Expression and function of the B and T lymphocyte attenuator (BTLA/CD272) on human T cells

Co-signal receptors provide crucial activating or attenuating signals for T cells. The B and T lymphocyte attenuator (BTLA/CD272) is a third member of co-inhibitory receptors, which belongs to the CD28 immunoglobulin-superfamily. Using monoclonal antibodies (mAbs) against human BTLA, we show that BTLA is constitutively expressed on most CD4+ and CD8+ T cells and its expression progressively decreases upon T cell activation. Polarized Th1 and Th2 cells contained both BTLA-positive and BTLA-negative populations, but the extended culture diminished BTLA expression. Cross-linking BTLA with an agonistic mAb inhibited T cell proliferation and the production of the cytokines IFN-gamma and IL-10 in response to anti-CD3 stimulation. BTLA-mediated inhibition of T cell activation occurred during both primary CD4+ T cell responses and secondary CD4+ and CD8+ T cell responses, suggesting that BTLA ligation sends a constitutive "off" signal to T cells and thus might play an important role in the maintenance of T cell tolerance.     

CD4-mediated signals induce T cell dysfunction in vivo.

Triggering of CD4 coreceptors on both human and murine T cells can suppress TCR/CD3-induced secretion of IL-2. We show here that pretreatment of murine CD4+ T cells with the CD4-specific mAb YTS177 inhibits the CD3-mediated activation of the IL-2 promoter factors NF-AT and AP-1. Ligation of CD4 molecules on T cells leads to a transient stimulation of extracellular signal-regulated kinase (Erk) 2, but not c-Jun N-terminal kinase (JNK) activity. Pretreatment with anti-CD4 mAb impaired anti-CD3-induced Erk2 activation. Costimulation with anti-CD28 overcame the inhibitory effect of anti-CD4 Abs, by induction of JNK activation. The in vivo relevance of these studies was demonstrated by the observation that CD4+ T cells from BALB/c mice injected with nondepleting anti-CD4 mAb were inhibited in their ability to respond to OVA Ag-induced proliferation and IL-2 secretion. Interestingly, in vivo stimulation with anti-CD28 mAb restored IL-2 secretion. Furthermore, animals pretreated with anti-CD4 elicited enhanced IL-4 secretion induced by OVA and CD28. These observations suggest that CD4-specific Abs can inhibit T cell activation by interfering with signal 1 transduced through the TCR, but potentiate those delivered through the costimulatory molecule CD28. These studies have relevance to understanding the mechanism of tolerance induced by nondepleting anti-CD4 mAb used in animal models for allograft studies, autoimmune pathologies, and for immunosuppressive therapies in humans.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells

The glucocorticoid-induced TNF receptor (GITR), which is a member of the TNF receptor family, is expressed preferentially at high levels on CD25+CD4+ regulatory T cells and plays a key role in the peripheral tolerance that is mediated by these cells. GITR is also expressed on conventional CD4+ and CD8+ T cells, and its expression is enhanced rapidly after activation. In this report we show that the GITR provides a potent costimulatory signal to both CD25+ and CD25- CD4+ T cells. GITR-mediated stimulation induced by anti-GITR mAb DTA-1 or GITR ligand transfectants efficiently augmented the proliferation of both CD25-CD4+ and CD25+CD4+ T cells under the limited dose of anti-CD3 stimulation. The augmentation of T cell activation was further confirmed by the enhanced cell cycle progression; early induction of the activation Ags, CD69 and CD25; cytokine production, such as IL-2, IFN-gamma, IL-4, and IL-10; anti-CD3-induced redirected cytotoxicity; and intracellular signaling, assessed by translocation of NF-kappaB components. GITR costimulation showed a potent ability to produce high amounts of IL-10, which resulted in counter-regulation of the enhanced proliferative responses. Our results highlight evidence that GITR acts as a potent and unique costimulator for an early CD4+ T cell activation.     

Human T cell activation by OKT3 is inhibited by a monoclonal antibody to CD44.

The CD44 molecule, also known as Hermes lymphocyte homing receptor, human Pgp-1, and extracellular matrix receptor III, has been shown to play a role in T cell adhesion and activation. Specifically, anti-CD44 mAb block binding of lymphocytes to high endothelial venules, inhibit T cell-E rosetting, and augment T cell proliferation induced by the CD2 or CD3-TCR pathways. We have characterized an anti-CD44 mAb (212.3) which immunoprecipitates a 90-kDa protein and is specific for CD44 as shown by peptide mapping and antibody competition studies. Interestingly, our studies with 212.3 demonstrate that this CD44-specific mAb completely inhibits T cell proliferation stimulated by the anti-CD3 mAb, OKT3. Inhibition is not a result of reduced cell viability, but is associated with 1) inhibition of IL-2 production, 2) inhibition of IL-2R expression, and 3) inhibition of OKT3-mediated increases in intracellular Ca2+ levels. In addition, 212.3 does not inhibit proliferation by the T cell mitogens PHA or PWM nor does it inhibit proliferation in a mixed lymphocyte reaction. Similar to other anti-CD44 mAb, 212.3 also augments T cell proliferation induced by mAb directed against the T11(2) and T11(3) epitopes of CD2. Thus, these studies describe a novel CD44-specific mAb (212.3) that inhibits T cell activation by OKT3 by blocking early signal transduction. Furthermore, these studies suggest that "receptor cross-talk" between the CD3-TCR complex and CD44 may regulate T cell activation.     

Cutting edge: stromal cell-derived factor-1 is a costimulator for CD4+ T cell activation

Stromal cell-derived factor (SDF)-1 is a chemoattractant for T cells, precursor B cells, monocytes, and neutrophils. SDF-1alpha was also found to up-regulate expression of early activation markers (CD69, CD25, and CD154) by anti-CD3-activated CD4+ T cells. In addition, SDF-1alpha costimulated proliferation of CD4+ T cells and production of IL-2, IFN-gamma, IL-4, and IL-10. Stimulation with SDF-1alpha alone did not induce activation marker expression, proliferation, or cytokine production by the CD4+ T cells. SDF-1alpha-mediated costimulation was blocked by anti-CXC chemokine receptor-4 mAb. RANTES also increased activation marker expression by anti-CD3-stimulated peripheral CD4+ T cells, but less effectively than SDF-1alpha did, and did not up-regulate IL-2 production and proliferation. These results indicate that SDF-1 and CXC chemokine receptor-4 interactions not only play a role in T cell migration but also provide potent costimulatory signals to Ag-stimulated T cells.     

The role of CD11a/CD18-CD54 interactions in human T cell-dependent B cell activation.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in human T cell and B cell collaboration was examined by studying the effect of mAb to these determinants on B cell proliferation and differentiation stimulated by culturing resting B cells with CD4+ T cells activated with immobilized mAb to the CD3 molecular complex. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) inhibited B cell responses significantly. The mAb did not directly inhibit B cell function, inasmuch as T cell-independent activation induced by formalinized Staphylococcus aureus and IL-2 was not suppressed. Moreover, DNA synthesis and IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells were not suppressed by the mAb to LFA-1 or ICAM-1. Although the mAb to LFA-1 inhibited enhancement of IL-2 production by co-culture of immobilized anti-CD3-stimulated CD4+ T cells with B cells, addition of exogenous IL-2 or supernatants of mitogen-activated T cells could not abrogate the inhibitory effects of the mAb to LFA-1 or ICAM-1 on B cell responses. Inhibition was most marked when the mAb were present during the initial 24 h in culture. Immobilized anti-CD3-stimulated LFA-1-negative CD4+ T cell clones from a child with leukocyte adhesion deficiency could induce B cell responses, which were inhibited by mAb to LFA-1 or ICAM-1. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the collaboration between activated CD4+ T cells and B cells necessary for the induction of B cell proliferation and differentiation, and for enhancement of IL-2 production by CD4+ T cells. Moreover, the data are consistent with a model of T cell-B cell collaboration in which interactions between LFA-1 on resting B cells and ICAM-1 on activated CD4+ T cells play a critical role in initial T cell-dependent B cell activation.     

CD63 as an activation-linked T cell costimulatory element

Dendritic cells (DC) are unique in their capacity to either stimulate or regulate T cells, and receptor/ligand pairs on DC and T cells are critically involved in this process. In this study we present such a molecule, which was discovered by us when analyzing the functional effects of an anti-DC mAb. This mAb, 11C9, reacted strongly with DC, but only minimally with lymphocytes. In MLR it constantly reduced DC-induced T cell activation. Therefore, we assumed that mAb 11C9 primarily exerts its functions by binding to a DC-structure. This does not seem to be the case, however. Preincubation of DC with mAb 11C9 before adding T cells had no inhibitory effect on T cell responses. Retroviral expression cloning identified the 11C9 Ag as CD63. This lysosomal-associated membrane protein (LAMP-3), is only minimally expressed on resting T cells but can, as we show, quickly shift to the surface upon stimulation. Cross-linkage of that structure together with TCR-triggering induces strong T cell activation. CD63 on T cells thus represents an alternative target for mAb 11C9 with its binding to activated T cells rather than DC being responsible for the observed functional effects. This efficient CD63-mediated costimulation of T cells is characterized by pronounced induction of proliferation, strong IL-2 production and compared with CD28 enhanced T cell responsiveness to restimulation. Particularly in this latter quality CD63 clearly surpasses several other CD28-independent costimulatory pathways previously described. CD63 thus represents an activation-induced reinforcing element, whose triggering promotes sustained and efficient T cell activation and expansion.     

Endothelial cell lymphocyte function-associated antigen-3 and an unidentified ligand act in concert to provide costimulation to human peripheral blood CD4+ T cells.

Our previous studies have demonstrated that cultured human endothelial cells (EC) provide costimulation to PHA-activated CD4+ T cells, measured as augmentation of IL-2 synthesis, through a cell contact-department pathway. Here we show that fixed and living EC provide comparable degrees of costimulation to CD4+ T cell populations, indicating that EC costimulation does not depend upon active metabolism. EC achieve these effects in part by utilizing lymphocyte function-associated antigen-3 (LFA-3) to interact with T cell CD2 as shown by observations that EC augmentation of IL-2 is partially (50-70%) blocked by eight of eight mAb tested which recognize LFA-3; that purified phosphatidylinositol-linked LFA-3 (PI-LFA-3) can also provide costimulation to CD4+ T cells; and that there is a delay of the EC effect on CD4+ T cells which express low levels of CD2 compared to those which express high levels of CD2. However, three lines of evidence suggest that EC also utilize at least one additional ligand. First, there is incomplete replacement of the EC effect by PI-LFA-3 such that the costimulatory ability of EC combined with PI-LFA-3 is additive at all concentrations of PI-LFA-3 tested. Second, costimulation by PI-LFA-3, but not by EC, is fully inhibited by anti-CD2 or anti-LFA-3 mAb. Finally, costimulation by PI-LFA-3, but not by EC, is completely suppressed by cyclosporine A. We have not formally identified the second ligand but it does not appear to be intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD44, or B7/BB1.     

LFA-1-mediated costimulation of CD8+ T cell proliferation requires phosphatidylinositol 3-kinase activity

LFA-1 binding to ICAM-I provides a costimulatory signal for CD8(+) T cell activation that results in increased IL-2 mRNA levels and protein production to support proliferation. CD28 binding to its B7 ligands has the same effect, and the two costimulatory receptors activate some of the same intracellular signaling events, including up-regulation of phosphatidylinositol (PI) 3-kinase activity. However, costimulation by LFA-1 depends upon the activity of this enzyme, whereas costimulation by CD28 does not, as evidenced by differential effects of specific inhibitors of PI 3-kinase. When cells are costimulated with ICAM-1 in the presence of the inhibitors wortmannin or LY294002, proliferation is blocked, but increases in IL-2 mRNA levels and protein production are not. Costimulation also results in increased surface expression of CD25, which is essential for formation of an active IL-2R. This is blocked by the PI 3-kinase inhibitors when costimulation is via LFA-1 but not when it is via CD28. Finally, IL-2-driven proliferation is not blocked by the inhibitors once CD25 surface expression has increased. Thus, the PI 3-kinase-dependent step in CD8 T cell costimulation by LFA-1 is up-regulation of IL-2R expression. In contrast, CD28 engagement also increases IL-2R surface expression, but the up-regulation does not depend upon PI 3-kinase activity.     

Patterns of costimulation of T cell clones by cross-linking CD3, CD4/CD8, and class I MHC molecules

The ability of mAb to class I MHC molecules, CD3, or CD4/CD8 to stimulate human T cell clones alone or in combination was examined. Cross-linking each of these surface Ag with appropriate mAb and goat anti-mouse Ig (GaMIg) resulted in a unique pattern of increase in intracellular free calcium ([Ca2+]i) and different degrees of functional activation. Cross-linking class I MHC molecules provided the most effective stimulus of IL-2 production and proliferation. Cross-linking more than one surface Ag induced a compound calcium signal with characteristics of each individual response. Cross-linking CD3 + HLA-A,B,C caused a rapid and prolonged increase in [Ca2+]i and synergistically increased IL-2 production and proliferation of all clones. Cross-linking CD3 + CD4/CD8 also generated a compound calcium signal and increased IL-2 production and DNA synthesis. Purposeful inclusion of CD3 was not required for costimulation as cross-linking HLA-A,B,C + CD4/CD8 also increased [Ca2+]i, IL-2 production, and proliferation. Cross-linking three surface Ag, CD3 + HLA-A,B,C + CD4/CD8, resulted in the greatest initial and sustained [Ca2+]i, IL-2 production, and DNA synthesis. Although there was a tendency for the various stimuli to increase both [Ca2+]i and functional responsiveness, neither the magnitude nor duration of the increased [Ca2+]i correlated with the amount of IL-2 produced or the ultimate proliferative response. To determine whether costimulation required that the various surface molecules were cross-linked together, experiments were carried out using isotype specific secondary antibodies. Augmentation of [Ca2+]i and costimulation of functional responses were noted when class I MHC molecules were cross-linked and CD3 was bound, but not cross-linked. Similarly, costimulation through CD3 and CD4/CD8 was observed when CD4/CD8 was cross-linked and the CD3 complex was engaged by an anti-CD3 mAb which was not further cross-linked. In contrast, costimulation by class I MHC molecules and CD4/CD8 was only observed when these molecules were cross-linked together. These data demonstrate that cross-linking class I MHC determinants or CD4/CD8 provides a direct signal to T cell clones that can be enhanced when CD3 is independently engaged. The results also indicate that T cell clones can be stimulated without engaging CD3 by the combination of signals delivered via class I MHC molecules and CD4/CD8, but only when these determinants were cross-linked together. These studies have demonstrated that these cell surface molecules differ in their capacity to deliver activation signals to T cell clones and also exhibit unique patterns of positive cooperativity in signaling potential.     

Maturation- and differentiation-dependent responsiveness of human CD4+ T helper subsets.

The human CD45R0+ (memory) CD4+ T cell population can be subdivided into a large (82%) CD27+ and a small (18%) CD27- subset. Within the CD45R0+CD27- subset, cells accumulate that have been persistently stimulated by Ag in vivo. As an apparent consequence, TLC with a differentiated functional phenotype, producing either high levels of IFN-gamma (Th1-like), high levels of IL-4 (Th2-like) or high amounts of both these cytokines (here referred to as Thx) can primarily be generated from the CD27- memory CD4+ T cell subset. In this study we examined the requirements for induction of proliferation of distinct CD4+CD45R0+ Th subsets. Immobilized CD3 mAb induced proliferation with comparable magnitude and kinetics in all types of TLC. However, interference with intracellular signaling pathways in this activation system, resulted in a strong inhibition of proliferation in TLC derived from CD27+ cells whereas, in contrast, TLC from CD27- cells were relatively resistant to elevation of [cAMP]i, inhibition of protein kinase C activation and the immunosuppressive effects of cyclosporin A. Stimulation with CD3 mAb in soluble form resulted in Il-4 secretion by Th2-like and Thx-type TLC but did not induce IFN-gamma or Il-2 secretion in any Th subset. Interestingly, Th2-like cells but not Thx-type cells were able to use endogenously produced Il-4 for proliferation. These data demonstrate a differential sensitivity of CD45R0+CD4+ Th subsets for immune activation and suppression, which correlated with their maturation stage, as reflected by CD27 membrane expression, as well as with their effector phenotype.     

Tumor-specific T cell activation by recombinant immunoreceptors: CD3 zeta signaling and CD28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3 zeta signaling receptor molecule.

Recombinant immunoreceptors with specificity for the carcinoembryonic Ag (CEA) can redirect grafted T cells to a MHC/Ag-independent antitumor response. To analyze receptor-mediated cellular activation in the context of CD28 costimulation, we generated: 1) CEA+ colorectal tumor cells that express simultaneously B7-1 and B7-2, and 2) CEA-specific immunoreceptors that harbor intracellularly the signaling moities either of CD28 (BW431/26-scFv-Fc-CD28), CD3zeta (BW431/26-scFv-Fc-CD3zeta), or FcepsilonRIgamma (BW431/26-scFv-Fc-gamma). By retroviral gene transfer, we grafted activated T cells from the peripheral blood with these immunoreceptors. T cells that express the FcepsilonRIgamma or CD3zeta signaling receptor lysed specifically CEA+ tumor cells and secreted high amounts of IFN-gamma upon receptor cross-linking, whereas anti-CEA-CD28 receptor-grafted T cells did not, indicating that CD28 signaling alone is not sufficient for efficient T cell activation. CD28 costimulation did not affect cytolysis by T cells equipped with gamma- or zeta-signaling receptors, but enhanced both IFN-gamma secretion and proliferation. CD28 costimulation, however, was required for efficient IL-2 secretion of anti-CEA-gamma receptor-grafted T cells. Both purified CD4+ and CD8+ T cells grafted with immunoreceptors required CD28 costimulation for complete T cell activation. We integrated both CD28 and CD3zeta signaling domains into one combined immunoreceptor molecule (BW431/26-scFv-Fc-CD28/CD3zeta) with dual signaling properties. T cells grafted with the combined CD28/CD3zeta signaling receptor secreted high amounts of IL-2 upon Ag binding without exogenous B7/CD28 costimulation, demonstrating that both MHC-independent cellular activation and CD28 costimulation for complete T cell activation can be delivered by one recombinant receptor molecule.     

Costimulation of T cell receptor/CD3-mediated activation of resting human CD4+ T cells by leukocyte function-associated antigen-1 ligand intercellular cell adhesion molecule-1 involves prolonged inositol phospholipid hydrolysis and sustained increase of intracellular Ca2+ levels.

Activation of resting human CD4+ T cells mediated by mAb ligation of the TCR/CD3 complex requires costimulatory signals to result in proliferation; these can be provided by intercellular cell adhesion molecule-1 (ICAM-1, CD54) a natural ligand of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18). We analyzed early signaling events involved in T cell activation to determine the contribution by the LFA-1/ICAM-1 interaction. We studied in detail the hydrolysis of phosphatidylinositol(4,5)bisphosphate and intracellular levels of free Ca2+ during stimulation with beads coated with the CD3 mAb OKT3 alone or in combination with purified ICAM-1 protein. Our investigations show no response to LFA-1/ICAM-1 alone, but that costimulation by LFA-1/CAM-1 interaction induces prolonged inositol phospholipid hydrolysis (up to 4 h), resulting in generation of both inositol(1,4,5)phosphate3 and inositol(1,3,4,5)phosphate4 and their derivatives. Based on studies with cycloheximide, this costimulatory effect of prolonged inositol phospholipid hydrolysis appears dependent in part on de novo protein synthesis. A sustained increase in intracellular levels of free Ca2+ level is also observed after LFA-1/ICAM-1 costimulation, which is at least partly dependent on extracellular sources of Ca2+. Kinetic studies indicate that costimulation requires a minimal period of 4 h of LFA-1/ICAM-1 interaction to provide maximal costimulation for OKT3-mediated T cell proliferation. Thus, the necessary costimulation required for OKT3-mediated proliferation in this model system may be provided by an extended LFA-1/ICAM-1 interaction that in combination with OKT3 mAb leads to signal-transducing events, resulting in prolonged phospholipase C activation and phosphatidylinositol(4,5)bisphosphate hydrolysis, and a sustained increase in intracellular levels of free Ca2+.     

Costimulation of proliferative responses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 with fibronectin or VLA-6 with laminin

Given prior evidence that adhesion molecules play critical roles in T cell recognition, it is important to identify new adhesion pathways and explore their role in T cell activation. Our studies of T cell proliferation complement concurrent studies of T cell adhesion; both demonstrate that resting CD4+ human T lymphocytes express the VLA integrins VLA-4, VLA-5, and VLA-6, and can use these receptors to interact with the extracellular matrix (ECM) proteins fibronectin (VLA-4 and VLA-5) and laminin (VLA-6). VLA-dependent interaction of resting human CD4+ T cells with fibronectin (FN) and laminin (LN) facilitates CD3-mediated T cell proliferation. Specifically, T cells do not proliferate in response to a wide range of concentrations of a CD3 mAb, OKT3, immobilized on plastic. However, coimmobilization with the CD3 mAb of FN or LN, but not other ECM proteins such as fibrinogen and collagen, consistently results in strong T cell proliferation. mAb blocking studies demonstrate that three VLA integrin receptor/ligand interactions mediate costimulation: VLA-4/FN, VLA-5/FN, and VLA-6/LN. VLA-5-dependent binding to FN but not costimulation by FN can be specifically blocked with peptides containing the RGD (arg-gly-asp) tripeptide sequence whereas VLA-4-dependent binding and costimulation can both be efficiently inhibited by a 12 amino acid peptide, LHGPEILDVPST (leu-his-gly-pro-glu-iso-leu-asp-val-pro-ser-thr), derived from the alternatively spliced IIICS region of FN. The costimulation provided by FN and LN in this system is stronger than and distinct from costimulatory signals provided by cytokines, such as IL-1 beta, IL-6,, and IL-7. These results suggest that, such as other adhesion molecules, T cell VLA integrins may also function in a dual capacity as adhesion and signalling molecules. In addition, they suggest that the interaction of T cells in vivo with ECM via VLA integrins plays a role not only in T cell migratory processes but may also influence Ag-specific T cell recognition.     

Ligation of B and T lymphocyte attenuator prevents the genesis of experimental cerebral malaria

B and T lymphocyte attenuator (BTLA; CD272) is a coinhibitory receptor that is predominantly expressed on T and B cells and dampens T cell activation. In this study, we analyzed the function of BTLA during infection with Plasmodium berghei ANKA. Infection of C57BL/6 mice with this strain leads to sequestration of leukocytes in brain capillaries that is associated with a pathology resembling cerebral malaria in humans. During the course of infection, we found an induction of BTLA in several organs, which was either due to up-regulation of BTLA expression on T cells in the spleen or due to infiltration of BTLA-expressing T cells into the brain. In the brain, we observed a marked induction of BTLA and its ligand herpesvirus entry mediator during cerebral malaria, which was accompanied by an accumulation of predominantly CD8+ T cells, but also CD4+ T cells. Application of an agonistic anti-BTLA mAb caused a significantly reduced incidence of cerebral malaria compared with control mice. Treatment with this Ab also led to a decreased number of T cells that were sequestered in the brain of P. berghei ANKA-infected mice. Our findings indicate that BTLA-herpesvirus entry mediator interactions are functionally involved in T cell regulation during P. berghei ANKA infection of mice and that BTLA is a potential target for therapeutic interventions in severe malaria.     

Human CD8+ T lymphocytes can be divided into CD45RA+ and CD45RO+ cells with different requirements for activation and differentiation.

The functional distinction between CD45RA+ and CD45RO+ cells within the human CD4+ T cell subset is well established. This study was undertaken to investigate whether a similar division can be made within the CD8+ T cell population. A quantitative comparison was made of the requirements for activation and differentiation of CD8+CD45RA+ and CD8+CD45RO+ cells. Stimulation of T lymphocytes with anti-CD3 mAb immobilized at high-density induced strong proliferation and CTL activity in both CD45RA+ and CD45RO+ cells. Suboptimal TCR/CD3 triggering, in contrast, induced substantially higher levels of proliferation and CTL activity in CD8+CD45RO+ cells compared with their CD45RA+ counterparts. Lymphokine secretion (i.e., Il-2 and TNF-alpha) was under any condition more readily induced in CD8+CD45RO+ cells. Markedly, proliferation of both CD8+CD45RA+ and CD8+CD45RO+ T cells initiated by anti-CD3 mAb immobilized at high densities was not inhibited by addition of anti-CD25 mAb, in contrast to proliferation induced by suboptimal anti-CD3 mAb concentrations. These findings show that a functional division between CD45RA+ and CD45RO+ T cells with distinct requirements for activation and differentiation may also be made in the CD8+ subset.     

CD4+ Leu-8+ T cell supernatant activity that inhibits Ig production.

The subpopulation of CD4+ T cells that expresses the Leu-8 peripheral lymph node homing receptor suppresses PWM-stimulated Ig synthesis. To determine the mechanism of this suppression, the immunoregulatory activity of culture supernatants obtained from peripheral blood CD4+ Leu-8+ T cells cultured with anti-CD3 mAb and PMA (Leu-8+ supernatant) was determined. Leu-8+ supernatant suppressed PWM-stimulated Ig synthesis in cultures containing non-T cells and CD4+ Leu-8- T cells. In contrast, the supernatant from CD4+ Leu-8- T cells did not suppress Ig synthesis. The inhibitory activity of CD4+ Leu-8+ T cell supernatants could not be accounted for by a deficiency or excess of IL-2, IL-4, IFN-gamma, IL-6, or PGE2. In studies examining the effect of CD4+ Leu-8+ supernatant on T cells, the supernatant did not alter either mitogen-induced proliferation or the helper function of CD4+ Leu-8- T cells. In studies examining the effect of CD4+ Leu-8+ supernatant on B cells, the supernatant inhibited Staphylococcus aureus Cowan I strain-induced B cell Ig secretion but not B cell proliferation. The suppressor activity of Leu-8+ supernatant was eliminated by protease treatment and was eluted by HPLC in two main peaks, with molecular sizes of 44 and 12 kDa. In summary, these studies indicate that supernatants from activated CD4+ Leu-8+ T cells directly suppress B cell Ig production.