CD4+ T cell activation and tolerance induction in B cell knockout mice.

B cell knockout mice microMT/microMT were used to examine the requirement for B cell antigen (Ag) presentation in the establishment of CD4+ T cell tolerance. CD4+T cells from microMT mice injected with exogenous protein Ag in adjuvant responded to in vitro challenge by transcription of cytokine mRNA, cytokine secretion, and proliferation. Peripheral tolerance could be established in microMT mice with a single dose of deaggragated protein. This tolerance was manifested by a loss of T cell proliferation and cytokine production (including both T helper cell type 1 [Th1]- and Th2-related cytokines), indicating that B cells are not required for the induction of peripheral T cell tolerance and suggesting that the dual zone tolerance theory is not applicable to all protein Ags and is not mediated through Ag presentation by B cells.

T cell differentiation and cytokine expression in late life.

Elderly humans are at significant risk with regard to the incidence and severity of many infectious diseases and cancers. Current theory holds that these late-life vulnerabilities arise, in part, through age-related changes in immune function, particularly in the T lymphocyte lineage. Herein, we discuss how such factors as thymic involution and ongoing T cell differentiation in the peripheral tissues contribute to progressive and irreversible shifts in the state of differentiation of the mature T cell pool. We propose that, by late life, these processes yield a T cell compartment with a suboptimal balance of naive and memory T cell subsets, each with altered, subset-specific programs for cytokine gene expression. As such, the T cell compartment in late life may be more prone to immune deficiency or cytokine-mediated dysregulation in response to new or previously encountered pathogens.

Membrane antigen phenotype of sensitized T lymphocytes mediating tuberculin-delayed hypersensitivity in rats.

The membrane antigen phenotype of immune lymph node cells (LNC) which mediate tuberculin-delayed hypersensitivity (DH) in Lewis rats was examined. The results show that the T-cell population which expresses the RT7.1 alloantigen defined by the BC 84A monoclonal antibody contained cells capable of transferring DH. Separation of the RT7.1-positive T-cell population with the monoclonal antibodies W3/25, MRC OX-8, or DS 4.23 (which defines the RT6.1 alloantigen) revealed that either the W3/25-positive or the RT6.1-negative T-cell subpopulations contained DH effector cells, whereas the corresponding MRC OX-8-positive or RT6.1-positive T-cell subpopulations did not. Moreover, when the W3/25-positive T-cell subpopulation was divided into either RT6.1-positive or RT6.1-negative T-cell subsets, only the W3/25-positive, RT6.1-negative subset transferred DH. These results indicate that the effector cells that mediate tuberculin DH are contained within the immune T-cell subset which bears both the RT7.1 and the W3/25 markers, but lacks both the MRC OX-8 and the RT6.1 markers.

Aging and lymphokine production by T cell subsets.

From sexual maturity to old age, the mammalian immune system undergoes progressive changes, some of which may predispose individuals to infectious, neoplastic and degenerative diseases. These age-associated changes are prominent in the T lymphocyte compartment and encompass both the CD4+ and CD8+ T cell subpopulations. In this review, we focus on the mouse model system and summarize current information on the existence of functionally distinct subsets within each of the CD4+ and CD8+ cell subpopulations. We describe how the representation of these subsets is altered during the aging process, with consequent changes in the lymphokine production repertoires and other functional attributes of the T cell pool. Lastly, we present evidence showing that similar changes occur in aging humans and discuss the potential impact of these changes on immune responsiveness in late life.

Retention of T cell reactivity to mitogens and alloantigens by Peyer's patch cells of aged mice.

Immune function generally declines with advancing age. However, recent evidence suggests that this decline may differentially affect the lymphoid compartments comprising the immune system. The effect of age on the T cell-mediated competency of cells from two different lymphoid compartments, Peyer's patch (PP) and spleen, was studied. PP cells of aged C57BL/6 mice retained a greater capacity to proliferate in response to concanavalin A, phytohemagglutinin, and alloantigen and to generate alloimmune cytotoxic cells than did splenocytes of aged mice when compared with their young counterparts. These results could not be explained by significant shifts in the response kinetics or in the proportions of T cells in individual tissues. These results support the concept that lymphoid compartments are not equally sensitive to the deleterious effects of the aging process.

Interleukin-10 production by splenic CD4+ cells and cell subsets from young and old mice.

We have examined whether aging is accompanied by changes in the capacity of CD4+ cells to produce IL-10, a potent immunoregulatory cytokine. Splenic CD4+ cells from young-adult and old C57BL/6NNia mice were stimulated in vitro with immobilized anti-CD3 epsilon mAb and were monitored for the release of IL-10 in short-term (3-day) cultures. In both age groups, detectable IL-10 accumulation in culture supernatants was stimulation dependent and reached a maximum level on Day 3. However, the peak IL-10 level in the old group was approximately 10-fold higher than that in the young-adult group. This age-associated difference in IL-10 production was also evident in the analysis of IL-10 mRNA levels in stimulated CD4+ cells. In contrast to these findings, the analysis of S-phase activity in the stimulated cell cultures revealed an age-related decline in this aspect of the cellular response. In studies on CD4+CD44lo and CD4+CD44hi subsets isolated from mice of various ages, we found that measurable IL-10 production segregated entirely with the CD44hi population, regardless of donor age. Taken together, our data suggest that the capacity for IL-10 synthesis by the splenic CD4+ cell pool is increased with age, and that the age-related shift toward a predominance of CD4+CD44hi cells in the peripheral tissues accounts for this quantitative change in IL-10 gene expression.

Transgenic thymocytes are refractory to transformation by the human T-cell leukemia virus type I tax gene.

Previous transgenic work demonstrated transforming activity of the human T-cell leukemia virus type I Tax protein in fibroblasts. In the present study, a Thy-1-based vector was used to express Tax in thymocytes. These mice developed no functional or neoplastic abnormalities of T cells but developed fibroblastic tumors with a longer latency than in the previous model.

Expression of membrane activation antigens on murine B lymphocytes stimulated with lipopolysaccharide.

The expression of two membrane glycoproteins, RL388 antigen and transferrin receptor (TfR), was examined on murine B cells stimulated with lipopolysaccharide (LPS) in vitro. Immunofluorescent staining with monoclonal antibodies and flow cytofluorometric analysis were used to monitor the expression of these markers as a function of the time in culture, the state of membrane Ia antigen expression, the position in cell cycle, and the degree of B-cell differentiation. Freshly explanted splenic B cells expressed low levels of RL388 antigen and TfR. Following LPS stimulation, increased expression of RL388 antigen was detectable by 8 to 12 hr of culture, a time span characterized by increased Ia antigen expression, blast transformation, and G0 to G1 phase transition. The increased expression of TfR was apparent later and correlated with entry into late G1 phase and the onset of S phase. LPS-stimulated cell cultures treated with actinomycin D (G0/G1 block) exhibited increased expression of Ia antigen, but neither RL388 antigen nor TfR, whereas hydroxyurea treatment (G1/S block) allowed expression of all three markers. These results indicate that hyperexpression of RL388 antigen and TfR occurs during G1 phase and that these events are subsequent to Ia antigen hyperexpression. Finally, B cells in late G1 through M phase of the cell cycle simultaneously express high levels of RL388 antigen and TfR. These findings suggest that the expression patterns of RL388 antigen and TfR might be useful parameters for defining compartments of the murine B-cell cycle.

In vivo tolerance induction and associated cytokine production by subsets of murine CD4+ T cells.

Male BXSB mice develop lupus-like disease and die early in life (4 to 5 mo) whereas female mice do not. Others have demonstrated that CD4+ cells from male mice support B cell resistance to tolerance induction to human gamma-globulin (HGG). In this study, male and female mice tolerized at 2 mo of age with deaggregated HGG and subsequently immunized with HGG in comparison with mice immunized only were tested for anti-HGG Ab responses. CD4+ cells from draining lymph nodes of these mice were tested in culture for proliferation and production of cytokine mRNA and protein in response to HGG plus APC. Tolerized male but not female mice produced anti-HGG Abs of both the IgG1 and IgG2a isotypes. HGG-stimulated CD4+ cells from immunized male and female mice that were not tolerized produced IL-2, IL-4, IL-5, IFN-gamma, and TNF-beta mRNA as well as IL-2 and IL-4 protein, whereas tolerized, immunized mice of both sexes failed to proliferate or produce either IL-2 or IL-4 or express any cytokine mRNA in response to HGG in vitro. A resistance in tolerance induction in male mice, as determined by anti-HGG Abs, was also observed at 3 mo of age. Although a resistance to tolerance was also seen in terms of proliferation in the 3-mo-old males, production of IL-2 or IL-4 protein was still not observed. Thus, all T cell subsets identified by cytokine expression profiles were tolerized not only from females but also from males, of which the latter appeared to show some resistance to tolerance induction.

Effects of tolerance induction on early cell cycle progression by Th1 clones.

Human gamma-globulin (HGG)-specific mouse Th1 clones exposed to tolerogenic signals provided by HGG-pulsed paraformaldehyde-fixed splenocytes (HGG-FAPC) were analyzed for antigen-induced progression through the early phases of the cell cycle. Exposure of Th1 clones to HGG-FAPC in primary cultures inhibits the ability of the clones to synthesize DNA in response to HGG and normal APC in secondary cultures. The Th1 clones in these secondary cultures were found to be blocked in G1a phase as evidenced by cell cycle analysis and by reduced numbers of cells expressing high levels of IL-2R and TfR. This cell cycle blockade of Th1 cells was not observed if the secondary cultures were stimulated with IL-2-containing Con A CM instead of antigen. These data suggest that in our system the inhibition in antigen-induced cell cycle progression associated with Th1 tolerance induction occurs at the G1a/G1b phase transition.

Evidence for a defective accumulation of protective T cells in old mice infected with Mycobacterium tuberculosis.

Mice infected with virulent Mycobacterium tuberculosis exhibit an age-related increase in susceptibility to disease. The basis of this susceptibility has previously been shown to reflect an inability of the aged host to generate protective CD4 T cells during the early course of the infection. The results of the present study, however, indicate that the emergence of interferon-gamma secreting protective CD4 T cells in such mice is not absent, but merely delayed. Furthermore, flow cytometric analysis of such cells accumulating in the spleens of intravenously infected 24-month-old animals revealed that a large percentage of CD4 cells initially had poor or negative expression of the cell surface markers L-selectin and CD11a, molecules that may be important in the movement of T cells across inflamed endothelial blood vessel surfaces. During the course of the tuberculosis infection the numbers of CD4 cells in the spleens of old mice expressing high levels of these molecules rose to levels similar to those observed in young mice, but by that time the numbers of bacilli in target organs had reached close to fatal levels. These data suggest that the capacity of CD4 cells to cross inflamed endothelial surfaces and home into sites of mycobacterial infection may be deficient in old mice, and hence support the hypothesis that the ensuing delay in accumulating such cells within infected lesions contributes to the increased susceptibility of these animals to disease.

Maturational changes in CD4+ cell subsets and lymphokine production in BXSB mice.

CD4+ cells are thought to play a significant role in the development of lupus-like disease in a variety of autoimmune disease-prone mouse strains. In one such strain, BXSB/MpJScR, male mice develop severe lupus-like symptoms early in life but females do not. In this study, splenic CD4+ cells from male and female BXSB mice were evaluated for age-related changes in: 1) membrane expression of CD4+ cell subset markers (1, 2, and 4 mo) and activation Ags (4 mo) and 2) the capacity to proliferate and produce cytokines (4 mo) in response to polyclonal stimuli. CD4+ cells from females of all age groups and from younger males were predominantly CD44lo, CD45RBhi, MEL-14hi, and 3G11hi (phenotypes associated with naive T cells). In contrast, 4-mo-old males were predominantly CD44hi, CD45RBlo, MEL-14lo, and 3G11lo (phenotypes associated with activated/memory T cells). Furthermore, an increased constitutive expression of the activation Ags RL388, IL-2R, and TfR was observed in CD4+ cells of 4-mo-old male BXSB mice in comparison with age-matched females. In 3-day cultures, purified CD4+ cells from 4-mo-old males proliferated significantly less than cells from age-matched females in response to plate-bound anti-CD3 epsilon (2C11i). The reduced proliferation was restored in large part by PMA and ionomycin. CD4+ cells from older males generally produced increased amounts of IFN-gamma and IL-4 and significantly less IL-2 than age-matched females in response to either stimulus (IL-2 mRNA was also decreased in response to 2C11i). Taken together, these studies suggest that profound phenotypic and functional changes occur with age in the CD4+ cells of male BXSB mice that are indicative of an activated state.

The age-associated increase in IFN-gamma synthesis by mouse CD8+ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, CD45RB, 3G11, and MEL-14 expression.

The mouse model system was used to evaluate age-associated changes in the subset composition and function of the splenic CD8+ T cell pool. In response to stimulation with plate-bound anti-CD3 epsilon mAb, CD8+ cells from old C57BL/6NNia mice produced greater levels of IFN-gamma than cells from young-adult controls. This age-associated difference was apparent at the levels of both IFN-gamma mRNA accumulation and cytokine release, and was established within the first major cell cycle in culture. In addition, the capacity to produce IFN-gamma appeared to increase gradually with age as evidenced by studies on CD8+ cells from intermediate aged mice. In contrast to these findings, the peak S-phase responses by stimulated CD8+ cells from old mice were significantly reduced relative to young-adult controls. Immunophenotypic analyses of membrane CD44, CD45RB, 3G11, and MEL-14 expression by splenic CD8+ cells from young-adult, intermediate-aged, and old mice revealed an age-associated decrease in the frequencies of cells that expressed low levels of CD44 and high levels of CD45RB, 3G11, and MEL-14, whereas the reciprocal phenotypes increased with age. The correlated analysis of all four subset markers identified a composite phenotype (CD44loCD45RBhiMEL-14hi3G11lo/hi) which, based on past functional studies, is a candidate phenotype for naive cells. This "naive" phenotype dominated the CD8+ cell pool of young-adult mice but decreased in frequency with age. In contrast to the CD44lo cell group, the CD44hi cell fraction, which is associated with preactivated/memory CD8+ cells, was found to be uniformly 3G11lo but expressed heterogeneous levels of CD45RB and MEL-14, perhaps defining multiple subsets within the memory population. All of these latter subsets increased in frequency with age. Finally, we found that when CD8+ cells were fractionated based on CD44 expression the capacity to release measurable levels of IFN-gamma segregated entirely with the CD44hi fraction, irrespective of donor age. Together, these findings support the hypothesis that aging is accompanied by dramatic shifts in the subset compositions of splenic CD8+ cell pools, which contribute significantly to their increased capacity to produce IFN-gamma at the population level.

Stimulation of murine T cell subsets with anti-CD3 antibody. Age-related defects in the expression of early activation molecules.

Splenocytes from young (3 to 4 mo) and aged (24 to 26 mo) C57BL/6 mice were stimulated with anti-CD3 epsilon mAb in vitro. At the time of peak DNA synthesis (day 2), cells from aged mice incorporated congruent to 60% less [3H]TdR than cells from young mice. This age-related defect was not attributable to gross differences in anti-CD3 does optima, response kinetics, accessory cell function, numbers of T cells cultured, CD4+:CD8+ cell ratios or surface levels of CD3 epsilon molecules. In an attempt to analyze pre-S phase events in these responses, we monitored CD4+ and CD8+ cells in splenocyte cultures for the time-dependent expression of three T cell activation markers: RL388 Ag and IL-2R and transferrin R. Parallel analyses of mean T cell size and cell cycle phase distributions were performed. Non-activated T cells from both age groups similarly expressed moderate levels of RL388 Ag, low levels of transferrin R, and undetectable levels of IL-2R. Analysis of stimulated T cells revealed, in both age groups: 1) detectable increases in expression of all three markers by 6 h of culture, and continued increases associated with blastogenesis and G1 phase transit and 2) a preferential stimulation of the CD8+ subset to a state of high level marker expression. Age group comparisons of activation marker expression over time suggested that the age-related defect reflects proportionally smaller fractions of CD4+ and CD8+ cells that respond normally, rather than a general defect in all T cells or a subset-specific defect. Finally, we found that supernatants from aged donor cell cultures stimulated with anti-CD3 contained less Il-2 than those of young controls. Addition of an IL-2 containing supernatant to aged donor cell cultures increased, but did not restore, the S phase response on day 2; however, the response on day 3 was comparable to the peak (day 2) response of young controls. These data suggest that exogenous IL-2 can improve the aged response, perhaps by expanding the fraction of normally reactive T cells.

Ability of tolerized Th1 and Th2 clones to stimulate B cell activation and cell cycle progression.

Tolerant and nontolerant murine Th1 and Th2 clones, specific for human gamma-globulin (HGG), were compared for their ability to promote cell cycle entry and progression by B cells in vitro. When stimulated with HGG, nontolerant Th1 and Th2 clones induced similar increases in B cell membrane MHC class II levels--a phenomenon associated with early B cell activation. Nontolerant Th1 and Th2 clones also induced B cell DNA synthesis, an event associated with subsequent G1 phase traversal, although Th2 cells were more efficient than Th1 cells in stimulating this activity. Exposure of Th clones to tolerogen in the form of HGG-pulsed chemically fixed APC inhibited the ability of Th1 clones, but not Th2 clones to promote polyclonal B cell DNA synthesis in HGG-stimulated secondary cultures. However, Th1 clones exposed to tolerogen did not lose their ability to increase the expression of MHC class II molecules on B cells in these cultures. These results indicate that tolerance induction does not inhibit the ability of Th1 clones promote B cell cycle progression. In contrast, exposure of Th2 cells to tolerogen does not inhibit significantly the ability of these cells to stimulate B cell cycle entry or progression.

Potentiation of the rat delayed-type hypersensitivity reaction by the Fc portion of human IgG1.

Fc fragments derived from a human IgG1 myeloma protein potentiate the rat delayed-type hypersensitivity (DTH) reaction to antigen challenge. Lewis rats immunized with heat-killed tubercle bacilli give augmented DTH reactions to the purified protein derivative of tuberculin when Fc fragments are included in the challenge dose. Similar potentiation of DTH by pFc' fragments indicates that the active site is located in the CH3 domain of IgG1. Histologic evaluation of the augmented reaction sites revealed predominantly mononuclear cell infiltrates characteristic of DTH reactions. Skin tests of tubercle bacilli-sensitized rats with an unrelated antigen and/or Fc fragments fail to elicit significant reactions. Augmentation of the DTH reaction to purified protein derivative is restricted to the Fc or pFc' region fragments since intact monomeric IgG1, Fab fragments, and bovine serum albumin were all shown not to be active potentiators. The DTH reaction of ovalbumin-sensitized rats was similarly augmented when Fc fragments were included with a challenge dose of ovalbumin, thus supporting the general nature of the phenomenon. These results support the concept of Ig molecules as multifunctional proteins that can not only serve effector functions but also participate in the regulation of immune responses.

Patterns of cytokine gene expression by CD4+ T cells from young and old mice.

We have analyzed the patterns of induced cytokine gene expression and cell cycle activity by CD4+ cells from mice, and have examined how these response patterns change during the aging process. CD4+ cells were isolated from spleens of young adult and old C57BL/6NNia mice and were stimulated in vitro with plate-bound anti-CD3 epsilon mAb. The cells were then assessed over time for the capacity to accumulate transcripts for IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IFN-gamma, TNF-alpha, and TNF-beta; to secrete IL-2, IL-3, IL-4, IL-5, IL-6, and IFN-gamma; and to progress through S phase. Before the first major cell division in culture (< 32 h), stimulated CD4+ cells of the old group contained similar peak levels of IL-2, TNF-alpha, and TNF-beta transcripts relative to young adult controls, whereas IL-3, IL-4, IL-5, and IFN-gamma transcripts accumulated to significantly higher peak levels in the old group. These findings were consistent with the patterns of cytokine secretion later in culture (24 to 72 h): the peak IL-2 levels were similar between age groups, but the old group exhibited an enhanced capacity to release IL-3, IL-4, IL-5, and IFN-gamma. In contrast, CD4+ cells of the young group were superior in the hyper-expression of the housekeeping gene, rpL32, before cell division and in the levels of S phase activity throughout 3-day cultures. Similar analyses of CD4+ cells from mice of intermediate ages showed that the alterations in cytokine profiles occurred gradually from young adulthood to old age, whereas the reductions in proliferative capacity were late life changes. Consistent with previous reports, we found that the splenic CD4+ cell group also underwent a progressive, age-dependent increase in the proportions of cells expressing high levels of membrane CD44 (a phenotype associated with memory or effector cells). Moreover, the analysis of IL-3, IL-5, and IFN-gamma production by isolated CD4+CD44lo and CD4+CD44hi cells revealed that the capacity to produce these cytokines segregated predominantly with the CD44hi subset, regardless of donor age. Taken together, our data suggest that gradual age-associated shifts in the subset composition of the splenic CD4+ cell pool underlie progressive changes in the patterns of cytokine gene expression by this cell group.