CD45 Isoforms Research Articles

Enhanced Formation and Survival of Regulatory T Cells in CLL.

Background. In healthy individuals it has been shown that regulatory T cells (Treg) differentiate from rapidly proliferating memory T cells. Rapid proliferation in combination with high susceptibility to apoptosis, make the Treg population highly dynamic. Tregs are thought to play an important role in immune evasion by malignancies. High Treg numbers in patients with malignancies are often associated with poor prognosis. An important question to be answered is whether tumor cells can promote the expansion and activation of Tregs. Recently it has been described that chronic lymphocytic leukemia (CLL) patients have increased numbers of Treg, with highest Treg frequencies in progressive patients1. The mechanism of this expansion however remains unknown. In the present study, we analysed the mechanism behind Treg expansion in CLL.Results. Neither analysis of the T cell receptor (TCR) repertoire nor CD45 isoform expression of Treg from CLL patients provided evidence for chronic (tumor) antigenic stimulation as a possible cause for Treg expansion in CLL. Furthermore, we found no correlation with CMV serology, which has been demonstrated to influence the CD8+ T cell repertoire in CLL. However, in line with recent studies demonstrating that CD70+ NHL cells are strong inducers of Treg, we found evidence for increased formation of Treg in CLL via CD70 costimulation. We observed that CD40 ligand activated CLL cells (which might be considered a model for lymph node CLL cells2) strongly induced CD70-dependent formation of Treg. RT-MLPA expression analysis of 34 apoptosis-regulating genes showed that in comparison to other CD4+ T cells, Treg of both healthy individuals and CLL patients had a high expression of pro-apoptotic Noxa and a low expression of anti-apoptotic Bcl-2. Strikingly, Bcl-2 levels of Treg in CLL patients were significantly higher than in healthy individuals. Finally, the different apoptotic profile resulted in differences at the functional level, since Treg from CLL patients were more resistant to drug-induced apoptosis than Treg from healthy individuals.Conclusion. Treg in CLL may accumulate both by increased formation, facilitated by CD27-CD70 interaction in the lymph node proliferation centres, and decreased sensitivity to apoptosis due to a shifted Noxa-Bcl-2 balance. Since high Treg numbers might negatively affect the course of disease, targeting these mechanisms may provide additional therapeutic approaches in CLL.

Expression of a B‐cell‐restricted isoform of CD45 is associated with maturity in rat serosal and connective‐tissue mast cells

Fas-associated protein with death domain/mediator of receptor induced toxicity (FADD/MORT1) was first described as a transducer of death receptor signalling but was later recognized also to be important for proliferation of T cells. B-cell lymphoma 3 (Bcl-3) is a relatively little understood member of the nuclear factor (NF)-kappaB family of transcription factors. We recently found that Bcl-3 is up-regulated in T cells from mice where FADD function is blocked by a dominant negative transgene (FADD-DN). To understand the importance of this, we generated FADD-DN/bcl-3(-/-) mice. Here, we report that T cells from these mice show massive cell death and severely reduced proliferation in response to T-cell receptor (TCR) stimulation in vitro. Transgenic coexpression of Bcl-2 (FADD-DN/bcl-3(-/-)/vav-bcl-2 mice) rescued the survival but not the proliferation of T cells. FADD-DN/bcl-3(-/-) mice had normal thymocyte numbers but reduced numbers of peripheral T cells despite an increase in cycling T cells in vivo. However, activation of the classical NF-kappaB and extracellular regulated kinase (ERK) pathways and expression of interleukin (IL)-2 mRNA upon stimulation were normal in T cells from FADD-DN/bcl-3(-/-) mice. These data suggest that FADD and Bcl-3 regulate separate pathways that both contribute to survival and proliferation in mouse T cells.

Trans-species activation of human T cells by rhesus macaque CD1b molecules

Despite crucial importance of non-human primates as a model of human infectious diseases, group 1 CD1 genes and proteins have been poorly characterized in these species. Here, we isolated CD1A, CD1B, and CD1C cDNAs from rhesus macaque lymph nodes that encoded full-length CD1 proteins recognized specifically by monoclonal antibodies to human CD1a, CD1b, and CD1c molecules, respectively. The monkey group 1 CD1 isoforms contained amino acid residues and motifs known to be critical for intramolecular disulfide bond formation, N-linked glycosylation, and endosomal trafficking as in human group 1 CD1 molecules. Notably, monkey CD1b molecules were capable of presenting a mycobacterial glycolipid to human CD1b-restricted T cells, providing direct evidence for their antigen presentation function. This also detects for the first time a trans-species crossreaction mediated by group 1 CD1 molecules. Taken together, these results underscore substantial conservation of the group 1 CD1 system between humans and rhesus macaque monkeys.

Polyarteritis Nodosa Mimicking Peripheral Vascular Disease

To the Editor:—With great interest I read the paper by De Golovine et al. 1, who reported on a case that illustrates the challenges in diagnosing polyarteritis nodosa (PAN). The characteristic sign, defined as arteritis in small and medium-sized arteries 2, needs a biopsy from a diseased artery. Since PAN can be life threatening 1, a delay of the correct diagnosis could be fatal. Therefore, a characteristic biomarker that can be easily screened in peripheral blood samples would be much more helpful than a biopsy. An interesting personal observation prompted me to present a male patient with a severe biopsy-proved PAN3 who fulfilled the American College of Rheumatology 1990 criteria 4. Flow cytometry with direct fluorochrome labelled monoclonal antibodies revealed increased absolute counts of total CD4+ cells and their CD45RO+RA- and CD45ROdim RAdim subsets that were absent in both healthy controls and patients with vasculitis others than PAN (Table 1). But there were no significant differences for total T-cytotoxic cells (CD3+CD8+), CD8+CD45ROdimCD45RAdim, CD8+CD45RO+CD45RA-, and CD8+CD45RO-CD45RA+ cells (Table 1). Table 1 Total T-helper (CD4) and T-cytotoxic (CD8) Cell Population and Their CD45RO CD45RA Subsets (in Cells/µl) in Twelve Healthy Controls, Eight Patients with Vasculitis, and in a Patient with PAN (Values Have Been Obtained by Flow Cytometric Analysis ... CD95 expression on total CD3+, CD3+CD4+, and CD3+CD8+ cells did not differ significantly among controls, PAN, and other inflammatory vessel diseases (data not shown). Upon the CD45 isoforms RO and RA T-helper cells can be devided into two subgroups namely CD45RO+CD45RA- (memory) and CD45RO-CD45RA (naive) cells 5. In healthy adults most of the CD4 cells are memory cells (~ 75%), while only a small proportion expresses the naive pattern. Concomitant loss of CD45RA expression and gain of CD45RO expression occurs during transition from naive to memory status 5. Thus, following in vitro activation of CD45RO-CD45RAbright T cells, a subset of transitional CD45ROdimCD45RAdim T cells is observed before conversion to a CD45RObrightCD45RA- phenotype is completed 5. Although all three of these phenotypic subsets are represented in the circulating human lymphocyte pool 5, increased values of intermediate expression pattern with CD45ROdim and CD45RAdim are typically for cord blood, representing the immature nature of cells 6. In postpartal life increases of double positive (CD45RO+RA+) cells have rarely been described 7–9. The presented data provide evidence for the notation that increased absolute counts of CD4 cells, memory T-helper cells (CD4+CD45RO+RA-), and the intermediate form of T-helper cells (CD4+CD45ROdimRAdim) in peripheral blood specimens seem to be associated with PAN. Although currently it is not clear what this finding means, it underscores the systemic nature of the disease. Since the results of the mentioned parameters are quickly available, they seem to be suitable as a screening test to make a rough estimate. Further investigations are warranted to further elucidate this exciting topic in larger patient groups in future.

Disease‐Associated Novel CD46 Splicing Variants and Pathologic Bone Remodeling in Otosclerosis

Otosclerotic bone is supposed to show unique CD46 expression pattern because otosclerosis is an organ-specific disease with viral etiology. Otosclerosis is a complex bone remodeling disorder of the human otic capsule, which is associated with persisting measles virus infection. The general cellular receptor of measles virus is the CD46, which has 14-known splicing isoforms. Nucleic acid was extracted from ankylotic stapes footplates (N = 99) removed during stapedectomies. Consecutive histological, CD46 specific immunohistologic analysis, and multiple polymerase chain reaction (PCR) amplifications were performed. Measles virus was detected by seminested reverse transcriptase-PCR. Splicing variants of CD46 were identified by nested reverse transcriptase-PCR and finally determined by mass sequencing of complementary DNA. Measles virus RNA was detectable only in histologically otosclerotic stapes footplates. Virus negative-fixed stapes represent degenerative disorders of variable histopathology. Otosclerosis is featured by an increased number of osteoclasts showing strong CD46 immunoreaction in contrast to nonotosclerotic stapes fixations. Normal and nonotosclerotic stapes footplates show consistent expression of "c," "d," "e," "f," and "l" CD46 splicing isoforms. In contrast, four novel CD46 splicing variants were additionally detected in otosclerosis: os1, os2, os3, and os4. Newly described CD46 isoforms have shorter or missing transmembrane domain and a rare cytoplasmic tail with pathological or uncommon signal transduction; however, virus binding ability remains equal and invariable. These changes may be responsible for the smooth virus replication. A special expression pattern and altered functions of CD46 could explain the organ-specific and virus-associated pathogenesis of otosclerosis.

IL-12-dependent activation of ERK1/2 in human T lymphoblasts

According to some authors, membrane compartmentalization is a key regulator of CD45 function. Indeed, it has been described that CD45 repositioning from raft microdomains to phospholipid-rich plasma membrane areas leads to the activation of extracellular signal-regulated kinases (ERKs). We have previously shown that interleukin-12 (IL-12) increases the expression of CD26, promoting the interaction of CD26 with CD45R0 (a CD45 isoform) and removing CD45R0 from lipid rafts. Thus, this IL-12-dependent removal of CD45RO from rafts could, hypothetically, fulfill functions like the activation of the ERK1/2 pathway. IL-12 is an important interleukin for T cells. Upon interaction with its receptor (interleukin-12 receptor; IL-12R), this cytokine triggers a signalling cascade, where the classical Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway and other additional routes participate. Due to the promitogenic effect of IL-12 and the influence of this cytokine on CD45RO compartmentalization, ERK kinases were likely candidates to be downstream of IL-12R. However, several research groups have rejected a role for these kinases. Now, results in this paper show that the IL-12R binding, similar to the stimulation via T cell receptor (TCR), promotes the activation of the Raf/MEK-1/ERK1/2 pathway. In addition, the IL-12R-associated Janus kinase JAK2, but not TYK2, seems upstream of this important pathway for the proliferation of human T cells. However, even though c-Myc is slightly up-regulated by IL-12 and partially mediates the proliferative effect of IL-12, this transcription factor was not found downstream of ERK1/2.

CD1a and MHC Class I Follow a Similar Endocytic Recycling Pathway

CD1 proteins are a family of major histocompatibility complex (MHC) class I-like antigen-presenting molecules that present lipids to T cells. The cytoplasmic tails (CTs) of all human CD1 isoforms, with the exception of CD1a, contain tyrosine-based sorting motifs, responsible for the internalization of proteins by the clathrin-mediated pathway. The role of the CD1a CT, which does not possess any sorting motifs, as well as its mode of internalization are not known. We investigated the internalization and recycling pathways followed by CD1a and the role of its CT. We found that CD1a can be internalized by a clathrin- and dynamin-independent pathway and that it follows a Rab22a- and ADP ribosylation factor (ARF)6-dependent recycling pathway, similar to other cargo internalized independent of clathrin. We also found that the CD1a CT is S-acylated. However, this posttranslational modification does not determine the rate of internalization or recycling of the protein or its localization to detergent-resistant membrane microdomains (DRMs) where we found CD1a to be enriched. We also show that plasma membrane DRMs are essential for efficient CD1a-mediated antigen presentation. These findings place CD1a closer to MHC class I in its trafficking and potential antigen-loading compartments among CD1 isoforms. Furthermore, we identify CD1a as a new marker for the clathrin- and dynamin-independent and DRM-dependent pathway of internalization as well as the Rab22a- and ARF6-dependent recycling pathway.

Group I CD1 isoforms (CD1a, CD1b and CD1c)

Group I CD1 isoforms (CD1a, CD1b and CD1c)

CD4+ T cell subsets defined by isoforms of CD45 in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune condition characterized by destruction of the intrahepatic bile ducts. Autoreactive CD4+ T cells have been reported both in the peripheral circulation and in the mononuclear cell infiltrate in the affected portal tracts. In this large study we have used two- and three-colour flow cytometry to determine the phenotypes of the CD4+ T cell subsets in the peripheral blood and liver-infiltrating lymphocytes of PBC patients (n = 43), normal controls (n = 19) and patients with alcoholic cirrhosis (n = 15), according to a novel classification based on the simultaneous expression of different isoforms of CD45. In PBC patients the proportion of peripheral blood CD4+ cells possessing the CD45ROhighRA- 'memory' phenotype was significantly increased, and the CD45RO-RAhigh 'naive' population was significantly decreased, compared with the two control groups. No significant differences in peripheral blood CD4+ T cell subsets were seen between patients with pre-cirrhotic and cirrhotic PBC. A similar, but more marked, shift towards the CD45ROhighRA- 'memory' phenotype was seen in the liver-infiltrating CD4+ T cells in PBC patients compared with alcoholic cirrhotics. Cells within the CD4+ memory subpopulation were further subgrouped according to expression of CD45RB, the level of expression of which has been associated with functional differences in the memory subset. In peripheral blood no differences were seen between PBC patients and controls with respect to the proportion of CD45ROhighRBhigh and CD45ROhighRBdim memory subsets. A statistically significant difference in the distribution of these memory subsets, with an increased memory-2/memory-1 ratio was observed in the liver-infiltrating CD4+ T cells of PBC patients compared with those from alcoholic cirrhotic patients. The potential implications of this observation are discussed.

Effect of HIV vertical transmission on the ontogeny of T cell antigens involved in the regulation of humoral immune response.

HIV infection causes progressive impairment of humoral immunity, including defective specific antibody production. To evaluate whether vertical HIV infection interferes with the expression on CD4+ lymphocytes of developmentally regulated molecules, that play a crucial role in the generation of immunological memory (CD45 isoforms) and in attainment of antibody responses (CD40L), 22 HIV-infected children and 36 seroreverted children born to HIV+ mothers were studied. The percentage of CD40L+ PBMC after activation in vitro with phorbol myristate acetate (PMA) plus ionomycin was lower in HIV-infected children than in controls (P < 0.004). This correlated with the depletion of CD4+ lymphocytes (r = 0.75; P < 0.001). CD40L expression rose progressively with age (r = 0.36; P = 0.03) in seroreverted children, but not in HIV-infected children, suggesting that while in normal children in vivo antigen stimulation results in progressive attainment of CD40L expression (and thus to effective T-B cell cooperation), this process is largely defective in HIV-infected children, contributing to the genesis of humoral immune deficiency. The proportion of CD4+ cells bearing the CD45RO isoform was increased among HIV-infected infants during the first years of life. However, the percentage of CD4+ CD45RO+ peripheral blood mononuclear cells (PBMC) progressively increased with age in controls (r = 0.69; P = 0.03), but not in HIV-infected children, showing that while vertical transmission of HIV does not prevent CD45RO expression early in life, it is associated with a disturbance of the physiological process of antigen priming, contributing to poor immunological memory to T cell-dependent antigens.

Variant CD45R expression with autosomal dominant inheritance affects both helper/inducer (CD4 +) and suppressor/cytotoxic (CD8 +) T cell populations

The differential expression of various membrane CD45R isoforms by normal lymphocyte populations is known to be closely associated with distinct immunoregulatory functions. Abnormal patterns of CD45R expression have been reported in patients with common variable immunodeficiency and HIV infection, and recent evidence has suggested the possibility that one type of variant CD45R expression may be inheritable. By multiple colour flow cytometry, we studied the immunological characteristics of CD4+ helper/inducer and CD8+ suppressor/cytotoxic T cells in a family with variant CD45R expression over three generations. This variant pattern of CD45R expression was shown to affect both CD4+ and CD8+ lymphocyte populations in individual family members and was immunologically characterized by a failure of the normal reciprocal expression of the CD45RA and CD45RO isoforms. Family studies also revealed that this trait had an autosomal dominant mode of inheritance and, in the heterozygous state, appeared not to be associated with major clinical abnormalities. The different isoforms of CD45 show distinct patterns of expression during lymphocyte ontogeny and activation, and these patterns appear to closely reflect function. Despite their diverse immunological roles, our finding that both CD4+ and CD8+ T cells in affected family members showed the same defect suggests a common regulatory mechanism(s) for both these lymphocyte populations. The recognition of this abnormality, particularly in homozygous individuals, will be of great importance in understanding the role of these molecules in immune function and disease.

A prospective study of CD45 isoform expression in haemophagocytic lymphohistiocytosis; an abnormal inherited immunophenotype in one family.

In a prospective study, CD45 isoform expression on T lymphocytes was analysed in seven patients with haemophagocytic lymphohistiocytosis (HLH) and their family members. Six patients, their parents and seven healthy age-matched controls showed the normal pattern of three subpopulations of CD45R expression. In one patient, his mother and one of his healthy brothers only two subpopulations could be identified, which might indicate that the maturation from a CD45RA/RO double-positive to a CD45RO single-positive phenotype does not occur in these individuals. It is not clear if this finding had any impact on the development of HLH in this patient and if the pattern of CD45RA and RO expression observed in this family might represent a predisposition to HLH. In another patient a marked increase of the CD45RO single-positive T cell population was observed during a period of increased disease activity. The clinical relevance of this observation and whether CD45RO expression could serve as a marker for disease activity, or in selected individuals as a susceptibility marker for HLH, remain unknown.

Co-expression of the CD45RA and CD45RO antigens on T lymphocytes in chronic arthritis.

The site of T lymphocyte activation in chronic arthritis is unknown. Peripheral blood (PB) lymphocytes from chronic arthritis patients are in a 'naive' or non-activated state, as defined by expression of the CD45RA antigen and lack of HLA class II expression. In contrast, most synovial fluid (SF) T lymphocytes express a 'memory' or activated phenotype, as defined by the CD45RO antigen and high HLA class II expression. Following stimulation, naive cells lose CD45RA and gain CD45RO expression to become memory cells with a transitional stage of dual CD45RA, CD45RO antigen expression. To localize where this change in phenotype occurs we used dual colour immunofluorescence labelling to compare the percentage of dual CD45RA, CD45RO-positive T lymphocytes in PB and SF from chronic arthritic patients and from normal PB, assuming this population would be increased at the primary site of T lymphocyte activation. Expression of the intermediate and late activation marker, HLA-DR, was also analysed using dual colour immunofluorescence labelling. The percentage of dual positive T lymphocytes was similar between arthritic PB, SF, and normal PB, as was the density of both CD45RA and CD45RO antigens. Thus, CD45 isoform expression did not indicate where T lymphocytes were activated. However, we identified a previously unreported population of CD45RA+ CD45RO+ HLA-DR- T lymphocytes in arthritic and normal PB. In SF, this population was absent, but a substantial number of dual CD45RA, CD45RO-positive HLA-DR+ T lymphocytes were identified. This population would not be predicted by the current model of T lymphocyte activation. Division of T lymphocytes into functional groups on the basis of CD45 isoform expression is likely to be more complicated than previously thought. Based on our findings we propose an alternative model of T lymphocyte differentiation.

CD45RB Is a Novel Molecular Therapeutic Target to Inhibit Aβ Peptide-Induced Microglial MAPK Activation

BackgroundMicroglial activation, characterized by p38 MAPK or p44/42 MAPK pathway signal transduction, occurs in Alzheimer's disease (AD). Our previous studies demonstrated CD45, a membrane-bound protein tyrosine phosphatase (PTP), opposed β-amyloid (Aβ) peptide-induced microglial activation via inhibition of p44/42 MAPK. Additionally we have shown agonism of the RB isoform of CD45 (CD45RB) abrogates lipopolysaccharide (LPS)-induced microglial activation.Methodology and ResultsIn this study, CD45RB modulation of Aβ peptide or LPS-activated primary cultured microglial cells was further investigated. Microglial cells were co-treated with “aged” FITC-Aβ1–42 and multiple CD45 isoform agonist antibodies. Data revealed cross-linking of CD45, particularly the CD45RB isoform, enhances microglial phagocytosis of Aβ1–42 peptide and inhibits LPS-induced activation of p44/42 and p38 pathways. Co-treatment of microglial cells with agonist CD45 antibodies results in significant inhibition of LPS-induced microglial TNF-α and IL-6 release through p44/42 and/or p38 pathways. Moreover, inhibition of either of these pathways augmented CD45RB cross-linking induced microglial phagocytosis of Aβ1–42 peptide. To investigate the mechanism(s) involved, microglial cells were co-treated with a PTP inhibitor (potassium bisperoxo [1,10-phenanthroline oxovanadate; Phen]) and Aβ1–42 peptides. Data showed synergistic induction of microglial activation as evidenced by TNF-α and IL-6 release; both of which are demonstrated to be dependent on increased p44/42 and/or p38 activation. Finally, it was observed that cross-linking of CD45RB in the presence of Aβ1–42 peptide, inhibits co-localization of microglial MHC class II and Aβ peptide; suggesting CD45 activation inhibits the antigen presenting phenotype of microglial cells.ConclusionIn summary, p38 MAPK is another novel signaling pathway, besides p44/42, in which CD45RB cross-linking negatively regulates microglial Aβ phagocytosis while increasing potentially neurotoxic inflammation. Therefore, agonism of CD45RB PTP activity may be an effective therapeutic target for novel agents to treat AD due to its Aβ lowering, and inflammation reducing, properties that are particularly targeted at microglial cells. Such treatments may be more effective with less potential to produce systemic side-effects than therapeutics which induce non-specific, systemic down-regulation of inflammation.

CD4+ T cell hyper-responsiveness in CD45 transgenic mice is independent of isoform

The CD45 tyrosine phosphatase is required for T cell development and function by virtue of its role as a positive regulator of src family kinase activity. In addition, recent data have highlighted that CD45 also acts as a negative regulator of Lck function by dephosphorylation of critical tyrosine residues. Lck functionality and TCR responsiveness are elevated in transgenic mice expressing the CD45RO isoform at 'intermediate' (10-40% of wild type) levels, indicating that the expression level of CD45 is critical in determining the sensitivity of T cells to TCR stimulation. However, it is unclear whether such a phenotype is specific for the CD45RO isoform, typically expressed by activated T cells. In the present work, the roles of three isoforms of CD45, RO, RB and RABC, in thymocyte development, T cell responses and TCR signalling pathways were directly compared. The data demonstrate that expression of CD45RB or CD45RABC at intermediate levels also results in CD4(+) T cell hyper-reactivity, as previously published for CD45RO. These data emphasize the dual functions of CD45 as both a positive and a negative regulators of TCR signalling irrespective of specific isoform expression.

A splenic marginal zone-like peripheral blood CD27+B220- B cell population is preferentially depleted in HIV type 1-infected individuals.

Peripheral blood CD27(+) B cells are reduced in HIV-1-infected individuals. In healthy individuals, the human peripheral blood CD27(+) B cell pool consists of two subsets defined by the expression, or lack thereof, of the CD45 isoform B220. We investigated the presence of circulating B220(+) and B220(-) memory B cells in HIV(+) individuals and found that the reduction in CD27(+) memory B cells occurs primarily among CD27(+)B220(-) B cells. Studies conducted using healthy controls indicate that CD27(+)B220(-) B cells have a splenic marginal zone like the immunophenotype IgM(hi)IgD(lo)CD21(+)CD23(-), express TLR9, and proliferate and secrete IgG and IgM in response to B cell-specific ODN. CD27(+)B220(+) B cells have the immunophenotype IgM(lo)IgD(hi)CD21(+)CD23(+), express activation-induced cytidine deaminase, and proliferate in response to SAC but do not secrete immunoglobulin. The AICD expression, along with CD86 expression, by CD27(+)B220(+) suggests these cells are of germinal center origin. The preferential depletion of CD27(+)B220(-) B cells mirrors alterations in spleen morphology and resident B cell populations due to HIV infection reported by other investigators and may play an important role in the defective B cell immunity against T-independent pathogens such as pneumococcus observed in HIV-1-infected individuals.

Altered signaling and split anergy in lupus T cells

MRL‐Faslpr/lpr (MRL‐lpr) and MRL‐Fas+/+ (MRL+/+) mice develop autoimmune disease that mimics human SLE. We have used these mice to address the role of T cells in lupus. We found that freshly isolated CD4+ T cells from the spleen of MRL‐lpr display features of in vivo antigen priming and activation as compared to MHC‐matched healthy strains (B10.BR and C3H). Autoimmune T cells also produce more IFN‐γ than control T cells. Surprisingly, however, MRL T cells exhibit reduced calcium flux, IL‐2 production, and proliferation. Such anergic phenotype is seen upon stimulation with anti‐CD3, but not when stimulated with PMA/ionomycin. Such split responsiveness (activated, but anergic) is particularly more profound in a subset of T cells, i.e., B220+ T cells that expand in MRL mice. B220 is an isoform of CD45, a transmembrane phosphatase that plays critical role in cell signaling. As compared to control strains, MRL T cells exhibit reduced expression of CD45RB and CD45RC isoforms, which correlates with altered expression of TCR signaling molecules, with enhanced constitutive phosphorylation of ZAP‐70 and ERK1/2. However, no further increase or a highly accelerated kinetics in p‐ZAP70 and p‐ERK1/2 is seen in MRL T cells as compared to controls. There was also a decrease in phosphorylation of a key target of CD45, Lck (Y505). Using p‐ERK1/2 as readout for T cell activation, we found that activation in MRL‐lpr T cells was inhibited at a relatively lower concentration of CD45 inhibitor as compared to control T cells. Inhibition of CD45 normalized the basal level of activation in MRL‐lpr T cells. These data lead us to hypothesize that CD45‐mediated regulation of TCR signaling is impaired in lupus T cells.Research Support: Supported in part by NIH.

PTPRC (CD45) variation and disease association studied using single nucleotide polymorphism tagging

CD45 is a haemopoietic tyrosine phosphatase, crucial for lymphocyte signalling. Two polymorphisms (C77G and A138G), which alter CD45 isoform expression, are associated with autoimmune and infectious diseases. Using HapMap data, we show that there is substantial linkage disequilibrium across the CD45 gene (PTPRC), with similar patterns in different populations. Employing a set of single nucleotide polymorphisms, correlated with a substantial proportion of variation across this gene, we tested for association with type 1 diabetes, Graves' disease in a Japanese population, hepatitis C in UK population and tuberculin response in a Chinese population. A limited number of common haplotypes was found. Most 138G alleles are present on only one haplotype, which is associated with Graves' disease, supporting previous data that A138G is a functionally important CD45 polymorphism.

Serial Assessment of Immune Status by Circulating CD8+Effector T Cell Frequencies for Posttransplant Infectious Complications

To clarify the role of CD8+ effector T cells for infectious complications, 92 recipients were classified according to the hierarchical clustering of preoperative CD8+CD45 isoforms: Group I was naive, Group II was effector memory, and Group III was effector (E) T cell-dominant. The posttransplant infection rates progressively increased from 29% in Group I to 64.3% in Group III recipients. The posttransplant immune status was compared with the pretransplant status, based on the measure (% difference) and its graphical form (scatter plot). In Groups I and II, both approaches showed a strong upward deviation from pretransplant status upon posttransplant infection, indicating an enhanced clearance of pathogens. In Group III, in contrast, both approaches showed a clear downward deviation from preoperative status, indicating deficient cytotoxicity. The % E difference and scatter plot can be used as a useful indicator of a posttransplant infectious complication.

CD45RO enriches for activated, highly mutated human germinal center B cells

AbstractTo date, there is no consensus regarding the influence of different CD45 isoforms during peripheral B-cell development. Examining correlations between surface CD45RO expression and various physiologic processes ongoing during the germinal center (GC) reaction, we hypothesized that GC B cells, like T cells, that up-regulate surface RO should progressively acquire phenotypes commonly associated with activated, differentiating lymphocytes. GC B cells (IgD−CD38+) were subdivided into 3 surface CD45RO fractions: RO−, RO+/−, and RO+. We show here that the average number of mutations per IgVH transcript increased in direct correlation with surface RO levels. Conjunctional use of RO and CD69 further delineated low/moderately and highly mutated fractions. Activation-induced cytidine deaminase (AID) mRNA was slightly reduced among RO+ GC B cells, suggesting that higher mutation averages are unlikely due to elevated somatic mutation activity. Instead, RO+ GC B cells were negative for Annexin V, comprised mostly (93%) of CD77− centrocytes, and were enriched for CD69+ cells. Collectively, RO+ GC B cells occupy what seems to be a specialized niche comprised mostly of centrocytes that may be in transition between activation states. These findings are among the first to sort GC B cells into populations enriched for live mutated cells solely using a single extracellular marker.