T-cell proliferation, augments natural killer (NK) cell lytic activity, and induces interferon-gamma (IFN-gamma) production in established type 1 T-helper (Th1) cells in the presence of anti-CD3 antibody. The in vitro induction of IFN-gamma by recombinant murine IGIF in these cells was more potent than that induced by murine interleukin-12 (IL-12) and occurred apparently independent of murine IL-12. Here we report that subcutaneous injection into mice of tumor cells transfected with murine IGIF complementary DNA (cDNA) resulted in > or = 10-fold increase of mitogen-stimulated IFN-gamma production in cultured splenocytes. In addition, IGIF-transfected Renca and K1735 tumor cells can be rejected in vivo. The IGIF antitumor effect was abrogated in mice that were sublethally irradiated or depleted of both CD4+ and CD8+ T cells but not in mice depleted of either subpopulation alone. The antitumor effect mediated by IGIF appears to be dependent on IFN-gamma production, because in vivo neutralization of IFN-gamma was accompanied by growth of IGIF-transfected tumors in 100% of the animals. Taken together, our results show that murine IGIF can elicit T-cell-dependent antitumor immunity associated with IFN-gamma induction.

Assessment of intracellular TAP-1 and TAP-2 in conjunction with surface MHC class I in plasma cells from patients with multiple myeloma.

Immunotherapy involving cytotoxic T lymphocytes (CTLs) is an attractive alternative for treatment of various malignancies, including multiple myeloma. For tumour cells to be recognized and killed by CTLs they must express cell surface major histocompatibility complex (MHC) class I molecules and the transporter associated with antigen processing (TAP). However, loss of MHC class I and the TAP protein are common among several types of solid tumours. This study assessed the expression of TAP protein (by intracellular flow cytometry) and cell surface MHC class I molecules in three human myeloma cell lines as well as the plasma cell population (CD38+ bright) in bone marrow specimens from 13 multiple myeloma patients. In all of the patients, 100% of the plasma cell population expressed both the TAP subunits and cell surface MHC class I molecules, but at varying intensities. Both TAP and MHC class I were also expressed in the three myeloma lines. Additionally, the function of the antigen transport machinery was evaluated by a peptide transporter assay in the three myeloma lines. TAP transporter activity was readily detectable in two out of three myeloma lines, whereas the diminished activity in the third cell line was completely restored by co-culturing with recombinant interferon-gamma (rIFN-gamma).

Detection of altered T helper 1 and T helper 2 cytokine production by peripheral blood mononuclear cells in patients with multiple sclerosis utilizing intracellular cytokine detection by flow cytometry and surface marker analysis.

Production of T helper 1 and T helper 2 cytokines was investigated in peripheral blood mononuclear cells (PBMCs) from multiple sclerosis (MS) patients by a newly described technique, detection of intracellular cytokines by flow cytometry in conjunction with immunophenotype analysis. T-cell gamma interferon (IFN-gamma) production and interleukin 10 (IL-10) production were examined after PBMC activation with T-cell mitogens at 5 and 24 h, and monocyte spontaneous production of IL-10 and production after PBMC activation with lipopolysaccharide (LPS) for 24 h were also examined. The data indicate that MS patients have decreased percentages of T cells capable of secreting IFN-gama compared with healthy controls, and this change is detectable at 5 and 24 h. the patients displaying decreased T-cell production of IFN-gamma were essentially confined to a group being treated with the newly approved drug Betaseron (berlex Labs, Cedar Knolls, N.J.), a recombinant form of IFN-beta (rIFN-beta 1b). By gating of the entire lymphocyte population, analysis of IFN-gama production in T cells (CD3+ versus that in non-T cells (CD3+) was possible. The percentage of IFN-gamma-producing lymphocytes that was made up of T cells was essentially unchanged between the Betaseron-treated patients, non-Betaseron-treated patients, and controls, indicating that the suppression of IFN-gamma production displayed by betaseron-treated MS patients was a nonspecific suppression of all IFN-gamma-producing lymphocytes as opposed to a suppression of T-cell production only. The data seem to indicate that treatment of MS with Betaseron corresponds to an inhibition of the lymphocyte's ability to produce IFN-gamma. No changes were detected in T-cell production of IL-10 at either time point. We also observed that MS patients in general appear to have small percentages of peripheral blood monocytes spontaneously producing slight but detectable levels of IL-10. No difference was seen regarding monocyte production of IL-10 after PBMC activation with LPS between MS patients and controls. Both populations responded with high percentages of monocytes producing IL-10. The data seem to indicate that treatment of MS with Betaseron, known to decrease the exacerbation rate of relapsing-remitting MS, corresponds to a suppression of peripheral blood lymphocyte production of IFN-gamma. Monocyte production of IL-10 may also play a role in regulating the disease process.

Alterations in peripheral blood mononuclear cell cytokine production in response to phytohemagglutinin in multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS). The disease is characterized by inflammatory lesions in the white matter of the CNS, consisting of a specific immune response to the myelin sheath. We investigated peripheral blood mononuclear cell (PBMC) cytokine production by enzyme-linked immunosorbent assays of 21 MS patients and 19 age-matched normal controls in response to the T-cell mitogen phytohemagglutinin (PHA). Peripheral blood mononuclear cells were cultured in medium alone or in medium with 5 micrograms of PHA per ml for 48 h, and culture supernatants were collected for analysis. Cytokines selected for study were interleukin-10 (IL-10), gamma interferon (IFN-gamma), IL-2, and IL-4. All cytokine activities described were expressed as concentrations per 500,000 cells. We found that 48% (10 of 21) of the MS patients produced small but detectable levels of IL-10 in medium alone, compared with 26% (5 of 18) of the controls. We found that the MS patients produced significantly higher quantities of IL-10 protein than the controls in response to PHA (mean supernatant concentrations of IL-10 for patients and controls, 421 and 204 pg/ml, respectively [P < 0.05]). No significant differences were detected in the production of IL-2, IFN-gamma, and IL-4 between patients and controls in response to PHA, although patients appeared to display a trend toward decreased production of IFN-gamma.

Alterations in levels of CD28-/CD8+ suppressor cell precursor and CD45RO+/CD4+ memory T lymphocytes in the peripheral blood of multiple sclerosis patients.

A comprehensive peripheral blood immunophenotype analysis of 16 multiple sclerosis (MS) patients was performed by three-color flow cytometric analysis, and the results were compared with those for age-matched healthy controls. The cell subsets quantified included T cells (CD3+), B cells (CD19+), NK cells (CD56+), CD4+ and CD8+ T cells, cytotoxic (CD28+) and suppressor precursor (CD28-) CD8+ T cells, CD45RA+ and CD45RO+ T cells (CD4+ and CD8+), and CD5+ T and B cells. Analysis of MS patients' peripheral blood revealed essentially normal levels of total T, B, and NK cells. In agreement with results obtained by other investigators, it was found that MS patients had an increased CD4/CD8 ratio, primarily due to a decrease in CD8+ T cells. MS patients were found to have a significantly decreased level of suppressor precursor (CD28-) CD8+ T cells compared with that of controls but to have normal levels of cytotoxic (CD28+) CD8+ T cells. These data indicate that MS patients do not have a general decrease in CD8+ T cells but that they have a specific decrease in the suppressor precursor subset only and normal levels of cytotoxic CD8+ T cells. MS patients also had a significant increase in memory (CD45RO+) CD4+ T cells and displayed a trend towards a decrease in naive (CD45RA+) T cells in the peripheral blood.