Mature dendritic cells (DCs) are stimulators of T-cell immune response, whereas immature DCs support T-cell tolerance. Murine B cells can inhibit the production of IL-12 by DCs and thereby hinder the inflammatory response. Notwithstanding the importance of this modulation, only a few studies are available in humans. Here, we have developed an in vitro model of cocultures to assess its significance. We establish that human activated B cells restrained the development of monocytes into immature DCs and their differentiation into mature DCs. In addition, they decreased the density of HLA-DR from mature DCs, the expression of CD80 and CD86 coactivation molecules, the production of IL-12p70 required for antigen presentation and Th1 differentiation, and inhibited the DC-induced T-cell proliferation. These modulations were mediated by CD19(+)IgD(low)CD38(+)CD24(low)CD27(-) B cells and needed direct cell-to-cell contacts that involved CD62L for the control of CD80 and CD86 expression and a soluble factor for the control of IL-12 production. Moreover, mature DCs from patients with systemic lupus erythematosus displayed insensitivity to the regulation of IL-12. Overall, it appears that human B cells can regulate DC maturation and function and that inefficient B-cell regulation may influence an improper balance between an effector inflammatory response and tolerance induction.

To develop a comprehensive catalogue of phenotypic and functional parameters of human CD4(+) T cell differentiation stages, we have performed microarray gene expression profiling on subpopulations of human thymocytes and circulating naive CD4(+) T cells, including CD3(-)CD4(+)CD8(-) intrathymic T progenitor cells, CD3(int)CD4(+)CD8(+) 'double positive' thymocytes, CD3(high)CD4(+)CD8(-) 'single positive' thymocytes, CD3(+)CD4(+)CD8(-) CD45RA(+)CD62L(+) naive T cells from cord blood and CD3(+)CD4(+)CD8(-) CD45RA(+)CD62L(+) naive T cells from adult blood. These subpopulations were sort-purified to >98% purity and their expressed RNAs were analyzed on Affymetrix Human Genome U133 arrays. Comparison of gene expression signals between these subpopulations and with early passage fetal thymic stromal cultures identify: (i) transcripts that are preferentially expressed in human CD4(+) T cell subpopulations and not in thymic stromal cells; (ii) major shifts in gene expression as progenitor T cells mature into progeny; (iii) preferential expression of transcripts at the progenitor cell stage with plausible relevance to the regulation of expansion and differentiation of these cells; and (iv) preferential expression of potential markers of recent thymic emigrants in naive-phenotype CD4(+) T cells from cord blood. Further evaluation of these findings may lead to a better definition of human thymopoiesis as well as to improved approaches to monitor and to augment the function of this important organ of T cell production.

In contrast to the ability of long-lived CD8(+) memory T cells to mediate protection against systemic viral infections, the relationship between CD4(+) T cell memory and acquired resistance against infectious pathogens remains poorly defined. This is especially true for T helper 1 (Th1) concomitant immunity, in which protection against reinfection coincides with a persisting primary infection. In these situations, pre-existing effector CD4 T cells generated by ongoing chronic infection, not memory cells, may be essential for protection against reinfection. We present a systematic study of the tissue homing properties, functionality, and life span of subsets of memory and effector CD4 T cells activated in the setting of chronic Leishmania major infection in resistant C57Bl/6 mice. We found that pre-existing, CD44(+)CD62L(-)T-bet(+)Ly6C+ effector (T(EFF)) cells that are short-lived in the absence of infection and are not derived from memory cells reactivated by secondary challenge, mediate concomitant immunity. Upon adoptive transfer and challenge, non-dividing Ly6C(+) T(EFF) cells preferentially homed to the skin, released IFN-γ, and conferred protection as compared to CD44(+)CD62L(-)Ly6C(-) effector memory or CD44(+)CD62L(+)Ly6C(-) central memory cells. During chronic infection, Ly6C(+) T(EFF) cells were maintained at high frequencies via reactivation of T(CM) and the T(EFF) themselves. The lack of effective vaccines for many chronic diseases may be because protection against infectious challenge requires the maintenance of pre-existing T(EFF) cells, and is therefore not amenable to conventional, memory inducing, vaccination strategies.

Induction of effective memory T cells is likely to be critical to the level and duration of protection elicited by novel live oral typhoid vaccines. Using cells from volunteers who ingested Salmonella Typhi vaccine strain CVD 909, we characterized the induction of interferon (IFN)-gamma-secreting central (T(CM), CD45RO(+)CD62L(+)) and effector (T(EM), CD45RO(+)CD62L(-)) memory T populations, and their gut-homing potential based on integrin alpha4/beta7 expression. Both CD4(+) T(EM) and T(CM) populations secreted IFN-gamma. However, although CD4(+) T(EM) expressed, or not, integrin alpha(4)/beta(7), CD4(+) T(CM) cells were predominantly integrin alpha(4)/beta(7)(+). In contrast, IFN-gamma-secreting CD8(+) cells were predominantly classical T(EM) and CD45RA(+) T(EM) (T(EMRA), CD45RO(-)CD62L(-)) subsets. However, although CD8(+) T(EM) expressed, or not, integrin alpha(4)/beta(7), CD8(+) T(EMRA) were predominantly integrin alpha(4)/beta(7)(+). This is the first demonstration that oral immunization of humans with S. Typhi elicits diverse IFN-gamma-secreting CD4(+) and CD8(+) T(CM) and T(EM) subsets able to migrate to the gut and other lymphoid tissues.

The interaction of CD8+CTL with epithelial layers is an important but poorly defined aspect of organ allograft rejection. We herein report that CD103 (formerly alpha E integrin), a known receptor for the epithelial cell-specific ligand E-cadherin, is expressed by a major subset of CD8 + CTL elicited in response to allogeneic renal epithelial cells (REC). In contrast, CD103 was expressed poorly on CD8 + CTL generated in the conventional manner by stimulation with allogeneic leukocytes, although expression could be dramatically up-regulated by supplementing cultures with REC or exogenous TGF-beta 1. That TGF-beta controls the expression of CD103 on CD8+ CTL was further supported by the capacity of anti-TGF-beta mAb to block the generation of such cells in anti-REC cultures. Clonal analyses of anti-REC cultures revealed that individual CD8+ CTL clones were discretely CD103+ or CD103-, nd maintained their respective phenotypes independently of the cell type used for clonal restimulation. In a mouse model of graft-vs-host disease, 16.4 +/- 2.7% of CD8 cells that infiltrated host kidneys were CD103+ (n = 4). CD8 kidney-infiltrating lymphocytes were predominantly of donor origin and displayed an activated/memory phenotype (CD62L-, CD44high), consistent with expression of CD103 on a CD8 effector subset elicited in vivo following allogeneic transplantation. Taken together, the present data demonstrate that CD103 identifies a novel CD8 effector subset and, moreover, that such cells may comprise a significant component of the response to allogeneic tissues. The potential for CD103+ CTL as an important effector mechanism in organ allograft rejection, and more generally, as a mechanistic basis for tissue-specific immune phenomena, is discussed.

Acetylcholine (ACh) regulates vital functions of T cells by acting on the nicotinic and muscarinic classes of cholinergic receptors, nAChR and mAChRs, respectively. This study was performed in murine splenic T cells. In freshly isolated CD4 and CD8 T cells, we detected mRNAs encoding α5, α9, α10, β1, β2, β4 nAChR subunits and M₁, M₃, M₄ and M₅ mAChR subtypes, whereas α2 was detected only in CD8 T cells. In vitro activation of CD4 T cells through T-cell receptor (TCR)/CD3 cross-linking was associated with the appearance of α4 and α7, upregulation of α5, α10, β4, M₁ and M₅ and downregulation of α9 and β2, whereas in vitro activation of CD8 T cells also featured the appearance of α4 and α7, as well as upregulation of α2, α5, β4, M₁ and M₄, and downregulation of α10, β1, β2 and M₃. In vitro polarization toward T helper (Th) 1 lineage was associated with a decrease of β2, β4 and M₃ expression; that toward Th2 cells with downregulation of α9 and M₃, and upregulation of M₁ and M₅; and that toward Th17 phenotype with downregulation of α9, α10, β2 and M₃ mAChR. Polarized T cells also expressed α4, but not α1, α2, α3, α6, β3 or M₂. To determine the role of cholinergic receptors in mediating the immunoregulatory action of autocrine/paracrine ACh, we analyzed the effects of nicotinic and muscarinic agonists±antagonists on cytokine production in the CD4+CD62L+ T cells co-stimulated via TCR/CD3 cross-linking. The nicotinergic stimulation upregulated interferon-γ (IFN-γ) and downregulated interleukin (IL)-17 secretion, whereas the muscarinic stimulation enhanced IL-10 and IL-17 and inhibited INF-γ secretion. These results demonstrated plasticity of the T-cell cholinergic system.

Enteric fever caused by Salmonella enterica serovar Paratyphi A infection has emerged as an important public health problem. Recognizing that in randomized controlled field trials oral immunization with attenuated S. enterica serovar Typhi live vaccine Ty21a conferred significant cross-protection against S. Paratyphi B but not S. Paratyphi A disease, we undertook a clinical study to ascertain whether humoral immune responses could explain the field trial results. Ty21a immunization of adult residents of Maryland elicited predominantly IgA antibody-secreting cells (ASC) that recognize S. Typhi lipopolysaccharide (LPS). Cross-reactivity to S. Paratyphi A LPS was significantly lower than that to S. Paratyphi B LPS. ASC producing IgG and IgA that bind LPS from each of these Salmonella serovars expressed CD27 and integrin α4β7 (gut homing), with a significant proportion coexpressing CD62L (secondary lymphoid tissue homing). No significant differences were observed in serum antibody against LPS of the different serovars. Levels of IgA B memory (B(M)) cells to S. Typhi LPS were significantly higher than those against S. Paratyphi A or B LPS, with no differences observed between S. Paratyphi A and B. The response of IgA B(M) to outer membrane proteins (OMP) from S. Typhi was significantly stronger than that to OMP of S. Paratyphi A but similar to that to OMP of S. Paratyphi B. The percentages of IgG or IgA B(M) responders to LPS or OMP from these Salmonella strains were similar. Whereas cross-reactive humoral immune responses to S. Paratyphi A or B antigens are demonstrable following Ty21a immunization, they cannot explain the efficacy data gleaned from controlled field trials.

Heightened proliferation and death of T lymphocytes may play a key role in human immunodeficiency virus (HIV) pathogenesis; however, the mechanism that mediates this effect and the phenotype of the proliferating T cells have not been clearly determined. We assessed S-phase cell frequencies and phenotype by ex vivo bromodeoxyuridine incorporation and flow-cytometric analysis in a group of 35 HIV-infected individuals. Frequencies of S-phase T cells were increased in HIV disease and were related to plasma HIV RNA levels but not to CD4 cell, total T cell, or total lymphocyte counts. S-phase cells were phenotypically defined as "central memory" cells (CD45RO+CD62L+CCR7+). Although activated (CD38+), S-phase cells lacked CD69 expression, rarely expressed CD25, and were not overrepresented among HIV-specific cells, as might have been expected if these cells had recently been activated by HIV antigens. Thus, in HIV infection, central memory T cells may be highly susceptible to bystander mechanisms of immune activation, leading to S-phase entry.

Foxp3(+) regulatory T cells (Tregs) express both ectoenzymes CD39 and CD73, which in tandem hydrolyze pericellular ATP into adenosine, an immunoinhibitory molecule that contributes to Treg suppressive function. Using Foxp3GFP knockin mice, we noted that the mouse CD4(+)CD39(+) T-cell pool contains two roughly equal size Foxp3(+) and Foxp3(-) populations. While Foxp3(+)CD39(+) cells are CD73(bright) and are the bone fide Tregs, Foxp3(-)CD39(+) cells do not have suppressive activity and are CD44(+)CD62L(-)CD25(-)CD73(dim/-), exhibiting memory cell phenotype. Functionally, CD39 expression on memory and Treg cells confers protection against ATP-induced apoptosis. Compared with Foxp3(-)CD39(-) naïve T cells, Foxp3(-)CD39(+) cells freshly isolated from non-immunized mice express at rest significantly higher levels of mRNA for T-helper lineage-specific cytokines IFN-gamma (Th1), IL-4/IL-10 (Th2), IL-17A/F (Th17), as well as pro-inflammatory cytokines, and rapidly secrete these cytokines upon stimulation. Moreover, the presence of Foxp3(-)CD39(+) cells inhibits TGF-beta induction of Foxp3 in Foxp3(-)CD39(-) cells. Furthermore, when transferred in vivo, Foxp3(-)CD39(+) cells rejected MHC-mismatched skin allografts in a much faster tempo than Foxp3(-)CD39(-) cells. Thus, besides Tregs, CD39 is also expressed on pre-existing memory T cells of Th1-, Th2- and Th17-types with heightened alloreactivity.

Infection of mice with murine gammaherpesvirus 68 (MHV-68) robustly activates CD8 T cells, but only six class I major histocompatibility complex (MHC)-restricted epitopes have been described to date for the widely used H-2(b) haplotype mice. To explore the specificity and kinetics of the cytotoxic T-lymphocyte response in MHV-68-infected C57BL/6 mice, we screened for H-2K(b)- and H-2D(b)-restricted epitopes using a set of 384 candidate epitopes in an MHC tetramer-based approach and identified 19 new epitopes in 16 different open reading frames. Of the six known H-2K(b)- and H-2D(b)-restricted epitopes, we confirmed a response against three and did not detect CD8 T-cell-specific responses for the remaining three. The peak of the CD8 T-cell response to most peptides occurs between 6 and 10 days postinfection. The respective MHC tetramer-positive CD8 T cells display an activated/effector phenotype (CD62L(lo) and CD44(hi)) and produce gamma interferon upon peptide stimulation ex vivo. MHV-68 infection in vivo elicits a response to multiple viral epitopes, derived from both early and late viral antigens, illustrating a far broader T-cell repertoire and more-rapid activation than those previously recorded.

In mice acutely infected with respiratory syncytial virus (RSV), more than 20% of pulmonary CD8(+) T cells, but only 2-3% of CD8(+) T cells in the draining lymph node secreted interferon-gamma in response to a single peptide. Surprisingly, the percentage of virus-specific T cells in the lung remained at these high levels long after the acute infection. Pulmonary memory T cells were further studied in a sensitive adoptive transfer system, which allows visualizing polyclonal CD4(+) and CD8(+) virus-specific memory T cell responses. Fifty days after infection, persisting RSV-specific pulmonary T cells remained CD69(hi) CD62L(lo), but had returned to a resting memory state according to functional criteria. In the absence of neutralizing antibodies reinfection first induced cell division among virus-specific memory T cells 3 days after infection predominantly in the local lymph node. However, divided cells then rapidly accumulated in the lung without significantly increasing in the lymph node. These results suggest rapid export of reactivated cells from the lymph node to the target organ. Thus, although memory T cells can be maintained in the infected organ after a localized virus infection, amplification of a recall response appears to be most effective in organized lymphoid tissue.

Immunization with radiation (gamma)-attenuated Plasmodia sporozoites (gamma-spz) confers sterile and long-lasting immunity against malaria liver-stage infection. In the P. berghei gamma-spz model, protection is linked to liver CD8+ T cells that express an effector/memory (T(EM)) phenotype, (CD44(hi)CD45RB(lo)CD62L(lo)), and produce IFN-gamma. However, neither the antigen presenting cells (APC) that activate these CD8+ T(EM) cells nor the site of their induction have been fully investigated. Because conventional (c)CD8alpha+ DC (a subset of CD11c+ DC) are considered the major inducers of CD8+ T cells, in this study we focused primarily on cCD8alpha+ DC from livers of mice immunized with Pb gamma-spz and asked whether the cCD8alpha+ DC might be involved in the activation of CD8+ T(EM) cells. We demonstrate that multiple exposures of mice to Pb gamma-spz lead to a progressive and nearly concurrent accumulation in the liver but not the spleen of both the CD11c+NK1.1(-) DC and CD8+ T(EM) cells. Upon adoptive transfer, liver CD11c+NK1.1(-) DC from Pb gamma-spz-immunized mice induced protective immunity against sporozoite challenge. Moreover, in an in vitro system, liver cCD8alpha(+) DC induced naïve CD8+ T cells to express the CD8+ T(EM) phenotype and to secrete IFN-gamma. The in vitro induction of functional CD8+ T(EM) cells by cCD8alpha+ DC was inhibited by anti-MHC class I and anti-IL-12 mAbs. These data suggest that liver cCD8alpha+ DC present liver-stage antigens to activate CD8+ T(EM) cells, the pre-eminent effectors against pre-erythrocytic malaria. These results provide important implications towards a design of anti-malaria vaccines.

Increases in catecholamines have been shown to induce changes in migration of lymphocytes, in particular NK cells. To analyze the mechanisms of catecholamine-induced NK cell trafficking, normal healthy male human subjects and splenectomized individuals were infused with either adrenaline (0.10 microgram/kg/min), noradrenaline (0.15 microgram/kg/min), or NaCl i.v. for 20 min. Lymphocyte subsets (CD3+, CD4+, CD8+) transiently increased after administration of both catecholamines, with most pronounced increases (up to 600%) in NK cell numbers (CD16+ or CD56+) after infusion of adrenaline. These changes in NK cell numbers and function were accompanied neither by alterations in expression of adhesion molecules (CD11a), CD11b, CD31, CD43, CD44, CD62L) on NK cells nor by changes in plasma concentrations of soluble (s) adhesion molecules (sVCAM-1, sICAM-1, sE-selectin). Comparable increases in lymphocyte subsets were observed in splenectomized subjects, suggesting lymphocyte recruitment from other sources than the spleen. Furthermore, catecholamine-induced increases in lymphocyte subsets could be inhibited by pretreatment with the nonselective beta-adrenoceptor antagonist propranolol, but not by the beta1-selective antagonist bisoprolol. These data demonstrate that adrenaline and noradrenaline modulate the migratory capacity of human NK cells via spleen-independent beta 2-adrenoceptor mechanism.

The peripheral tolerance mechanism prevents effective antitumor immunity, even though tumor cells possess recognizable tumor-associated Ags. Recently, it has been elucidated that regulatory T cells (Treg) play a critical role in maintaining not only self-tolerance, but also tolerance of tumor cells. However, because the Treg that maintain self-tolerance arise naturally in the thymus and are thought to be anergic in peripheral, it is still unclear where and when Treg for tumor cells are generated. In this study we analyze tumor-draining lymph nodes (LNs) and demonstrate that both antitumor effector T cells and Treg capable of abrogating the antitumor reactivity of the effector T cells are primed in the same LNs during tumor progression. The regulatory activity generated in tumor-draining LNs exclusively belonged to the CD4(+) T cell subpopulation that expresses both CD25 and a high level of CD62L. Forkhead/winged helix transcription factor gene expression was detected only in the CD62L(high)CD4(+)CD25(+) T cells. CD62L(high)CD4(+)CD25(+) Treg and CD62L(low)CD4(+)CD25(+) T cells, which possess effector T cell functions, had comparable expression of LFA-1, VLA-4, CTLA-4, lymphocyte activation gene-3, and glucocorticoid-induced TNFR. Thus, only CD62L expression could distinguish regulatory CD4(+)CD25(+) cells from effector CD4(+)CD25(+) cells in draining LNs as a surface marker. The Treg generated in tumor-draining LNs possess the same functional properties as the Treg that arise naturally in the thymus but recognize tumor-associated Ag. CD62L(high)CD4(+)CD25(+) Treg contained a subpopulation that expressed CD86. Blocking experiments revealed that ligation of CTLA-4 on effector T cells by CD86 on Treg plays a pivotal role in regulating CD4(+) effector T cells.

To investigate if transporter associated with antigen processing (TAP)-1 is required for CD8(+) T cell-mediated control of Toxoplasma gondii in vivo, we compared the resistance of TAP-1(-/-), CD8(-/-), and wild-type (WT) mice to infection with the parasite. Unexpectedly, TAP-1(-/-) mice displayed greater susceptibility than CD8(-/-), beta(2)-microglobulin(-/-) (beta(2)m(-/-)), or WT mice to infection with an avirulent parasite strain. The decreased resistance of the TAP-1(-/-) mice correlated with a reduction in the frequency of activated (CD62L(low) CD44(hi)) and interferon (IFN)-gamma-producing CD4(+) T cells. Interestingly, infected TAP-1(-/-) mice also showed reduced numbers of IFN-gamma-producing natural killer (NK) cells relative to WT, CD8(-/-), or beta(2)m(-/-) mice, and after NK cell depletion both CD8(-/-) and WT mice succumbed to infection with the same kinetics as TAP-1(-/-) animals and displayed impaired CD4(+) T cell IFN-gamma responses. Moreover, adoptive transfer of NK cells obtained from IFN-gamma(+/+), but not IFN-gamma(-/-), animals restored the CD4(+) T cell response of infected TAP-1(-/-) mice to normal levels. These results reveal a role for TAP-1 in the induction of IFN-gamma-producing NK cells and demonstrate that NK cell licensing can influence host resistance to infection through its effect on cytokine production in addition to its role in cytotoxicity.

Lymphocytes from normal subjects or patients with chronic lymphocytic leukemia are known to possess receptors for extracellular ATP termed P2Z purinoceptors whose physiological role is undefined. Addition of extracellular ATP (50-500 microM) to both normal and leukemic lymphocytes caused loss of binding of monoclonal antibodies to L-selectin (CD62L) on the cell surface. UTP, ADP, and adenosine (all at 500 microM) had no effect on L-selectin expression. Several features of the ATP-induced loss of L selectin indicate that this effect is mediated by lymphocyte P2Z purinoceptors. First the loss was attenuated in isotonic NaCl medium compared to 150 mM KCl medium. Second the loss of L-selectin was immediately halted by addition of Mg2+ ions in molar excess of ATP. The most potent nucleotide causing L-selectin loss was benzoylbenzoic ATP >> 10 microM) which is also the most potent agonist for the P2Z purinoceptor. Finally preincubation of lymphocytes with oxidized ATP, an irreversible inhibitor of P2Z purinoceptors, also inhibited ATP induced loss of L-selectin. Extracellular ATP is known to open an ion channel associated with the P2Z purinoceptor on B-lymphocytes which allows influx of Ca2+. However, ATP-induced loss of L-selectin did not require extracellular Ca2+. Moreover addition of the calcium ionophore, ionomycin, had minimal effect on L-selectin expression. Staurosporine (500 nM), an inhibitor of protein kinase C, inhibited only 10% of ATP induced loss of L-selectin but completely inhibited the loss of L-selectin caused by 50 nM PMA. Thus extracellular ATP interacts with lymphocyte P2Z purinoceptors which leads to shedding of L-selectin via a pathway which requires neither Ca2+ influx nor activation of protein kinase C. ATP may have a physiological role in the loss of L-selectin which occurs during the interactions of lymphocytes with other cells.

Selective depletion of alloreactive T cells from allogeneic stem cell grafts can reduce graft-versus-host disease (GVHD) while preserving beneficial effects of T cells including facilitation of engraftment, protection against opportunistic infection, and reduced relapse risk. Memory T cells (CD62L(-)) represent a population of T cells that have previously encountered pathogens and may contain fewer T cells capable of recognizing neoantigens including recipient allogeneic antigen (aAg). We investigated whether human naive (CD62L(+)) or memory (CD62L(-)) T cells had different capacities to respond to aAg by assessing their ability to proliferate in response to and lyse HLA-mismatched Epstein-Barr virus-transformed B cells. Freshly sorted and in vitro expanded CD62L(-) memory T cells were less responsive to aAg stimulation than were CD62L(+) naive T cells but contained higher levels of cytomegalovirus (CMV)-specific T cells. Analysis of T cell receptor (TCR) repertoire showed restricted TCR diversity in the memory T-cell population possibly due to selection associated with chronic exposure to common pathogens. Memory T cells may represent a donor cell subpopulation suitable for enhancing immune reconstitution without increasing the risk of GVHD.

Although recent studies have focused on CD4(+) regulatory T cells (Treg), CD8(+) Treg have also been reported to play important roles in the induction and maintenance of immune tolerance. Adoptive transfer of CD8(+) Treg in rodents or induction of CD8(+) Treg in humans can prevent or treat allograft rejection and autoimmune diseases. However, no approaches have been reported for the generation of human Ag-specific CD8(+) Treg at a practical scale for clinical use. Here, we found that two novel CD8(+) T cell subsets with different levels of CD8 surface expression, CD8(high) and CD8(low), could be induced from naive CD8(+) precursors in vitro by allogeneic CD40-activated B cells, whereas only CD8(high) T cells were alloantigen-specific Treg with relatively poor alloantigen-specific cytotoxicity. Importantly, alloantigen-specific CD8(high) Treg could be induced and expanded from naive CD8(+)CD25(-) T cells at a large scale after 3 wk of culture without exogenous cytokines. These induced alloantigen-specific Treg were CD45RO(+) and CCR7(-) memory cells, and they expressed Foxp3, CD25, CD27, CD28, and CD62L. The induction and expansion of CD8(high) Treg by CD40-activated B cells were dependent on endogenously expressed IFN-gamma, IL-2, IL-4, and CTLA-4. This approach may facilitate the clinical application of CD8(+) Treg-based immunotherapy in transplantation and autoimmune diseases.

Multiple studies have established that the ability of CD8(+) T cells to act as cytolytic effectors and produce gamma interferon is important in mediating resistance to the intracellular parasite Toxoplasma gondii. To better understand the generation of the antigen-specific CD8(+) T-cell responses induced by T. gondii, mice were immunized with replication-deficient parasites that express the model antigen ovalbumin (OVA). Class I tetramers specific for SIINFEKL were used to track the OVA-specific endogenous CD8(+) T cells. The peak CD8(+) T-cell response was found at day 10 postimmunization, after which the frequency and numbers of antigen-specific cells declined. Unexpectedly, replication-deficient parasites were found to induce antigen-specific cells with faster kinetics than replicating parasites. The generation of optimal numbers of antigen-specific CD8(+) effector T cells was found to require CD4(+) T-cell help. At 7 days following immunization, antigen-specific cells were found to be CD62L(low), KLRG1(+), and CD127(low), and they maintained this phenotype for more than 70 days. Antigen-specific CD8(+) effector T cells in immunized mice exhibited potent perforin-dependent OVA-specific cytolytic activity in vivo. Perforin-dependent cytolysis appeared to be the major cytolytic mechanism; however, a perforin-independent pathway that was not mediated via Fas-FasL was also detected. This study provides further insight into vaccine-induced cytotoxic T-lymphocyte responses that correlate with protective immunity to T. gondii and identifies a critical role for CD4(+) T cells in the generation of protective CD8(+) T-cell responses.

To explore whether any co-stimulatory receptor(s) for TCR signaling is involved in the age-associated decline in T-cell function, we analyzed changes in these receptors in freshly isolated mouse CD4(+) T cells during aging. Both the mRNA and protein expression levels of CTLA-4 and PD-1, negative co-stimulatory receptors, increase with aging. No such changes are observed for CD28, a positive regulatory receptor. PD-1 is highly expressed on the surface of old, but not young, mouse T cells, while the level of surface-expressed CTLA-4 is very low regardless of age. PD-1 is preferentially expressed on the surface of effector-memory (CD44(hi)CD62L(lo)) T cells, a subset that increases with aging. CD4(+)PD-1(+) T cells from old mice exhibit proliferative hyporesponsiveness. These results suggest that the up-regulation of surface-expressed PD-1 may cause the age-dependent functional decline in effector-memory T cells.

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic stem cell transplantation. Effector memory T cells (T(EM)) do not cause GVHD but engraft and mount immune responses, including graft-versus-tumor effects. One potential explanation for the inability of T(EM) to cause GVHD is that T(EM) lack CD62L and CCR7, which are instrumental in directing naive T cells (T(N)) to lymph nodes (LN) and Peyer patches (PP), putative sites of GVHD initiation. Thus T(EM) should be relatively excluded from LN and PP, possibly explaining their inability to cause GVHD. We tested this hypothesis using T cells deficient in CD62L or CCR7, transplant recipients lacking PNAd ligands for CD62L, and recipients without LN and PP or LN, PP, and spleen. Surprisingly, CD62L and CCR7 were not required for T(N)-mediated GVHD. Moreover, in multiple strain pairings, GVHD developed in recipients that lacked LN and PP. Mild GVHD could even be induced in mice lacking all major secondary lymphoid tissues (SLT). Conversely, enforced constitutive expression of CD62L on T(EM) did not endow them with the ability to cause GVHD. Taken together, these data argue against the hypothesis that T(EM) fail to induce GVHD because of inefficient trafficking to LN and PP.

Defining the cellular composition of the memory T cell pool has been complicated by an inability to distinguish effector and memory T cells. We present here an activation profile assay, using anti-CD3 and antigenic stimuli, that clearly distinguishes effector and memory CD4 T cells and defines subsets of long-lived memory CD4 T cells based on CD62 ligand (CD62L) expression. The CD62L(low) memory subset functionally resembles effector cells, exhibiting hyper-responsiveness to antigenic and anti-CD3 mediated stimuli, high proliferative capacity, and rapid activation kinetics. The CD62L(high) memory subset functionally resembles resting memory cells, exhibiting hyporesponsiveness to anti-CD3 stimuli, lower proliferative capacity, and slower activation kinetics. Our results indicate that the memory CD4 T cell pool is heterogeneous, consisting of persisting effectors and resting memory T cells.

An acute bout of exercise evokes mobilisation of lymphocytes into the bloodstream, which can be largely attributed to increases in CD8+ T lymphocytes (CD8TLs) and natural killer (NK) cells. Evidence further suggests that, even within these lymphocyte subsets, there is preferential mobilisation of cells that share certain functional and phenotypic characteristics, such as high cytotoxicity, low proliferative ability, and high tissue-migrating potential. These features are characteristic of effector-memory CD8TL subsets. The current study therefore investigated the effect of exercise on these newly-identified subsets. Thirteen healthy and physically active males (mean+/-SD: age 20.9+/-1.5 yr) attended three sessions: a control session (no exercise); cycling at 35% Watt(max) (low intensity exercise); and 85% Watt(max) (high intensity exercise). Each bout lasted 20 min. Blood samples were obtained before exercise, during the final min of exercise, and +15, and +60 min post-exercise. CD8TLs were classified into naïve, central memory (CM), effector-memory (EM), and CD45RA+ effector-memory (RAEM) using combinations of the cell surface markers CCR7, CD27, CD62L, CD57, and CD45RA. In parallel, the phenotypically distinct CD56(bright) 'regulatory' and CD56(dim) 'cytotoxic' NK subsets were quantified. The results show a strong differential mobilisation of CD8TL subsets (RAEM>EM>CM>naïve); during high intensity exercise the greatest increase was observed for RAEM CD8Tls (+450%) and the smallest for naïve cells (+84%). Similarly, CD56(dim) NK cells (+995%) were mobilised to a greater extent than CD56(bright) (+153%) NK cells. In conclusion, memory CD8TL that exhibit a high effector and tissue-migrating potential are preferentially mobilised during exercise. This finding unifies a range of independent observations regarding exercise-induced phenotypic and functional changes in circulating lymphocytes. The selective mobilisation of cytotoxic tissue-migrating subsets, both within the NK and CD8TL population, may enhance immune-surveillance during exercise.

Naive T cell responses are eroded with aging. We and others have recently shown that unimmunized old mice lose ≥ 70% of Ag-specific CD8 T cell precursors and that many of the remaining precursors acquire a virtual (central) memory (VM; CD44(hi)CD62L(hi)) phenotype. In this study, we demonstrate that unimmunized TCR transgenic (TCRTg) mice also undergo massive VM conversion with age, exhibiting rapid effector function upon both TCR and cytokine triggering. Age-related VM conversion in TCRTg mice directly depended on replacement of the original TCRTg specificity by endogenous TCRα rearrangements, indicating that TCR signals must be critical in VM conversion. Importantly, we found that VM conversion had adverse functional effects in both old wild-type and old TCRTg mice; that is, old VM, but not old true naive, T cells exhibited blunted TCR-mediated, but not IL-15-mediated, proliferation. This selective proliferative senescence correlated with increased apoptosis in old VM cells in response to peptide, but decreased apoptosis in response to homeostatic cytokines IL-7 and IL-15. Our results identify TCR as the key factor in differential maintenance and function of Ag-specific precursors in unimmunized mice with aging, and they demonstrate that two separate age-related defects--drastic reduction in true naive T cell precursors and impaired proliferative capacity of their VM cousins--combine to reduce naive T cell responses with aging.