Multiple reports implicated the function of G protein-coupled receptor (GPR)-30 with nongenomic effects of estrogen, suggesting that GPR30 might be a G-protein coupled estrogen receptor. However, the findings are controversial and the expression pattern of GPR30 on a cell type level as well as its function in vivo remains unclear. Therefore, the objective of this study was to identify cell types that express Gpr30 in vivo by analyzing a mutant mouse model that harbors a lacZ reporter (Gpr30-lacZ) in the Gpr30 locus leading to a partial deletion of the Gpr30 coding sequence. Using this strategy, we identified the following cell types expressing Gpr30: 1) an endothelial cell subpopulation in small arterial vessels of multiple tissues, 2) smooth muscle cells and pericytes in the brain, 3) gastric chief cells in the stomach, 4) neuronal subpopulations in the cortex as well as the polymorph layer of the dentate gyrus, 5) cell populations in the intermediate and anterior lobe of the pituitary gland, and 6) in the medulla of the adrenal gland. In further experiments, we aimed to decipher the function of Gpr30 by analyzing the phenotype of Gpr30-lacZ mice. The body weight as well as fat mass was unchanged in Gpr30-lacZ mice, even if fed with a high-fat diet. Flow cytometric analysis revealed lower frequencies of T cells in both sexes of Gpr30-lacZ mice. Within the T-cell cluster, the amount of CD62L-expressing cells was clearly reduced, suggesting an impaired production of T cells in the thymus of Gpr30-lacZ mice.

OBJECTIVE

To compare the impact of HIV infection and healthy ageing on monocyte phenotype and function and determine whether age-related changes induced by HIV are reversed in antiretroviral treated individuals.

METHODS

A cross sectional study of monocyte ageing markers in viremic and virologically suppressed HIV-positive males aged 45 years or less and age-matched and elderly (≥65 years) HIV-uninfected individuals.

METHODS

Age-related changes to monocyte phenotype and function were measured in whole blood assays ex vivo on both CD14(++)CD16(-) (CD14(+)) and CD14(variable)CD16(+) (CD16(+)) subsets. Plasma markers relevant to innate immune activation were measured by ELISA.

RESULTS

Monocytes from young viremic HIV-positive males resemble those from elderly controls, and show increased expression of CD11b (P < 0.0001 on CD14(+) and CD16(+)subsets) and decreased expression of CD62L and CD115 (P = 0.04 and 0.001, respectively, on CD14(+) monocytes) when compared with young uninfected controls. These changes were also present in young virologically suppressed HIV-positive males. Innate immune activation markers neopterin, soluble CD163 and CXCL10 were elevated in both young viremic (P < 0.0001 for all) and virologically suppressed (P = 0.0005, 0.003 and 0.002, respectively) HIV-positive males with levels in suppressed individuals resembling those observed in elderly controls. Like the elderly, CD14(+) monocytes from young HIV-positive males exhibited impaired phagocytic function (P = 0.007) and telomere-shortening (P = 0.03) as compared with young uninfected controls.

CONCLUSIONS

HIV infection induces changes to monocyte phenotype and function in young HIV-positive males that mimic those observed in elderly uninfected individuals, suggesting HIV may accelerate age-related changes to monocytes. Importantly, these defects persist in virologically suppressed HIV-positive individuals.

Themis (thymocyte-expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis had high expression in thymocytes between the pre-T cell antigen receptor (pre-TCR) and positive-selection checkpoints and low expression in mature T cells. Themis-deficient thymocytes showed defective positive selection, which resulted in fewer mature thymocytes. Negative selection was also impaired in Themis-deficient mice. A greater percentage of Themis-deficient T cells had CD4(+)CD25(+)Foxp3(+) regulatory and CD62L(lo)CD44(hi) memory phenotypes than did wild-type T cells. In support of the idea that Themis is involved in TCR signaling, this protein was phosphorylated quickly after TCR stimulation and was needed for optimal TCR-driven calcium mobilization and activation of the kinase Erk.

In humans, CD56, CD161, CD16, CD94 or CD57 represent prototypic markers of NK cells, although they are also found on a subset of CD8+ T cells. We propose here that the expression of NK receptors on CD8+ T cells can be considered a marker of cytotoxic effector T cells that are expanded in vivo after antigenic activation leading to extensive proliferation. The persistence of antigen will lead to loss of co-stimulatory molecules, telomere shortening and defective IL-2 production, changes that define the state replicative senescence in T lymphocytes. The majority of these "effector/senescent" T lymphocytes are CD8+, CD45RA+, CD11a(bright), CD28-, CD27-, CD62L- and CCR7-. They are cytotoxic T cells with strong expression of intracytoplasmic perforin and granzyme, but with low proliferative capacity and defective IL-2 production. Many of these characteristics are shared by the recently defined "effector/memory" T cells, being mainly distinguished by the absence of CD45RA expression on the memory cells. The expression of NK receptor in these effector cells will probably contribute to the regulation of their cytotoxic function. Expansion of cells with these characteristics can be found not only in the elderly but also in other clinical conditions involving chronic activation of the immune system such as viral infections, rheumatic and autoimmune diseases or tumors. Another subset of T cells that expresses the NK receptors is the alpha-galactosyl-ceramide specific T cell subset defined by the expression of canonical Valpha24JalphaQ TCR, recognition of CD1d and secretion of high amounts of IL-4 and IFN-gamma. However, the changes observed in the expression of NK-R on T cells associated to immunosenescence can not be attributed to expansion of this particular T cell subset, although alterations in the number and function of these cells have been demonstrated in some autoimmune diseases.

Naive T lymphocytes acquire a phenotype similar to Ag-experienced memory T cells as a result of proliferation under lymphopenic conditions. Such "memory-like" T (T(ML)) cells constitute a large fraction of the peripheral T cell pool in patients recovering from T cell ablative therapies, HIV patients under highly active antiretroviral therapy, and in the elderly population. To generate a model that allows characterization of T(ML) cells without adoptive transfer, irradiation, or thymectomy, we developed genetically modified mice that express diphtheria toxin A under control of a loxP-flanked stop cassette (R-DTA mice). Crossing these mice to CD4Cre mice resulted in efficient ablation of CD4 single-positive thymocytes, whereas double-positive and CD8 single-positive thymocytes were only partially affected. In the periphery the pool of naive (CD44(low)CD62L(high)) T cells was depleted. However, some T cells were resistant to Cre activity, escaped deletion in the thymus, and underwent lymphopenia-induced proliferation resulting in a pool of T(ML) cells that was similar in size and turnover to the pool of CD44(high)CD62L(low) "memory phenotype" T cells in control mice. CD4Cre/R-DTA mice remained lymphopenic despite the large available immunological "space" and normal Ag-induced T cell proliferation. CD4Cre/R-DTA mice showed a biased TCR repertoire indicating oligoclonal T cell expansion. Infection with the helminth Nippostrongylus brasiliensis resulted in diminished effector cell recruitment and impaired worm expulsion, demonstrating that T(ML) cells are not sufficient to mediate an effective immune response.

We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor alpha (IL-15Ralpha) in the same cell allows for the intracellular interaction of the two proteins early after translation, resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15Ralpha, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15Ralpha expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver, and spleen. The presence of IL-15Ralpha also increased the number of CD44(high) memory cells with effector phenotype (CD44(high)CD62L-). Thus, mutual stabilization of IL-15 and IL-15Ralpha leads to remarkable increases in production, stability, and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor alpha either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for coordinate expression of IL-15 and IL-15Ralpha in the same cell and suggest that the IL-15Ralpha is part of the active IL-15 cytokine rather than part of the receptor.

Measuring the magnitudes and specificities of antiviral CD8 T-cell responses is critical for understanding the dynamics and regulation of adaptive immunity. Despite many excellent studies, the accurate measurement of the total CD8 T-cell response directed against a particular infection has been hampered by an incomplete knowledge of all CD8 T-cell epitopes and also by potential contributions of bystander expansion among CD8 T cells of irrelevant specificities. Here, we use several techniques to provide a more complete accounting of the CD8 T-cell response generated upon infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV). Eight days following infection, we found that 85 to 95% of CD8 T cells exhibit an effector phenotype as indicated by granzyme B, 1B11, CD62L, CD11a, and CD127 expression. We demonstrate that CD8 T-cell expansion is due to cells that divide >7 times, whereas heterologous viral infections only elicited <3 divisions among bystander memory CD8 T cells. Furthermore, we found that approximately 80% of CD8 T cells in spleen were specific for ten different LCMV-derived epitopes at the peak of primary infection. These data suggest that following a single LCMV infection, effector CD8 T cells divide>> or =15 times and account for at least 80%, and possibly as much as 95%, of the CD8 T-cell pool. Moreover, the response targeted a very broad array of peptide major histocompatibility complexes (MHCs), even though we examined epitopes derived from only two of the four proteins encoded by the LCMV genome and C57BL/6 mice only have two MHC class I alleles. These data illustrate the potential enormity, specificity, and breadth of CD8 T-cell responses to viral infection and demonstrate that bystander activation does not contribute to CD8 T-cell expansion.

Transcription factors that regulate the quiescence, proliferation and homing of lymphocytes are critical for effective immune system function. Here we demonstrate that the transcription factor ELF4 directly activated the tumor suppressor KLF4 'downstream' of T cell antigen receptor signaling to induce cell cycle arrest in naive CD8(+) T cells. Elf4- and Klf4-deficient mice accumulated CD8(+)CD44(hi) T cells during steady-state conditions and generated more memory T cells after immunization. The homeostatic population expansion of CD8(+)CD44(hi) T cells in Elf4-null mice resulted in a redistribution of cells to nonlymphoid tissue because of lower expression of the transcription factor KLF2 and the surface proteins CCR7 and CD62L. Our work describes the combinatorial effect of lymphocyte-intrinsic factors on the homeostasis, activation and homing of T cells.

Upon antigen recognition, T cells form either static (synapses) or migratory (kinapses) contacts with antigen-presenting cells. Addressing whether synapses and kinapses result in distinct T cell receptor (TCR) signals has been hampered by the inability to simultaneously assess T cell phenotype and behavior. Here, we introduced dynamic in situ cytometry (DISC), a combination of intravital multiphoton imaging and flow cytometry-like phenotypic analysis. Taking advantage of CD62L shedding as a marker of early TCR signaling, we examined how T cells sense TCR ligands of varying affinities in vivo. We uncovered three modes of antigen recognition: synapses with the strongest TCR signals, kinapses with robust signaling, and kinapses with weak signaling. As illustrated here, the DISC approach should provide unique opportunities to link immune cell behavior to phenotype and function in vivo.

Several populations of memory T cells have been described that differ in their migration and function. In this study, we have identified a unique subset of memory T cells, which we have named recirculating memory T cells (T(RCM)). By exposing Kaede transgenic mouse skin to violet light, we tracked the fate of cutaneous T cells. One population of memory CD4(+) T cells remained in the skin. A second population migrated from the skin into draining lymph nodes (LNs) in a CCR7-dependent manner. These migrating CD4(+) T cells expressed a novel cell surface phenotype (CCR7(int/+)CD62L(int)CD69(-)CD103(+/-) E-selectin ligands(+)) that is distinct from memory T cell subsets described to date. Unlike memory T cell subsets that remain resident within tissues long-term, or that migrate either exclusively between lymphoid tissues or into peripheral nonlymphoid sites, CD4(+) T(RCM) migrate from the skin into draining LNs. From the draining LNs, CD4(+) T(RCM) reenter into the circulation, distal LNs, and sites of non-specific cutaneous inflammation. In addition, CD4(+) T(RCM) upregulated CD40L and secreted IL-2 following polyclonal stimulation. Our results identify a novel subset of recirculating memory CD4(+) T cells equipped to deliver help to both distal lymphoid and cutaneous tissues.

We previously demonstrated that L-selectin (CD62L)-dependent T cell homing to lymph nodes (LN) is required for tolerance induction to alloantigen. To explore the mechanisms of this observation, we analyzed the development and distribution of regulatory T cells (Treg), which play an important protective role against allograft rejection in transplantation tolerance. Alloantigen-specific tolerance was induced using either anti-CD2 plus anti-CD3 mAbs, or anti-CD40L mAbs plus donor-specific transfusion, in fully mismatched (BALB/c donor, C57BL/6 recipient) vascularized cardiac allografts. An expansion of CD4(+)CD25(+)CD62L(high) T cells was observed specifically within the LN of tolerant animals, but not in other anatomic sites or under nontolerizing conditions. These cells exhibited a substantial up-regulation of Foxp3 expression as measured by real-time PCR and by fluorescent immunohistochemistry, and possessed alloantigen-specific suppressor activity. Neither LN nor other lymphoid cells expressed the regulatory phenotype if recipients were treated with anti-CD62L mAbs, which both prevented LN homing and caused early allograft rejection. However, administration of FTY720, a sphingosine 1-phosphate receptor modulator that induces CD62L-independent T cell accumulation in the LNs, restored CD4(+)CD25(+) Treg in the LNs along with graft survival. These data suggest that alloantigen-specific Foxp3(+)CD4(+)CD25(+) Treg develop and are required within the LNs during tolerization, and provide compelling evidence that distinct lymphoid compartments play critical roles in transplantation tolerance.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease.

Adoptive immunotherapy using cultured T cells holds promise for the treatment of cancer and infectious disease. Ligands immobilized on surfaces fabricated from hard materials such as polystyrene plastic are commonly employed for T cell culture. The mechanical properties of a culture surface can influence the adhesion, proliferation, and differentiation of stem cells and fibroblasts. We therefore explored the impact of culture substrate stiffness on the ex vivo activation and expansion of human T cells. We describe a simple system for the stimulation of the TCR/CD3 complex and the CD28 receptor using substrates with variable rigidity manufactured from poly(dimethylsiloxane), a biocompatible silicone elastomer. We show that softer (Young's Modulus [E] < 100 kPa) substrates stimulate an average 4-fold greater IL-2 production and ex vivo proliferation of human CD4(+) and CD8(+) T cells compared with stiffer substrates (E>> 2 MPa). Mixed peripheral blood T cells cultured on the stiffer substrates also demonstrate a trend (nonsignificant) toward a greater proportion of CD62L(neg), effector-differentiated CD4(+) and CD8(+) T cells. Naive CD4(+) T cells expanded on softer substrates yield an average 3-fold greater proportion of IFN-γ-producing Th1-like cells. These results reveal that the rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter influencing T cell activation, proliferation, and Th differentiation. Substrate rigidity should therefore be a consideration in the development of T cell culture systems as well as when interpreting results of T cell activation based upon solid-phase immobilization of TCR/CD3 and CD28 ligands.

TGF-beta induces Foxp3 expression in stimulated T cells. These Foxp3 cells (induced regulatory T cells (iTreg)) share functional and therapeutic properties with thymic-derived Foxp3 regulatory T cells (natural regulatory T cells (nTreg)). We performed a single-cell analysis to better characterize the regulation of Foxp3 in iTreg in vitro and assess their dynamics after transfer in vivo. TGF-beta up-regulated Foxp3 in CD4(+)Foxp3 T cells only when added within a 2- to 3-day window of CD3/CD28 stimulation. Up to 90% conversion occurred, beginning after 1-2 days of treatment. Foxp3 expression strictly required TCR stimulation but not costimulation and was independent of cell cycling. Removal of TGF-beta led to a loss of Foxp3 expression after an approximately 4-day lag. Most iTreg transferred into wild-type mice down-regulated Foxp3 within 2 days, and these Foxp3 cells were concentrated in the blood, spleen, lung, and liver. Few of the Foxp3 cells were detected by 28 days after transfer. However, some Foxp3 cells persisted even to this late time point, and these preferentially localized to the lymph nodes and bone marrow. CXCR4 was preferentially expressed on Foxp3 iTreg within the bone marrow, and CD62L was preferentially expressed on those in the lymph nodes. Like transferred nTreg and in contrast with revertant Foxp3 cells, Foxp3 iTreg retained CD25 and glucocorticoid-induced TNFR family-related gene. Thus, Foxp3 expression in naïve-stimulated T cells is transient in vitro, dependent on TGF-beta activity within a highly restricted window after activation and continuous TGF-beta presence. In vivo, a subset of transferred iTreg persist long term, potentially providing a lasting source for regulatory activity after therapeutic administration.

The chromosomal locus encoding the early secreted antigenic target, 6 kDa (ESAT-6) secretion system 1 of Mycobacterium tuberculosis, also referred to as "region of difference 1 (RD1)," is absent from Mycobacterium bovis bacillus Calmette-Guerin (BCG). In this study, using low-dose aerosol infection in mice, we demonstrate that BCG complemented with RD1 (BCG::RD1) displays markedly increased virulence which albeit does not attain that of M. tuberculosis H37Rv. Nevertheless, phenotypic and functional analyses of immune cells at the site of infection show that the capacity of BCG::RD1 to initiate recruitment/activation of immune cells is comparable to that of fully virulent H37Rv. Indeed, in contrast to the parental BCG, BCG::RD1 mimics H37Rv and induces substantial influx of activated (CD44highCD45RB(-)CD62L(-)) or effector (CD45RB(-)CD27(-)) T cells and of activated CD11c(+)CD11bhigh cells to the lungs of aerosol-infected mice. For the first time, using in vivo analysis of transcriptome of inflammatory cytokines and chemokines of lung interstitial CD11c+ cells, we show that in a low-dose aerosol infection model, BCG::RD1 triggered an activation/inflammation program comparable to that induced by H37Rv while parental BCG, due to its overattenuation, did not initiate the activation program in lung interstitial CD11c+ cells. Thus, products encoded by the ESAT-6 secretion system 1 of M. tuberculosis profoundly modify the interaction between mycobacteria and the host innate and adaptive immune system. These modifications can explain the previously described improved protective capacity of BCG::RD1 vaccine candidate against M. tuberculosis challenge.

A CD4(+) T cell subpopulation defined by the expression levels of a particular cell surface molecule (e.g. CD5, CD45RB, CD25, CD62L or CD38) bears an autoimmune-preventive activity in various animal models. Here we show that the expression of CD25 is highly specific, when compared with other molecules, in delineating the autoimmune-preventive immunoregulatory CD4(+) T cell population. Furthermore, although CD25 is an activation marker for T cells, the following findings indicate that immunoregulatory CD25(+)CD4(+) T cells are functionally distinct from activated or anergy-induced T cells derived from CD25(-)CD4(+) T cells. First, the former are autoimmune-preventive in vivo, naturally unresponsive (anergic) to TCR stimulation in vitro and, upon TCR stimulation, able to suppress the activation/proliferation of other T cells, whereas the latter scarcely exhibit the in vivo autoimmune-preventive activity or the in vitro suppressive activity. Second, such activated or anergy-induced CD25(-) spleen cells produce various autoimmune diseases when transferred to syngeneic athymic nude mice, whereas similarly treated normal spleen cells, which include CD25(+)CD4(+) T cells, do not. Third, upon polyclonal T cell stimulation, CD25(+)CD4(+) T cells express CD25 at higher levels and more persistently than CD25(-)CD4(+) T cell-derived activated T cells; moreover, when the stimulation is ceased, the former revert to the original levels of CD25 expression, whereas the latter lose the expression. These results collectively indicate that naturally anergic and suppressive CD25(+)CD4(+) T cells present in normal naive mice are functionally and phenotypically stable, distinct from other T cells, and play a key role in maintaining immunologic self-tolerance.

BACKGROUND

Suboptimal CD4+ T cell recovery during antiretroviral therapy (ART) is a common clinical dilemma.

METHODS

We analyzed viral and immunologic predictors of CD4+ T cell recovery in 116 human immunodeficiency virus type 1 (HIV-1)-infected subjects who had suppressed viremia (< or = 50 copies/mL) while receiving ART. Successive measurements of T cell immunophenotypes and cellular HIV-1 DNA levels were obtained before and during receipt of ART. On the basis of increases in the CD4+ T cell count, subjects were classified as immunologically concordant (demonstrating an increase of>> or = 100 CD4+ T cells/mm3) or discordant (demonstrating an increase of <100 CD4+ T cells/mm3) after 48 weeks of ART.

RESULTS

In adjusted analyses, CD4+ and CD8+ T cell activation at baseline was negatively associated with immunologic concordance at week 48 of ART (odds ratio [OR], 0.80 [P = .04] and 0.67 [P = .02], respectively). High memory (CDRA(-)CD62L-) CD8+ T cell counts at baseline (OR, 0.33 [P = .05]) predicted less CD4+ T cell recovery, whereas increased naive CD4+ T cell counts were associated with higher increases in CD4+ T cells (OR, 1.19 [P = .052]). Neither the cell-associated HIV-1 DNA level at baseline (P = .32) nor the cell-associated HIV-1 DNA level at week 48 of ART (P = .42) was associated with immunologic concordance during ART.

CONCLUSIONS

These results support the potential clinical usefulness of the baseline determination of immune activation and maturation subsets in the prediction of CD4+ T cell recovery during viral suppression. Furthermore, identification of individuals with reduced potential for CD4+ T cell recovery during ART may provide a rationale for the initiation of early therapy for some patients.

Several studies have demonstrated roles for eosinophils during innate and adaptive immune responses to helminth infections. However, evidence that eosinophils are capable of initiating an immune response to parasite antigens is lacking. The goal of the present in vitro study was to investigate the potential of eosinophils to serve as antigen-presenting cells (APC) and initiate an immune response to parasite antigens. Purified eosinophils were exposed to soluble Strongyloides stercoralis antigens, and the expression of various surface markers involved in cell activation was examined. Antigen-exposed eosinophils showed a sixfold increase in expression levels of CD69 and major histocompatibility complex (MHC) class II, a fourfold increase in levels of T-cell costimulatory molecule CD86, and a twofold decrease in levels of CD62L compared to eosinophils cultured in medium containing granulocyte-macrophage colony-stimulating factor. The ability of eosinophils to present antigen to T cells was determined by culturing them with T cells in vitro. Eosinophils pulsed with antigen stimulated antigen-specific primed T cells and CD4+ T cells to increase interleukin-5 (IL-5) production. The blocking of MHC class II expression on eosinophils inhibited their ability to induce IL-5 production by CD4+ T cells in culture. Antigen-pulsed eosinophils were able to prime naïve T cells and CD4+ T cells in culture and polarized them into Th2 cells producing IL-5 similar to that induced by antigen-loaded dendritic cells. These results demonstrate that eosinophils are capable of activating antigen-specific Th2 cells inducing the release of cytokines and assist in the priming of naïve T cells to initiate Th2 responses against infection. This study highlights the potential of eosinophils to actively induce immune responses against infection by amplifying antigen-specific Th2-cell responses.

The bacille Calmette-Guérin vaccine provides very efficient protection in standard animal models of Mycobacterium tuberculosis challenge. We show in this article that although bacille Calmette-Guérin controlled M. tuberculosis growth for 7 wk of infection, the protection was gradually lost as the infection entered the chronic phase. The regrowth of M. tuberculosis coincided with an almost complete disappearance of IL-2-producing CD4 T cells. Booster vaccination with a subunit vaccine (Ag85B-ESAT-6+CAF01) expanded IL-2(+) CD4(+) T cell coexpressing either TNF-α or TNF-α/IFN-γ, and the maintenance of this population in the late stage of infection was associated with enhanced control of bacterial growth. The IL-2(+) CD4(+) T cell subsets were KLRG1(-) (nonterminally differentiated), were found to be CD62L(high), and further maintained a pronounced proliferative and cytokine-producing potential in the draining lymph nodes, when the animals were challenged 2 y postvaccination. These results suggest that the CD4(+) KLRG1(-) IL-2-secreting subsets are central memory T cells with the potential to continuously replenish the T cells at the site of infection and prevent attrition and functional exhaustion.

Chronic lymphocytic leukemia (CLL) cells are thought to have diminished cell-cycling capacity, a view challenged by their phenotypic resemblance to activated human B lymphocytes. The present study addresses the cell-cycling status of CLL cells, focusing on those leukemic cells expressing CD38, a molecule involved in signaling and activation that also serves as a prognostic marker in this disease. CD38(+) and CD38(-) members of individual CLL clones were analyzed for coexpression of molecules associated with cellular activation (CD27, CD62L, and CD69), cell-cycle entry (Ki-67), signaling (ZAP-70), and protection from apoptosis (telomerase and Bcl-2). Regardless of the size of the CD38(+) fraction within a CLL clone, CD38(+) subclones are markedly enriched for expression of Ki-67, ZAP-70, human telomerase reverse transcriptase, and telomerase activity. Although the percentage of cells (approximately 2%) entering the cell cycle as defined by Ki-67 expression is small, the absolute number within a clone can be sizeable and is contained primarily within the CD38(+) fraction. Despite these activation/proliferation differences, both CD38(+) and CD38(-) fractions have similar telomere lengths, suggesting that CD38 expression is dynamic and transient. These findings may help explain why high percentages of CD38(+) cells within clones are associated with poor clinical outcome.

Aging is usually accompanied by diminished immune protection upon infection or vaccination. Although aging results in well-characterized changes in the T cell compartment of long-lived, outbred, and pathogen-exposed organisms, their relevance for primary Ag responses remain unclear. Therefore, it remains unclear whether and to what extent the loss of naive T cells, their partial replacement by oligoclonal memory populations, and the consequent constriction of TCR repertoire limit the Ag responses in aging primates. We show in this study that aging rhesus monkeys (Macaca mulatta) exhibit poor CD8 T cell and B cell responses in the blood and poor CD8 responses in the lungs upon vaccination with the modified vaccinia strain Ankara. The function of APCs appeared to be maintained in aging monkeys, suggesting that the poor response was likely intrinsic to lymphocytes. We found that the loss of naive CD4 and CD8 T cells, and the appearance of persisting T cell clonal expansions predicted poor CD8 responses in individual monkeys. There was strong correlation between early CD8 responses in the transitory CD28+ CD62L- CD8+ T cell compartment and the peak Ab titers upon boost in individual animals, as well as a correlation of both parameters of immune response to the frequency of naive CD8+ T cells in old but not in adult monkeys. Therefore, our results argue that T cell repertoire constriction and naive cell loss have prognostic value for global immune function in aging primates.

Interstitial pneumonia (IP) is a severe organ manifestation of cytomegalovirus (CMV) disease in the immunocompromised host, in particular in recipients of bone marrow transplantation (BMT). Diagnostic criteria for the definition of CMV-IP include clinical evidence of pneumonia together with CMV detected in bronchoalveolar lavage or lung biopsy. We have used the model of syngeneic BMT and simultaneous infection of BALB/c mice with murine CMV for studying the pathogenesis of CMV-IP by controlled longitudinal analysis. A disseminated cytopathic infection of the lungs with fatal outcome was observed only when reconstituting CD8 T cells were depleted. Neither CD8 nor CD4 T cells mediated an immunopathogenesis of acute CMV-IP. By contrast, after efficient hematolymphopoietic reconstitution, viral replication in the lungs was moderate and focal. The histopathological picture was dominated by preferential infiltration of CD8 T cells confining viral replication to inflammatory foci. Notably, after clearance of acute infection, CD62L(lo) and CD62L(hi) subsets of CD44(+) memory CD8 T cells were found to persist in lung tissue. One can thus operationally distinguish an early CMV-positive IP (phase 1) and a late CMV-negative IP (phase 2). According to the definition, phase 2 histopathology would not be diagnosed as a CMV-IP and could instead be misinterpreted as a CMV-induced immunopathology. We document here that phase 1 as well as phase 2 pulmonary CD8 T cells are capable of exerting effector functions and are effectual in protecting against productive infection. We propose that antiviral "stand-by" memory-effector T cells persist in the lungs to prevent virus recurrence from latency.

Dendritic cells (DCs) comprise heterogeneous subsets of professional antigen-presenting cells, linking innate and adaptive immunity. Analysis of DC subsets has been hampered by a lack of specific DC markers and reliable quantitation assays. We characterised the immunophenotype and functional characteristics of psoriatic arthritis (PsA)-derived and rheumatoid arthritis (RA)-derived myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to evaluate their potential role in arthritis. Circulating peripheral blood (PB) pDC numbers were significantly reduced in PsA patients (P = 0.0098) and RA patients (P = 0.0194), and mDCs were significantly reduced in RA patients (P = 0.0086) compared with healthy controls. The number of circulating mDCs in RA PB was significantly inversely correlated to C-reactive protein (P = 0.021). The phenotype of both DC subsets in PsA PB and RA PB was immature as compared with healthy controls. Moreover, CD62L expression was significantly decreased on both mDCs (PsA, P = 0.0122; RA, P = 0.0371) and pDCs (PsA, P = 0.0373; RA, P = 0.0367) in PB. Both mDCs and pDCs were present in PsA synovial fluid (SF) and RA SF, with the mDC:pDC ratio significantly exceeding that in matched PB (PsA SF, P = 0.0453; RA SF, P = 0.0082). pDCs isolated from RA SF and PsA SF displayed an immature phenotype comparable with PB pDCs. RA and PsA SF mDCs, however, displayed a more mature phenotype (increased expression of CD80, CD83 and CD86) compared with PB mDCs. Functional analysis revealed that both SF DC subsets matured following toll-like receptor stimulation. pDCs from PB and SF produced interferon alpha and tumour necrosis factor alpha on TLR9 stimulation, but only SF pDCs produced IL-10. Similarly, mDCs from PB and SF produced similar tumour necrosis factor alpha levels to TLR2 agonism, whereas SF mDCs produced more IL-10 than PB controls. Circulating DC subset numbers are reduced in RA PB and PsA PB with reduced CD62L expression. Maturation is incomplete in the inflamed synovial compartment. Immature DCs in SF may contribute to the perpetuation of inflammation via sampling of the inflamed synovial environment, and in situ presentation of arthritogenic antigen.

CD103 (alphaEbeta7) has been shown to be an excellent marker for identifying in vivo-activated FoxP3(+)CD4(+) regulatory T (Treg) cells. It is unknown whether reinfusion of in vivo-activated donor-type CD103(+) Treg cells from recipient can ameliorate ongoing chronic graft-versus-host disease (GVHD). Here, we showed that, in a chronic GVHD model of DBA/2 (H-2(d)) donor to BALB/c (H-2(d)) recipient, donor-type CD103(+) Treg cells from recipients were much more potent than CD25(hi) natural Treg cells from donors in reversing clinical signs of GVHD and tissue damage. Furthermore, in contrast to CD25(hi) natural Treg cells, CD103(+) Treg cells expressed high levels of CCR5 but low levels of CD62L and directly migrated to GVHD target tissues. In addition, the CD103(+) Treg cells strongly suppressed donor CD4(+) T-cell proliferation; they also induced apoptosis of in vivo-activated CD4(+) T and B cells and significantly reduced pathogenic T and B cells in GVHD target tissues. These results indicate that CD103(+) Treg cells from chronic GVHD recipients are functional, and reinfusion of the CD103(+) Treg cells can shift the balance between Treg cells and pathogenic T cells in chronic GVHD recipients and ameliorate ongoing disease.