Elma Z. Tchilian

A Deletion in the Gene Encoding the CD45 Antigen in a Patient with SCID

SCID is a heterogeneous group of hereditary diseases. Mutations in the common γ-chain (γc) of cytokine receptors, including those for IL-2, IL-4, IL-7, IL-9, and IL-15, are responsible for an X-linked form of the disease, while mutations of several other genes, including Janus-associated kinase-3, may cause autosomal recessive forms of SCID. We investigated the first SCID patient to be described with minimal cell surface expression of the leukocyte common (CD45) Ag. CD45 is an abundant transmembrane tyrosine phosphatase, expressed on all leukocytes, and is required for efficient lymphocyte signaling. CD45-deficient mice are severely immunodeficient and have very few peripheral T lymphocytes. We report here that a homozygous 6-bp deletion in the gene encoding CD45 (PTPRC, gene map locus 1q31–32), which results in a loss of glutamic acid 339 and tyrosine 340 in the first fibronectin type III module of the extracellular domain of CD45, is associated with failure of surface expression of CD45 and SCID. Molecular modeling suggests that tyrosine 340 is crucial for the structural integrity of CD45 protein. This is the second description of a clinically relevant CD45 mutation, provides direct evidence for the importance of CD45 in immune function in humans, and suggests that abnormalities in CD45 expression are a possible cause of SCID in humans.

Does 77C-->G in PTPRC modify autoimmune disorders linked to the major histocompatibility locus?

A 77G allele of the gene encoding CD45, also known as the protein tyrosine phosphatase receptor-type C gene (PTPRC), has been associated with multiple sclerosis (MS). Here we determine allele frequencies in large numbers of MS patients, primary immunodeficiencies linked to the major histocompatibility complex (MHC) locus and over 1,000 controls to assess whether aberrant splicing of PTPRC caused by the 77C→G polymorphism results in increased susceptibility to these diseases. Our results show no difference in the frequency of the 77G allele in patients and controls and thus do not support a causative role for the polymorphism in the development of disorders with a strong autoimmune component in etiology.

Combinations of CD45 Isoforms Are Crucial for Immune Function and Disease

Expression of the CD45 Ag in hemopoietic cells is essential for normal development and function of lymphocytes, and both mice and humans lacking expression exhibit SCID. Human genetic variants of CD45, the exon 4 C77G and exon 6 A138G alleles, which alter the pattern of CD45 isoform expression, are associated with autoimmune and infectious diseases. We constructed transgenic mice expressing either an altered level or combination of CD45 isoforms. We show that the total level of CD45 expressed is crucial for normal TCR signaling, lymphocyte proliferation, and cytokine production. Most importantly, transgenic lines with a normal level, but altered combinations of CD45 isoforms, CD45RABC/+ and CD45RO/+ mice, which mimic variant CD45 expression in C77G and A138G humans, show more rapid onset and increased severity of experimental autoimmune encephalomyelitis. CD45RO/+ cells produce more TNF-α and IFN-γ. Thus, for the first time, we have shown experimentally that it is the combination of CD45 isoforms that affects immune function and disease.

CD45 negatively regulates tumour necrosis factor and interleukin-6 production in dendritic cells

CD45 is known to regulate signalling through many different surface receptors in diverse haemopoietic cell types. Here we report for the first time that CD45–/– bone marrow dendritic cells (BMDC) are more activated than CD45+/+ cells and that tumour necrosis factor (TNF) and interleukin‐6 (IL‐6) production by BMDC and splenic dendritic cells (sDC), is increased following stimulation via Toll‐like receptor (TLR)3 and TLR9. Nuclear factor‐κB activation, an important downstream consequence of TLR3 and TLR9 signalling, is also increased in CD45–/– BMDC. BMDC of CD45–/– mice also produce more TNF and IL‐6 following stimulation with the cytokines TNF and interferon‐α. These results show that TLR signalling is increased in CD45–/– dendritic cells and imply that CD45 is a negative regulator of TLR and cytokine receptor signalling in dendritic cells.

A CD45 polymorphism associated with abnormal splicing is absent in African populations

Abstract. The CD45 antigen is essential for normal antigen receptor-mediated signalling in lymphocytes, and different patterns of splicing of CD45 are associated with distinct functions in lymphocytes. Abnormal CD45 splicing has been recognized in humans, caused by a C77G transversion in the gene encoding CD45 (PTPRC). Recently the C77G polymorphism has been associated with multiple sclerosis and increased susceptibility to HIV-1 infection. These studies suggest that the regulation of CD45 splicing may be critical for the proper function of the immune system. Because of these data we examined the frequency of the C77G allele in African and Asian populations from countries with high or low prevalence of HIV infection. Here we report that the variant CD45 C77G allele is absent in African populations. We further show that populations living in the Pamir mountains of Central Asia have a very high prevalence of the C77G variant.

Altered CD45 expression in C77G carriers influences immune function and outcome of hepatitis C infection

Background: A polymorphism in exon 4 (C77G) of CD45 that alters CD45 splicing has been associated with autoimmune and infectious diseases in humans. Objective: To investigate the effect of C77G in hepatitis C virus (HCV) infected individuals and study the phenotype and function of peripheral blood mononuclear cells (PBMC) from healthy and hepatitis C infected C77G carriers. Results: C77G individuals showed an increased proportion of primed CD45RA and effector memory CD8 T cells and more rapid activation of the lymphocyte specific protein tyrosine kinase (Lck) following CD3 stimulation. Transgenic mice with CD45 expression mimicking that in human C77G variants had more activated/memory T cells, more rapid proliferative responses, and activation of Lck. Conclusions: Changes in CD45 isoform expression can alter immune function in human C77G variants and CD45 transgenic mice. The C77G allele may influence the outcome of HCV infection.

The Exon A (C77G) Mutation Is a Common Cause of Abnormal CD45 Splicing in Humans

The leukocyte common (CD45) Ag is essential for normal T lymphocyte function and alternative splicing at the N terminus of the gene is associated with changes in T cell maturation and differentiation. Recently, a statistically significant association was reported in a large series of human thymus samples between phenotypically abnormal CD45 splicing and the presence of the CC chemokine receptor 5 deletion 32 (CCR5del32) allele, which confers resistance to HIV infection in homozygotes. We show here that abnormal splicing in these thymus samples is associated with the presence of the only established cause of CD45 abnormal splicing, a C77G transversion in exon A. In addition we have examined 227 DNA samples from peripheral blood of healthy donors and find no association between the exon A (C77G) and CCR5del32 mutations. Among 135 PBMC samples, tested by flow cytometric analysis, all those exhibiting abnormal splicing of CD45 also showed the exon A C77G transversion. We conclude that the exon A (C77G) mutation is a common cause of abnormal CD45 splicing and that further disease association studies of this mutation are warranted.

Novel perforin mutation in a patient with hemophagocytic lymphohistiocytosis and CD45 abnormal splicing

Hemophagocytic lymphohistiocytosis (HLH) composes a group of rare heterogenous disorders characterized by uncontrolled accumulation and infiltration of activated T lymphocytes and macrophages. Cytotoxic T and natural killer cell activity is significantly reduced or absent in these patients. Mutations in the important mediator of lymphocyte cytotoxicity perforin were identified in a number of HLH individuals. Here we report a novel missense mutation thr435met in the conserved Ca2+ binding domain of perforin in a patient with HLH. Prediction of the 3‐dimensional structure of the thr435met perforin mutant using comparative molecular modeling indicates that the proteins ability to bind Ca2+, and therefore its cytolytic function, would be strongly compromised. In addition, this patient exhibited abnormal CD45 splicing caused by a C77G mutation in the gene encoding CD45 (PTPRC). Our findings suggest a combined role for perforin mutation and abnormal CD45 splicing as significant contributory factors in the pathogenesis of HLH.

CD45 is required for type I IFN production by dendritic cells

CD45 is a leukocyte tyrosine phosphatase, essential for normal immune responses. We have studied the function of splenic dendritic cells of CD45+/+, CD45–/–, CD45RABC and CD45RO transgenic mice. We show that there are increased numbers of plasmacytoid dendritic cells in CD45–/– mice. DC of all mice are capable of responding to lymphocytic choriomeningitis virus (LCMV) infection by up‐regulation of MHC and costimulatory molecules. DC of CD45–/– mice have an impaired capacity to produce typeu2004I interferons in response to LCMV infection in vivo. These data indicate that lack of CD45 expression in DC has a profound effect on their function. This is largely restored by CD45RABC or CD45RO transgenes.

Abnormal Cell Surface Antigen Expression in Individuals with Variant CD45 Splicing and Histiocytosis

Hemophagocytic lymphohistiocytosis (HLH) and Langerhans cell histiocytosis (LCH) are members of a group of rare heterogenous disorders, the histiocytoses, characterized by uncontrolled accumulation of pleomorphic infiltrates of leukocytes. The etiology of these diseases is mainly unknown. CD45 is a hemopoietic cell specific tyrosine phosphatase essential for antigen receptor mediated signaling in lymphocytes and different patterns of CD45 splicing are associated with distinct functions. Recently a polymorphism (C77G) in exon 4 of CD45 causing abnormal CD45 splicing and a point mutation affecting CD45 dimerization were implicated in multiple sclerosis in humans and lymphoproliferation and autoimmunity in mice respectively. Here we show that two patients with HLH exhibited abnormal CD45 splicing caused by the C77G variant allele, while a further 21 HLH patients have normal CD45. We have also examined 62 LCH patients and found three to have the C77G mutation. Peripheral blood thymus-derived (T) CD8+ cells from normal individuals carrying the C77G mutation show a significant decrease in the proportion of cells expressing L-selectin and increased frequency of cells with LFA-1hi expression. It remains to be established whether C77G is a contributing factor in these histiocytic disorders.