Activation pathways implicate anti-HLA-DP and anti-LFA-1 antibodies as lead candidates for intervention in chronic berylliosis

CD4(+) T cells play a key role in granulomatous inflammation in the lung of patients with chronic beryllium disease. The goal of this study was to characterize activation pathways of beryllium-responsive bronchoalveolar lavage (BAL) CD4(+) T cells from chronic beryllium disease patients to identify possible therapeutic interventional strategies. Our results demonstrate that in the presence of APCs, beryllium induced strong proliferation responses of BAL CD4(+) T cells, production of superoptimal concentrations of secreted proinflammatory cytokines, IFN-gamma, TNF-alpha,and IL-2, and up-regulation of numerous T cell surface markers that would promote T-T Ag presentation. Ab blocking experiments revealed that anti-HLA-DP or anti-LFA-1 Ab strongly reduced proliferation responses and cytokine secretion by BAL CD4(+) T cells. In contrast, anti-HLA-DR or anti-OX40 ligand Ab mainly affected beryllium-induced proliferation responses with little impact on cytokines other than IL-2, thus implying that nonproliferating BAL CD4(+) T cells may still contribute to inflammation. Blockade with CTLA4-Ig had a minimal effect on proliferation and cytokine responses, confirming that activation was independent of B7/CD28 costimulation. These results indicate a prominent role for HLA-DP and LFA-1 in BAL CD4(+) T cell activation and further suggest that specific Abs to these molecules could serve as a possible therapy for chronic beryllium disease.     

Up-regulation of programmed death-1 expression on beryllium-specific CD4+ T cells in chronic beryllium disease

Chronic beryllium disease (CBD) is caused by workplace exposure to beryllium and is characterized by the accumulation of memory CD4+ T cells in the lung. These cells respond vigorously to beryllium salts in culture by producing proinflammatory Th1-type cytokines. The presence of these inflammatory cytokines leads to the recruitment of alveolar macrophages, alveolitis, and subsequent granuloma development. It has been shown that chronic exposure to conventional Ags leads to up-regulation in the expression of negative regulators of T cells such as programmed death-1 (PD-1). Due to the persistence of beryllium in the lung after the cessation of exposure, aberrant regulation of the PD-1 pathway may play an important role in CBD development. In the present study, PD-1 expression was measured on blood and bronchoalveolar lavage (BAL) CD4+ T cells from beryllium-sensitized and CBD subjects. PD-1 expression was significantly higher on BAL CD4+ T cells compared with those cells in blood, with the highest expression on the beryllium-specific T cell subset. In addition, the expression of PD-1 on BAL CD4+ T cells directly correlated with the severity of the T cell alveolitis. Increased expression of the PD-1 ligands, PD-L1 and PD-L2, on BAL CD14+ cells compared with blood was also seen. The addition of anti-PD-1 ligand mAbs augmented beryllium-induced CD4+ T cell proliferation, and an inverse correlation was seen between PD-1 expression on beryllium-specific CD4+ T cells and beryllium-induced proliferation. Thus, the PD-1 pathway is active in beryllium-induced disease and plays a key role in controlling beryllium-induced T cell proliferation.     

4-1BB costimulation promotes human T cell adhesion to fibronectin

CD28 and 4-1BB (CD137) are costimulatory molecules for T cells. In this study we investigated the role of 4-1BB in T cell adhesion to fibronectin (FN). Unlike CD28, 4-1BB is present in only a small subset of T cells prepared from fresh human peripheral blood mononuclear cells, but was induced after prolonged TCR/CD28 activation in vitro. 4-1BB-expressing T cells were characteristically unique in their strong responsiveness to FN. Anti-4-1BB cross-linking synergized CD28 costimulation by lowering the threshold of CD3 signal required for CD28-mediated maximal proliferative response. In addition to increasing proliferative responses, 4-1BB promoted T cell adhesion to FN in the presence of CD28 costimulation. 4-1BB-mediated cell adhesion to FN was blocked by anti-beta1 integrin, suggesting that 4-1BB mediates beta1 integrin activation. The role of 4-1BB in inducing CD4(+) T cell adhesion to FN was confirmed by showing that the human leukemic CD4(+) T cell line, Jurkat, when transfected with cDNA encoding 4-1BB, became adherent to FN with anti-4-1BB stimulation. Taken together, our results suggest that 4-1BB-promoted T cell adhesion to extracellular matrix proteins is an important postactivation process for T cell migration.     

Differential expression and costimulatory effect of 4-1BB (CD137) and CD28 molecules on cytokine-induced murine CD8(+) Tc1 and Tc2 cells

In this study we report that the relative expression of 4-1BB (CD137) and CD28 molecules can differentially be modulated on CD8(+) T cells by combinations of various cytokines and anti-cytokine antibodies. During allostimulation of naive CD8(+) T cells in the presence of IL-2, IFN-gamma, IL-12, and anti-IL-4, they evolved into IL-2, IFN-gamma-producing Tc1 cells and showed inability to upregulate 4-1BB expression but not CD28. On the other hand, the Tc2 cells, generated in the presence of allogeneic APCs, IL-2, IL-10, IL-4, and anti-IFN-gamma, demonstrated intact and elevated 4-1BB and CD28 molecules. Activation of Tc1 and Tc2 cells with anti-CD3 and plate-bound anti-4-1BB and anti-CD28 mAbs revealed differential proliferative and cytokine secretory patterns. The 4-1BB signaling in the context of anti-CD3 as first signal led to the increased secretion of IL-4 by the Tc2 cells and not by Tc1 cells, while CD28 triggering produced IL-4 from Tc2 and IL-2 and IFN-gamma from Tc1 cells. Flow cytometric analysis of cell surface expression on Tc1 and Tc2 cells strengthened our observation that 4-1BB expression but not CD28 is poorly expressed on Tc1 cells. Both of the polarized CD8(+) T cell subsets exhibited comparable cytotoxic abilities and perforin and granzyme expression. The regeneration of 4-1BB expression is possible on Tc1 cells when back cultured in a Tc2 cytokine environment, but its expression could not be significantly altered on the Tc2 population unless IL-12 was included in the system.     

Homeostatic expansion occurs independently of costimulatory signals.

Naive T cells undergo homeostatic proliferation in lymphopenic mice, a process that involves TCR recognition of specific self peptide/MHC complexes. Since costimulation signals regulate the T cell response to foreign Ags, we asked whether they also regulate homeostatic expansion. We report in this study that homeostatic expansion of CD4 and CD8 T cells occurs independently of costimulation signals mediated through CD28/B7, CD40L/CD40, or 4-1BB/4-1BBL interactions. Using DO11.10 TCR transgenic T cells, we confirmed that CD28 expression was dispensable for homeostatic expansion, and showed that the presence of endogenous CD4(+)CD25(+) regulatory cells did not detectably influence homeostatic expansion. The implications of these findings with respect to regulation of T cell homeostasis and autoimmunity are discussed.     

Beryllium skin patch testing to analyze T cell stimulation and granulomatous inflammation in the lung

Chronic beryllium disease (CBD) is characterized by granulomatous inflammation and the accumulation of CD4(+) T cells in the lung. Patch testing of CBD patients with beryllium sulfate results in granulomatous inflammation in the skin. We investigated whether the T cell clonal populations present in the lung of CBD patients would also be present in the involved skin of a positive beryllium patch test and thus mirror the granulomatous process in the lung. CBD patients with clonal TCR expansions in bronchoalveolar lavage (BAL) were selected for study. All three CBD patients studied had a positive response to beryllium sulfate application and a negative patch test to normal saline. Immunohistochemistry showed extensive infiltration with CD4(+) T cells and few, if any, CD8(+) T cells both at 3 days and at later times when granulomas were apparent. T cell infiltration early after skin testing appeared to be nonspecific with the TCR repertoire of infiltrating T cells being distinct from that present in BAL. At later times when granulomas were present, T cell clones in skin overlapped with those in BAL in all patients tested. Total TCR matches in skin and BAL were as high as 40% in selected Vbeta T cell subsets. Studies of peripheral blood T cells before and after patch testing provided evidence for mobilization of large numbers of pathogenic beryllium-reactive T cells into the circulating pool. These studies using skin patch testing provide new insight into the dynamics of T cell influx and mobilization during granulomatous inflammation.     

Genetic characterization of strain differences in the ability to mediate CD40/CD28-independent rejection of skin allografts

Simultaneous blockade of the CD40 and CD28 T cell costimulatory pathways effectively promotes skin allograft survival in C3H/HeJ mice, extending median survival times (MSTs) beyond 100 days. This strategy is markedly less effective in C57BL/6 mice, with MSTs ranging between 20 and 30 days. In this study, we investigate the underlying genetic causes of these distinct phenotypes. Using H-2 congenic mice, we show that the genetic basis for the varied responses between these two strains is independent of the H-2 locus and T cell precursor frequency. C57BL/6 mice treated with costimulation blockade are able to generate allospecific CTL- and IFN-gamma-producing T cells within 3-4 wk posttransplant, whereas mice with a C3H background generate neither CTL- nor IFN-gamma-producing cells. Thus, differences appear to be in the generation of the immune response and not T cell homing. Strain differences in costimulation blockade-induced hyporesponsiveness persist in the absence of CD4(+) T cells, implying a direct effect on CD8(+) T cells. We demonstrate that genetic differences are important in cells of hemopoietic origin and that the costimulation blockade-resistant phenotype is dominant. Analysis of BXH recombinant inbred strains indicates that multiple loci contribute to the phenotype, and that the blockade resistance loci are preliminarily linked to 17 markers on four chromosomes. We conclude that strain variation in allograft MSTs following CD40/CD28 blockade results from the ability of CD8(+) T cells in some strains to use alternative modes of costimulation to mount an effective alloresponse.     

Ligation of 4-1BB (CDw137) regulates graft-versus-host disease, graft-versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients

4-1BB is expressed on activated CD4(+) and CD8(+) T cells; its ligand, 4-1BB ligand is expressed on APCs. Despite expression on both T cell subpopulations, 4-1BB has been reported to predominantly affect CD8(+) T cell responses. By quantifying graft-vs-host disease alloresponses in vivo, we demonstrate that both CD4(+) and CD8(+) T cell-mediated alloresponses are regulated by 4-1BB/4-1BB ligand interactions to approximately the same extent. 4-1BB receptor-facilitated CD4(+) T cell-mediated alloresponses were partly CD28 independent. In two distinct marrow graft rejection systems, host CD8(+) and CD4(+) T cells each separately contributed to host anti-donor T cell-mediated allograft rejection. alpha 4-1BB mAb increased the graft-vs-leukemia effect of a suboptimal number of donor splenocytes given later post bone marrow transplantation by bolstering allogeneic responses resulting in leukemia elimination. In summary, 4-1BB ligation is a potent regulator of CD4(+) and CD8(+) T cell-mediated allogeneic responses in vivo. Modifying the ligation of 4-1BB represents a new approach to altering the graft-vs-host disease and graft-vs-leukemia effects of allogeneic T cells post bone marrow transplantation.     

4-1BB is superior to CD28 costimulation for generating CD8+ cytotoxic lymphocytes for adoptive immunotherapy

Artificial APCs (aAPCs) genetically modified to express selective costimulatory molecules provide a reproducible, cost-effective, and convenient method for polyclonal and Ag-specific expansion of human T cells for adoptive immunotherapy. Among the variety of aAPCs that have been studied, acellular beads expressing anti-CD3/anti-CD28 efficiently expand CD4+ cells, but not CD8+ T cells. Cell-based aAPCs can effectively expand cytolytic CD8+ cells, but optimal costimulatory signals have not been defined. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently up-regulated by all CD8+ T cells following activation. We compared expansion of human cytolytic CD8+ T cells using cell-based aAPCs providing costimulation via 4-1BB vs CD28. Whereas anti-CD3/anti-CD28 aAPCs mostly expand naive cells, anti-CD3/4-1BBL aAPCs preferentially expand memory cells, resulting in superior enrichment of Ag-reactive T cells which recognize previously primed Ags and efficient expansion of electronically sorted CD8+ populations reactive toward viral or self-Ags. Using HLA-A2-Fc fusion proteins linked to 4-1BBL aAPCs, 3-log expansion of Ag-specific CD8+ CTL was induced over 14 days, whereas similar Ag-specific CD8+ T cell expansion did not occur using HLA-A2-Fc/anti-CD28 aAPCs. Furthermore, when compared with cytolytic T cells expanded using CD28 costimulation, CTL expanded using 4-1BB costimulation mediate enhanced cytolytic capacity due, in part, to NKG2D up-regulation. These results demonstrate that 4-1BB costimulation is essential for expanding memory CD8+ T cells ex vivo and is superior to CD28 costimulation for generating Ag-specific products for adoptive cell therapy.     

Cooperation between 4-1BB and ICOS in the immune response to influenza virus revealed by studies of CD28/ICOS-deficient mice

CD28, ICOS, and 4-1BB each play distinct roles in the CD8 T cell response to influenza virus. CD28-/- mice are severely impaired in primary CD8 T cell expansion and fail to mount a secondary response to influenza. Influenza-specific CD8 T cells expand normally in ICOS-/- mice, with only a small and transient defect late in the primary response and an unimpaired secondary response. Conversely, 4-1BB/4-1BBL interaction is dispensable for the primary CD8 T cell response to influenza, but maintains CD8 T cell survival and controls the size of the secondary response. Previous results showed that a single dose of agonistic anti-4-1BB Ab at priming allowed partial restoration of primary CD8 T cell expansion and full recovery of the secondary CD8 T cell responses to influenza in CD28-/- mice. In this study we show that anti-4-1BB fails to correct the CD8 T cell defect in CD28-/-ICOS-/- mice, suggesting that ICOS partially compensates for CD28 in this model. In support of this hypothesis, we found that anti-4-1BB enhances ICOS expression on both T cell subsets and that anti-4-1BB and anti-ICOS can synergistically activate CD4 and CD8 T cells. Furthermore, ICOS and 4-1BB can cooperate to directly stimulate isolated CD28-/- CD8 T cells. These results reveal a novel interaction between the ICOS and 4-1BB costimulatory pathways as well as unexpected redundancy between CD28 and ICOS in primary CD8 T cell expansion. These findings have implications for costimulation of human T cell responses in diseases such as AIDS or rheumatoid arthritis, in which CD28- T cells accumulate.     

The 4-1BB costimulation augments the proliferation of CD4+CD25+ regulatory T cells

The thymus-derived CD4(+)CD25(+) T cells belong to a subset of regulatory T cells potentially capable of suppressing the proliferation of pathogenic effector T cells. Intriguingly, these suppressor cells are themselves anergic, proliferating poorly to mitogenic stimulation in culture. In this study, we find that the 4-1BB costimulator receptor, best known for promoting the proliferation and survival of CD8(+) T cells, also induces the proliferation of the CD4(+)CD25(+) regulatory T cells both in culture and in vivo. The proliferating CD4(+)CD25(+) T cells produce no detectable IL-2, suggesting that 4-1BB costimulation of these cells does not involve IL-2 production. The 4-1BB-expanded CD4(+)CD25(+) T cells are functional, as they remain suppressive to other T cells in coculture. These results support the notion that the peripheral expansion of the CD4(+)CD25(+) T cells is controlled in part by costimulation.     

Polarized type 1 cytokine profile in bronchoalveolar lavage T cells of patients with hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is characterized by an inflammatory lymphocytic alveolitis comprised of both CD8+ and CD4+ T cells. Animal models suggest that HP is facilitated by overproduction of IFN-gamma, and that IL-10 ameliorates severity of the disease, indicating a Th1-type response. To determine whether a Th1 phenotype in HP also exists clinically, bronchoalveolar lavage (BAL) and peripheral blood (PB) T cells were obtained from HP individuals and analyzed for Th1 vs Th2 cytokine profiles. It was determined that soluble OKT3-stimulated BAL T cells cocultured with alveolar macrophages produced more IFN-gamma and less IL-10 than PB T cells cocultured with monocytes, but no difference was observed in IL-4 production. The monocytic cells did not account for this difference, as CD80 and CD86 expressions were similar, and coculturing PB T cells with alveolar macrophages resulted in no difference in IFN-gamma production. Similarly, there was no difference in IL-12 production between stimulated BAL or PB T cells; however, addition of rIL-12 significantly increased production of IFN-gamma by BAL T cells, but not by PB T cells. This effect was due to a difference in IL-12R expression. High affinity IL-12R were only present in association with BAL T cells. These studies indicate that clinical HP is characterized by a predominance of IFN-gamma-producing T cells, perhaps resulting from a reduction in IL-10 production and an increase in high affinity IL-12R compared with blood T cells.     

Role of TNF receptor-associated factor 2 and p38 mitogen-activated protein kinase activation during 4-1BB-dependent immune response

4-1BB is a costimulatory member of the TNFR family, expressed on activated CD4(+) and CD8(+) T cells. Previous results showed that 4-1BB-mediated T cell costimulation is CD28-independent and involves recruitment of TNFR-associated factor 2 (TRAF2) and activation of the stress-activated protein kinase cascade. Here we describe a role for the p38 mitogen-activated protein kinase (MAPK) pathway in 4-1BB signaling. Aggregation of 4-1BB alone induces p38 activation in a T cell hybridoma, whereas, in normal T cells, p38 MAPK is activated synergistically by immobilized anti-CD3 plus immobilized 4-1BB ligand. 4-1BB-induced p38 MAPK activation is inhibited by the p38-specific inhibitor SB203580 in both a T cell hybridoma and in murine T cells. T cells from TRAF2 dominant-negative mice are impaired in 4-1BB-mediated p38 MAPK activation. A link between TRAF2 and the p38 cascade is provided by the MAPK kinase kinase, apoptosis-signal-regulating kinase 1. A T cell hybrid transfected with a kinase-dead apoptosis-signal-regulating kinase 1 fails to activate p38 MAPK in response to 4-1BB signaling. To assess the role of p38 activation in an immune response, T cells were stimulated in an MLR in the presence of SB203580. In a primary MLR, SB203580 blocked IL-2, IFN-gamma, and IL-4 secretion whether the costimulatory signal was delivered via 4-1BB or CD28. In contrast, following differentiation into Th1 or Th2 cells, p38 inhibition blocked IL-2 and IFN-gamma without affecting IL-4 secretion. Nevertheless, IL-4 secretion by Th2 cells remained costimulation-dependent. Thus, critical T cell signaling events diverge following Th1 vs Th2 differentiation.     

A novel costimulation pathway via the 4C8 antigen for the induction of CD4+ regulatory T cells

CD4(+)CD25(+) regulatory T (Treg) cells naturally occur in mice and humans, and similar Treg cells can be induced in vivo and in vitro. However, the molecular mechanisms that mediate the generation of these Treg cell populations remain unknown. We previously described anti-4C8 mAbs that inhibit the postadhesive transendothelial migration of T cells through human endothelial cell monolayers. We demonstrate in this work that Treg cells are induced by costimulation of CD4(+) T cells with anti-CD3 plus anti-4C8. The costimulation induced full activation of CD4(+) T cells with high levels of IL-2 production and cellular expansion that were comparable to those obtained on costimulation by CD28. However, upon restimulation, 4C8-costimulated cells produced high levels of IL-10 but no IL-2 or IL-4, and maintained high expression levels of CD25 and intracellular CD152, as compared to CD28-costimulated cells. The former cells showed hyporesponsiveness to anti-CD3 stimulation and suppressed the activation of bystander T cells depending on cell contact but not IL-10 or TGF-beta. The suppressor cells developed from CD4(+)CD25(-)CD45RO(+) cells. The results suggest that 4C8 costimulation induces the generation of Treg cells that share phenotypic and functional features with CD4(+)CD25(+) T cells, and that CD25(-) memory T cells may differentiate into certain Treg cell subsets in the periphery.     

IL-12 augments CD8+ T cell development for contact hypersensitivity responses and circumvents anti-CD154 antibody-mediated inhibition.

During sensitization with dinitrofluorobenzene for contact hypersensitivity (CHS) responses, hapten-specific CD8(+) T cells develop into IFN-gamma-producing cells, and CD4(+) T cells develop into IL-4/IL-5-producing cells. Administration of IL-12 during sensitization skews CD4(+) T cell development to IFN-gamma-producing cells, resulting in exaggerated CHS responses. In the current report we tested the role of IL-12 on CD8(+) T cell development during sensitization and elicitation of CHS to dinitrofluorobenzene. Administration of IL-12 during hapten sensitization induced the expression of IL-12Rbeta2 on both CD4(+) and CD8(+) T cells, augmented IFN-gamma production by these T cell populations, and increased the magnitude and duration of the CHS response to hapten challenge. CHS responses were virtually identical in wild-type and IL-12 p40(-/-) mice. Since engagement of CD40 on APC may stimulate IL-12 production, we also tested the role of CD40-CD154 interactions on the development of IFN-gamma-producing CD4(+) and CD8(+) T cells following hapten sensitization. Development of IFN-gamma-producing CD4(+) T cells during hapten sensitization was absent in wild-type mice treated with anti-CD154 mAb or in CD154(-/-) mice. In contrast, the absence of CD40-CD154 signaling had little or no impact on the development of IFN-gamma-producing CD8(+) T cells. These results demonstrate that the development of hapten-specific Th1 effector CD4(+) T cells in CHS requires both CD40-CD154 interactions and IL-12, whereas the development of IFN-gamma-producing effector CD8(+) T cells can occur independently of these pathways.     

In vivo triggering through 4-1BB enables Th-independent priming of CTL in the presence of an intact CD28 costimulatory pathway

Triggering of 4-1BB, a member of the TNFR family, through in vivo administration of agonistic anti-4-1BB Ab delivers a powerful costimulatory signal to CTL. We found this signal to effectively replace the need for CD4(+) T cell help in the cross-priming of tumor-specific CTL immunity. Furthermore, 4-1BB Ab can convert an otherwise tolerogenic peptide vaccine into a formulation capable of efficient CTL priming. Initial activation of naive CTL can occur in the absence of 4-1BB costimulation, but this signal permits increased survival of Ag-stimulated CTL. Because naive CTL do not express 4-1BB at their surface, susceptibility to 4-1BB triggering depends on prior up-regulation of this receptor. We show that this requires both stimulation of the TCR and CD28-dependent costimulation. Accordingly, blockade of the CD28-costimulatory pathway abrogates the capacity of agonistic anti-4-1BB Ab to trigger Th-independent CTL immunity. In conclusion, our data reveal that the 4-1BB-mediated survival signal is positioned downstream of Ag-specific TCR triggering and CD28-dependent costimulation of naive CTL. The powerful effects of 4-1BB triggering on the induction, amplification, and persistence of CTL responses provide a novel strategy for increasing the potency of vaccines against cancers.     

Recombinant HLA-DP2 binds beryllium and tolerizes beryllium-specific pathogenic CD4+ T cells

Chronic beryllium disease is a lung disorder caused by beryllium exposure in the workplace and is characterized by granulomatous inflammation and the accumulation of beryllium-specific, HLA-DP2-restricted CD4+ T lymphocytes in the lung that proliferate and secrete Th1-type cytokines. To characterize the interaction among HLA-DP2, beryllium, and CD4+ T cells, we constructed rHLA-DP2 and rHLA-DP4 molecules consisting of the alpha-1 and beta-1 domains of the HLA-DP molecules genetically linked into single polypeptide chains. Peptide binding to rHLA-DP2 and rHLA-DP4 was consistent with previously published peptide-binding motifs for these MHC class II molecules, with peptide binding dominated by aromatic residues in the P1 pocket. 9Be nuclear magnetic resonance spectroscopy showed that beryllium binds to the HLA-DP2-derived molecule, with no binding to the HLA-DP4 molecule that differs from DP2 by four amino acid residues. Using beryllium-specific CD4+ T cell lines derived from the lungs of chronic beryllium disease patients, beryllium presentation to those cells was independent of Ag processing because fixed APCs were capable of presenting BeSO4 and inducing T cell proliferation. Exposure of beryllium-specific CD4+ T cells to BeSO4 -pulsed, plate-bound rHLA-DP2 molecules induced IFN-gamma secretion. In addition, pretreatment of beryllium-specific CD4+ T cells with BeSO4-pulsed, plate-bound HLA-DP2 blocked proliferation and IL-2 secretion upon re-exposure to beryllium presented by APCs. Thus, the rHLA-DP2 molecules described herein provide a template for engineering variants that retain the ability to tolerize pathogenic CD4+ T cells, but do so in the absence of the beryllium Ag.     

Contribution of antigen-primed CD8+ T cells to the development of airway hyperresponsiveness and inflammation is associated with IL-13

The role of Th2/CD4 T cells, which secrete IL-4, IL-5, and IL-13, in allergic disease is well established; however, the role of CD8(+) T cells (allergen-induced airway hyperresponsiveness (AHR) and inflammation) is less clear. This study was conducted to define the role of Ag-primed CD8(+) T cells in the development of these allergen-induced responses. CD8-deficient (CD8(-/-)) mice and wild-type mice were sensitized to OVA by i.p. injection and then challenged with OVA via the airways. Compared with wild-type mice, CD8(-/-) mice developed significantly lower airway responsiveness to inhaled methacholine and lung eosinophilia, and exhibited decreased IL-13 production both in vivo, in the bronchoalveolar lavage (BAL) fluid, and in vitro, following Ag stimulation of peribronchial lymph node (PBLN) cells in culture. Reconstitution of sensitized and challenged CD8(-/-) mice with allergen-sensitized CD8(+) T cells fully restored the development of AHR, BAL eosinophilia, and IL-13 levels in BAL and in culture supernatants from PBLN cells. In contrast, transfer of naive CD8(+) T cells or allergen-sensitized CD8(+) T cells from IL-13-deficient donor mice failed to do so. Intracellular cytokine staining of lung as well as PBLN T cells revealed that CD8(+) T cells were a source of IL-13. These data suggest that Ag-primed CD8(+) T cells are required for the full development of AHR and airway inflammation, which appears to be associated with IL-13 production from these primed T cells.     

CD30 expression identifies the predominant proliferating T lymphocyte population in human alloimmune responses

CD30 is an inducible member of the TNFR superfamily that is expressed on activated T and B cells and some lymphoid malignancies. We have previously shown that human CD30(+) T cells elicited with allogeneic APC are a major source of IFN-gamma and IL-5 production. In the present study we have used alloantigen, as well as anti-CD3 plus anti-CD28 mAb stimulation, to further characterize human CD30(+) T cells with respect to function and the expression of other activation-dependent cell surface molecules, including the related TNFR family members OX-40 and 4-1BB (CD137). Our results indicate that human CD30(+) T cells are a subset of activated T cells that also express CD25 and CD45RO. Moreover, we observed that allogeneic APC consistently induced a greater proportion of CD30(+) cells within the activated T cell population than did stimulation with plate-bound anti-CD3 plus anti-CD28 mAb or stimulation with soluble anti-CD3 plus anti-CD28 and autologous APC. The enhanced induction of CD30 expression by alloantigen was not common to other inducible TNFR family members because anti-CD3 plus anti-CD28 mAbs were far more effective in inducing expression of 4-1BB and OX-40. Furthermore, CD30 expression marked the predominant proliferating T cell population induced by alloantigen as determined by CFSE staining and flow cytometry. These results indicate that CD30, but not 4-1BB or OX-40, is preferentially induced by alloantigen, suggesting that CD30 may be important in human alloimmune responses.     

Ex vivo expansion of CD4+CD25+FoxP3+ T regulatory cells based on synergy between IL-2 and 4-1BB signaling

Naturally occurring CD4(+)CD25(+)FoxP3(+) T regulatory (Treg) cells require three distinct signals transduced via TCR, CD28, and IL-2R for their development and maintenance. These requirements served as the basis for several recently developed ex vivo expansion protocols that relied on the use of solid support-bound Abs to CD3 and CD28 in the presence of high dose IL-2. We report in this study that Treg cells up-regulate the expression of inducible costimulatory receptor 4-1BB in response to IL-2, and stimulation using this receptor via a novel form of 4-1BB ligand (4-1BBL) fused to a modified form of core streptavidin (SA-4-1BBL) was effective in expanding these cells up to 110-fold within 3 wk. Expanded cells up-regulated CD25, 4-1BB, and membranous TGF-beta, suppressed T cell proliferation, and prevented the rejection of allogeneic islets upon adoptive transfer into graft recipients. Importantly, SA-4-1BBL rendered CD4(+)CD25(-) T effector cells refractive to suppression by Treg cells. This dual function of signaling via 4-1BB, vis-à-vis Treg cell expansion and licensing T effector cells resistant to Treg cell suppression, as well as the up-regulation of 4-1BB by IL-2 may serve as important regulatory mechanisms for immune homeostasis following antigenic challenge. Stimulation using a soluble form of SA-4-1BBL represents a novel approach to expand Treg cells with potential therapeutic applications in autoimmunity and transplantation.