Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity

Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4⁺ T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4⁺ T cell epitopes relevant in health and disease.Human Leukocyte Antigen (HLA)¹ class II molecules on professional antigen presenting cells such as dendritic cells (DC) expose peptide fragments derived from exogenous and endogenous proteins to be screened by CD4⁺ T cells (1, 2). The activation and recruitment of CD4⁺ T cells recognizing disease-related peptide antigens is critical for the development of efficient antipathogen or antitumor immunity. Furthermore, the presentation of self-peptides and their interaction with CD4⁺ T cells is essential to maintain immunological tolerance and homeostasis (3). Knowledge of the nature of HLA class II-presented peptides on DC is of great importance to understand the rules of antigen processing and peptide binding motifs (4), whereas the identity of disease-related antigens may provide new knowledge on immunogenicity and leads for the development of vaccines and immunotherapy (5, 6).Mass spectrometry (MS) has proven effective for the analysis HLA class II-presented peptides (4, 7, 8). MS-based ligandome studies have demonstrated that HLA class II molecules predominantly present peptides derived from exogenous proteins that entered the cells by endocytosis and endogenous proteins that are associated with the endo-lysosomal compartments (4). Yet proteins residing in the cytosol, nucleus or mitochondria can also be presented by HLA class II molecules, primarily through autophagy (9–11). Multiple studies have mapped the HLA class II ligandome of antigen presenting cells in the context of infectious pathogens (12), autoimmune diseases (13–17) or cancer (14, 18, 19), or those that are essential for self-tolerance in the human thymus (3, 20). Notwithstanding these efforts, and certainly not in line with the extensive knowledge on the HLA class I ligandome (21), the nature of the HLA class II-presented peptide repertoire and particular its relationship to the cellular source proteome remains poorly understood.To advance our knowledge on the HLA-DR ligandome on activated DC without having to deal with limitations in cell yield from peripheral human blood (12, 21, 22) or tissue isolates (3), we explored the use of MUTZ-3 cells. This cell line has been used as a model of human monocyte-derived DCs. MUTZ-3 cells can be matured to act as antigen presenting cells and express then high levels of HLA class II molecules, and can be propagated in vitro to large cell densities (23–25). We also evaluated the performance of complementary and hybrid MS fragmentation techniques electron-transfer dissociation (ETD), electron-transfer/higher-energy collision dissociation (EThcD) (26), and higher-energy collision dissociation (HCD) to sequence and identify the HLA class II ligandome. Together this workflow allowed for the identification of an unprecedented large set of about 14 thousand unique peptide sequences presented by DC derived HLA-DR molecules, providing an in-depth view of the complexity of the HLA class II ligandome, revealing underlying features of antigen processing and surface-presentation to CD4⁺ T cells.     

LFA-1 Regulates CD8+ T Cell Activation via T Cell Receptor-mediated and LFA-1-mediated Erk1/2 Signal Pathways

LFA-1 regulates T cell activation and signal transduction through the immunological synapse. T cell receptor (TCR) stimulation rapidly activates LFA-1, which provides unique LFA-1-dependent signals to promote T cell activation. However, the detailed molecular pathways that regulate these processes and the precise mechanism by which LFA-1 contributes to TCR activation remain unclear. We found LFA-1 directly participates in Erk1/2 signaling upon TCR stimulation in CD8⁺ T cells. The presence of LFA-1, not ligand binding, is required for the TCR-mediated Erk1/2 signal pathway. LFA-1-deficient T cells have defects in sustained Erk1/2 signaling and TCR/CD3 clustering, which subsequently prevents MTOC reorientation, cell cycle progression, and mitosis. LFA-1 regulates the TCR-mediated Erk1/2 signal pathway in the context of immunological synapse for recruitment and amplification of the Erk1/2 signal. In addition, LFA-1 ligation with ICAM-1 generates an additional Erk1/2 signal, which synergizes with the existing TCR-mediated Erk1/2 signal to enhance T cell activation. Thus, LFA-1 contributes to CD8⁺ T cell activation through two distinct signal pathways. We demonstrated that the function of LFA-1 is to enhance TCR signaling through the immunological synapse and deliver distinct signals in CD8⁺ T cell activation.Leukocyte function-associated antigen-1 (LFA-1)² plays an important role in regulating leukocyte adhesion and T cell activation (1, 2). LFA-1 consists of the αL (CD11a) and β2 (CD18) subunits. The ligands for LFA-1 include intercellular adhesion molecular-1 (ICAM-1), ICAM-2, and ICAM-3 (3). LFA-1 participates in the formation of the immunological synapse, which regulates T cell activation synergistically with TCR engagement. The immunological synapse is a specialized structure that forms between the T cell and the APC or target cell (1, 2, 4). The function of the immunological synapse is to facilitate T cell activation and signal transduction. Mice deficient in LFA-1 (CD11a KO) have defects in leukocyte adhesion, lymphocyte proliferation, and tumor rejection (5–7).Upon TCR stimulation, the nascent immunological synapse is initiated with surface receptor clustering and cytoskeleton rearrangement, then followed by mature synapse formation after prolonged stimulation (8, 9). In the mature immunological synapse, LFA-1 forms a ring-like pattern at the peripheral supramolecular activation cluster (pSMAC), which surrounds the central supramolecular activation cluster (cSMAC) containing TCR/CD3/lipid rafts (10, 11). The structure of the mature synapse is stable for hours and thought to be important for sustained TCR signaling (12–14). LFA-1 functions via pSMAC to stabilize the cSMAC and is associated with the induction of T cell proliferation, cytokine production, and lytic granule migration toward cSMAC (1, 15). Although LFA-1-containing pSMAC is self-evident in lipid bilayer systems and cell lines, whether it is required for T cell activation under physiological conditions remains controversial (15).TCR stimulation rapidly induces the functional activity of LFA-1, which then provides unique LFA-1-dependent signals to promote T cell activation (16). The process can be divided into two steps. First, the intracellular signaling from TCR regulating LFA-1 activation is known as “inside-out” signaling; second, activated LFA-1, as a signaling receptor, can feedback to transduce the intracellular signal, the “outside-in” signaling (1, 17). It is widely accepted that TCR stimulation activates LFA-1 through affinity and/or avidity regulation, as supported by increased adhesion to ICAM-1 and pSMAC formation (16, 17). The “inside-out” signal process has been investigated extensively (18–21). The TCR proximal signal molecules, Lck, ZAP-70, and PI3K, are known to be important for TCR signaling to LFA-1 activation (22–26). The molecular mechanisms of LFA-1 “outside-in” signaling have been explored only recently. Perez et al. (27) have demonstrated that LFA-1 and ICAM-1 ligation activates the downstream Erk1/2 MAPK signaling pathway upon TCR stimulation, which ultimately leads to the qualitative modulation of CD4⁺ T cell activation through distinct LFA-1-dependent signals. Another recent study provided compelling evidence that LFA-1 reshapes the Ras MAPK pathway downstream of TCR (28). However, the detailed molecular pathways that regulate these processes are poorly defined. Especially, the evidence in support of a distinctive role for LFA-1 in the T cell signaling pathway has lagged behind; whether the function of LFA-1 is to enhance TCR signaling through the immunological synapse and/or deliver distinct signal in T cell activation and whether LFA-1 is indispensable for or merely assists the existing TCR signal pathway. Furthermore, whether and how TCR proximal signal molecules regulate LFA-1 function remains unknown. Further studies are required to understand the LFA-1 and TCR signaling network.In this study, we found that LFA-1 directly participates in CD8⁺ T cell activation. Upon TCR stimulation, LFA-1 regulates both TCR-mediated and LFA-1-mediated Erk1/2 signal pathways. First, the presence of LFA-1, not ligand binding, is required for the sustained Erk1/2 signaling and TCR/CD3 clustering on the surface of CD8⁺ T cells, subsequently leading to MTOC reorientation, cell cycle progression, and mitosis. Second, LFA-1 ligation with ICAM-1 enhances Erk1/2 signaling, which promotes T cell activation with increased IL-2 production and cell proliferation. This LFA-1-mediated Erk1/2 signal pathway integrates with the existing TCR-mediated Erk1/2 signal pathway to enhance T cell activation.     

Vaccine-Induced,Pseudorabies Virus-Specific,Extrathymic CD4+CD8+ Memory T-Helper Cells in Swine

Pseudorabies virus (PRV; suid herpesvirus 1) infection causes heavy economic losses in the pig industry. Therefore, vaccination with live attenuated viruses is practiced in many countries. This vaccination was demonstrated to induce extrathymic virus-specific memory CD4⁺CD8⁺ T lymphocytes. Due to their major histocompatibility complex (MHC) class II-restricted proliferation, it is generally believed that these T lymphocytes function as memory T-helper cells. To directly prove this hypothesis, 15-amino-acid, overlapping peptides of the viral glycoprotein gC were used for screening in proliferation assays with peripheral blood mononuclear cells of vaccinated d/d haplotype inbred pigs. In these experiments, two naturally processed T-cell epitopes (T1 and T2) which are MHC class II restricted were identified. It was shown that extrathymic CD4⁺CD8⁺ T cells are the T-lymphocyte subpopulation that responds to epitope T2. In addition, we were able to show that cytokine secretion can be induced in these T cells through recall with inactivated PRV and demonstrated that activated PRV-primed CD4⁺CD8⁺ T cells are able to induce PRV-specific immunoglobulin synthesis by PRV-primed, resting B cells. Taken together, these results demonstrate that the glycoprotein gC takes part in the priming of humoral anti-PRV memory responses. The experiments identified the first T-cell epitopes so far known to induce the generation of virus-specific CD4⁺CD8⁺ memory T lymphocytes and showed that CD4⁺CD8⁺ T cells are memory T-helper cells. Therefore, this study describes the generation of virus-specific CD4⁺CD8⁺ T cells, which is observed during vaccination, as a part of the potent humoral anti-PRV memory response induced by the vaccine.Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, is the causative agent of Aujeszky’s disease. This disease is lethal to young pigs and causes important economic losses (52). Therefore, vaccination of pigs is practiced in many countries.Several humoral immune system effector mechanisms are involved in the protection of pigs from PRV infection. Virus-neutralizing antibodies, antibodies mediating antibody-dependent cell-mediated cytotoxicity, and antibodies mediating complement-mediated lysis of PRV-infected target cells have been demonstrated (22, 23, 53, 54). The main targets of this humoral immune response were shown to be the viral glycoproteins (3, 45), and passive immunization with monoclonal antibodies (MAbs) against gB, gC, and gD protects pigs from a lethal challenge (20, 49).The protection conferred through cell-mediated immunity is poorly understood. An increase in major histocompatibility complex (MHC)-unrestricted cell-mediated cytotoxicity against uninfected and PRV-infected cells has been detected after infection or vaccination of pigs with PRV (16, 53, 54), and specific cellular immune responses to PRV infections could be demonstrated by stimulation of proliferation and lymphokine secretion of porcine PRV-immune lymphocytes (10, 17, 42, 43, 51) as well as by the detection of PRV-specific cytotoxic lymphocytes (21, 56).There are some difficulties in defining more precisely the impact of cell-mediated immune effector mechanisms to protection from PRV-infection and their interplay with the observed humoral immune response. Considerably fewer porcine than human or mouse differentiation markers are available (34). In addition, the immune system of swine differs considerably from that of humans and mice. The pig has a substantial number of CD4⁻CD8⁻ T lymphocytes in the peripheral blood (4, 6, 12, 36, 39). In young animals, this subpopulation of T lymphocytes comprises up to 60% of the T lymphocytes and contains mainly γδ T lymphocytes. The pig is also the only species so far known to contain a substantial number of resting extrathymic CD4⁺CD8⁺ T lymphocytes (28, 36, 39). This T-lymphocyte population shows morphologically the phenotype of mature T lymphocytes (40) and increases with age to up to 60% of peripheral T lymphocytes (29, 35, 39, 55). Further, it was demonstrated that CD4⁺CD8⁺ T lymphocytes comprise memory T cells which proliferate upon stimulation with recall antigen (43, 55). Since the observed proliferative response was shown to be MHC class II-restricted, it was speculated that the porcine CD4⁺CD8⁺ T-cell subset contains memory T-helper lymphocytes (43). However, the ability of these T lymphocytes to secrete cytokines or to provide help to B cells has so far not been demonstrated.To gain a better understanding of immune effector mechanisms conferring protection from PRV infection, the function of these unusual extrathymic T-lymphocyte subsets has to be elucidated. In the present study, we identified two T-cell epitopes on glycoprotein gC which are primed during vaccination of d/d haplotype inbred pigs (41) against PRV and demonstrated that MHC class II-restricted, peripheral CD4⁺CD8⁺ memory T lymphocytes are the responding T lymphocytes. We were further able to show that PRV-specific, extrathymic CD4⁺CD8⁺ T lymphocytes are able to secrete cytokines and have the capacity to stimulate the secretion of PRV-specific immunoglobulins (Ig) by PRV-primed B cells. These results demonstrate that porcine CD4⁺CD8⁺ T lymphocytes can function as memory T-helper cells and can direct humoral anti-PRV memory responses.     

The Cell Tropism of Human Immunodeficiency Virus Type 1 Determines the Kinetics of Plasma Viremia in SCID Mice Reconstituted with Human Peripheral Blood Leukocytes

Most individuals infected with human immunodeficiency virus type 1 (HIV-1) initially harbor macrophage-tropic, non-syncytium-inducing (M-tropic, NSI) viruses that may evolve into T-cell-tropic, syncytium-inducing viruses (T-tropic, SI) after several years. The reasons for the more efficient transmission of M-tropic, NSI viruses and the slow evolution of T-tropic, SI viruses remain unclear, although they may be linked to expression of appropriate chemokine coreceptors for virus entry. We have examined plasma viral RNA levels and the extent of CD4⁺ T-cell depletion in SCID mice reconstituted with human peripheral blood leukocytes following infection with M-tropic, dual-tropic, or T-tropic HIV-1 isolates. The cell tropism was found to determine the course of viremia, with M-tropic viruses producing sustained high viral RNA levels and sparing some CD4⁺ T cells, dual-tropic viruses producing a transient and lower viral RNA spike and extremely rapid depletion of CD4⁺ T cells, and T-tropic viruses causing similarly lower viral RNA levels and rapid-intermediate rates of CD4⁺ T-cell depletion. A single amino acid change in the V3 region of gp120 was sufficient to cause one isolate to switch from M-tropic to dual-tropic and acquire the ability to rapidly deplete all CD4⁺ T cells.The envelope gene of human immunodeficiency virus type 1 (HIV-1) determines the cell tropism of the virus (11, 32, 47, 62), the use of chemokine receptors as cofactors for viral entry (4, 17), and the ability of the virus to induce syncytia in infected cells (55, 60). Cell tropism is closely linked to but probably not exclusively determined by the ability of different HIV-1 envelopes to bind CD4 and the CC or the CXC chemokine receptors and initiate viral fusion with the target cell. Macrophage-tropic (M-tropic) viruses infect primary cultures of macrophages and CD4⁺ T cells and use CCR5 as the preferred coreceptor (2, 5, 15, 23, 26, 31). T-cell-tropic (T-tropic) viruses can infect primary cultures of CD4⁺ T cells and established T-cell lines, but not primary macrophages. T-tropic viruses use CXCR4 as a coreceptor for viral entry (27). Dual-tropic viruses have both of these properties and can use either CCR5 or CXCR4 (and infrequently other chemokine receptors [25]) for viral entry (24, 37, 57). M-tropic viruses are most frequently transmitted during primary infection of humans and persist throughout the duration of the infection (63). Many, but not all, infected individuals show an evolution of virus cell tropism from M-tropic to dual-tropic and finally to T-tropic with increasing time after infection (21, 38, 57). Increases in replicative capacity of viruses from patients with long-term infection have also been noted (22), and the switch to the syncytium-inducing (SI) phenotype in T-tropic or dual-tropic isolates is associated with more rapid disease progression (10, 20, 60). Primary infection with dual-tropic or T-tropic HIV, although infrequent, often leads to rapid disease progression (16, 51). The viral and host factors that determine the higher transmission rate of M-tropic HIV-1 and the slow evolution of dual- or T-tropic variants remain to be elucidated (4).These observations suggest that infection with T-tropic, SI virus isolates in animal model systems with SCID mice grafted with human lymphoid cells or tissue should lead to a rapid course of disease (1, 8, 44–46). While some studies in SCID mice grafted with fetal thymus and liver are in agreement with this concept (33, 34), our previous studies with the human peripheral blood leukocyte-SCID (hu-PBL-SCID) mouse model have shown that infection with M-tropic isolates (e.g., SF162) causes more rapid CD4⁺ T-cell depletion than infection with T-tropic, SI isolates (e.g., SF33), despite similar proviral copy numbers, and that this property mapped to envelope (28, 41, 43). However, the dual-tropic 89.6 isolate (19) caused extremely rapid CD4⁺ T-cell depletion in infected hu-PBL-SCID mice that was associated with an early and transient increase in HIV-1 plasma viral RNA (29). The relationship between cell tropism of the virus isolate and the pattern of disease in hu-PBL-SCID mice is thus uncertain. We have extended these studies by determining the kinetics of HIV-1 RNA levels in serial plasma samples of hu-PBL-SCID mice infected with primary patient isolates or laboratory stocks that differ in cell tropism and SI properties. The results showed significant differences in the kinetics of HIV-1 replication and CD4⁺ T-cell depletion that are determined by the cell tropism of the virus isolate.     

An Important Role for Major Histocompatibility Complex Class I-Restricted T Cells,and a Limited Role for Gamma Interferon,in Protection of Mice against Lethal Herpes Simplex Virus Infection

Herpes simplex virus (HSV) inhibits major histocompatibility complex (MHC) class I expression in infected cells and does so much more efficiently in human cells than in murine cells. Given this difference, if MHC class I-restricted T cells do not play an important role in protection of mice from HSV, an important role for these cells in humans would be unlikely. However, the contribution of MHC class I-restricted T cells to the control of HSV infection in mice remains unclear. Further, the mechanisms by which these cells may act to control infection, particularly in the nervous system, are not well understood, though a role for gamma interferon (IFN-γ) has been proposed. To address the roles of MHC class I and of IFN-γ, C57BL/6 mice deficient in MHC class I expression (β2 microglobulin knockout [β2KO] mice), in IFN-γ expression (IFN-γKO mice), or in both (IFN-γKO/β2KO mice) were infected with HSV by footpad inoculation. β2KO mice were markedly compromised in their ability to control infection, as indicated by increased lethality and higher concentrations of virus in the feet and spinal ganglia. In contrast, IFN-γ appeared to play at most a limited role in viral clearance. The results suggest that MHC class I-restricted T cells play an important role in protection of mice against neuroinvasive HSV infection and do so largely by mechanisms other than the production of IFN-γ.

Two gene products of herpes simplex virus (HSV) block presentation of viral proteins by class I major histocompatibility complex (MHC) molecules: the viral host shutoff protein (vhs), which is present in the viral particle, and the immediate-early protein ICP47 (1, 14, 41, 42). Through the sequential action of these proteins, antigen presentation by MHC class I is inhibited early in the viral replication cycle. ICP47 binds to human transporter associated with antigen-processing proteins (TAP), thereby inhibiting peptide loading on MHC class I and recognition by HSV-specific, MHC class I-restricted, CD8⁺ T cells (1, 14, 42, 43). This effect is greatest in nonhematopoietic cells in which the abundance of MHC class I and TAP are lower than in antigen-presenting cells (41). As a consequence, HSV is more likely to impair recognition of infected target cells in the tissues than to block the generation of antigen-specific CD8⁺ T cells. Consistent with this, recent studies indicate that HSV antigen-specific CD8⁺ cytotoxic-T-lymphocyte (CTL) precursors can be readily detected in the blood and cutaneous lesions of HSV-infected individuals (16, 31, 32). However, NK cells and HSV antigen-specific CD4⁺ T cells are detected earlier than antigen-specific CD8⁺ T cells in lesions of humans with recurrent HSV-2 disease (16). This finding has led to the proposal that gamma interferon (IFN-γ) produced by infiltrating NK and CD4⁺ T cells overrides the inhibitory effects of HSV on TAP function and MHC class I expression (22, 41), thereby allowing the eradication of virus by CD8⁺ T cells, whose numbers increase in lesions around the time of viral clearance (16, 31). In patients with AIDS, a lower frequency in the blood of HSV antigen-specific CD8⁺ CTL precursors is associated with more frequent and severe recurrences of genital disease (32). These correlative data suggest that CD8⁺ T cells may play an important role in the clearance of HSV in humans, at least from mucocutaneous lesions.ICP47 inhibits murine TAP poorly (1, 42), which may explain the greater ease with which anti-HSV CD8⁺ CTLs have been detected in mice than in humans (3, 8, 28, 34, 35). Despite the weak interaction of ICP47 with murine TAP, results of a recent study (12) suggested that ICP47 impairs CD8⁺ T-cell-dependent viral clearance from the nervous system: CD8⁺ T cells protected susceptible BALB/c or A/J mice from lethal, nervous system infection with an HSV mutant lacking ICP47 but did not appear to protect against infection with wild-type HSV or to contribute to clearance of either virus from the eye. These findings are consistent with data suggesting that CD8⁺ T cells limit persistence of HSV in the spinal ganglia and decrease spread to the central nervous system (35, 36). However, other studies have concluded that CD4⁺ T cells but not CD8⁺ T cells play the critical role in viral clearance and protection from lethal primary infection with wild-type HSV (20, 23, 24) or that either CD4⁺ or CD8⁺ T cells are sufficient for protection (26, 37). Since the effects of ICP47 are likely to be greater in humans than in mice, if MHC class I-restricted CD8⁺ T cells do not play an important role in protection of mice from lethal, neuroinvasive infection due to wild-type HSV, an important role in humans would be unlikely.The mechanisms by which T cells may limit the spread of infection in the nervous system are not clearly understood. Studies by Simmons and colleagues suggested that CD8⁺ T cells may lyse infected Schwann cells or satellite cells but that they probably do not lyse infected neurons (31, 32). They and others have proposed that CD8⁺ T cells protect neurons through the production of cytokines, in particular IFN-γ (35, 36). IFN-γ contributes to the clearance of HSV from mucocutaneous sites (4, 24, 25, 37, 44). However, the role of IFN-γ in protection from lethal, neuroinvasive infection is uncertain and may vary with the strain of mice, method used to inhibit IFN-γ function, and route of inoculation (4, 5, 24, 37, 44). IFN-γ is produced in the ganglia of mice with acute or latent HSV infection (5, 13, 19). Both CD4⁺ and CD8⁺ T cells (and NK cells) produce IFN-γ, but CD4⁺ T cells appear to be the predominant source of IFN-γ following intravaginal infection with HSV (24, 25). Thus, it is possible that the disparity in results regarding the relative importance of CD4⁺ and CD8⁺ T cells in protection from lethal, neuroinvasive HSV infection reflects their redundant roles in production of this cytokine or that IFN-γ and CD8⁺ T cells contribute independently to control of infection in the nervous system.To address in parallel the contributions of MHC class I-restricted T cells and of IFN-γ to protection of mice from HSV, MHC class I and CD8⁺ T-cell-deficient β2 microglobulin knockout (β2KO) mice, IFN-γ knockout (IFN-γKO) mice, and mice deficient in both MHC class I and IFN-γ expression (IFN-γKO/β2KO) were studied. The results indicated that loss of MHC class I expression in β2KO mice substantially increased their susceptibility to HSV, whereas the loss of IFN-γ expression had a much more limited effect. These findings indicate that MHC class I-restricted T cells play an important role in protection against neuroinvasive HSV infection in mice and that they do so largely by mechanisms other than the production of IFN-γ. Though MHC class I expression is more severely impaired in β2KO mice than in human cells infected with wild-type HSV, these findings support the notion that inhibition of MHC class I expression is an important factor in the virulence of this virus.     

Inhibition and Activation by CD244 Depends on CD2 and Phospholipase C-��1

Regulation by the NK and T cell surface receptor CD244 in mice and humans depends both on engagement at the cell surface by CD48 and intracellular interactions with SAP and EAT-2. Relevance to human disease by manipulating CD244 in mouse models is complicated by rodent CD2 also binding CD48. We distinguish between contributions of mouse CD244 and CD2 on engagement of CD48 in a mouse T cell hybridoma. CD2 and CD244 both contribute positively to the immune response as mutation of proline-rich motifs or tyrosine motifs in the tails of CD2 and CD244, respectively, result in a decrease in antigen-specific interleukin-2 production. Inhibitory effects of mouse CD244 are accounted for by competition with CD2 at the cell surface for CD48. In humans CD2 and CD244 are engaged separately at the cell surface but biochemical data suggest a potential conserved intracellular link between the two receptors through FYN kinase. We identify a novel signaling mechanism for CD244 through its potential to recruit phospholipase C-γ1 via the conserved phosphorylated tyrosine motif in the tail of the adaptor protein EAT-2, which we show is important for function.The CD2 family of cell surface receptors is differentially expressed on immune cells (1, 2) and is involved in regulating both innate and adaptive immunity (3). These receptors have related extracellular immunoglobulin superfamily domains and interact either homophilically or heterophilically within the CD2 family (1, 2). The CD2 family contains a subgroup of receptors termed the SLAM family that have a conserved tyrosine signaling motif in their cytoplasmic region TXYXX(I/V) referred to as an immunoreceptor tyrosine-based switch motif (ITSM).² The SLAM family of receptors include CD244 (2B4), NTB-A (Ly-108), CD319 (CRACC, CS-1), CD150 (SLAM), CD84, and CD229 (Ly-9). Defects in signaling and aberrant expression of these receptors have been implicated in several immunodeficiency and autoimmune disorders in humans and mice (4–8). Within the SLAM family, CD244 is unusual in that it shares its ligand CD48 with the receptor CD2 in rodents, whereas in humans CD2 has evolved to interact with CD58 (9). The affinity of CD244 for CD48 in rodents is 6–9-fold higher than the still functionally relevant CD2/CD48 interaction (10). CD244 and CD2 have different cytoplasmic regions comprised of tyrosine motifs or proline-rich motifs, respectively.CD244 is predominantly found on NK cells and cytotoxic T cells and primarily characterized as an activating receptor (11–15). CD2 is found on the same cells as CD244 but is also expressed on all T cells, both activated and resting, and has an activating or costimulatory function upon engagement of ligand (9). The tyrosine motifs found in the cytoplasmic tail of CD244 have been shown to bind the SH2 domains of cytoplasmic adaptor proteins SAP and EAT-2 and FYN kinase (16–18) and are important to its function (5, 19–21). In contrast to SH2 interactions of CD244, several SH3 domain-mediated interactions have been reported for the cytoplasmic region of CD2 including CD2AP/CMS, CIN85, FYN, and LCK (22–26).The activating function of CD244 was called into question when a study using cells from a CD244 knock-out mouse showed that CD244 had an inhibitory effect as loss of CD244 resulted in enhanced NK killing of target cells (27). This suggested that previous results in mice where positive effects were seen may have been due to blocking CD244 ligand engagement as opposed to cross-linking with antibodies against CD244 (27). This has led to proposals that there are differences in function between mouse and human CD244 as there is more evidence to suggest that human CD244 is a positive regulator enhancing cytotoxicity and cytokine production (13, 15, 28). However, other more recent studies have shown the mouse CD244/CD48 interaction to be important for cytokine production and effector functions such as cytotoxicity against tumor targets in CD244-deficient mice (29). Long and short forms of CD244 have been cloned from mice with the short form being described as activating and the long form inhibitory (27, 30). Only the long form of CD244 is present in humans and is regarded as activating (14).Positive signaling by CD244 has been attributed to the recruitment of SAP (18), which is a signaling adaptor molecule comprised of a single SH2 domain encoded by the SH2D1A gene and has the ability to recruit the kinase FYN by binding its SH3 domain (31, 32). Loss of the SAP/FYN interaction can lead to X-linked lymphoproliferative disease in humans (17). The molecular basis of in vitro inhibitory effects observed with CD244 in mice on ligation with mAb or ligand remains elusive (33). Protein tyrosine and inositol phosphatases have been reported to associate with CD244 (18, 19, 34) but our studies using surface plasmon resonance found them to be very weak and unlikely to bind competitively compared with the SAP family of adaptors or FYN (16). The SAP-related adaptor EAT-2 has been reported to have an active inhibitory effect that is dependent on tyrosine motifs in the tail of EAT-2 (35) but its mechanism is not understood. The only interaction reported for the tail of EAT-2 is with FYN kinase and studies overexpressing EAT-2 in a T cell hybridoma resulted in increased IL-2 production upon antigen stimulation (16).The conservation between mouse and human CD244 cytoplasmic regions and associated adaptors suggests that both function in a similar way. We have explored the main difference between mouse and human CD244, which is the extracellular interaction through CD48 ligation in the mouse. This has revealed that inhibitory effects of CD244 ligation in mice can be due to competition between CD244 and CD2 for CD48. We have also found that the adaptor protein EAT-2 binds PLCγ1 providing a molecular basis for the important role CD244 plays in regulating cellular cytotoxicity (13, 36). We demonstrate that there is a potentially shared signaling mechanism through the FYN kinase that links CD2 and CD244 intracellularly even though in humans CD2 and CD244 no longer share a cell surface ligand.     

CD38/cADPR/Ca2+ Pathway Promotes Cell Proliferation and Delays Nerve Growth Factor-induced Differentiation in PC12 Cells

Intracellular Ca²⁺ mobilization plays an important role in a wide variety of cellular processes, and multiple second messengers are responsible for mediating intracellular Ca²⁺ changes. Here we explored the role of one endogenous Ca²⁺-mobilizing nucleotide, cyclic adenosine diphosphoribose (cADPR), in the proliferation and differentiation of neurosecretory PC12 cells. We found that cADPR induced Ca²⁺ release in PC12 cells and that CD38 is the main ADP-ribosyl cyclase responsible for the acetylcholine (ACh)-induced cADPR production in PC12 cells. In addition, the CD38/cADPR signaling pathway is shown to be required for the ACh-induced Ca²⁺ increase and cell proliferation. Inhibition of the pathway, on the other hand, accelerated nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells. Conversely, overexpression of CD38 increased cell proliferation but delayed NGF-induced differentiation. Our data indicate that cADPR plays a dichotomic role in regulating proliferation and neuronal differentiation of PC12 cells.Mobilization of intracellular Ca²⁺ stores is involved in diverse cell functions, including fertilization, cell proliferation, and differentiation (1–4). At least three endogenous Ca²⁺-mobilizing messengers have been identified, including inositol trisphosphate (IP₃),³ nicotinic adenine acid dinucleotide phosphate (NAADP), and cyclic adenosine diphosphoribose (cADPR). Similar to IP₃, cADPR can mobilize calcium release in a wide variety of cell types and species, from protozoa to animals. The cADPR-mediated Ca²⁺ signaling has been indicated in a variety of cellular processes (5–7), from abscisic acid signaling and regulation of the circadian clock in plants, to mediating long-term synaptic depression in hippocampus.Ample evidence shows that the ryanodine receptors are the main intracellular targets for cADPR (1, 2, 8). Ryanodine receptors (RyRs) are intracellular Ca²⁺ channels widely expressed in various cells and tissues, including muscles and neurons. It is the major cellular mediator of Ca²⁺-induced Ca²⁺ release (CICR) in cells. There are three isoforms of ryanodine receptors: RyR1, RyR2, and RyR3, all of which have been implicated in the cADPR signaling (1, 2, 8). However, evidence regarding cADPR acting directly on the receptors is lacking (9). It has been suggested that accessory proteins, such as calmodulin and FK506-binding protein (FKBP), may be involved instead (10–15).cADPR is formed from nicotinamide adenine dinucleotide (NAD) by ADP-ribosyl cyclases. Six ADP-ribosyl cyclases have been identified so far: Aplysia ADP-ribosyl cyclase, three sea urchin homologues (16, 17), and two mammalian homologues, CD38 and CD157 (18). CD38 is a membrane-bound protein and the main mammalian ADP-ribosyl cyclase. As a novel multifunctional enzyme, CD38 catalyzes the synthesis and hydrolysis of both cADPR and NAADP, two structurally and functionally distinct Ca²⁺ messengers. Virtually all mammalian tissues ever examined have been shown to express CD38. CD38 knock-out mice exhibit multiple physiological defects, ranging from impaired immune responses, metabolic disturbances, to behavioral modifications (1, 6, 18).CD38 was originally identified as a lymphocyte differentiation antigen (18). Indeed, CD38/cADPR has been linked to cell differentiation (5). For example, in human HL-60 cells, CD38 expression and the consequential accumulation of cADPR play a causal role in mediating granulocytic differentiation (19). In addition, expression of CD38 in HeLa and 3T3 cells not only increased intracellular Ca²⁺ concentration but also induced cell proliferation by significantly reducing the S phase duration, leading to shortened cell doubling time (20). The ability of cADPR to increase cell proliferation has also been observed in human T cells (21), human hemopoietic progenitors (22), human peripheral blood mononuclear cells (23), human mesenchymal stem cells (24), and murine mesangial cells (25).The PC12 cell line was derived from rat adrenal medulla and has been used extensively as a neuronal model, since it exhibits many of the functions observed in primary neuronal cultures (26). Most importantly, PC12 cells can be induced by nerve growth factor (NGF) to differentiate into cells with extensive neurite outgrowths, resembling neuronal dendritic trees (26, 27). In contrast to NGF, numerous growth factors and neurotransmitters can induce the proliferation of PC12 cells instead (26). Both IP₃ receptor- and ryanodine receptor-mediated Ca²⁺ stores have been shown to be present in PC12 cells (28–31). The type 2 ryanodine receptor is expressed in PC12 cells and activation of the NO/cGMP pathway in PC12 cells results in calcium mobilization, which is mediated by cADPR and similar to that seen in sea urchin eggs (32). It has been demonstrated that NAADP, another Ca²⁺-mobilizing messenger, is also a potent neuronal differentiation inducer in PC12 cells, while IP₃ exhibits no such role (33, 34). Whether cADPR is involved in the proliferation and differentiation of PC12 cells is unknown.Here we show that activation of the CD38/cADPR/Ca²⁺ signaling is required for the ACh-induced proliferation in PC12 cells, while inhibition of the pathway accelerates NGF-induced neuronal differentiation. Our data indicate that cADPR is important in regulating cell proliferation and neuronal differentiation in PC12 cells.     

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na⁺/nucleoside and H⁺/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H⁺ dependence (all mutants), shift in Na⁺:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na⁺-specific hCNT1, uncoupled Na⁺ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)³ proteins for transport into or out of cells (1–4). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (3–6). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1–3, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na⁺-specific and transport pyrimidine and purine nucleosides, respectively (11–13). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na⁺- but not H⁺-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H⁺-coupled. Na⁺:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H⁺:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na⁺-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na⁺- and H⁺-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX₃NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (20–30). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX₃NEFVA(Y/M/F) motif.     

Efficient Class II Major Histocompatibility Complex Presentation of Endogenously Synthesized Hepatitis C Virus Core Protein by Epstein-Barr Virus-Transformed B-Lymphoblastoid Cell Lines to CD4+ T Cells

The induction of an efficient CD4⁺ T-cell response against hepatitis C virus (HCV) is critical for control of the chronicity of HCV infection. The ability of HCV structural protein endogenously expressed in an antigen-presenting cell (APC) to be presented by class II major histocompatibility complex molecules to CD4⁺ T cells was investigated by in vitro culture analyses using HCV core-specific T-cell lines and autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) expressing structural HCV antigens. The T- and B-cell lines were generated from peripheral blood mononuclear cells derived from HCV-infected patients. Expression and intracellular localization of core protein in transfected cells were determined by immunoblotting and immunofluorescence. By stimulation with autologous B-LCLs expressing viral antigens, strong T-cell proliferative responses were induced in two of three patients, while no substantial stimulatory effects were produced by B-LCLs expressing a control protein (chloramphenicol acetyltransferase) or by B-LCLs alone. The results showed that transfected B cells presented mainly endogenously synthesized core peptides. Presentation of secreted antigens from adjacent antigen-expressing cells was not enough to stimulate a core-specific T-cell response. Only weak T-cell proliferative responses were generated by stimulation with B-LCLs that had been pulsed beforehand with at least a 10-fold-higher amount of transfected COS cells in the form of cell lysate, suggesting that presentation of antigens released from dead cells in the B-LCL cultures had a minimal role. Titrating numbers of APCs, we showed that as few as 10⁴ transfected B-LCL APCs were sufficient to stimulate T cells. This presentation pathway was found to be leupeptin sensitive, and it can be blocked by antibody to HLA class II (DR). In addition, expression of a costimulatory signal by B7/BB1 on B cells was essential for T-cell activation.Hepatitis C virus (HCV) has been known as a major etiologic agent of posttransfusion and sporadic community-acquired non-A, non-B hepatitis. Like the other members in the family Flaviviridae, HCV contains a single, positive-strand RNA genome with a single long open reading frame (ORF) coding for a polyprotein precursor of about 3,000 amino acids (aa) (12). HCV infection is frequently persistent in the majority of patients and is closely associated with the later development of liver cirrhosis and hepatocellular carcinoma (3, 12, 16, 32). The effective control of HCV infection has been limited by the high frequency of viral genetic heterogeneity (7), the low rate of response to alpha interferon (46), and inadequate production of protective immunity (44, 45). These features strongly suggest that there is a great need to establish a new, highly effective therapy.CD4⁺ T cells are considered to play a central role in the generation of protective immunity against infections, because they can provide help to B cells for antibody production (42) and to cytotoxic precursor T cells for their maturation to effectors (21). Some CD4⁺ T cells may also act as cytotoxic effectors (30). It has been recognized that CD4⁺ T-cell response to HCV antigens is important for determining the clinical course of HCV infection (17, 37). Generally, T-cell proliferation is more frequent and stronger in patients with a benign course (6, 17, 20, 33, 37) that is accompanied by the normalization of serum alanine aminotransferase and, in some cases, the clearance of viral RNA (17, 37). In contrast, patients who have a poor T-cell response tend to develop persistent infection (17, 37). These findings support the hypothesis that a sufficient CD4⁺ T-lymphocyte response is critical for limiting HCV infection.Activation of T lymphocytes depends on the recognition of processed viral peptides, but not native antigens, in the context of major histocompatibility complex (MHC) molecules that are presented by antigen-presenting cells (APCs) (56). The B cell is an important professional APC, and its role in mediating antigen-specific immune response has been described extensively (11). Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells are frequently used as APCs in in vitro analyses for antigen processing and presentation to T cells. These cells are characterized by high-level expression of class I and class II MHC molecules, along with accessory molecules such as ICAM-1, B7/BB1, and LFA-3, known to be important costimulatory molecules for T-cell activation (9, 15, 24). Importantly, transfected EBV-immortalized B cells expressing a tumor antigen have been shown to be capable of eliciting both T-helper and cytotoxic-T-lymphocyte (CTL) responses following in vivo inoculation (40). Nevertheless, dendritic cells have been shown to be critical for initiating responses by naive T cells (53), and in some situations presentation by B cells has been suggested to be toleragenic (35). To date, the role of B cells in processing and presenting HCV antigens has not been studied in detail and the mechanisms underlying T-cell–B-cell interaction are still being worked out.In the present study, EBV-transformed B-lymphoblastoid cell lines (B-LCLs) established from HCV-infected patients were transfected with an expression vector coding for the whole structural region and part of the NS2 region of the HCV genome. The capacity of transfected B-LCL APCs for presenting intracellularly synthesized peptides was assessed by in vitro induction of the HCV-specific lymphoproliferative response of autologous T-cell lines. Our results indicated that core protein was properly expressed and efficiently presented by B-LCL APCs to CD4⁺ T cells. We demonstrated that the endogenous core peptides were presented through the class II MHC pathway and that they need B7/BB1 for providing costimulatory signals.     

Characterization of and Functional Antigen Presentation by Central Nervous System Mononuclear Cells from Mice Infected with Theiler’s Murine Encephalomyelitis Virus

We examined the phenotype and function of cells infiltrating the central nervous system (CNS) of mice persistently infected with Theiler’s murine encephalomyelitis virus (TMEV) for evidence that viral antigens are presented to T cells within the CNS. Expression of major histocompatibility complex (MHC) class II in the spinal cords of mice infected with TMEV was found predominantly on macrophages in demyelinating lesions. The distribution of I-A^s staining overlapped that of the macrophage marker sialoadhesin in frozen sections and coincided with that of another macrophage/microglial cell marker, F4/80, by flow cytometry. In contrast, astrocytes, identified by staining with glial fibrillary acidic protein, rarely expressed detectable MHC class II, although fibrillary gliosis associated with the CNS damage was clearly seen. The costimulatory molecules B7-1 and B7-2 were expressed on the surface of most MHC class II-positive cells in the CNS, at levels exceeding those found in the spleens of the infected mice. Immunohistochemistry revealed that B7-1 and B7-2 colocalized on large F4/80⁺ macrophages/microglia in the spinal cord lesions. In contrast, CD4⁺ T cells in the lesions expressed mainly B7-2, which was found primarily on blastoid CD4⁺ T cells located toward the periphery of the lesions. Most interestingly, plastic-adherent cells freshly isolated from the spinal cords of TMEV-infected mice were able to process and present TMEV and horse myoglobin to antigen-specific T-cell lines. Furthermore, these cells were able to activate a TMEV epitope-specific T-cell line in the absence of added antigen, providing conclusive evidence for the endogenous processing and presentation of virus epitopes within the CNS of persistently infected SJL/J mice.Theiler’s murine encephalomyelitis virus (TMEV) is a picornavirus that induces a lifelong persistent central nervous system (CNS) infection leading to a chronic CNS demyelinating disease when inoculated intracerebrally into susceptible strains of mice. Infected mice develop progressive symptoms of gait disturbance, spastic hind limb paralysis, and urinary incontinence (39), histologically related to perivascular and parenchymal mononuclear cell infiltration and demyelination of white matter tracts within the spinal cord (8, 9, 38). Several lines of evidence have demonstrated that demyelination is immunologically mediated. These include the ability of nonspecific immunosuppression with cyclophosphamide (37), antithymocyte serum (36), and anti-CD4 or anti-major histocompatibility complex (MHC) class II monoclonal antibodies (MAbs) (14, 16, 63) to inhibit or prevent disease and the ability of TMEV-specific tolerance to prevent induction of disease (28). In the highly susceptible SJL/J mouse strain, current evidence indicates that the myelin damage is initiated by TMEV-specific CD4⁺ T cells targeting virus antigen (16, 28, 45, 46, 54), while the chronic stage of the disease also involves CD4⁺ myelin epitope-specific T cells primed via epitope spreading (48). Thus, the immune response itself may be deleterious to CNS function, as exemplified in humans by multiple sclerosis (MS), for which TMEV infection serves as a model.The identity of the cells responsible for initiating and sustaining immune responses in the CNS remains controversial. The CNS lacks normal lymphatic circulation and tissue and is shielded from the systemic circulation by a specialized continuous vascular endothelium (6). There are specialized cells within the CNS with the potential to present antigens to T cells. In vitro, astrocytes (11, 59) and microglia (3, 13), particularly when treated with gamma interferon (IFN-γ), are capable of expressing MHC class II and presenting antigens to T cells. However, studies such as these have relied on the ability to isolate and continuously culture cells from neonatal or embryonic brain and have assumed that such cells are representative of the adult populations in vivo. Antigen presentation by neonatal cells in long-term culture may not faithfully reproduce the in vivo state in adult animals, as the ability of microglia directly isolated from adult rats to present myelin basic protein (MBP) to T-cell lines in vitro was found to differ from that of neonatally derived microglia (12). In addition, studies using allogeneic bone marrow chimeras between strains of mice or rats have generally supported the idea that cells of hematopoietic origin, i.e., microglia and macrophages, are the principal antigen-presenting cells (APCs) in the CNS active during the initiation of experimental autoimmune encephalomyelitis (EAE) (20, 22, 50). Although they are much more abundant than microglia, astrocytes are less potent when inducing EAE in chimeras (50).The role of antigen presentation in the CNS during TMEV-induced demyelination has not been addressed directly. We previously showed that a relatively large fraction of the CD4⁺, but not CD8⁺, T cells isolated from the spinal cords of TMEV-infected mice expressed high-affinity interleukin-2 (IL-2) receptor (IL-2R), a marker of recent T-cell activation. In addition, TMEV-specific CD4⁺ T cells could be demonstrated in the spinal cord infiltrates of TMEV-infected mice (54). This finding raises the possibility that T cells are locally activated within the target tissue and participate directly in the pathogenesis of disease. Macrophages (5, 41, 56), astrocytes (7, 56), and oligodendroglia (55, 56) in TMEV-infected mice contain virus and conceivably could present viral antigens to pathogenic CD4⁺ T cells within the CNS. Isolated microglia (34) and astrocytes (17) have been shown to support persistent viral infection in vitro, and astrocytes derived from neonatal mice have been shown to present TMEV to T cells in vitro (2). To examine whether CNS cells present viral antigens and participate in the pathogenesis of TMEV-induced demyelination, the expression of MHC class II and B7 costimulatory molecules was examined in detail. Based on our previous results showing that a large proportion of CD4⁺ T cells isolated from the CNS of TMEV-infected mice bear markers of recent activation, we also asked if mononuclear cells isolated from the CNS of TMEV-infected mice were capable of presenting viral antigens leading to the functional activation of Th1 lines in vitro.     

The Role of In Vitro-Induced Lymphocyte Apoptosis in Feline Immunodeficiency Virus Infection: Correlation with Different Markers of Disease Progression

Human immunodeficiency virus infection is characterized by a progressive decline in the number of peripheral blood CD4⁺ T lymphocytes, which finally leads to AIDS. This T-cell decline correlates with the degree of in vitro-induced lymphocyte apoptosis. However, such a correlation has not yet been described in feline AIDS, caused by feline immunodeficiency virus (FIV) infection. We therefore investigated the intensity of in vitro-induced apoptosis in peripheral blood lymphocytes from cats experimentally infected with a Swiss isolate of FIV for 1 year and for 6 years and from a number of long-term FIV-infected cats which were coinfected with feline leukemia virus. Purified peripheral blood lymphocytes were either cultured overnight under nonstimulating conditions or stimulated with phytohemagglutinin and interleukin-2 for 60 h. Under stimulating conditions, the isolates from the infected cats showed significantly higher relative counts of apoptotic cells than did those from noninfected controls (1-year-infected cats, P = 0.01; 6-year-infected cats, P = 0.006). The frequency of in vitro-induced apoptosis was inversely correlated with the CD4⁺ cell count (P = 0.002), bright CD8⁺ cell count (P = 0.009), and CD4/CD8 ratio (P = 0.01) and directly correlated with the percentage of bright major histocompatibility complex class II-positive peripheral blood lymphocytes (P = 0.004). However, we found no correlation between in vitro-induced apoptosis and the viral load in serum samples. Coinfection with feline leukemia virus enhanced the degree of in vitro-induced apoptosis compared with that in FIV monoinfected cats. We concluded that the degree of in vitro-induced apoptosis was closely related to FIV-mediated T-cell depletion and lymphocyte activation and could be used as an additional marker for disease progression in FIV infection.Feline immunodeficiency virus (FIV) infection is a naturally occurring infection, and disease progression in infected cats is associated with a decline in the number of CD4⁺ cells (2, 6, 22, 23, 36), a loss of bright CD8⁺ cells in the advanced stage of the disease (22), an increased number of activated T cells (39, 41), and a changed cytokine production, i.e., decreased production of interleukin-2 (IL-2) and concomitantly increased production of tumor necrosis factor alpha (TNF-α) (25, 26). The increased production of TNF-α has been reported to induce apoptosis in chronically FIV-infected cells (38). Apoptosis, a controlled mode of cell death (34), plays an important role in the regulation of immune responses (5, 14). As described for FIV (23), the hallmark of human immunodeficiency virus (HIV)-induced disease is the loss of T-helper cells (31, 43). Theoretically, cell loss can be caused by decreased production of cells, increased destruction, or a combination of the two mechanisms. Findings of an early infection of thymocytes followed by pathologic changes in the thymus support the model of decreased T-helper cell production triggered by HIV (13) and FIV (52). The destruction model is supported by findings of an increased number of peripheral blood T cells undergoing apoptosis upon HIV (20, 32) and FIV (11, 21, 33) infection. However, increased CD4⁺-T-cell turnover may not be the main cause of the observed T-helper cell decline in HIV-1 infection, as reviewed by others (44, 51). In addition, the degree of HIV-induced apoptosis correlates with the T-helper cell decline and disease progression (19, 40). However, such a relationship has not yet been described for FIV. It has been reported that cross-linking of CD4 molecules by HIV gp160 triggers apoptosis in noninfected CD4⁺ T cells (1). Investigation of this aspect in the feline system is especially interesting since FIV does not use the feline homologue of CD4 (50).The aim of the present study was to compare the degree of in vitro-induced lymphocyte apoptosis in FIV-infected cats with normal and decreased T-helper cell counts. We used two different culture conditions to trigger apoptosis in vitro: cells were either cultured overnight under nonstimulating conditions and in the absence of growth factors or cultured for 60 h in the presence of phytohemagglutinin, IL-2, and fetal calf serum. We additionally examined cats which were coinfected with the feline leukemia virus (FeLV). This coinfection is known to accelerate the progression toward feline AIDS (23) by an unknown mechanism (8).     

Fas Ligand-Mediated Lysis of Self Bystander Targets by Human Papillomavirus-Specific CD8+ Cytotoxic T Lymphocytes

Mouse cytotoxic T lymphocytes (CTL) reactive with a H-2D^b-presented 9-mer peptide of the human papillomavirus type 16 protein E7^49-57 (RAHYNIVTF) were generated from the spleen cells of wild-type C57BL/6 (B6) or B6 perforin-deficient (B6.P⁰) mice. CD8⁺ B6 CTL displayed peptide-specific perforin- and Fas-mediated lysis of E7-transfected mouse RMA lymphoma cells (RMA-E7), while CD8⁺ CTL from B6.P⁰ mice lysed RMA-E7 cells via Fas ligand (FasL) exclusively. Rapid and efficient lysis of syngeneic bystander B6 blasts or RMA cells by either B6 or B6.P⁰ Ag-activated CTL was mediated by a FasL-Fas mechanism. Fas-resistant bystanders were not lysed, nor were allogeneic Fas-sensitive C3H/HeJ (H-2^k) or BALB/c (H-2^d) bystander blasts. Interestingly, however, phorbol myristate acetate-ionomycin preactivation of B6.P⁰ effectors enabled lysis of allogeneic H-2^k and H-2^d bystanders even in the absence of antigenic stimulation. Lysis of syngeneic bystander cells was always FasL-Fas dependent and required effector-bystander contact and, in particular, an interaction between CTL LFA-1 and bystander ICAM-1. Thus, in the context of major histocompatibility complex class I molecule-peptide ligation of the T-cell receptors of CD8⁺ CTL, neighboring bystander cells that are syngeneic and Fas sensitive and express the adhesion molecule ICAM-1 are potential targets of CTL attack.With the dissection of two basic cytolytic mechanisms of cytotoxic T lymphocytes (CTL) (10, 14, 20, 34), it has become possible to delineate the important criteria that determine direct (Ag-restricted) and bystander cytotoxicity. CTL use complementary cytotoxic mechanisms, one based on the granule exocytosis of a calcium-dependent pore-forming protein, perforin (8, 26), and granzymes (35) and another that depends on a calcium-independent interaction of effector T-cell tumor necrosis factor or Fas ligand (TNF or FasL) and target cell TNF receptor (TNFR) or Fas (22, 33). The function of the granule exocytosis pathway appears to be largely in non-major histocompatibility complex (MHC)-restricted NK lysis of class I molecule-defective tumor cells and in direct CTL-mediated immunity against tumor cells (37) or virus-infected cells (11, 19, 39). By contrast, the FasL-Fas and TNF-TNFR interactions are important for the maintenance of T-cell homeostasis following exposure to foreign Ag (5, 42) and Th-1 FasL-mediated B-cell apoptosis (27, 28). Blockage of both TNF and FasL is required to abrogate T-cell death: TNF mediates the death of most CD8⁺ T cells, whereas FasL mediates the death of most CD4⁺ T cells (42). While FasL-dependent lysis appears to be the primary mechanism used by CD4⁺ Th-1 effectors, CD8⁺ CTL use FasL or TNF secondarily in the absence of perforin-mediated lysis (10, 14, 20).After T-cell activation, a functional role for FasL is not apparent for several days until the T cell becomes Fas sensitive and hence susceptible to autocrine T-cell suicide (1, 5, 38). However, by using alloreactive CTL cultures or clones, it has recently become apparent that in the presence of Ag-bearing target cells (i.e., upon T-cell receptor [TCR] activation) CTL can also lyse Ag-free bystander cells via a FasL-Fas interaction (13, 34). While the specificity of CTL toward Ag-bearing target cells has been considered a hallmark of an efficient immune response, CTL do not appear to spare Ag-free bystander cells during lysis of specific Ag-bearing target cells. In this study, we have generated CD8⁺ CTL from both wild-type and perforin-deficient (P⁰) mice reactive with a high-affinity H-2D^b-binding peptide of human papillomavirus type 16 protein E7. These peptide-specific CTL have been employed to demonstrate the requirements for CD8⁺ CTL-mediated lysis of Ag-free bystander cells and in particular the different properties of CTL activated by antigen versus a nonspecific stimulus.