Characterization of host immunity during persistent vaginal colonization by Group B Streptococcus.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterium, which colonizes the vaginal tract in 10-30% of women. Colonization is transient in nature, and little is known about the host and bacterial factors controlling GBS persistence. Gaining insight into these factors is essential for developing therapeutics to limit maternal GBS carriage and prevent transmission to the susceptible newborn. In this work, we have used human cervical and vaginal epithelial cells, and our established mouse model of GBS vaginal colonization, to characterize key host factors that respond during GBS colonization. We identify a GBS strain that persists beyond a month in the murine vagina, whereas other strains are more readily cleared. Correspondingly, we have detected differential cytokine production in human cell lines after challenge with the persistent strain vs. other GBS strains. We also demonstrate that the persistent strain more readily invades cervical cells compared with vaginal cells, suggesting that GBS may potentially use the cervix as a reservoir to establish long-term colonization. Furthermore, we have identified interleukin-17 production in response to long-term colonization, which is associated with eventual clearance of GBS. We conclude that both GBS strain differences and concurrent host immune responses are crucial in modulating vaginal colonization.

T cell senescence.

The aging of the immune system, referred to as immunosenescence, is associated with a dramatic reduction in responsiveness as well as functional dysregulation. This deterioration of immune function with advancing age contributes to the increased incidence among the elderly of morbidity and mortality from infectious disease, and possibly autoimmunity and cancer. In mammals, the defense for fighting infectious agents is composed of the innate and adaptive immune systems. Macrophages, granulocytes, and natural killer cells are the major components of the innate system whereas T and B lymphocytes comprise the adaptive system. Although both compartments are affected, adaptive immunity is most susceptible to the deleterious effects of aging. Innate immunity functions immediately after birth and manifests little change throughout life. In contrast, adaptive immunity is immature at birth, peaks at puberty and progressively declines thereafter. Though marginal alterations in B lymphocytes are apparent, the dramatic decline in humoral and cell-mediated responses is predominantly the consequence of senescent T cells. The following review focuses on the aging effect on T cells as reflected in altered function, subset representation, development, lifespan and activation. Age-associated alterations in antigen presenting cells are also discussed since these cells are required for T cell activation and may impact T cell function.

Aging affects the regeneration of the CD8+ T cell compartment in bone marrow transplanted mice.

A chimeric mouse model has been used to determine the effect of aging on the differentiation of CD8+ T cells and on the regeneration capacity of the mature peripheral T cell pool after radiation induced depletion. Bone marrow cells from Thy 1.1+ mice were transplanted into lethally irradiated young or aged mice (Thy 1.2+). After 6 weeks, splenic CD8+ T cells were subjected to phenotypic and functional examinations by flow cytometry. Both young and aged mice were able to develop donor derived (Thy 1.1+) CD8+ T cells. Although the absolute number of T cells was reduced in aged recipients, the ratio of CD4+ to CD8+ T cells of donor-origin was the same in young Thy 1.1+ control mice as it was in both young and aged chimeric mice, indicating that aging has no effect on the ratio of CD4+ to CD8+ T cells produced by the thymus. However, the percentage of CD8+ cells in the total Thy 1.2+ (host-origin) T cell population was significantly higher in young chimeric mice than in age-matched Thy 1.2+ control mice (P < 0.01), suggesting that a significant over expansion of the Thy 1.2+ CD8+ subset occurred in young mice during regeneration. The Thy 1.1+ CD8+ T cells that developed in young hosts were of a naive phenotype with a majority of cells expressing a low level of CD44. In contrast, the majority of those that developed in the aged host displayed a memory phenotype with a high percentage of cells being CD44hi. In addition, the production of IL-4 and IFN-gamma by Thy 1.1+ CD8+ T cells was affected by the age of the host. A greater fraction of aged Thy 1.1+ CD8+ T cells could be induced to produce either IFN-gamma or IL-4 than young CD8+ T cells. These results suggested that the aged microenvironment has a significant effect on newly developed CD8+ T cells and that the age of the microenvironment also influences the regeneration capacity of CD8+ T cells.

The age-dependent cytokine production by murine CD8+ T cells as determined by four-color flow cytometry analysis.

Flow cytometry has been used to simultaneously examine intracellular cytokine production and expression of CD44 and CD45RB by murine CD8+ T cells derived from young (2-3 months) or aged (18-22 months) mice. Cytokine production by aged CD8+ T cells differs from that by CD8+ T cells derived from young animals in that a significantly higher percentage of the aged can be triggered to produce interleukin (IL)-4, interferon (IFN)-gamma, and tumor necrosis factor alpha (TNF alpha). Conversely, a greater fraction of young CD8+ T cells produce IL-2. Aged mice not only have a higher percentage of CD8+/CD44hi T cells, but also a larger fraction of these cells are IFN-gamma+ and IL-4+, while a lower fraction are IL-2+ than is observed in young CD8+/CD44hi T cells. In terms of relative contribution to total cytokine synthesis, a greater fraction of CD8+ T cells produce IFN-gamma and IL-4 than in CD4+ T cells, whereas CD4+ T cells are the major producers of IL-2.

Maturation of CD4+ lymphocytes in the aged microenvironment results in a memory-enriched population.

With advancing age the CD4+ T lymphocyte compartment becomes enriched for memory cells in both humans and experimental animals. Although it has been assumed that the shift from a naive to a memory-dominant population is due to a lifetime of antigenic exposure and selection as well as a loss of naive cell input due to reduced thymopoiesis, the present data suggest that the aged microenvironment influences the maturation of newly produced CD4+ T cells. In two models, aged and young mice were compared for the ability to reconstitute their peripheral CD4+ T cell pools following depletion, and both age groups were found to be competent to renew this population. However, the phenotype and lymphokine profile of populations arising in aged animals were distinctly different from those in the young mice. In contrast to the expectation that depletion and reconstitution might give rise to a naive-dominant T cell pool, aged mice reconstituted a population nearly indistinguishable from that found in control age-matched individuals. The majority of the CD4+ pool were CD44(high) CD45RB(low) Mel-14(low) and upon activation with anti-CD3 these CD4+ T cells produced mRNA for IL-2, IL-4, IL-5, and IFN-gamma. In aged bone marrow-transplanted mice, the same phenotypic profile and cytokine mRNA pattern were found in CD4+ T cells of host and donor origin. In contrast, the majority of CD4+ T cells in young reconstituted mice were CD44(low) CD45RB(high) Mel-14(high). These lymphocytes, when activated, produced high levels of mRNA for IL-2, with little or no IL-4, IL-5, or IFN-gamma mRNA.

Effects of the aged microenvironment on CD4+ T cell maturation.

Contributing to the functional alterations of the aged immune system is the accumulation of memory CD4+ T lymphocytes and decline in the proportion of naive cells occurring with advancing age. During attempts to alter the naive to memory ratio of CD4+ cells in aged mice, it was observed that regeneration of the peripheral T cell compartment resulted in a population which possessed the same memory cell-enriched characteristics as the unmanipulated age-matched controls. Thymopoiesis in aged mice does not appear to be altered in such a way as to give rise to emigrants with 'memory-like' characteristics. The aged peripheral microenvironment does, however, cause the accelerated maturation of mature, naive CD4+ T cells to the memory state.

Transforming growth factor-beta 1 induces antigen-specific unresponsiveness in naive T cells.

Transforming growth factor-beta 1 (TGF-beta 1) is a cytokine with complex immunomodulatory effects including the ability to inhibit the onset or severity of autoimmune disease. This study was designed to test the possibility that one mechanism by which TGF-beta 1 exerts its immunosuppressive effects is by inducing antigen (Ag)-specific unresponsiveness in CD4+ cells. TGF-beta 1 was shown here to inhibit the Ag-specific proliferation of naive CD4+ cells from T cell receptor (TCR) transgenic mice. More importantly, the naive CD4+ cells exposed to TGF-beta 1 and Ag, but not to TGF-beta 1 alone, in primary cultures were unable to proliferate or secrete IL-2 in response to a subsequent Ag challenge following removal of TGF-beta 1 from the cultures. Anti-CD28 mAb partially blocked the Ag-specific inactivation induced by TGF-beta 1 in naive CD4+ cells. The inhibitory effects of TGF-beta 1 on CD4+ cells are not mediated by alterations in APC costimulation since TGF-beta 1 did not inhibit the Ag-induced expression of MHC class II molecules, CD80 or CD86 on splenic APC. Taken together, the results suggest that the immunosuppressive activities of TGF-beta 1 encompass direct induction of Ag-specific unresponsiveness in naive CD4+ cells.

Early steps in T cell development are affected by aging.

Involution of the thymus accompanies aging, a process in which the organ diminishes in size and cellularity and becomes disorganized. The rate of T cell emigration from the thymus is markedly reduced with age, and phenotypic analyses have identified alterations in the relative proportions of the major thymocyte subpopulations. The present studies made use of the capacity of the thymus to regenerate following irradiation from an intrathymic radio-resistant precursor population. By analysis of the differentiation of this "wave" of thymocytes, it was determined that aging most severely affects the earliest developmental transitions. While the overall rate of differentiation does not appear to be affected in older mice, fewer thymic progenitors initiate differentiation. The reduced expansion of late pre-T cells in the middle-aged is due to the smaller pool size of these cells.

Anti-human kappa opioid receptor antibodies: characterization of site-directed neutralizing antibodies specific for a peptide kappa R(33-52) derived from the predicted amino terminal region of the human kappa receptor.

Site-directed polyclonal Abs specific for a synthetic peptide with sequence homology to the predicted N-terminal sequence of the human kappa opioid receptor [anti-kappa R-(33-52)] are capable of binding to normal human cells and cell lines expressing mRNA specific for the human kappa receptor. Flow cytometric analysis of 1) a neuronal cell line (NT2), 2) blood-derived CD14+ monocytes, 3) monocyte-like cell lines (U937 and THP 1), 4) blood-derived CD3+ T cells and a T cell line, and 5) human B cell lines bound anti-kappa R-(33-52) in a specific manner. Anti-kappa R-(33-52) was also found to specifically neutralize the immunosuppressive activities associated with the kappa R-selective agonist U50,488H. This antiserum was found to block U50,488H-mediated inhibition of 1) Staphylococcus aureus Cowen strain I-induced B and T lymphocyte proliferation, 2) PHA-induced T lymphocyte proliferation, and 3) S. aureus Cowen strain I-induced IgG production. However, this antiserum failed to neutralize mu R-selective agonist (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol)-mediated suppression of IgG synthesis. Finally, the kappa R-selective antagonist nor-binaltorphimine hydrochloride inhibits the binding of anti-kappa R-(33-52) to the U937 cell line. These results suggest that anti-kappa R-(33-52) specifically interacts with the human kappa R molecule. Studies conducted with anti-kappa R-(33-52) indicated that this antiserum effectively blocked U50,488H-mediated immunosuppression, but by itself did not enhance or suppress lymphocyte activation. These data suggest that anti-kappa R-(33-52) 1) does not interact with the effector binding site of the receptor, but sterically interferes with U50,488H binding to the receptor; and/or 2) the antiserum interacts with a secondary binding site that is important for ligand binding, but may not be involved in signal transduction.

The pattern of T lymphocyte differentiation is altered during thymic involution.

Thymic involution is likely to be a significant factor in the alteration of peripheral T lymphocyte function with age. The process of thymic involution involves the progressive loss of normal organ architecture and cellular composition, and a significant reduction in the output of mature T lymphocytes. The present study assesses the impact of thymic involution on the T cell differentiation process by quantitating the number and percent representation of various phenotypically distinguishable T cell developmental intermediates in C57BL/6 mice of various ages. The results suggest that several distinct sites in the developmental sequence are impacted by aging. By middle-age (14-17 months), significant perturbations in the frequencies of several CD4-CD8- (DN) subpopulations have occurred. These include a shift towards an increased percentage of Pgp-1+ IL-2R- DN cells, the earliest thymic progenitors, and a decreased percentage and total number of Pgp-1- IL-2R+ DN cells. Furthermore there is a threefold increase in the percentage of DN cells which express CD3 (from 16.6% to 45.5%) which occurs between 4 and 14 months of age. By 24-27 months of age, the percentage of the total DN population increases two- to threefold over that of young (2-3 months) animals, while the fraction of CD4+CD8+ (DP) is significantly reduced. These alterations are consistent with the possibility that thymic involution results in one or more 'developmental' blocks which limit key differentiative transitions within the DN population, and furthermore, the marked increase in the frequency of DN cells displaying CD3 argues that an alternative T cell differentiation pathway plays an increasingly significant role with advancing age.

The expression of CD45RB on antigen-responsive CD4+ lymphocytes: mouse strain polymorphism and different responses to distinct antigens.

The levels of CD45RB expression by HGG-specific CD4+ cells residing in the Ag-draining lymph nodes of HGG-primed CBA/CaJ mice were analyzed. When sorted populations of CD4+, CD45RBhi, and CD4+, CD45RBlo cells were cultured with HGG and Ag-presenting cells, the majority of the proliferative response was found in the CD45RBlo fraction early after in vivo priming (Day 6), and this pattern remained stable through 12 days postpriming. To determine whether this segregation of responsiveness was consistent in other mouse strains, HGG-primed C57BL/6J mice were similarly analyzed. In contrast to findings with the CBA/CaJ strain, the CD4+, CD45RBhi cell fraction obtained from C57BL/6J mice was the predominant responding population early after in vivo priming (Day 6); however, there was a parallel increase in responsiveness of CD4+, CD45RBhi, and CD4+, CD45RBlo cells by Day 12. Thus, there was not a decrease in CD45RBhi expression with a concommitant increase in CD45RBlo expression in CD4+ cells proliferating to HGG. Despite the heterogeneity in CD45RB expression by the primed CD4+ cells of the two strains, the entire proliferative response to HGG early after priming resided in the fraction bearing high levels of membrane CD44, thus arguing for the existence of CD45RBhi, CD44hi and CD45RBlo, CD44hi cells during the early phase of the response. In both mouse strains the CD4+, CD45RBhi subset of primed lymph node cells produced significant levels of IL-2 in response to HGG and APC, whereas no significant IL-2 or IL-4 production was detectable in HGG-stimulated CD45RBlo cells of either strain. The CD4+, CD45RBhi subset also proliferated more vigorously in response to polyclonal activation than the CD4+ CD45RBlo fraction. To examine whether the patterns of CD45RB expression on HGG-primed cells from C57BL/6J mice were common to other antigens, the response profiles were examined after in vivo priming with a second antigen, KLH. In contrast to studies with HGG as the Ag, the proliferative response to KLH in C57BL/6J mice was evenly divided among the CD45RBhi and CD45RBlo fractions on Day 8 after priming, but shifted markedly to the CD45RBlo fraction by Day 12 after priming. Taken together, these data show that the patterns of CD45RB expression on primed populations of CD4+ cells can exhibit mouse strain polymorphism and can differ depending on the choice of antigen for immunization.

Cell proliferation and cytokine production by CD4+ cells from old mice.

Splenocytes from young adult or old C57BL/6NNia mice were stimulated in vitro with the anti-CD3 epsilon mAb, 145-2C11, in either soluble (2C11s) or plate-bound (2C11i) form. In the young group, each mode of cell activation resulted in peak DNA synthesis at approximately 48 h of culture; at this time point, the old group exhibited response levels to 2C11s or 2C11i that were approximately 40% of those in the young group. However, in the presence of 2C11i, splenocytes from old donors showed a delayed peak response which approached the peak levels attained in the young group. To analyze the responsiveness of the CD4+ T cell subpopulation, this cell type was isolated from spleens of young or old mice and was stimulated in vitro with 2C11s or 2C11i, in the presence or absence of added accessory cells (T cell-depleted, irradiated splenocytes). The induction of DNA synthesis by 2C11s was accessory cell dependent, and the response in the old group were markedly reduced in comparison to those in the young group. In contrast, stimulation of DNA synthesis with 2C11i was relatively accessory cell independent, resulted in higher response levels in both age groups, and lessened the disparity between age groups. The analysis of IL-2 and IL-4 secretion by stimulated CD4+ cells revealed that, in response to 2C11s and accessory cells, only IL-2 accumulation was detectable and the levels in the young group were approximately 10-fold higher than the IL-2 levels in the old group. However, stimulation of CD4+ cells with 2C11i and accessory cells yielded improved IL-2 production and a detectable IL-4 response in the old group, whereas the young group exhibited a response profile similar to that induced by 2C11s. Further analysis of the IL-2, IL-4, and IFN gamma mRNA levels in 2C11i-stimulated CD4+ cells revealed that old donor cells accumulated similar levels of IL-2 transcripts, but higher levels of IL-4 and IFN gamma transcripts, than young donor CD4+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired responsiveness of IL-2 receptor-expressing T lymphocytes from aged mice.

IL-2 receptor-bearing splenic T lymphocytes derived from aged C57BL6/J mice (22-24 months) display a relative inability to respond to IL-2 when compared to similar cells from young (2-3 months) animals. As a population the aged cells incorporate less [3H]thymidine and fewer are able to undergo vigorous clonal growth. Both the CD4+ and CD8+ subsets display these defects. The clonal assay indicates that aged T cells, in addition to having longer cell cycle transit time, also have a higher frequency of cell cycle arrest than similarly activated young T cells. This defect in IL-2 responsiveness is distinct from those in early signal transduction which limit aged T lymphocyte entry into cycle and cannot be corrected by phorbol myristate acetate or ionomycin.

Induction of human B cell differentiation by Fc region activators. II. Stimulation of IL-6 production.

Fc region fragments derived from the enzymatic cleavage of human IgG have been shown to induce human peripheral blood-derived B cells to differentiate into Ig secreting cells (ISC). The synthetic peptide p23, corresponding to residues 335 to 357 in the Fc region of human IgG1, represents a region of the molecule responsible for stimulation of ISC formation. Fc region-induced ISC formation requires at least two signals; one supplied by Fc region activators and one supplied by a T cell-derived factor(s). In this report we show that the coculture of human PBMC with pFc' or p23, results in the release of factor(s) that resemble IL-6 in its pattern of biologic activity. This conclusion is based on the observations that supernatants from Fc region-stimulated PBMC cultures contained increased levels of elements that scored as positive in two assays for IL-6: the B9.9 hybridoma growth and the CESS cell differentiation assays. Moreover, RNA from Fc region-stimulation PBMC contained increased levels of IL-6 cDNA-hybridizable elements. Finally, it was observed that rabbit anti-IL-6 inhibited the ability of supernatants derived from Fc region-stimulated PBMC cultures to induce B9.9 cell proliferation as well as p23-induced ISC formation in intact PBMC cultures. Fc region fragments induce both monocytes and T cells to produce IL-6. Taken together, these results indicate that IL-6 is produced in Fc region-stimulated PBMC cultures and is involved in B cell activation by these activators.

Toxicokinetics of Bordetella pertussis.

Pertussis vaccine immunization is required in most of the 50 states in the United States. Since it is required or recommended it is imperative that the clinician give this vaccine, crude as it is, in the safest possible environment. Giving specific active (verbal) as well as passive (written) instructions to the parent or caretakers is mandatory. If in doubt, defer or delete the pertussis portion.

Partial restoration of Con A-induced proliferation, IL-2 receptor expression, and IL-2 synthesis in aged murine lymphocytes by phorbol myristate acetate and ionomycin.

The activation by concanavalin A (Con A) of murine lymphocytes derived from aged animals is impaired, as demonstrated by lower IL-2 synthesis, display of fewer IL-2 receptors, and less [3H]thymidine incorporation relative to similarly treated cells from young animals. The ability of the phorbol ester, phorbol 12-myristate 13-acetate (PMA), and the calcium ionophore, ionomycin, in conjunction with Con A, to improve these parameters of activation has been assessed. When splenic lymphocytes derived from aged mice are cultured with Con A plus ionomycin, IL-2 synthesis and IL-2 receptor expression are increased over the values obtained in parallel cultures triggered by Con A alone up to levels equal to that obtained in Con A-activated young cultures. The proliferative response is less sensitive to the augmenting effect of ionomycin. PMA is much less effective in improving these parameters of Con A activation. PMA plus ionomycin, in the absence of Con A, triggers cell cycle transition of lymphocytes derived from both aged and young animals. Both IL-2 synthesis and IL-2 receptor expression induced by PMA plus ionomycin reach levels equal or near equal that found in parallel cultures of cells from young animals; however, proliferation is lower than in young adult cultures.