Ag-bearing Cells Research Articles

Siglecs induce tolerance to cell surface antigens by BIM-dependent deletion of the antigen-reactive B cells.

Infusion of blood cells from a donor can induce humoral tolerance in a recipient and increase the probability of successful organ transplant, a clinical method defined as donor-specific transfusion (DST). Despite the clinical success of DST, the immunological mechanisms by which blood cells displaying a foreign Ag induce tolerance remain poorly understood. Based on recent findings showing that the B cell siglecs, CD22 and Siglec-G, can promote tolerance to Ags presented on the same surface as their ligands, we speculated that the B cell siglecs are key players in tolerance induced by DST. Using a variety of chemical and genetic approaches, we show that the B cell siglecs mediate tolerance to cell surface Ags by initiating an inhibitory signal that culminates in elimination of the Ag-reactive B cell. CD22 and Siglec-G are recruited to the immunological synapse by sialic acid ligands on the Ag-bearing cells, producing a tolerogenic signal involving Lyn and the proapoptotic factor BIM that promotes deletion of the B cell and failure of mice to develop Abs to the Ag upon subsequent challenge. We speculate that this tolerogenic mechanism is a contributing factor in DST and a mechanism of peripheral B cell tolerance to cell surface autoantigens.

P-D13 Vaginal type-II mucosa acts as an inductive site for the generation of primary CD8+ T cell mucosal immune responses

It is widely believed that primary immune T cell induction in type-II mucosa (found in vagina, glans penis & esophagus) occurs only in the draining lymph nodes (DLNs) due to a lack of mucosa-associated lymphoid tissue (MALT). In this process, the naïve T cells in DLNs are primed by the antigen (Ag)-bearing dendritic cells (DCs) migrating from the Ag-exposed mucosa and differentiate into memory T cells that are then able to traffic back to mucosal sites through the bloodstream. Contrary to this believe, here we show that the vaginal type-II mucosa itself could act as an inductive site for the generation of primary CD8+ T cell mucosal immunity. As evidence, we found that naive CD8+ T cells routinely migrated to the female genital tract and that vaginal Ag-specific CD8+ T cells could be generated in the LN-deficient mice after intravaginal (Ivag) immunization. Further, adoptively transferred naïve OT-1 CD8+ T cells were activated in the vaginal mucosa but not in the DLNs 24 hours after Ivag immunization under conditions where the T cells from the circulation or DLNs could not reach the vaginal mucosa. More importantly, the immune priming could occur on the naive OT-I CD8+ T cells that naturally migrate into the physiologically intact vaginal mucosa before any priming occurs in the DLNs. In addition, the Ag-bearing cells isolated from immunized vaginal mucosa were able to stimulate naive OT-I cells to secret IFN-gamma and undergo proliferation. Finally, vaginal mucosa largely supported the expansion of Ag-specific CD8+ T cells. In conclusion, we present evidence for a new paradigm for primary CD8+ T cell immune induction in type-II mucosa of vagina, 1 that occurs locally without the help of DLNs or MALT.

Vaginal type-II mucosa acts as an inductive site during the generation of primary CD8+ T cell mucosal immune responses (P3186)

Abstract It is widely believed that primary immune induction in type-II mucosa (vagina, mouth & cornea) occurs in the draining LNs due to a lack of mucosa-associated lymphoid tissue. In this process, naïve T cells located in the draining LNs are primed by antigen (Ag)-bearing dendritic cells migrating from the Ag-exposed mucosa. Primed T cells then travel to the mucosal site through the bloodstream. In contrast to this paradigm, we show that vaginal mucosa itself can act as an immune inductive site for generation of primary CD8+ T cell mucosal immunity. As evidence, we found that naïve CD8+ T cells routinely migrated to the female reproductive tract and that Ag-specific CD8+ T cells could be generated in the LN-deficient mice after intravaginal immunization. Further, the adoptively transferred naïve OT-1 CD8+ T cells were activated in the vaginal mucosa but not in the draining LNs at 24 hours after intravaginal immunization, even in the presence of FTY720, a drug blocking the egress of T cells from LNs. In addition, the Ag-bearing cells isolated from immunized vaginal mucosa were able to stimulate naïve TCR-transgenic RT-1 CD8+ T cells to secret IFN-γ and undergo proliferation. Finally, vaginal mucosa largely supported the expansion of Ag-specific CD8+ T cells. In conclusion, we present evidence for a new paradigm for primary CD8+ T cell immune induction in type-II mucosa of the vagina, one that occurs locally without the help of draining LNs or mucosa-associated lymphoid tissue.

Soluble Proteins Induce Strong CD8+ T Cell and Antibody Responses through Electrostatic Association with Simple Cationic or Anionic Lipopeptides That Target TLR2

The low immunogenicity exhibited by most soluble proteins is generally due to the absence of molecular signatures that are recognized by the immune system as dangerous. In this study, we show that electrostatic binding of synthetic branched cationic or anionic lipopeptides that contain the TLR-2 agonist Pam(2)Cys markedly enhance a protein's immunogenicity. Binding of a charged lipopeptide to oppositely charged protein Ags resulted in the formation of stable complexes and occurs at physiologic pH and salt concentrations. The induction of cell-mediated responses is dependent on the electrostatic binding of lipopeptide to the protein, with no CD8(+) T cells being elicited when protein and lipopeptide possessed the same electrical charge. The CD8(+) T cells elicited after vaccination with lipopeptide-protein Ag complexes produced proinflammatory cytokines, exhibited in vivo lytic activity, and protected mice from challenge with an infectious chimeric influenza virus containing a single OVA epitope as part of the influenza neuraminidase protein. Induction of a CD8(+) T cell response correlated with the ability of lipopeptide to facilitate Ag uptake by DCs followed by trafficking of Ag-bearing cells into draining lymph nodes. Oppositely charged but not similarly charged lipopeptides were more effective in DC uptake and trafficking. Very high protein-specific Ab titers were also achieved by vaccination with complexes composed of oppositely charged lipopeptide and protein, whereas vaccination with similarly charged constituents resulted in significant but lower Ab titers. Regardless of whether similarly or oppositely charged lipopeptides were used in the induction of Ab, vaccination generated dominant IgG1 isotype Abs rather than IgG2a.

Identification of a Unique Population of Tissue-Memory CD4+ T Cells in the Airways after Influenza Infection That Is Dependent on the Integrin VLA-1

During the immune response to influenza infection, activated T cells are distributed to both lymphoid and extralymphoid tissues, including the infected airways where direct recognition of viral Ag-bearing cells takes place. The collagen-binding alpha(1)beta(1) integrin VLA-1 is essential for the development of memory CD8(+) T cells in the airways, and although expressed by some CD4(+) T cells, its significance has not been demonstrated. We investigated the role of VLA-1 on virus-specific CD4(+) T cells during and after primary or secondary influenza infection of mice. The proportion of CD4(+) cells expressing CD49a (alpha(1) integrin) was low in all tissues sampled during primary infection but increased in the airways after viral clearance. Furthermore, during the first 24 h of a secondary influenza challenge, the majority of IFN-gamma-secreting effector CD4(+) T cells from the airways was in the CD49a(+) population. Airway CD49a(+)CD4(+) cells also expressed reduced markers of apoptosis compared with CD49a(-) cells, and fewer memory or effector CD4(+) cells could be recovered from airways of alpha(1)(-/-) mice, although lymphoid tissues appeared unaffected. These data suggest VLA-1 expression defines a population of tissue memory CD4(+) T cells that act as rapid effectors upon reinfection, and VLA-1 expression is integral to their accumulation in the airways.

The distribution of antigen in lymphoid tissues following its injection into the anterior chamber of the rat eye.

Injection of Ag into the anterior chamber (AC) of the eye induces deviant immune responses. It has been proposed that Ag internalized by ocular APCs is presented in a tolerogenic fashion in the spleen. However, the nature and distribution of the Ag-bearing cells in the lymphoid organs remain unclear. Fluorescent-labeled Ag (dextran, BSA) injected into the AC of Lewis rats was detected in the subcapsular sinus of the right submandibular lymph nodes (LNs) and cervical LNs, the marginal zone of the spleen, and the medulla of the mesenteric LNs. In the spleen, Ag-bearing cells were CD1(+), CD11b(+), ED1(+), ED2(low), ED3(+), CD86(low), OX6(+), CD11c(-), ED5(-) and in the LNs were CD4(+), CD8(+), CD80(+), and OX41(+) suggesting these were lymphoid organ resident macrophages. These Ag-bearing macrophages were located adjacent to CD4(+) cells, CD8(+) cells, and NK cells in the LNs and spleen and to marginal zone B cells in the spleen. No interaction with gamma delta T cells was observed. The data demonstrates that Ag derived from the AC of the eye is mainly internalized by resident macrophages in the LNs and spleen which are ideally placed to interact with cells involved in the induction of deviant ocular immune responses. The extensive distribution of Ag in LNs draining the upper airway and gastrointestinal tracts, together with the phenotype of Ag-bearing cells in the lymphoid organs, suggests that Ag leaves the eye predominantly in a soluble form and implies other mechanisms of tolerance may contribute to ocular-specific immune responses.

Local Retention of Soluble Antigen by Potential Antigen-Presenting Cells in the Anterior Segment of the Eye

To determine the capacity of bone marrow-derived cells in the anterior segment of the eye to capture a fluorescence-labeled antigen (Ag) injected into the anterior chamber (AC). Uveal tract and corneoscleral tissues from Lewis rats were cultured in vitro, with or without FITC-dextran (4 microg/mL final concentration), for 48 hours and examined by confocal microscopy. To investigate antigen uptake in vivo 2 microL (20 microg) of Cascade Blue-labeled dextran (CB-Dx) was injected into the right AC of Lewis rats. The density of Ag-positive cells in the iris at 1, 3, 5, or 12 days after injection was examined by in vivo video fluorescence microscopy. The distribution and phenotype of Ag-positive cells in frozen and paraffin-embedded sections of ocular tissues and in iris wholemounts from animals killed at 24 hours and day 7 were analyzed by fluorescence and confocal microscopy. In organ culture conditions numerous cells in the iris, ciliary body, choroid, and corneal limbus were capable of capturing fluorescence-labeled Ag. In vivo observations and microscopic examination of experimental eyes at days 1 and 7 after AC injection revealed Ag-positive cells within the iris, iridocorneal angle, the suprachoroidal space and around limbal-episcleral vessels. Ag-bearing cells in the iris express combinations of macrophage markers but rarely expressed major histocompatibility complex (MHC) class II molecules. A reduced number of Ag-bearing cells were still present in the iris at day 12. Potential antigen-presenting cells (APCs) in the iris and ciliary body are capable of internalizing intracameral Ag. The characteristics of these cells in the iris are consistent with a predominantly macrophage phenotype. These observations also suggest that the Ag leaving the eye through both the conventional and nonconventional aqueous outflow pathways may be captured by potential APCs in the episcleral tissues.

In situ beta cell death promotes priming of diabetogenic CD8 T lymphocytes.

CTLs are important mediators of pancreatic beta cell destruction in the nonobese diabetic mouse model of type 1 diabetes. Cross-presentation of Ag is one means of priming CTLs. The death of Ag-bearing cells has been implicated in facilitating this mode of priming. The role of beta cell death in facilitating the onset of spontaneous autoimmune diabetes is unknown. Here, we used an adoptive transfer system to determine the time course of islet-derived Ag presentation to naive beta cell-specific CD8 T cells in nonobese diabetic mice and to test the hypothesis that beta cell death enhances the presentation of beta cell autoantigen. We have determined that beta cell death enhances autoantigen presentation. Priming of diabetogenic CD8 T cells in the pancreatic lymph nodes was negligible before 4 wk, progressively increased until 8 wk of age, and was not influenced by gender. Administration of multiple low doses of the beta cell toxin streptozotocin augmented in situ beta cell apoptosis and accelerated the onset and magnitude of autoantigen presentation to naive CD8 T cells. Increasing doses of streptozotocin resulted in both increased pancreatic beta cell death and significantly enhanced T cell priming. These results indicate that in situ beta cell death facilitates autoantigen-specific CD8 T cell priming and can contribute to both the initiation and the ongoing amplification of an autoimmune response.

Donor MHC class II antigen is essential for induction of transplantation tolerance by bone marrow cells.

Posttransplant infusion of donor bone marrow cells (BMC) induces tolerance to allografts in adult mice, dogs, nonhuman primates, and probably humans. Here we used a mouse skin allograft model and an allogeneic radiation chimera model to examine the role of MHC Ags in tolerance induction. Infusion of MHC class II Ag-deficient (CIID) BMC failed to prolong C57BL/6 (B6) skin grafts in ALS- and rapamycin-treated B10.A mice, whereas wild-type B6 or MHC class I Ag-deficient BMC induced prolongation. Removal of class II Ag-bearing cells from donor BMC markedly reduced the tolerogenic effect compared with untreated BMC, although graft survival was significantly longer in mice given depleted BMC than that in control mice given no BMC. Infusion of CIID BMC into irradiated syngeneic B6 or allogeneic B10.A mice produced normal lymphoid cell reconstitution including CD4+ T cells except for the absence of class II Ag-positive cells. However, irradiated B10.A mice reconstituted with CIID BMC rejected all B6 and a majority of CIID skin grafts despite continued maintenance of high degree chimerism. B10.A mice reconstituted with B6 BMC maintained chimerism and accepted both B6 and CIID skin grafts. Thus, expression of MHC class II Ag on BMC is essential for allograft tolerance induction and peripheral chimerism with cells deficient in class II Ag does not guarantee allograft acceptance.

Leukocyte function-associated antigen-1-dependent lysis of Fas+ (CD95+/Apo-1+) innocent bystanders by antigen-specific CD8+ CTL.

Exquisite specificity toward Ag-bearing cells (cognate targets) is one of the most important properties of CD8+ CTL-mediated cytotoxicity. Using highly Ag-specific CD8+ CTL lines and clones, which spare noncognate, Ag-free targets, we found that in the presence of Ag-bearing targets the CTL acquire the ability to lyse noncognate target cells (bystanders). It is shown that the unexpectedly rapid and efficient lysis of bystanders by Ag-activated CTL is mediated by a Fas ligand (FasL)/Fas-based mechanism and does not depend on perforin. The CTL lysed Fas-expressing bystanders, but spared the Fas-negative or anti-Fas mAb-resistant bystander cells. Accordingly, the FasL-deficient gld/gld CTL did not kill bystanders, while perforin-deficient CTL did. Unlike anti-Fas mAb-induced cell death, the lysis of bystanders was not only FasL/Fas dependent but also required adhesion molecule LFA-1 on the surface of the activated CTL. Lysis of bystanders is viewed as acceptable "collateral" damage, but the persistent presence of activated CTL could result in immunopathologies involving functional Fas-expressing tissues.

Perforin, Fas/Fas ligand, and TNF-alpha pathways as specific and bystander killing mechanisms of hepatitis C virus-specific human CTL.

In chronic hepatitis C, Fas expression is up-regulated in the hepatocytes, especially near liver-infiltrating lymphocytes, and Fas ligand is expressed on the lymphocytes. The presence of hepatitis C virus (HCV)-specific CTLs has been demonstrated both in peripheral blood and among liver-infiltrating lymphocytes of patients with chronic hepatitis C. We studied the killing mechanisms of HCV-specific human CTLs using target cells that were sensitive or resistant to agonistic anti-Fas Abs and TNF-alpha. We show that HCV-specific CTL clones kill non-Ag-bearing bystander cells as well as Ag-bearing cells, although the bystander killing is less efficient than the specific target cell killing, and the efficacy of the bystander killing of anti-Fas- and soluble TNF-alpha-sensitive cells is greater than that of resistant cells. We also show that the killing of Ag-presenting, sensitive cells is mediated by Fas ligand and TNF-alpha as well as perforin, although the latter plays a major role in the killing at a low E:T ratio, and that the killing of sensitive bystander cells is primarily mediated by Fas ligand and TNF-alpha on CTLs expressed upon specific Ag stimulation, which may be relevant to the bystander lysis by HCV-specific CTLs of uninfected hepatocytes, in which Fas expression is up-regulated. Activated CTLs also kill bystander cells by the perforin-based mechanism, although it requires a high E:T ratio. The effective bystander killing requires a close intercellular contact between CTLs and target cells, although TNF-alpha released from the CTLs mediates lysis of the bystander cells without a close cell-cell contact.

Down-regulation of CD8 on mature antigen-reactive T cells as a mechanism of peripheral tolerance.

Previously we have shown that intravenous injection of male B6 lymphocytes containing CD8+ cells into B6 female anti-HY TCR transgenic mice results at 6 wk in the disappearance of the majority of male Ag-reactive T cells (TghighCD8+) from the periphery. Here we investigate the process in more detail. B6 female anti-HY TCR transgenic mice were intravenously injected with viable lymphocytes from male B6 normal, CD4-/-, CD8-/- or CD8-/- carrying a CD8 transgene lacking its cytoplasmic tail ("CD8 tail-less") mice. The fate of TghighCD8+ cells was followed in vivo. There was always a large (at least twofold) expansion of these cells in the periphery 4 days after encountering male Ag. Their subsequent fate differed, however, depending on whether or not the injected male lymphocytes contained normal CD8-expressing cells. If the injected male lymphocytes contained normal CD8 cells, at 6 wk there was a large drop in the number of TghighCD8+ cells associated with a rise in the number of TghighCD8- cells. If the injected male lymphocytes lacked CD8 cells or expressed only tail-less CD8, TghighCD8+ cell numbers returned to normal by 6 wk, while TghighCD8- cell numbers remained unchanged. The TghighCD8- cells, although carrying high levels of the male specific TCR, did not react to male Ag. In addition, their ability to respond in anti-CD3-induced activation, which does not require CD8 as a co-receptor, was significantly impaired. Our data suggest that down-regulation of CD8 on the Ag-reactive T cells accounts, at least partially, for the disappearance of HY-reactive T cells from the periphery. Further, some step in the process requires signaling through the cytoplasmic domain of CD8 on the injected Ag-bearing cells.

Antigen recognition by T cells. Quantitative effects of augmentation by antibodies providing accessory interactions.

Although engagement of the TCR via antibody can be sufficient to trigger T cells, responses to Ag-bearing cells require additional "accessory" interactions in many cases. A method has been developed which allows preparation of surfaces bearing both purified class I alloantigen and coimmobilized antibodies. With this approach, it is possible to mimic such "accessory" interactions and to examine their quantitative effects on triggering via TCR-Ag interaction. Experiments are described which use this approach to examine triggering of degranulation by cloned, allogeneic CTL lines. Coimmobilization of antibodies specific for any of a variety of CTL surface proteins, including CD8, class I MHC proteins, CD45 (T200) and Thy-1, had the effect of decreasing the critical threshold density of Ag necessary to trigger responses, and decreasing by an order of magnitude the density required to stimulate a half-maximal response. Furthermore, in comparison with the 30-min lag seen with Ag alone, response was initiated immediately when an antibody specific for a CTL surface component was present. These results are consistent with the hypothesis that any CTL surface molecule having sufficient affinity for a component of the target surface can contribute to activation via the Ag-specific TCR; and at low Ag density could determine whether any response occurs.

T cell recognition of nonpolymorphic determinants on H-2 class I molecules.

Recognition of polymorphic determinants on class I or class II MHC Ag is required for T lymphocyte responses. Using cell-size artificial membranes (pseudocytes) bearing H-2 class I Ag it is demonstrated that T cells can, in addition, recognize nonpolymorphic determinants on class I proteins. Pseudocytes bearing class I alloantigen stimulate in vitro generation of secondary allogeneic CTL responses. At a suboptimal alloantigen surface density, incorporation of class I molecules identical to those of the responder cells (self-H-2) or from third-party cells resulted in dramatically enhanced responses, whereas incorporation of class II proteins had no effect. The receptor that mediates recognition of conserved class I determinants has not been identified, but results of antibody blocking studies are consistent with the Lyt-2/3 complex of CTL having this role. Thus, class I proteins on Ag-bearing cells can have two distinct roles in T cell activation, one involving recognition of polymorphic determinants by the Ag-specific receptor and the other involving recognition of conserved determinants.

Monoclonal antibody H9/25 reacts with functional subsets of T and B cells: killer, killer precursor and plaque-forming cells.

Monoclonal antibody (McAb) H9/25 has previously been shown to react with an alloantigen expressed on subpopulations of mouse lymphocytes. We here investigated the expression of the antigen (H9/25 Ag) on functional subsets of T and B cells. Lymphocytes were depleted of H9/25 Ag-bearing cells by complement-dependent cytolysis or by the affinity to immobilized McAb H9/25, and the residual immunological functions were tested. In these tests, killer T cells generated in mixed lymphocyte cultures and their precursors were found to express H9/25 Ag. In contrast, the activity and frequency of helper T cells specific to keyhole limpet hemocyanin carrier were not decreased by the depletion of H9/25 Ag-bearing cells. On the other hand, IgG and IgM anti-TNP plaque-forming cells were found to express H9/25 Ag, while it was not detected on unprimed B cells, as tested by using TNP-Ficoll and TNP-lipopolysaccharide. Only a small proportion of TNP memory B cells seemed to express the antigen. These results revealed significant differences between H9/25 Ag and other known alloantigens in the distribution among functional subsets of lymphocytes.