Harnessing local and systemic immunity for vaccines against tuberculosis.

The lung is the portal of entry for Mycobacterium tuberculosis (Mtb) and animal experimental evidence indicates that local immune defense mechanisms are crucial for protective immunity. Immunization via the lower respiratory tract efficiently induces a dividing, activated, antigen-dependent, lung-resident, memory T-cell population, which is partly recoverable by bronchoalveolar lavage. These cells can inhibit the growth of Mtb in the lungs immediately after infection. Delivery of appropriate signals to the lung innate immune system is critical for induction of effective local immunity. In contrast after parenteral immunization, antigen-specific cells may be found in lung tissue but few are recoverable by lavage and inhibition of mycobacterial growth is delayed. Harnessing both local and systemic immunity can provide highly effective protection in animal models and the evidence suggests that taken in aggregate, multiple animal models may predict the success of novel vaccine strategies in humans.

Immune memory: the basics and how to trigger an efficient long-term immune memory.

Immunological memory consists of expanded clones of T and B lymphocytes that show an increased rate of cell division and shortened telomeres compared with naïve cells. However, exhaustion of clones is delayed by kinetic heterogeneity within clones and altered survival and up-regulation of telomerase. Prolonged maintenance of protective B-cell immunity is T-cell dependent and requires a balance between plasma cells and memory B cells. Protective T-cell immunity also requires correct quality of T cells and that they are located appropriately.

Unusual case presentations associated with the CD45 C77G polymorphism.

CD45, the leucocyte common antigen, is a haematopoietic cell specific tyrosine phosphatase. Human polymorphic CD45 variants are associated with autoimmune and infectious diseases and alter the phenotype and function of lymphocytes, establishing CD45 as an important regulator of immune function. Here we report four patients with diverse diseases with unusual clinical features. All four have the C77G polymorphism of CD45 exon 4, which alters the splicing and CD45RA/CD45R0 phenotype of lymphocytes. We suggest that C77G may be a contributing factor in these unusual cases.

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.

Immunology of vaccination.

An ideal vaccine is relatively easy to define, but few real vaccines approach the ideal and no vaccines exist for many organisms, for which a vaccine is the only realistic protective strategy in the foreseeable future. Many difficulties account for the failure to produce these vaccines. All micro-organisms deploy evasion mechanisms that interfere with effective immune responses and, for many organisms, it is not clear which immune responses provide effective protection. However, recent advances in methods for studying immune response to pathogens have provided a better understanding of immune mechanisms, including immunological memory, and led to the realisation that the initiation of immune responses is a key event requiring triggering through 'danger' signals. Based on these findings, the development of novel adjuvants, vectors and vaccine formulations allowing stimulation of optimal and prolonged protective immunity should lead to the introduction of vaccines for previously resistant organisms.

Vaccine programmes and policies.

Management of an effective national vaccine strategy necessitates careful planning. In the face of budgetary constraints and the likely development of many new vaccines over the next few years, a rational choice of which vaccines to use and how best to use them will depend on first class disease surveillance, economic analysis of cost effectiveness and mathematical modelling to ensure optimal vaccine delivery. Effective immunisation programmes require strategic planning that integrates the outputs of these parameters with available health facilities with the least possible disruption. At the present time, the greatest threat to vaccination is resistance to continuing vaccination in the face of declining prevalence of many infectious diseases and heightened fears over vaccine safety. Re-assurance of the public that vaccines are safe demands effective detection of vaccine-related side-effects and rigorous investigation of any safety concerns.

Lipopolysaccharide modulation of dendritic cells is insufficient to mature dendritic cells to generate CTLs from naive polyclonal CD8+ T cells in vitro, whereas CD40 ligation is essential.

Many cytotoxic CD8+ T cell responses are dependent on the interactions between CD40 ligand on the helper CD4+ T cell and CD40 on the APC. Although CD40 triggering of dendritic cells (DC) has been shown to mature the DC by increasing the level of expression of costimulatory molecules and inducing IL-12 secretion, the precise mechanisms by which CD40-CD40 ligand interactions allow DC to drive CTL responses remain unknown. We have used an in vitro model in which naive polyclonal CD8+ T cells can be activated by bone marrow-derived DC to investigate factor(s) that are responsible for this CD40-dependent generation of CTLs. DC modulated with agonistic anti-CD40 mAb (aCD40) are able to generate Ag-specific CTL responses while DC modulated with the microbial stimulus LPS alone do not. We compared the Ag-presenting capacity, levels of costimulatory molecules, and release of cytokines and chemokines of DC modulated with aCD40 to that of DC modulated by LPS. None of the factors assayed account for the unique capacity of anti-CD40-matured DC to drive CTL but this model provides a simplified system for further investigation. Although we attempted to use an LPS-free system for these studies, we are unable to rule out the possibility that very low levels of endotoxin (<20 pg/ml) may synergize with CD40 ligation in the generation of CTLs.

A point mutation in CD45 may be associated with an increased risk of HIV-1 infection.

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. Here we show that abnormal CD45 splicing caused by a C77G transversion in exon A of the gene encoding CD45 (PTPRC) is associated with increased susceptibility to HIV-1 infection.

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 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.

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.

Analysis of lymphocyte diversity in the elderly: heteroduplex analysis and alternative techniques.

Heteroduplex analysis allows global analysis of T cell receptor clonality. This review outlines the method, compares it to other available techniques for the study of clonality and reviews current literature on how these are being used to investigate alterations in the T cell repertoire within elderly individuals.

Structural and functional analysis of the human CD45 gene (PTPRC) upstream region: evidence for a functional promoter within the first intron of the gene.

Expression of the leucocyte common antigen (CD45) in mammals is restricted to the nucleated lineages of haematopoietic cells. It appears in early progenitors in the bone marrow and is expressed at the surface of these cells throughout their differentiation. However, at least in T cells, the pattern of expression switches between different isoforms during the successive stages of differentiation in the thymus and after activation in the periphery. In order to understand the mechanisms controlling the transcription of the human CD45 gene, 2.7 kbp of the 5'-flanking region were sequenced and analysed for their ability to direct expression of a reporter gene. The only region with promoter activity was localized within the first intron of the gene. This promoter shows no tissue specificity but could be enhanced by a heterologous enhancer. Mobility shift assays showed complex but specific protein binding. The sequence in this region lacks similarity with known promoters or initiators but is highly conserved in evolution. No transcription initiation could be detected within or downstream of this region, suggesting that this might be a new type of RNA polymerase II promoter able to drive transcription from an upstream sequence. An additional exon was also found upstream of exon 1. The two exons 1 (1a and 1b) are mutually exclusive and both are spliced to exon 2. This makes the structure of the 5' region of the human CD45 gene identical to its mouse homologue.

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 gamma-chain (gamma(c)) 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.

Clonal expansions in acute EBV infection are detectable in the CD8 and not the CD4 subset and persist with a variable CD45 phenotype.

We have applied a sensitive global analysis of TCR heterogeneity to compare clonal dynamics of CD4(+) and CD8(+) T cells in acute infectious mononucleosis. Using this approach, we are able to identify a broad representation of the total virus-specific population without the bias of in vitro culture and then to track their phenotype and fate by their unique molecular footprint. We demonstrate a large number of Ag-driven clones using different TCRs in the acute phase, all CD8(+). The diverse large clones generated in the CD8 subset in response to this virus contrast with the complete lack of detectable clonal expansion in the CD4 compartment. Many of the same clones remain detectable in directly ex vivo CD8(+) T cells for at least a year after resolution of infectious mononucleosis, although the clone size is reduced. Thus, memory CD8 cells following EBV infection persist at relatively high circulating frequency and represent a subset of the large range of clonotypes comprising the acute effectors. Separation of samples into CD45RA (naive) and CD45RO (memory) fractions shows the accumulation of identical CDR3 region defined clonotypes in both CD45RO and CD45RA fractions and sequencing confirms that dominant long-lived monoclonal expansions can reside in the CD45RA pool.

Genomic structure and sequence of the leukocyte common antigen (CD45) from the pufferfish Fugu rubripes and comparison with its mammalian homologue.

The leukocyte common antigen (CD45) is a transmembrane protein tyrosine phosphatase expressed only in nucleated hematopoietic cells. It can be expressed as different isoforms depending on the cell type and the state of activation or differentiation and it is known to play a crucial role in the maturation and differentiation of B and T lymphocytes. However, the regulation of CD45 expression and function has been difficult to study due to the complexity of the gene in mammals. In this paper, we report the isolation and characterization of a CD45 orthologue gene from the Japanese pufferfish Fugu rubripes (Fugu). The Fugu CD45 cDNA sequence contains an open reading frame of 1,246 amino acids with a variable extracellular region as a result of the alternative splicing of two exons. The intracellular region is organized into two highly conserved tyrosine phosphatase domains. The extracellular region is not conserved except in some structural domains. The Fugu CD45 gene has a similar exon/intron organization to that of mammals except in the 5' end where some exons are missing or fused together. By contrast, the gene is ten times smaller in Fugu due to the small size of the introns. These studies show a greater flexibility to evolve at the 5' end of the gene and provide clues to the functionally important domains of the molecule. In addition, the lower complexity of this gene in Fugu should allow easier mapping of its regulatory sequences.

Differences in the regulation of CD4 and CD8 T-cell clones during immune responses.

The functional units of immune response are lymphocyte clones. Analysis of lymphocyte life span in vivo shows that the overall turnover of CD4 and CD8 lymphocytes does not differ greatly. Recently, molecular methods have been developed which allow a global analysis of T-cell clones responding to an antigen in vivo. We have used a sensitive, modified heteroduplex analysis to follow T-cell clones responding to Epstein-Barr virus in acute infectious mononucleosis (AIM). Strikingly, all the many large clones detected in freshly isolated AIM blood were found within the CD8 fraction. CD4 clonal populations responding to the soluble recall antigen tetanus toxoid could only be detected after in vitro re-stimulation. These data imply that CD4 responses may be more polyclonal than those of CD8 cells and that the size of CD4 clones is more tightly regulated. Several molecular mechanisms may contribute to this. Up-regulation of telomerase allows very large expansions of CD8 cells to occur without exhaustion of proliferative capacity.

Regulation of alternative splicing of CD45 by antagonistic effects of SR protein splicing factors.

CD45 is a transmembrane glycoprotein possessing tyrosine phosphatase activity, which is involved in cell signaling. CD45 is expressed on the surface of most leukocytes and can be alternatively spliced by the inclusion or skipping of three variable exons (4, 5, and 6 or A, B, and C) to produce up to eight isoforms. In T cells, the splicing pattern of CD45 isoforms changes after activation; naive cells express high m.w. isoforms of CD45 which predominantly express exon A (CD45RA), whereas activated cells lose expression of exon A to form low m.w. isoforms of CD45 including CD45RO. Little is known about the specific factors controlling the switch in CD45 splicing which occurs on activation. In this study, we examined the influence of the SR family of splicing factors, which, like CD45, are expressed in tissue-specific patterns and have been shown to modulate the alternative splicing of a variety of transcripts. We show that specific SR proteins have antagonistic effects on CD45 splicing, leading either to exon inclusion or skipping. Furthermore, we were able to demonstrate specific changes in the SR protein expression pattern during T cell activation.

Regulation of apoptosis and replicative senescence in CD8+ T cells from patients with viral infections.

Activated T lymphocytes are generated during an immune response. The induction of T lymphocyte proliferation is one way in which cell numbers can be controlled. However, once generated, the increased numbers of cells must be removed in order to re-establish cellular homoeostasis within the immune system. In this paper we describe how the numbers of activated T cells can be regulated by two distinct mechanisms, namely apoptosis and replicative senescence. In addition, we suggest that the regulation of cell clearance, as opposed to cell persistence, after an immune response is intimately involved in the generation of immune memory.

CD7 expression distinguishes subsets of CD4(+) T cells with distinct functional properties and ability to support replication of HIV-1.

Enrichment of a subset of CD4(+)CD45R0(+)CD7(-) T cells has been observed in HIV-infected individuals. We have investigated the ability of CD7(+) and CD7(-) T cells to support replication of HIV and show that virus replicates preferentially in CD7(+) cells. Several possible mechanisms that may underlie such differences in susceptibility to HIV were studied. Our data demonstrate that mitogen stimulation induces poor expression of CD25 and IL-2 in CD7(-) compared with CD7(+) cells. We also show that uninfected CD7(-) cells are more resistant to mitogen-induced apoptosis than CD7(+) cells. Our data support the view that the CD7(-) subset is inherently resistant to HIV replication and that this is due in part to reduced CD25 expression and IL-2 production.

Is immune senescence reversible?

Many genes have been shown to be involved in the decline in immune function of the elderly. However, normal numbers of myeloid and lymphoid colonies can be grown from elderly bone marrow under optimal conditions and some thymic function is preserved well into adult life. It may also be possible to reverse partially declining thymic function by IL-7 treatment. Peripheral B and T cells show evidence of dysregulation with production of large clones, changes in subset distribution and altered signalling and cytokine production, particularly decreased IL-2 production in the mouse. The identification of these defects may lead to relatively simple procedures to improve vaccination for the elderly.