CD45RB Status of CD8+ T Cell Memory Defines T Cell Receptor Affinity and Persistence

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A shared epitope of the interphotoreceptor retinoid-binding protein recognized by the CD4+ and CD8+ autoreactive T cells

We previously demonstrated that cultures of rat uveitogenic T cells rapidly become dominated by CD4+ cells, but activation of CD8+ autoreactive T cells also occurred during the in vitro culture of in vivo-primed T cells. In the present study, we show that the commonly used uveitogenic peptide, interphotoreceptor retinoid-binding protein (IRBP) 1-20, generated both CD4+ and CD8+ autoreactive T cells in the C57BL/6 (B6) mouse and that this 20-mer contains at least two distinct antigenic epitopes. To determine whether the CD8 response was Ag-specific and whether CD4+ and CD8+ IRBP1-20-specific T cells recognize distinct antigenic epitopes, we prepared highly purified CD4+ and CD8+ T cells from IRBP1-20-primed mice and tested their proliferative response to a large panel of truncated peptides derived from IRBP1-20. The results showed that both CD4+ and CD8+ T cells recognized the same spectrum of peptides. In addition, peptides P10-18 were found to bind effectively to CD8+ IRBP1-20-specific T cells when complexed with recombinant H-2K(b) and also stimulate the proliferation and cytokine production of CD4+ IRBP1-20-specific T cells. Our results document for the first time that CD8+ and CD4+ autoreactive T cells display characteristic epitope recognition and they both recognize the same core epitope.     

Activation of T cells through a T cell-specific epitope of CD45.

The 180- and 190-kDa isoforms of CD45 are preferentially expressed on the helper inducer (memory) subset of CD4 cells. In order to generate monoclonal antibodies against the extracellular domains of these isoforms and determine whether they could regulate the function and activation of these cells, we developed a mAb, anti-4H2D, by immunizing Balb/c mice with an isogenic mouse pre-B cell line expressing the human 190-kDa CD45 isoform. Anti-4H2D reacts with approximately 60% of T cells, 70% of CD4 cells, and 60% of CD8 cells. The CD4 cell population defined by this mAb corresponds functionally and phenotypically to that defined by the CD45RO+CD29+ subset. Western blotting demonstrated that anti-4H2D reacts primarily with the 190-kDa isoform of CD45 and to a minor extent, the 205- and 180-kDa CD45 isoforms. Interestingly, this mAb reacted with only a subpopulation of mature thymocytes and peripheral T cells, despite the fact that the 190-kDa CD45 isoform, as well as CD45RO and CD29, is more widely distributed on cells of hematopoietic origin. The 4H2D epitope was neuraminidase sensitive, indicating that anti-4H2D reacts with a carbohydrate epitope which is present on only a subset of the T cells containing the 190-kDa CD45 isoform epitopes. Functional studies showed that soluble anti-4H2D augmented T cell proliferation induced by the CD2 and CD3 pathways, and treatment of T cells with this mAb up-regulated [Ca2+]i flux induced by both anti-CD2 and anti-CD3 mAbs. These results suggest that the 190-kDa CD45 isoform on human CD4 cells is heterogeneous and that the 190-kDa isoform recognized by anti-4H2D regulates the function and activation of CD4 helper T cells.     

Specific CD45 isoforms differentially regulate T cell receptor signaling.

Multiple isoforms of T cell CD45 tyrosine phosphatase are expressed as a result of alternative RNA splicing among extracellular exons. To discern the presence and identity of distinct functions among CD45 isoforms, we compared thymic T cell activation responses by elevating expression of two CD45 isoforms normally found on quiescent T cells. We report that CD45RABC significantly increased CD4+ thymic T cell proliferation in both a mixed lymphocyte reaction and following anti-T cell receptor (TCR) antibody stimulation. Additionally, CD45RABC enhanced Ca2+ mobilization and phosphotyrosine accumulation, and suppressed the inhibitory effect of anti-CD4 antibodies. By contrast, CD45R0 did not enhance TCR signaling or phosphotyrosine levels in CD4+ thymic T cells and required a TCR co-stimulus to augment cellular proliferation. These studies provide genetic evidence that alternative CD45 isoforms are functionally distinct and disclose a unique mechanism by which T cell immunologic responsiveness can be modified.     

Requirement for CD4 T cell help in maintenance of memory CD8 T cell responses is epitope dependent

CD4 Th cells play critical roles in stimulating Ab production and in generating primary or maintaining memory CTL. The requirement for CD4 help in generating and maintaining CTL responses has been reported to vary depending on the vector or method used for immunization. In this study, we examined the requirement for CD4 T cell help in generating and maintaining CTL responses to an experimental AIDS vaccine vector based on live recombinant vesicular stomatitis virus (VSV) expressing HIV Env protein. We found that primary CD8 T cell responses and short-term memory to HIV Env and VSV nucleocapsid (VSV N) proteins were largely intact in CD4 T cell-deficient mice. These responses were efficiently recalled at 30 days postinfection by boosting with vaccinia recombinants expressing HIV Env or VSV N. However, by 60 days postinfection, the memory/recall response to VSV N was lost in CD4-deficient mice, while the recall response HIV Env was partially maintained in the same animals for at least 90 days. This result indicates that there are epitope-specific requirements for CD4 help in the maintenance of memory CD8 T cell responses. Our results also suggest that choice of epitopes might be critical in an AIDS vaccine designed to protect against disease in the context of reduced or declining CD4 T cell help.     

The novel carbohydrate epitope L3 is shared by some neural cell adhesion molecules

The monoclonal L3 antibody reacts with an N-glycosidically linked carbohydrate structure on at least nine glycoproteins of adult mouse brain. Three out of the L3 epitope-carrying glycoproteins could be identified as the neural cell adhesion molecules L1 and myelin-associated glycoprotein, and the novel adhesion molecule on glia. Expression of the L3 carbohydrate epitope is regulated independently of the protein backbone of these three glycoproteins. Based on the observation that out of three functionally characterized L3 epitope-carrying glycoproteins three fulfill the operational definition of an adhesion molecule, we would like to suggest that they form a new family of adhesion molecules that is distinct from the L2/HNK-1 carbohydrate epitope family of neural cell adhesion molecules. Interestingly, some members in each family appear to be unique to one family while other members belong to the two families.     

Development of CD8+ effector T cells is differentially regulated by IL-18 and IL-12

We investigated the effects of IL-18 on the development of CD8+ effector T cells in DBA/2 anti-BDF1 whole spleen cell MLC and compared the results with those of IL-12. Addition of IL-18 to the MLC resulted in a twofold increase in CD8/CD4 ratios compared with the control cultures when cells were expanded in IL-2-containing medium following MLC. Purified CD8+ T cells recovered from the IL-18-stimulated MLC produced 20- to 30-fold more IFN-gamma after secondary stimulation with C57BL/6 spleen cells or anti-CD3 mAb, and exhibited strong allospecific CTL activity. Neither IL-18 nor IL-18-supplemented culture supernatants from DBA/2 anti-BDF1 MLC induced type I CD8+ effector T cells when purified CD8+ T cells were used as responder cells in primary MLC. Furthermore, CD4+ T cell depletion from the responder cells abrogated the IL-18-induced increase in secondary IFN-gamma production by CD8+ T cells, suggesting that IL-18-induced type I effector CD8+ T cell development was CD4+ T cell dependent. In marked contrast, adding IL-12 to primary MLC decreased CD8/CD4 ratios by 50% and suppressed secondary IFN-gamma production and CTL activity by CD8+ T cells regardless of concentration, whereas Th1 development was promoted by IL-12. Moreover, both IL-12 and IL-18 efficiently induced type I CD8+ effector T cells in C57BL/6 anti-BDF1 MLC. These findings show that IL-18 plays an important role in the generation of type I CD8+ effector T cells, and further suggest that functional maturation of CD8+ T cells is differentially regulated by IL-18 and IL-12.     

Reversal of CD45R isoform switching in CD8+ T cells.

Both CD4+ and CD8+ T cells express either CD45RA or CD45R0 isoform of CD45R in an exclusive way. Recent reports have shown that CD45RA+ T cells lose CD45RA and gain CD45R0 upon activation. This switching has been suggested to be irreversible although more recently, examples of reversal of CD45R isotype switching in CD4+ T cells have been reported. We report here that freshly isolated unprimed CD8+ T cells, when activated with PHA, temporarily lose CD45RA but reexpress an intermediate level of CD45RA 2-3 weeks after activation with PHA. This reversal seems to take place much more slowly in unprimed CD4+ T cells: the majority of CD4+ T cells that had lost CD45RA and gained CD45R0 remained CD45RA-CD45R0+ in 3 weeks after the stimulation. Also, long-term CD8+ CD45RA+ T cell lines stimulated with PHA or OKT3 showed even more rapid recovery of CD45RA while PPD-specific CD4+ T cell clones retained the original CD45R0 phenotype 3 weeks after stimulation with PPD or PHA.     

High IL-4 production is a stable phenotype of CD8CD45RACD27 T cells

CD27^neg T cells are found only among CD4^pos-CD45RO^pos T cells and represent a T cell subset functionally distinct from CD27^pos T cells. We examined CD4^posCD45RO^pos T cells that were sorted into CD27^neg and CD27^pos populations for their cytokine production in response to different activation pathways. We found that CD27^neg T cells are characterized by high IL-4 and low IL-2 production, regardless of whether the cells were activated through CD3 plus CD28, CD2 plus CD28, or PHA plus PMA. However, subpopulation-specific patterns of cytokines were the clearest demonstrable following CD2 plus CD28 stimulation. We conclude from these data that high IL-4 production is a stable phenotype of CD27^neg T cells.     

Cleavage of the cell-surface O-sialoglycoproteins CD34, CD43, CD44, and CD45 by a novel glycoprotease from Pasteurella haemolytica.

The study of structural/functional characteristics of the cell-surface glycoproteins of leukocytes has led to a better understanding of the differentiation and maturation of hematopoietic cells. We have assessed the ability of a unique metalloprotease that is secreted by the bovine fibrinous pneumonia pathogen Pasteurella haemolytica, to cleave cell-surface glycoproteins expressed on human leukocytes. Biochemical analysis shows that the O-glycosylated cell surface Ag CD34, CD43 (leukosialin), CD44 (hyaluronic acid receptor), and CD45 (leukocyte common Ag), are all cleaved by this protease. Although these enzyme-sensitive structures contain N-linked glycans, they are all extensively glycosylated with O-linked carbohydrates, which are especially abundant on CD34 and CD43. In contrast, the glycoproteins CD18/11a,b,c (leukocyte integrins), CD71 (transferrin receptor), HLA class I, and 8A3 Ag, which contain N-linked glycans but no O-sialo-glycans, were resistant to the action of the enzyme. Inasmuch as previous studies using glycophorin A had indicated that the substrate specificity of this enzyme may be uniquely restricted to the cleavage of O-sialoglycoproteins, we have designated this activity, P. haemolytica glycoprotease. Immunofluorescence analysis with a variety of antibodies to different epitopes of the P. haemolytica glycoprotease-sensitive structures indicate that this enzyme may have widespread applications in epitope-mapping studies, and represents a novel tool with which to study structure/function relationships for O-sialoglycosylated cell-surface proteins. However, most significantly these results suggest that the P. haemolytica glycoprotease may be of use in the affinity purification and recovery of clinically important leukocyte subsets, such as primitive hematopoietic progenitors that express CD34.     

Human virus-specific CD8+ CTL clones revert from CD45ROhigh to CD45RAhigh in vivo: CD45RAhighCD8+ T cells comprise both naive and memory cells.

It has been generally believed that human CD8+ memory cells are principally found within the CD45ROhigh population. There are high frequencies of CD8+ memory CTL specific for the human CMV tegument phosphoprotein pp65 in PBMC of long-term virus carriers; the large population of memory CTL specific for a given pp65 peptide contains individual CTL clones that have greatly expanded. In this study, we found high frequencies of pp65 peptide-specific memory CTL precursors in the CD45ROhighCD45RA- population, but also appreciable frequencies in the CD45RAhigh subpopulation. Because the majority of CD8+ T cells in PBMC are CD45RAhigh, more of the total pp65-specific memory CTL pool is within the CD45RAhigh than in the CD45ROhigh compartment. Using clonotypic oligonucleotide probes to quantify the size of individual pp65-specific CTL clones in vivo, we found the CD45RAhigh population contributed 6- to 10-fold more than the CD45ROhigh population to the total virus-specific clone size in CD8+ cells. During primary CMV infection, an individual virus-specific CTL clone was initially CD45ROhigh, but after resolution of infection this clone was detected in both the CD45ROhigh and the CD45RAhigh populations. We conclude that CD45RA+ human CD8+ T cells do not solely comprise naive cells, but contain a very significant proportion of memory cells, which can revert from the CD45ROhigh to CD45RAhigh phenotype in vivo.     

A novel approach of direct ex vivo epitope mapping identifies dominant and subdominant CD4 and CD8 T cell epitopes from Listeria monocytogenes

We used a novel approach for the direct ex vivo identification and characterization of T cell epitopes based on the screening of peptide spot libraries with freshly isolated splenocytes in a sensitive enzyme-linked immunospot (ELISPOT) assay. This technique was applied for the analysis of splenocytes from Listeria monocytogenes-infected BALB/c and C57BL/6 mice. The screening of peptide spot libraries covering the whole listeriolysin O and p60 of L. monocytogenes confirmed all known CD4 and CD8 T cell epitopes of these proteins and additionally revealed six new H-2(d) and six new H-2(b)-restricted T cell epitopes. New epitopes were categorized into CD4 and CD8 T cell epitopes by ex vivo ELISPOT analysis with separated T cell populations. The quantitative analysis of cells reactive with these CD4 and CD8 T cell epitopes revealed the existence of dominant and subdominant CD4 and CD8 T cell populations during L. monocytogenes infection. As a consequence of these data we suggest that ELISPOT-based screening of peptide spot libraries could be a general approach for the rapid identification and characterization of pathogen-specific T cell populations during various infectious diseases.     

Liver damage by infiltrating CD8+ T cells is Fas dependent.

Ag stimulation of CD8+ lymphocytes in vivo results in their migration to various tissues as well as the activation of a cytolytic program involving perforin, TNF-alpha, and Fas ligand. The liver is one of the main sites for infiltration by activated CD8+ T cells, and this is followed by the death of hepatocytes. The contribution of the various cytolytic components to this process is unclear. Hepatocyte damage by CD8+ T cells was studied using the MHC class I-restricted OVA-specific TCR transgenic mouse (OT-1) to examine the contribution of Fas to hepatocyte death. Activated CD8+ T cells from both OT-1 and Fas-deficient OT-1lpr mice migrated to the liver in similar numbers after OVA administration, but only in OT-1 mice was there evidence of significant hepatocyte damage histologically and by elevation of serum aspartate transaminase. These differences were not the result of inefficient induction of cytolytic activity in OT-1lpr liver T cells, since they were as cytolytic in vitro as OT-1 liver T cells. This was supported by findings of similar high levels of message for perforin, TNF-alpha, and Fas ligand in liver lymphocytes from both mice. These findings demonstrate that following Ag activation, infiltrating liver CD8+ T lymphocytes induce hepatocyte damage in a Fas-dependent manner.     

Addition of a prominent epitope affects influenza A virus-specific CD8+ T cell immunodominance hierarchies when antigen is limiting

A reverse genetics strategy was used to insert the OVA peptide (amino acid sequence SIINFEKL; OVA(257-264)) into the neuraminidase stalk of both the A/PR8 (H1N1) and A/HKx31 (H3N2) influenza A viruses. Initial characterization determined that K(b)OVA257 is presented on targets infected with PR8-OVA and HK-OVA without significantly altering D(b) nucleoprotein (NP)366 presentation. There were similar levels of K(b)OVA257- and D(b)NP366-specific CTL expansion following both primary and secondary intranasal challenge. Interestingly, while variable, the presence of the immunodominant K(b)OVA257-specific response resulted in diminished D(b) acidic polymerase224- and K(b) basic polymerase subunit 1(703)-, but not D(b)NP366-specific responses and didn't alter endogenous influenza A virus-specific immunodominance hierarchies. However, challenging PR8-OVA-primed mice with HK-OVA via the i.p. route, and thereby limiting Ag dose, led to a reduction in the magnitude of all the influenza A virus-specific responses measured. A similar reduction in CTL response to native epitopes was also seen following primary respiratory HK-OVA infection of mice that received substantial numbers of K(b)OVA257-specific TCR transgenic T cells. Thus, during the course of infection, the generation of individual virus-specific CTL responses is independently regulated. However, in cases in which Ag is limiting, or high precursor frequency, the presence of immunodominant CTL responses can impact on the magnitude of other specific populations. Therefore, depending on both the size of the T cell precursor pool and the mode of Ag presentation, the addition of a major epitope can diminish the size of endogenous, influenza-specific CD8+ T cell responses, although never to the point that these are totally compromised.     

Promiscuous malaria peptide epitope stimulates CD45Ra T cells from peripheral blood of nonexposed donors.

PBL from individuals with no history of malaria exposure, as well as cord blood lymphocytes, were tested for proliferation to T cell epitopes from the malaria circumsporozoite proteins of Plasmodium falciparum and Plasmodium vivax. Cells from many individuals proliferated in response to these peptides, but for two peptides (P. vivax317-336 and P. falciparum CS331-350) the response rate ranged from 64 to 93%, with the specific stimulation indices reaching as high as 38. The phenotype of the cells responding to PfCS331-350 was predominantly CD4+,CD8-,CD45Ra+,CD45Ro-, which was the inverse of the phenotype of the cells responding to tetanus toxoid with respect to CD45 isoforms. T cell clones from different individuals specific for PfCS331-350 were restricted by at least four different HLA-DR molecules and there was no evidence that the peptide was a "superantigen." Overlapping peptides were used to demonstrate that clones had different fine specificities although the peptide specificities of the DR4-restricted and DR11-restricted clones were similar. Although the individuals tested here have had no history of malaria exposure, these data demonstrate that they have T cells specific for malaria sequences present in high frequency that proliferate as intensely as some memory responses. Although one clone from an individual with a history of BCG vaccination did react strongly with PPD, the phenotype of these cells suggests that they are not classical memory cells for a cross-reactive recall Ag. Such cells may affect the induction or expression of malaria immunity.     

A second subunit of CD8 is expressed in human T cells.

The CD8 glycoprotein plays important functions in T cell development and in T cell activation. In rodents, CD8 is a heterodimer, consisting of an alpha-chain (Lyt2) and a beta-chain (Lyt3). In humans, only the alpha-chain has been detected, and it has been thought that CD8 consists of homodimers of this protein. We have isolated functional cDNA clones encoding human CD8 beta, and show that the CD8 beta protein is expressed on the surface of CD8+ human T cells. cDNA clones encoding multiple forms of the human CD8 beta-chain have been isolated and characterized. These structural variants, which are likely to arise by alternative splicing, differ in the sequences encoding the cytoplasmic domain, which can consist of 19, 30, or 52 amino acids. One of the cDNAs lacks nucleotide sequences corresponding to a hydrophobic transmembrane domain, and may encode a secreted CD8 beta protein. The protein product of the human CD8 beta gene can be detected by a recently described anti-CD8 monoclonal antibody, 597. Expression of the epitope recognized by this antibody requires co-expression of the CD8 alpha and CD8 beta gene products. About 90% of human CD8 alpha positive thymocytes and peripheral blood lymphocytes express CD8 beta at the cell surface. Expression of the CD8 beta chain is thus conserved between human and rodents, and the variant CD8 beta polypeptides may have distinct roles in T cell function and development.     

The antiviral efficacy of simian immunodeficiency virus-specific CD8+ T cells is unrelated to epitope specificity and is abrogated by viral escape

CD8(+) T lymphocytes appear to play a role in controlling human immunodeficiency virus (HIV) replication, yet routine immunological assays do not measure the antiviral efficacy of these cells. Furthermore, it has been suggested that CD8+ T cells that recognize epitopes derived from proteins expressed early in the viral replication cycle can be highly efficient. We used a functional in vitro assay to assess the abilities of different epitope-specific CD8+ T-cell lines to control simian immunodeficiency virus (SIV) replication. We compared the antiviral efficacies of 26 epitope-specific CD8+ T-cell lines directed against seven SIV epitopes in Tat, Nef, Gag, Env, and Vif that were restricted by either Mamu-A*01 or Mamu-A*02. Suppression of SIV replication varied depending on the epitope specificities of the CD8+ T cells and was unrelated to whether the targeted epitope was derived from an early or late viral protein. Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T-cell lines were consistently superior at suppressing viral replication compared to the other five SIV-specific CD8+ T-cell lines. We also investigated the impact of viral escape on antiviral efficacy by determining if Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T-cell lines could suppress the replication of an escaped virus. Viral escape abrogated the abilities of Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T cells to control viral replication. However, gamma interferon (IFN-gamma) enzyme-linked immunospot and IFN-gamma/tumor necrosis factor alpha intracellular-cytokine-staining assays detected cross-reactive immune responses against the Gag escape variant. Understanding antiviral efficacy and epitope variability, therefore, will be important in selecting candidate epitopes for an HIV vaccine.     

Leaf respiration is differentially affected by leaf vs. stand-level night-time warming

Plant respiration is an important physiological process in the global carbon cycle serving as a major carbon flux from the biosphere to the atmosphere. Respiration is sensitive to temperature providing a link between environmental variability, climate change and the global carbon cycle. We measured leaf respiration in Populus deltoides after manipulating the air temperature surrounding part of a single leaf, and compared this to the temperature response of the same leaves after manipulating the temperature of the stand. The short‐term temperature response of respiration (Q₁₀– change in the respiration rate with a 10 °C increase in leaf temperature) was 1.7 when the leaf temperature was manipulated, but 2.1 when the stand‐level temperature was changed. As a result, total night‐time carbon release during the five‐day experiment was 21% lower when using the Q₁₀ estimates from the tradition leaf manipulation compared to the stand‐level manipulation. We conclude that the temperature response of leaf respiration is related to whole plant carbon and energy demands, and that appropriate experimental procedures are required in examining respiratory CO₂ release under variable temperature conditions.