Antigen-pulsed Antigen-presenting Cells Research Articles

CD45 pre-exclusion from the tips of T cell microvilli prior to antigen recognition

The tyrosine phosphatase CD45 is a major gatekeeper for restraining T cell activation. Its exclusion from the immunological synapse (IS) is crucial for T cell receptor (TCR) signal transduction. Here, we use expansion super-resolution microscopy to reveal that CD45 is mostly pre-excluded from the tips of microvilli (MV) on primary T cells prior to antigen encounter. This pre-exclusion is diminished by depleting cholesterol or by engineering the transmembrane domain of CD45 to increase its membrane integration length, but is independent of the CD45 extracellular domain. We further show that brief MV-mediated contacts can induce Ca2+ influx in mouse antigen-specific T cells engaged by antigen-pulsed antigen presenting cells (APC). We propose that the scarcity of CD45 phosphatase activity at the tips of MV enables or facilitates TCR triggering from brief T cell-APC contacts before formation of a stable IS, and that these MV-mediated contacts represent the earliest step in the initiation of a T cell adaptive immune response.

Ratios of effector to central memory antigen‐specific CD4+ T cells vary with antigen exposure in HIV+ patients

The OX40/CD25 assay is a novel technique that assesses antigen-specific CD4(+) T-cell responses. To unequivocally demonstrate that responding cells are memory cells that become activated after secondary stimulation, naïve CD45RA(+) and memory CD45RO(+) populations were stimulated with cytomegalovirus (CMV) lysate and the combined expression of CD25 and OX40 measured. As expected, the naïve population showed very little response, whereas there was a higher response from the memory counterpart. To further elucidate CD4(+) memory T-cell subsets involved in recall responses, CD4(+) T cells were separated into central memory (Tcm) and effector memory (Tem) subsets and stimulated with antigen-pulsed antigen-presenting cells (APCs). CMV responses in healthy donors showed a Tem-dominant response with a Tem/Tcm ratio of 1.2, whereas the tetanus toxoid responses were dominated by a Tcm response with a Tem/Tcm ratio of 0.35. To determine memory response in the chronic of HIV infection, patient samples were used. A similar pattern to healthy donors was observed in seven chronic HIV+ patients at week 4 after anti-retroviral therapy who responded to CMV with a larger response coming from Tem. The pattern was similar after 48 weeks of therapy but the responses were lower in magnitude. In chronic HIV+ patients who respond to Gag peptides, following institution of therapy there was an inversion of the ratio of the responding memory subsets compared with week 4, with a greater response from Tcm at week 48. This result was concordant with reduction in antigen load. As immune activation decreased there was also a decrease in the percentage of responding effector memory cells and maintenance of long-term central memory.

Antigen-Specific Transfer of Functional Programmed Death Ligand 1 From Antigen Presenting Cells Onto Human CD8+ T Cells Via Trogocytosis

AbstractAbstract 584Specific T-cell responses are initiated by T-cell receptor (TCR) recognition of peptide-MHC-complexes on antigen presenting cells (APCs). Upon specific interaction of T cells with APCs T cells capture membrane fragments and surface molecules of APCs in a process termed trogocytosis. Exchange of membrane molecules/antigens between immune cells has been observed for a long time, but the mechanisms and functional consequences of these transfers remain unclear. Here, we demonstrate that human antigen-specific CD8+ T cells do acquire the co-inhibitory molecule programmed death ligand 1 (PD-L1) from mature monocyte-derived dendritic cells (mDC) and tumor cells in an antigen-specific manner. The kinetics of PD-L1 transfer revealed a maximal PD-L1 expression on antigen-specific T cells within 3–4 hours after co-incubation with antigen-pulsed APCs, being detectable up to 72 hours. Antigen-pulsed immature DCs were less effective in transfering surface molecules such as PD-L1 onto CD8+ T cells after antigen-specific recognition. Using a transwell system we could show that the acquisition of PD-L1 requires cell-cell contact. Furthermore, PD-L1 cannot be acquired by T cells from a lysate of mDCs. The transfer process is impaired after pretreatment of T cells with concanamycin A, a specific inhibitor of vacuolar ATPases, playing an important role in membrane trafficking. Moreover, fixation of DCs with glutaraldehyde completely abrogated the acquisition of PD-L1 on T cells suggesting that an active interaction between APCs and T cells is required for trogocytosis. Of importance, CD8+ T cells which acquired PD-L1 complexes, were able to induce apoptosis of neighbouring PD-1 expressing CD8+ T cells, that could be completely blocked by an anti-PD-L1 antibody. In summary our data demonstrate for the first time that human antigen-specific CD8+ T cells take up functionally active PD-L1 from APCs in an antigen-specific fashion, leading to apoptosis of PD-1 expressing T cells. The transfer of functionally active co-inhibitory molecules from APCs onto human CD8+ T cells may serve to limit clonal expansion of antigen-specific T-cell responses but may also play a major role for T-cell exhaustion in chronic infection and tumor immunosurveillance. Disclosures:No relevant conflicts of interest to declare.

Human Naive CD4 T-Cell Clones Specific for HIV Envelope Persist for Years In Vivo in the Absence of Antigenic Challenge

To study the persistence of HIV-specific human naive CD4-lymphocytes in vivo in the absence of antigenic stimulation, we identified 2 HIV-seronegative low-risk subjects carrying CD4-cells specific for gp120 that could be expanded in vitro. CD4 T-cell lines specific for gp120 were generated by stimulation cycles with antigen-pulsed antigen-presenting cells. Clonal analysis was performed by spectratyping and by sequencing of the CDR3 regions of the BV and AV-T-cell receptor (TCR) genes. HIV-specific T cells were expanded in vitro in 1989 and 2004. These lines were generated from naive precursors. Analysis of TCR-BV gene family use and sequencing of the TCR-BV22 hypervariable region revealed a BV22 clonotype in the 1989 line. The BV22-CDR3-based polymerase chain reaction primer confirmed that the 1989 and 2004 T-cell lines contained the same clonotype. In addition, the 1989 and 2004 T cells used the same TCR-AV38 gene family and identical CDR3-AV regions, confirming clonal identity. Similar data for a persistent clonotype defined by BV CDR3 sequencing were obtained from the second subject. In conclusion, naive CD4-cells specific for an HIV antigen not encountered in vivo persisted for more than 10 to 15 years. An extended lifespan, homeostatic proliferation, or the ability of the thymus to issue the same CD4 T-cell clone reiteratively might account for the phenomenon.

Disruption of the interaction of T cells with antigen‐presenting cells by the active leflunomide metabolite teriflunomide: Involvement of impaired integrin activation and immunologic synapse formation

Leflunomide, a potent disease-modifying antirheumatic drug of the isoxazole class, exhibits antiinflammatory, antiproliferative, and immunosuppressive effects by largely unknown mechanisms, although alterations of pyrimidine synthesis have been proposed. Successful immune responsiveness requires T cell activation by interaction with antigen-presenting cells (APCs), and integrin activation and formation of an immunologic synapse (IS). In this study, we evaluated the impact of the active leflunomide metabolite teriflunomide on T cell integrin activation, evolution of the IS, and antigen-specific formation of stable T cell/APC conjugates. Effects of pharmacologic concentrations of teriflunomide on CD3/CD28- and lymphocyte function-associated antigen 1-induced signal transduction and activation of primary human T cells were investigated. Furthermore, T cells were stimulated with superantigen- and antigen-pulsed APCs to study relocalization of molecules to the IS and T cell/APC conjugate formation. Teriflunomide inhibited T cell receptor (TCR)/CD3-mediated calcium mobilization, but other critical T cell signaling events, including activation of MAPK and NF-kappaB, remained unaltered. In contrast, inhibition of TCR/CD3-triggered beta1,2 integrin avidity and integrin-mediated costimulation (outside-in signaling) by teriflunomide revealed a striking interference with integrin function that was independent of altered pyrimidine synthesis. Moreover, teriflunomide abolished molecule relocalization to the IS and induction of T cell/APC conjugates. These data show that the active metabolite of leflunomide prevents the interaction of T cells with APCs to form an IS. Since IS formation is crucial for eliciting an immune response, this novel mechanism could underlie the beneficial effects of leflunomide in immune-mediated disorders such as rheumatoid arthritis.

Terapias emergentes en artritis reumatoide

The use of biological agents such as etanercept, infliximab, adalimumab and anakinra has been recently approved for the treatment of rheumatoid arthritis. All are effective controlling signs and symptoms and inhibiting disease progression. To overcome the problems generated by their high costs and possible participation in reactivating latent infections, other therapeutic tools are being developed. Gene therapy using expression vectors carrying genes coding for specific proteins, may interfere in key points involved in the pathogenesis of the disease. Intra-articular administration of cDNA coding for soluble TNF receptors, IL-1, or IL-1Ra decreases signs of the disease in animal models. Vectors, expressing inhibitors of signal transduction pathways involving to NF-kB and JAK-STAT-3, are effective in modulating joint inflammation in mice. The use of antigen-pulsed antigen presenting cells or dendritic cells (DC) bound to apoptosis-inducing molecules, specifically eliminates autoreactive T cells. Other novel approach attempts the development of T regulatory-inducing tolerogenic DC-based vaccines that inhibit autoreactive T cells, through the secretion of suppressing cytokines or by other mechanisms to be elucidated. Oral tolerance induction to auto-antigens is also a successful experimental strategy under study. Current research aims to control peripheral tolerance in rheumatoid arthritis patients.

Repeated stimulation of CD4 effector T cells can limit their protective function

Chronic infections often result in CD8 T-cell deletion or functional nonresponsiveness. However, to date no definitive studies have attempted to determine the impact of repeated T cell receptor stimulation on CD4 effector T cell generation. We have determined that when antigen presentation is limited to 2 d, optimum in vitro CD4 effector generation is achieved. Alternatively, repeated stimulation results in decreased CD4 effector expansion, decreased cytokine production, and altered migration. Similarly, functionally impaired effectors develop in vivo when antigen-pulsed antigen-presenting cells are replenished every 24 h during a primary immune response. CD4 effectors that are generated with repeated stimulation provide no protection during influenza infection, and have an impaired ability to provide cognate help to B cells. These results suggest that duration of antigen presentation dictates CD4 effector function, and repeated T cell receptor stimulation in vitro and in vivo that exceeds an optimal threshold results in effectors with impaired function.

Interactions of Haemophilus ducreyi and purified cytolethal distending toxin with human monocyte-derived dendritic cells, macrophages and CD4 + T cells

To evaluate the early stages of the host response to chancroid bacterium Haemophilus ducreyi, we investigated the in vitro responses of monocyte-derived dendritic cells (DCs) and macrophages (MQs) to this pathogen and Haemophilus influenzae. The phagocytic activities and pro-inflammatory cytokine secretion profiles of the antigen-presenting cells (APCs) were analyzed after exposure to gentamycin-killed bacteria, H. ducreyi lipooligosaccharide (LOS), and purified cytolethal distending toxin (HdCDT). T-cell proliferation and cytokine release were examined after co-culturing isolated autologous CD4 + T cells with antigen-pulsed APCs. Both the DCs and MQs phagocytosed H. ducreyi and H. influenzae, as estimated by flow cytometry. All of the strains induced APC secretion of TNF-α, IL-6, IL-8, and IL-12, as measured by ELISA. Other human cells, particularly endothelial cells and fibroblasts, also produced cytokines when stimulated with these bacteria. Purified LOS at concentration 1 μg/ml induced two to threefold lower levels of cytokines than the whole bacteria, which indicates that other components are involved in immune activation. HdCDT inhibited partially the production of the aforementioned cytokines. High levels of IFN-γ, but not of IL-4 and IL-13, were secreted by T cells after activation by either DCs or MQs that were pre-exposed to bacteria, indicating the Th1 nature of the immune response. The levels of T-cell proliferation induced by H. ducreyi were lower than those induced by H. influenzae. HdCDT-treated APCs did not display cytokine responses or T-cell proliferation. These results indicate that HdCDT intoxication, which results in progressive apoptosis of APCs, may hamper early stage immune responses.

Itk Functions to Control Actin Polymerization at the Immune Synapse through Localized Activation of Cdc42 and WASP

Actin polymerization at the immune synapse is required for T cell activation and effector function; however, the relevant regulatory pathways remain poorly understood. We showed previously that binding to antigen presenting cells (APCs) induces localized activation of Cdc42 and Wiskott-Aldrich Syndrome protein (WASP) at the immune synapse [1]. Several lines of evidence suggest that Tec kinases could interact with WASP-dependent actin regulatory processes [2]. Since T cells from Rlk−/−, Itk−/−, and Rlk−/− × Itk−/− mice have defects in signaling and development [3], we asked whether Itk or Rlk function in actin polymerization at the immune synapse. We find that Itk−/− and Rlk−/− × Itk−/− T cells are defective in actin polymerization and conjugate formation in response to antigen-pulsed APCs. Itk functions downstream of the TCR, since similar defects were observed upon TCR engagement alone. Using conformation-specific probes, we show that although the recruitment of WASP and Arp2/3 complex to the immune synapse proceeds normally, the localized activation of Cdc42 and WASP is defective. Finally, we find that the defect in Cdc42 activation likely stems from a requirement for Itk in the recruitment of Vav to the immune synapse. Our results identify Itk as a key element of the pathway leading to localized actin polymerization at the immune synapse.

Rap1 functions as a key regulator of T-cell and antigen-presenting cell interactions and modulates T-cell responses.

Activation of T cells by antigen requires adhesive interactions with antigen-presenting cells (APC) in which leukocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecules (ICAMs) are important. However, it is not well understood what signaling molecules regulate this process and how the modulation of adhesive events influences T-cell activation. Here we show that Rap1 is activated in T cells in an antigen-dependent manner and accumulated at the contact site of T-cell and antigen-loaded APC. Inhibition of Rap1 activation by a dominant-negative Rap1 or SPA-1, a Rap1 GTPase-activating protein, abrogates LFA-1-ICAM-1-mediated adhesive interactions with antigen-pulsed APC and the subsequent T-cell-receptor triggering and interleukin-2 production. Conversely, augmented antigen-dependent Rap1 activation by the expression of wild-type Rap1 enhances these responses but culminates in apoptosis by Fas and FasL. Thus, Rap1 functions as a key regulator of T-cell and APC interactions and modulates T-cell responses from productive activation to activation-induced cell death by regulating the strength of adhesive interactions. Moreover, constitutive Rap1 activation rendered T cells unresponsive with accumulation of p27(Kip1). Our study indicates that the activation state of Rap1 has a decisive effect on the T-cell response to antigen.

Autologous Mixed Lymphocyte Responses in Experimentally-induced Arthritis of the Lewis Rat

Lewis rats develop immune-mediated arthritis following injection with a variety of agents including bovine type II collagen (bCII), mycobacteria, muramyl dipeptide and CP20961. Since susceptibility to experimentally-induced arthritis has been linked to the genes encoding the major histocompatibility complex, it is hypothesized that antigen presentation to autoreactive T-cells is a critical event in the pathogenesis of disease. T-cells, isolated from Lewis rats immunized with bCII or mycobacteria, were co-cultured with splenic or thymic antigen presenting cells (APC) and proliferative responses to antigen were assessed by 3 H-thymidine incorporation. T-cell proliferation was observed upon culture with APC without requiring the addition of antigen. T-cells from rats injected with non-immunogenic adjuvants also demonstrated an increased autologous MLR compared to T-cells from non-injected animals. In contrast, T-cells from animals immunized with non-arthritogenic antigens, including ovalbumin or tetanus toxoid, proliferated only when co-cultured with specific antigen-pulsed APC. These results suggest that immunization with arthritogens activates a population of self-reactive T-cells, which respond in an autologous MLR. We propose that these autoreactive T-cells recognize endogenously-derived self peptides rather than peptides derived from a joint autoantigen.

T cell lines generated with type II collagen proliferate in an autologous mixed lymphocyte response.

Collagen-induced arthritis (CIA) is a T cell-dependent disease induced in susceptible rodents by immunizing with bovine type II collagen (bCII). In order to study T cell responses, a programme to generate bCII-specific T cell lines from arthritic rats was initiated. Lymph node cells from bCII-immune WA/KIR/kcl rats were cultured with bCII in vitro, and the T cells were isolated and restimulated with bCII-pulsed antigen presenting cells (APC) (thymus cells or splenic low density cells). However, T cells, generated initially to bCII, subsequently proliferated upon co-culture with syngeneic APC even in the absence of bCII. This suggests that exposure to bCII resulted in the activation of a population of self-reactive T cells which proliferate in an autologous mixed lymphocyte response. In contrast, short-term T cell lines generated to ovalbumin, heat-denatured bCII and the collagen peptide bCII(184-198) proliferated in response to specific antigen-pulsed APC without demonstrating self-reactivity. Since denatured bCII and bCII(184-198) peptide are not arthritogenic and failed to generate self reactivity in vitro, this suggests that the native triple helical conformation of bCII is required for stimulating autoreactive T cell responses.

The adjuvant monophosphoryl lipid A increases the function of antigen-presenting cells.

The induction of immune responses in vivo is typically performed with antigens administered in external adjuvants, like alum, complete Freund's adjuvant, LPS and, more recently, monophosphoryl lipid A (MPL). However, the role of the adjuvant is still poorly defined. The aim of this study was to test whether the MPL affects the function of antigen-presenting cells (APC) in vitro and in vivo. Antigen-pulsed APC [including macrophages, B cells and dendritic cells (DC)] were incubated or not with MPL, and their ability to sensitize naive T cells was tested in vitro and in vivo. The data show that MPL enhances the ability of macrophages and B cells to sensitize naive T cells, and confers to them the capacity to induce the development of T(h)1 and T(h)2. Administration of MPL i.v. in mice results in the redistribution of fully mature DC in the T cell area of the spleen. These observations suggest that MPL may induce an antigen-specific primary immune response by provoking the migration and maturation of DC that are the physiological adjuvant of the immune system.

Priming to mycobacterial antigen in vivo using antigen-pulsed antigen presenting cells generated in vitro is influenced by the dose and presence of IL-4 in APC cultures.

Antigen presenting cells (APC) similar to immature dendritic cells can be generated in vitro from bone marrow precursors. The authors have compared the yield, the phenotype and the function of murine bone marrow cells cultured for 7 or 11 days in either granulocyte macrophage colony stimulating factor alone (GM BMAPC) or in combination with interleukin-4 (GM/IL-4 BMAPC). The results showed that GM/IL-4 BMAPC expressed the highest levels of MHC Class 2 molecules, CD86/B7-2 and CD80/B7-1 co-stimulatory molecules and the lowest levels of F4/80 macrophage marker. However, when these APC were pulsed with BCG culture filtrate antigen or PPD they were not correspondingly more effective at stimulating activated T lymphocytes in vitro or priming naive T lymphocytes in vivo. Also, in contrast to GM BMAPC, high backgrounds recorded following injections of GM/IL-4 BMAPC without antigen were not consistently reduced by lowering the dose and irradiating the cells prior to administration. The authors conclude that the degree of maturity of BMAPC varies with culture conditions and that this may be an important consideration where BMAPC are to be used in vivo in immunotherapeutic regimens.

CD4-mediated and CD8-mediated cytotoxic and proliferative immune responses to Toxoplasma gondii in seropositive humans.

Both CD4+ and CD8+ cytotoxic T lymphocytes (CTL) are part of the human immune response to Toxoplasma gondii infection. To further our understanding of Toxoplasma immunity, we investigated factors influencing stimulation of CD4+ or CD8+ human T. gondii-specific immune cells. Both antigen-pulsed and Toxoplasma-infected antigen-presenting cells (APC) induced cell proliferation. Toxoplasma-infected APC elicited strong proliferation of CD4+ cells, but little or no proliferation of CD8+ cells, unless high antigen loads were used. Toxoplasma-infected APC stimulated specific cytotoxicity poorly or not at all, owing to death of stimulated cultures, whereas antigen-pulsed APC strongly elicited specific cytotoxicity. Cytotoxicity elicited by either type of APC resided exclusively in CD4+ T cells in polyclonal cultures. Thus, Toxoplasma-infected APC elicited stronger CD4-mediated than CD8-mediated cell proliferation and generated CD4+ CTL more readily than CD8+ CTL. Nonetheless, specific CD8+ memory cells were demonstrated, and rare CD8+ Toxoplasma-specific CTL were subcloned. Fixed Toxoplasma-infected APC (which induce CD8+ CTL) also elicited cell proliferation, but polyclonal cultures stimulated with these infected APC did not die. Unfixed Toxoplasma-infected APC strongly inhibited phytohemagglutinin-induced cell proliferation, whereas fixed APC did not. These data suggested that infected APC were inhibitory or lethal to some immune cells. Further investigations into interactions between immune cells and Toxoplasma-infected cells likely will help elucidate factors involved in the immunopathogenesis of Toxoplasma infection. As other intracellular parasites, including Plasmodium spp. and Leishmania spp., also elicit CD4+ CTL, such work may help establish paradigms governing immunity to intracellular parasites.

Regulation of CD40 ligand expression on naive CD4 T cells: a role for TCR but not co-stimulatory signals.

We have investigated the roles of TCR and accessory co-stimulatory signals in the induction of CD40 ligand (CD40L) on CD4 cells. Using naive T cells form TCR transgenic mice, specific for a peptide of pigeon cytochrome c, we show that in contrast to IL-2 secretion, CD40L expression is regulated primarily by signalling through the TCR, is enhanced by accessory molecule interactions, but co-stimulatory signals play little if any role. CD40L was induced at high levels on naive T cells, peaking at 5 h, by class II MHC+ fibroblast antigen-presenting cells (APC) which expressed either ICAM-1, B7-1 or both molecules, whereas only low levels were induced by fibroblasts which did not express any accessory molecules. Differences in intensity and duration of expression were seen following stimulation with ICAM- and B7-expressing APC, with the presence of ICAM resulting in greater and longer expression, although both molecules together were most efficient. The involvement of co-stimulatory signals delivered from accessory molecules was investigated in systems where there was no effect on TCR signalling from adhesive interactions. Anti-CD3, or antigen-pulsed APC lacking accessory molecules, were used to provide the TCR signal, with co-stimulus from either anti-CD28 or accessory molecule-expressing fibroblasts not presenting antigen. Anti-CD3 in the absence of co-stimuli induced high CD40L expression but no IL-2 production and provision of co-stimulatory signals, although inducing large quantities of IL-2, did not increase CD40L expression. In addition, low CD40L expression induced by antigen presented in the absence of accessory molecules was not enhanced by co-stimulation, although IL-2 was strongly up-regulated. These studies suggest that efficient expression of CD40L on naive CD4 cells does require accessory molecules on APC. However, the role of these molecules for CD40L induction, as opposed to IL-2 secretion, is not one of co-stimulation but one of adhesion, presumably allowing stronger or more prolonged signals to be generated through the TCR. The synergistic role of ICAM and B7 during naive CD4 activation was confirmed using dendritic cells as APC, with nearly complete inhibition of CD40L expression as well as IL-2 secretion being seen when both CTLA-4-Ig and anti-LFA-1 were used to block these molecules.

Processing and presentation of type II collagen, a fibrillar autoantigen, by H-2q antigen-presenting cells.

Native type II collagen (CII) is a high molecular-weight fibrillar molecule which induces a chronic polyarthritis in mice expressing the H-2q haplotype. The present study was initiated to analyze the processing and the presentation of this nonglobular protein by H-2q antigen-presenting cells (APC). Efficiency of presentation was assessed by the ability of antigen-pulsed APC to activate collagen-specific CD4+ T cell hybridomas. Fixation of APC or the presence of chloroquine completely blocked the reactivity of the T cell hybrids to native, denatured and cyanogen bromide (CB) degraded CII, thus indicating the requirement of intracellular processing for adequate presentation of CII peptides to T cells. In the presence of various processing inhibitors (brefeldin A, leupeptin and N-tosyl-L-phenylalanine chloromethylketone) stimulation of T hybrids by CII-pulsed APC was reduced, pointing to the need of newly synthesized class II molecules, the use of several intracellular compartments and the implications of different proteases in the generation of CII peptides. Peritoneal macrophages and, to a lesser extent, total spleen cells, presented native and denatured CII with higher efficiency than purified splenic dendritic cells, naive or even immune B cells from CII-primed mice. In contrast, these dendritic and B cells were fully competent to present intact ovalbumin to a specific T cell hybrid. The stimulation by dendritic cells and immune B cells was greater when CB peptides of CII were added instead of the native molecule. Similarly, the cleavage of CII was an absolute requirement for its presentation by epidermal cells and B cell lymphomas to the T cell hybridomas. Taken together, these findings emphasize the crucial role of intracellular processing for recognition of soluble CII, similar to most antigens. However, in contrast to ovalbumin, the size and fibrillar nature of the native CII molecule influences its capture by the APC, thus limiting the type of APC able to present this antigen.

Normal HBsAg presentation and T-cell defect in the immune response of nonresponders.

Lymphocytes from nonresponders to HBsAg fail to proliferate in vitro in the presence of HBsAg-pulsed antigen presenting cells. We studied four pairs of major histocompatibility complex (MHC)-matched, mixed lymphocyte reaction-negative individuals discordant for HBsAg response. For each pair, responder lymphocytes proliferated in the presence of nonresponder antigen-pulsed antigen presenting cells. Responder and nonresponder antigen presenting cells were equally effective. There was no evidence for inhibition of responder T-cell proliferation by nonresponder lymphocytes or antigen presenting cells. The defect is thus in the helper T cells of nonresponders and not in the antigen processing or binding of processed peptides to MHC molecules on antigen presenting cells.

Ocular immune privilege in the immunosuppressive intraocular microenvironment

Immune privilege exists in the normal eye (anterior chamber, vitreous cavity, subretinal space): when foreign tissue/cellular grafts are placed in the eye, rejection is largely, if not completely, avoided. The ineffectiveness of the immune response in this circumstance results from eye-dependent alterations in (a) the induction of systemic immunity to graft antigens, and (b) the expression of cell-mediated immunity within the eye. Recent evidence indicates that eye-derived factors, present in the intraocular microenvironment, modify the afferent and efferent limbs of the immune response. Studies of aqueous humor as a prototype of the intraocular microenvironment have demonstrated that this fluid profoundly inhibits antigen-driven T cell activation such that proliferation and lymphokine production are curtailed. Moreover, aqueous humor modifies the functional properties of conventional antigen presenting cells (APC) such that exogenous antigens are processed uniquely. When antigen-pulsed APC exposed to aqueous humor are injected intravenously, they induce Anterior Chamber Associated Immune Deviation (ACAID). Several factors in aqueous humor have been implicated as immunosuppressants: transforming growth factorbeta, alpha-melanocyte stimulating hormone, vasoactive intestinal peptide, calcitonin gene related peptide, and hydrocortisone (because Cortisol binding globulin is virtually absent). In certain experimentally-induced ocular disorders, resident ocular cells fail to secrete immunosuppressive factors constitutively, and this may explain why immune privilege is also lost.

Truncation of the A alpha chain of MHC class II molecules results in inefficient antigen presentation to antigen-specific T cells.

Antigen presenting cells (APC) expressing MHC class II molecules composed of chains with part or all of the cytoplasmic domains deleted are inefficient at presenting hen egg lysozyme peptides to antigen specific T cell hybrids compared with APC that express wild-type MHC class II molecules. This effect is most apparent for mutants in which the alpha chain has been truncated. The inefficiency in antigen presentation can be amplified by pulsing the APC for 4 h with peptide rather than having peptide present throughout the presentation assay. Fixation of antigen-pulsed APC improves the capacity of APC with truncated class II molecules to stimulate T cell hybrids. Fixation of APC prior to exposure to antigen also leads to significant improvement in antigen presentation by the truncated class II molecules. Because the inefficiency of a given hybrid for antigen presentation does not correlate with its ability to transduce a signal as measured by protein kinase C translocation, we suggest that defects in this pathway are not the only cause of impaired antigen presentation. However, because previous studies have demonstrated the need for an intact cytoskeleton for successful antigen presentation, we propose that the carboxy truncated class II molecules are inefficient in antigen presentation because they are unable to generate the signal that ultimately leads to their interaction with the cytoskeleton. These observations underscore the complexity of the events that are required for achieving effective interactions between MHC class II molecules and TCR, and suggest, with regard to efficient antigen presentation, that the physical state of the class II molecules is at least as important as their signal transducing capacity.