Immunodeficiencies Flashcards
Define primary immunodeficiency
groups of disorders caused by inherited or genetic defects in the cells and tissues of the immune system. Can be due to gene mutations in a single gene, and can be spontaneous or inherited.
Define secondary immunodeficiency
when the immune system is compromised due to an environmental factor
What are the initial tests done if someone is presenting with the hallmark of ID?
• The ‘hallmark’ of an ID is recurrent infections – this is the first sign of a potential ID
• There are two types of infection, each mainly using one of 2 different arms of the immune system:
− Bacterial → antibody (humoral)
− Viral/fungal → T cell (cell-mediated)
• If someone presents with recurrent infections, they would have their blood count tested (CBC) to see if certain blood cells are missing.
Viral fungal infections:
• As well as CBC, do a DTH response test or HIV test
• If HIV+ → you know they have an acquired immunodeficiency
• If patient has lymphopenia or absent DTH, you can isolate the lymphocytes and do functional assays
Bacterial infections:
• As well as CBC, can do a test for specific antibodies
• You would look to see if antibodies are normal with a normal response
• Can also do functional assays
→ If you have done all of these, can also do genetic testing.
Describe the use of targeted mouse mutants in ID
- Way to identify the genes that lead to ID
- Hard to look in patients directly, as need to compare patient to patient and with their family to identify the gene that is causative
- Easier and faster to create mouse knockouts, and see if they develop the predicted immunodeficiency, then see if you can rescue the gene defect in mice by adding the functional human gene.
Why are IDs much more common in boys?
- Many immunodeficiencies are X-linked, and hence a lot more common in boys
- This is because girls are usually heterozygous, and due to stochastic X chromosome inactivation, they often have the X that is carrying the mutation silenced
- However, in boys, they only have 1 X chromosome, so if they have an X chromosome with the Btk mutation, there is no compensation anymore
Describe the general features of XLA.
X-linked agammaglobulinaemia → Mutations in Bruton’s tyrosine kinase (btk) gene.
• No mature B cells (agammalgobulin = absence of globulin = absence of antibodies)
• Recurrent haemophilus and pneumococcus infections
• Most common form of inherited agammaglobulinaemia – accounts for 85% of all cases
• Mutant Btk:
− BTK plays a crucial role in B cell maturation (proliferation, differentiation and survival of early B cell lineages) and mast cell activation through the IgE receptor
− Cytoplasmic, non-receptor tyrosine kinase expressed at all stages of B cell development, with the exception of long-lived plasma cells.
− Many other haematopoetic cells express BTK, but the functional impairment seems confined to B cells
• Btk mutant mice have the same phenotype, and if you introduce the human Btk gene into Btk mutant mice, you get normal B cell development
• Patients with XLA have normal pre-B cell populations, but there is a developmental block manifested at the transition between the pro-B and pre-B cell stages.
Describe the two treatments for XLA.
Pooled immunoglobulins:
• Sterile, purified IgG products manufactured from pooled plasma and typically contain >95% unmodified IgG, with has infact Fc-dependent effector functions and only trace amounts of IgA or IgM
• Can be given IV or subcut.
• Given prophylactically – needed to maintain the levels
• Also need generous admin. of antibiotics
• If the donor has an infection, it can be transmitted via the pooled Ig, eg) HIV and Hep C can be a problem, although donors are screened. There is also the risk of vCJD due to prion protein transmission
• Expensive
• Considerably improves quality of life, but patients still suffer chronic infections and life expectancy is still reduced.
Splice-correcting oligonucleotides – Bestas et al,:
• Splicing defects identified as an important cause of genetic disease – mutations may disrupt regular splice sites, which can result in the inclusion of intronic sequences or loss of part of the exons
• An A to T transition in intron 4 of the BTK gene generates a novel 5’splice site, resulting in the inclusion of a cryptic exon → this changes the reading frame and completely abolishes BTK expression.
• This prompted investigations into using splice-correcting antisense oligonucleotides
• A key feature of XLA is that BTK is expressed in all B-lineage stages, except for mature plasma cells – so if a transient correction could be restored at the pro-B cell stage, this could result in long-term effect.
• Using both a mouse mouse model and primary cells from XLA patients, they were able to use BTK-specific SCOs to correct aberrant spliced BTK in B lymphocytes. This restored expression of the functional protein.
• It may therefore be feasible to obtain pro-B cells, carry out the treatment ex-vivo and return the splice-corrected cells to the patients.
What are the features of DGS/VCFS?
Deletion of chromosome 22q11
• Wide range of developmental anomalies in the heart, glands and facial structures (underdeveloped chin, eyes with heavy eyelids)
• Thymic aplasia → very few T cells
• Patients with DGS develop autoimmune disease at a rate that is higher than in the general population – the most common being idiopathic thrombocytopenia, autoimmune haemolytic anaemia and autoimmune arthritis.
• Clinical features highly variable in penetrance, even in twins with identical deletions
• Autosomal dominant
• Approx 90 % occurs spontaneously
• The TBX1 transcription factor has been found to be important (Jerome & Papaloannou):
− Recently, aortic arch defects have been reported in mice heterozygous for a deletion that includes the TBX1 gene
− TBX2 identified as a candidate for this syndrome both due to its chromosomal location and by its expression in the head mesenchyme
− TBX1 knockout mice display a spectrum of phenotypic effects encompassing most of the common DGS/VCFS malformations.
• No cure, but there are some therapies aimed at correcting the defects in affected organs eg) corrective heart surgery.
• For the ID, just involved monitoring overall immune system
− Complete lack of T cells is called ‘complete DGS’, and is potentially fatal and similar to SCID → need a thymus transplant (only available on a research basis) or stem cells transplant to reconstitute the T cells.
− In some patients, the T-lymphocyte defect is enough to cause the B-lymphocytes to fail to make sufficient antibody.
Describe the features of ADA SCID.
• T- B- NK-
• Autosomal recessive
• ADA is a key enzyme in the purine pathway, and is ubiquitously expressed
• Mutation leads to accumulation of lymphotoxic deoxyadenosine → purine metabolite, accumulates in plasma and cells.
• Results in depletion of ATP, inhibits proliferation of T and B lymphocytes
− Accordingly, broad lymphopenia, recurrent infections and failure to thrive are the prominent features
− Systemic accumulation of purine metabolites can cause alterations in several organs inc. skeleton, lung, liver, GI tract and CNS.
Cassani et al, :
• CD4+ T cells from ADA-SCID patients have severely compromised TCR/CD28 driven proliferation and cytokine production
• Impairment is associated with intrinsically reduced ZAP-70 phosphorylation, Ca2+ influx and ERK1/2 signalling
• Exposure to deoxyadenosine results in stronger inhibition of T cell activation
How is ADA SCID treated?
- HSC transplant is curative, but dependent on a good donor match
- Enzyme replacement therapy with PEG-adenosine deaminase bovine (PEG-ADA)
− Used in over 150 patients worldwide
− Allowed stabilization of patients awaiting more definitive treatment
− Affords metabolic deotoxification but immune reconstitution is suboptimal, and may not be long lived
− Gives partial restoration of T cell immunity - Gene therapy
− First disease to be treated with gene therapy (4 year old girl in 1990)
− Remove blood and grow T cells, add inactivated retrovirus carrying functional ADA gene, infuse transduced cells back into patient
− T cell function found ot be restored for 2 years
Describe the features and treatment of Omenn Syndrome SCID
- Oligoclonal T cells
- Autosomal recessive
Genetics:
• Associated with hypomorphic missense mutations in immunologically relevant genes of T and B cells, eg, RAG genes, IL-7R, ZAP-70, DNA-ligase
• Partial loss of RAG gene function is common, loss of this ability means cant perform recombination of TCR and BCR.
• For Omenn syndrome, it is the loss of T cell function that is important → results in only a few T cell clones
• Leads to engraftment of maternal lymphocytes in the feoteus and a co-existence of autologous and maternal lymphocytes.
Symptoms:
• Similar to GvHD → the T cells present have limited levels of recombination, and have specific affinity for self-antigen found in the thymus and periphery
• Characteristic symptom is chronic inflammation of the skin, appearing as a red rash
• Eosinophilia
• Failure to thrive
• Swollen lymph nodes and spleen
• Low Ig levels
Treatment:
• Only treatment is HSC transplant , which is curative - without this, it is rapidly fatal.
Describe the features and diagnosis of X-linked SCID.
- T- B+ NK-
- 50-60% of SCIDs
- X-linked recessive
- Although B cells are present, in the absence of T cell help they will become defective
- No C/M demarcation in thymus
- Hypoplastic lymphoid tissue
Symptoms:
• Infections before 3 months of age due to decreased IgG in the infant
• Followed by viral infections such as pneumonitis
• Tell-tale sign of SCID is candidiasis
• Recurrent eczema-like rashes common
• Other common infections include diarrhea and sepsis
• Failure to thrive, gut problems, skin problems and muscle hypotonia
• Syptoms may not appear for the first 6 months due to passive immunity from the mother
Genetics:
• Mutation in the xq13.1 locus of the X chromosome
• Only tends to affect males, as recessive – males only have 1 X whereas females have 2.
• Mutates the gene for the common y chain of the IL-2RG → shared between the receptors for IL-2, 4,7,9,15 and 21
− IL-7 important for the development of lymphocytes
− Il-15 required for development of NK cells
− IL-21 needed to promote Th17 response
− Il-4 needed for Th2 and Ig production
• Usually passed on through mother being a carrier, but can also arise through de novo mutations in a germ cell.
− Since only 1/3rd SCID patients have a positive family history of SCID, it is thought that de novo mutations account for a significant percentage of cases.
- Absence of thymic shadow on chest X rays
- Genetic testing for IL2RG mutations
Cell count:
Normal Approx 5000,
Describe bone marrow transplant as a treatment for X-linked SCID.
− First successfully reported for SCID in 1968
− Results in full immune reconstitution if the treatment is successful because bone marrow contains multipotent HSCs
− Major problem is GvHD
− BMT requires HLA match between donor & recipient
− Autologous BMT involves full HLA match, allogeneic can be full or half HLA match
− In allogeneic BMT, the chances of GvHD is high if the match is not close enough
− By this, we mean that there are various HLA haplotypes that can result in mismatch, and some are worse than others
− In the case of mismatch, donor bone marrow attack the patients cells.
− BM donor and recipient must share at least one haplotype to restore immune function, eg)
➢ HLA a/b donor & HLA c/d recipient → HLAc or d restricted T cells cannot be activated by HLA a/b APCs.
➢ HLA a/b donor and HLA b/d recipient → HLAb-restricted T cells can be activated by HLAa/b APCs and therefore recognize HLAb/d infected cells.
Symptoms GvHD include rash, high serum bilirubin and diarrhea.
− Depletion of donor T cells and close HLA match will reduce the chance of GvHD, however still not 100% chance of successful cure. In haploidentical twins, still 20% chance of death.
− Other sources of HSCs include:
− Peripheral blood – but can be difficult to get enough cells
− Umbilical cord → the best source
− Would otherwise be discarded
− Rich in stem cells
− Quicker engraftment
− Lower risk of GvHD
− No donor discomfort
− In families where a diagnosis has been previously made (ie, have other ID children), in utero bone marrow transplantation may be done. This has been successful in a number of cases.
Discuss gene therapy as a treatment for X-SCID
− Available only for clinical trials
− Monogenic disorder, so gene therapy can theoretically easily replace mutated IL2RG
− HSCs can be isolated for SCID gene therapy – needs to be in a stem cell because this cell will repopulate the niche. Wouldn’t get long term reconstitution if did in a differentiated cell
− Viral vectors can be employed
− Insert normal gene into the virus
− Infect bone marrow cell with the virus
− Viral DNA inserts into the chromosome
− Inject cells into the patient
− Potential treatment option for many of the SCID classifications (already discussed ADA SCID)
− Cavazzana-Calvo et al:
− 10 children treated with gene therapy at infancy for X SCID
− 9/10 cured
− However, 3 years after treatment, 2 children developed T cell leukemia due to insertion of the IL2RG gene near the LMO2 gene (a known oncogene), resulting in its activation → insertional mutagenesis.
− Insertional mutategenesis is a problem in X-SCID and CGD, but not in ADA SCID.
− Research into the use if insulator and suicide genes is warranted as this may prevent cancer from development
− Insulator genes → inhibit the activation of adjacent genes
− Suicide gene → stimulated when a tumour begins to form, and will result in the deactivation of the therapeutic gene.
− Restriction enzymes such as the zinc-finger nuclease allows the research to choose the site of gene integration
− Vectors that self-inactivate the promoter and enhancer are being research
− CRISPR/Cas9 may also prove highly effective, as will bypass the need for retroviral vectors.
Gene therapy may not always be long-term:
− In X-linked and ADA SCID, the cells that have the repaired gene have a cellular advantange. Repairing both genes allows the cells to proliferate, so they grow over the endogenous cells – so it is long term.
− In CGD
Describe the features of JAK3-SCID.
• T- B+ NK- SCID
• JACK3 is a tyrosine kinase functionally coupled to cytokine receptors sharing the common gamma chain
• Mediates the signal transduction from the cytokines that share the common gamma chain, so mutation affects multiple immune cells:
− IL-7 important for the development of lymphocytes
− Il-15 required for development of NK cells
− IL-21 needed to promote Th17 response
− Il-4 needed for Th2 and Ig production
• Autosomal recessive
• Patients present with the classical features of SCID – respiratory infections, diarrhea, thrush and failure to thrive
• Rare, only 6% of all cases of SCID
Describe the features of RAG1/2 SCID.
- B- NK+ SCID
• Results in defects in TCR and BCR gene rearrangements
• Normal myeloid cells and innate immunity
• Pro-B cell present, but preB cell absent (precursors are present, but not any cells with rearranged gene segments).
• RAG1/2 KO mice show same phenotype
Describe the features of ZAP70 SCID
- (CD4+ CD8-) T, B+ NK+ SCID
- Autosomal recessive
- Characteirsed by the selective absence of CD8+ T cells, in the presence of normal or elevated CD4+ T cells
- ZAP70 is the complex that is recruited to the TCR to form the ZAP70/SLP76 signalosome to mediate intracellular T cell signaling.
- Symptoms present in first year of life – recurrent opportunistic infections, diarrhea, failure to thrive, candidiasis common.
- Affected children usually do not survive past 2 years without HSCT
Diagnosis:
• Lymphocyte counts
• Lymphocyte function testine
• ZAP70 protein expression or molecular genetic testing
Management:
• Treatment of manifestations → immediate IVIG, antibacterials/virals/fungals to reduce infection
• Prevention of manifestations → allogeneic HSCT to reconstitute immune system, preferably within first 3 months
• Prevention of secondary complications → Use of irradiated, CMV&EBV negative blood products, deferment of immunisations until immune reconstitution, formula feed instead of breast feed unitl CMV status of mother known.
• Surveillance → Following successful HSCT, monitoring of the following every 6-12 months: immune status, liver and renal function, CBC, growth and psychomotor development
• Agents to avoid → Non-irradiated blood products, live vaccinations
Genetic counselling:
• Each sibling of an infected individual has an 25% of being affected, a 50% chance of being a carrier and a 25% chance of being unaffected.
What is the long quest for neonatal screening of SCID?
- Early recognition of SCID is a pediatric emergency, before live vaccines or non-irradiated blood products are given.
- Need for newborns screening has been recognized for the past 15 years
- However, required an assay for T cell lymphopenia that could be performed on dried blood spots routinely collected from newborns → was accomplished 6 years ago and have already been 7 successful pilot studies
- Recommendation to add screening to routine panel was approved in 2010
Describe the features of MHC-II deficiency (BLS)
• Absence of MHC-II due to deficiencies in the TFs required for MHC-II expression
• Low T cell numbers, mainly CD4
• Affected genes encode RFXANK, RFX5, RFXAP and CIITA – four regulatory factors highly specific and essential for MHCII genes.
− The first 3 are subunits of RFX – a trimeric complex that binds to MHCII promoters.
− CIITA is a coactivate that functions as the master control factor for MHCII expression
• Clinically similar to SCID, however doesn’t result in decreased B and T cell counts as the development of these cells is not impaired
• There is also a type I BLS, called HLA Class I deficiency → much more rare, associated with TAP1, TAP2 or TAP binding proteins involved in transporting antigens from the proteasome to the MHC-I
• Although gene therapy is attractive, currently BMT is the only treatment
Describe the features of LAD
- Defect in beta-2 integrin
- Diminished cell adhesion and migration of leukocytes to inflamed sites
- A problem in race horses and Holstein cattle (all bred from one bull that had LAD)
Genetics:
• LAD result of mutations in the B2 subunit of CD11 and CD18 integrins LFA-1, Mac-1, p150, p95 and adb2
• Located on chromosome 21
• CD18 KO mouse has similar problems to human LAD, but also suffers from dermatitis not seen in man
• Majority of LAD cases involved a single point missense mutation, giving an altered precursor protein which fails to bind to the alpha subunit
• Other diseases resulting from mutations of integrins include Glanzmanns thrombasthenia, and epidermolysis bullosa
Normal function of integrins:
• LFA-1 and Mac-1 operate together with selectins to aid leukocyte migration across blood vessels into injured tissue.
• LFA-1 involved in adhesion and signaling at the immunological synapse
• LFA-1 involved in clustering of proliferating lymphocytes
• LFA-1 involved in targeting of cytotoxic T cells to their targets
• Neutrophils use Mac-1 as a phagocytic receptor
Symptoms:
• Range of disease severity
• Recurring necrotic soft tissue infections
• Impaired wound healing with lack of pus
• Severe gingivitis
• Disorder viewed primarily as a failure of neutrophil function, as they are the first to arrive at sites of tissue injury, and rely the most heavily on B2 integrins.
New types of LAD:
• Hallmark of LAD is reduced expression of B2 integrins, but new variants have been described which are exceptions
• One patient had moderately severe phenotype despite expressing 60% normal level of B2 integrins → patient found to have mutations that allowed normal expression but no function.
Treatment:
• BMT successful
• Should be possible to do prenatal diagnosis based on lack of integrin expression levels at 20 weeks gestation
Describe the general features of CGD
• Failure to generate the oxidative burst due to defects in components of the NADPH oxisase system
− Macrophages phagocytose, but cant digest
− Defective at generating the microbicidal reactive oxidant superoxide anion and its metabolites hydrogen peroxide, hydroxyl anion and hypopalous acid.
• Patients suffer from recurrent life-threatening bacterial and fungal infections
− Repeat infections with catalase +ve organisms, eg) Staphylococcus
− The bactericidal defect is not global, but quite specific.
− Killing of Staph. aureus is defective, but killing of Streptococci normal → Streptococci do not degrade their own H2O2 with catalase, therefore providing H2O2 which interacts with myeloperoxidase in the phagocytic vacuoles.
• CGD also characterized by excessive inflammatory responses leading to granuloma formation → granulomas form when the immune system attempts to wall off substances it perceives as foreign, but is unable to eliminate.
• Causes multiple abcesses in the LN, liver, bone, gut wall (→ diarrhea)
• Usually fatal by 7 years
• Affects 1 in 200,000 people
What is the genetics of CGD, and how is it diagnosed?
- X-linked → gp91 phox (most common form)
- Autosomal recessive → p22 phox, p47 phox, p67 phox
- Female carriers of X-linked CGD have been found to suffer from recurring mouth ulcers and skin rashes
- No health issues associated with being an autosomal recessive carrier
Diagnosis of CGD:
• 95% diagnosed by age 5
• The nitroblue-tetrazolium (NBT) test most widely known
− Depends upon the reduction of NBT to the insoluble blue compound formazan by NADPH oxidase
− → negative in CGD (doesn’t turn blue), as the higher the blue socre, the better a cell is at producing ROS.
• Cytochrome c reduction assay can tell physicians how much superoxide is being produced
• Once diagnosis established, can undergo genetic testing to determine which mutation is the underlying cuase
How is CGD treated?
- Aggressive and prolonged admin of antibiotics and prednisone
- Treatment for inflammatory component of CGD problematic, as post treatments are immunosuppressive
- Long-term broad-spectrum antibiotics
- Granulocyte transfusions from G-CSF and dexamethasome stimulated donors
- HSCT may be considered as an early treatment option
- Gene therapy → correction of neutrophil oxidase activity in vivo
What are the general features of CVID and how is it diagnosed?
- Spectrum of diseases, less severe than SCIDs
- Characterised by deficiencies in one or more antibody classes
- Present in late childhood or early adulthood → because it is milder, takes more time to see the symptoms
- Occurs in 1 in 75,000 live births → most frequent symptomatic primary immunodeficiency in adults
- Selective IgA deficiency the most common
- Most are sporadic, but some inherited.
Common Symptoms:
• Characteristic histological lesions inc. granulomatous disorder, nodular lymphoid hyperplasia of the gut, nodular regenerative hyperplasia of the liver or pneumonitis of the lungs.
Disease also exists in:
• Inner ear → repetitive ear infection, loss of hearing
• Thyroid → hormone production disturbed
• Lung → localized, irreversible dilation of part of the lungs
• Spleen → splenomegaly as the body is trying to compensate
• Autoimmune aneamia
Diagnosis:
• Difficult, according to ESID criteria, CVID is a diagnosis of exlusion
• 2014 revised diagnostic criteria for CVID:
− At least one of:
− Incresed infection
− Autoimmune manifestations
− Granulomatous disease
− And marked decrease of IgG and IgG
− And at least one of:
− Poor antibody response to vaccines
− Low switched memory B cells
− And secondary cases of hypogammaglobulinaemia
− And diagnosis established after the fourth year of life
− And evidence of profound T cell deficiency
Describe the features of X-linked hyper-IgM syndrome
- Class switching involves recombination between specific switch signals
- It is controlled by CD4+ T helper cells and cytokines
- CD40/CD40 ligand interactions is essential for class switch → without it, cells only make IgM
- If either CD40 or CD40L are mutated, T cell help isn’t provided to the B cell, so plasma cells, class switching and memory cell production is affected.
- Patients deficiency in CD40L have high IgM, little or no IgG, IgA or IgE and cannot form germinal centres
Kroczek et al, :
• Molecular genetic analysis of HIgM was started in 1987
• Gene was mapped to Xq24-27 → so occurs more in males (occurs in 1 in 2 million newborn boys).
• TRAP expressed on the surface of activated T cells is a ligand for CD40 on B cells, and has been mapped to this chromosomal region, suggesting a relationship with HIgM
• Further work demonstrated that failure of TRAP/CD40L to interact with CD40 on B cells is responsible for the ineffeicient T cell help observed in HIgM
• The availability of reagents for the testing of TRAP expression provides tools for early diagnosis of patients
• Will also provide a basis for future attempts at gene therapy
Symptoms: • Sinus and respiratory infections • Ear infections • Neutropenia • Lung damage • Liver dysfuncton
Describe the features of ICOS defective CVID
- Expressed on T cells, realted on CD28 but is only expressed on activated T cells
- Interacts with ICOS ligand on B cells
- Patients can be female, suggesting autosomal recessive inheritance
- CD29 and CTLA-4 are on the same chromosome as ICOS – these are still present, so the defect is unlikely to be due to a large deletion.
- Patients have normal differential blood counts
- T cell proliferation and cytokine responses are normal → the only T cell defect is failure to express ICOS
- Memory B cells reduced
- Serum Ig Reduced
- → All features of fault T/B cell cooperation
Symptoms:
• Bacterial infections
• Splenomegaly
• Autoimmune neutrophenia
Describe the features of CD19 defective CVID
• CD19 forms a complex with CD21, CD81 and CD225 in the membrane of mature B cells → together with the BCR, this signals the B cell to decrease its thereshold for activation
Symptoms: • Recurrent childhood bacterial infections • Normal B cell numbers • Low Ab levels • CD19 KO mice have the same phenotype
Van Zelm et al:
• Evaluated four patients from two unrelated fmailies who had hypogammaglobulinaemia and normal numbers of mature B cells
• Found a mutation in CD19 in all 4 patients
• Composition of precursor B cells normal, but reduced memory cells.
• Response of the B cells to in vitro stimulation was impaired, and Ab response to rabies vaccination was poor
• Conclusions → CD19 mutation results in a type of hypogammaglobulinaemia in which the response of mature B cells to antigenic stimulation is defective.
What are the causes and symptoms of familial haemophagocytic lymphohistiocytosis?
• Lack of perforin function, so cant kill the target cell
Function of perforin:
• CD8+ T cells and NK cells kill by two mechanisms, Fas pathway and granule exocytosis pathway
• The principle mechanism is calcium-dependent release of specialized cytotoxic granules upon recognition of antigen on a target cell
• How do these granules cause death?
1. Serglycin acts as a scaffold
2. Perforin forms pores in the membrane
3. The pores deliver granzyme into the target cell → triggers apoptosis in the target cell by activating caspases.
• These are carefully targed just to the cells that need killing due to the formation of immunological synapses → deficient in LAD due to defective integrin
- Leads to a failure to resolve viral infections
- Leads to uncontrolled proliferation and activation of T cells → they are overcompensating to try and kill the cells, but futile as they don’t have the correct effector mechanisms.
- Life threatening disease of severe inflammation
- Cytokine storm syndrome → potentially fatal immune reaction consisting of a positive feedback loop between cytokines and white blood cells.
- There are primary and secondary (occurring after strong immunologic activation, such as systemic infection, immunodeficiency or malignancy) forms.
- Occurs under the age of 1 in 70% of cases
Symptoms: • Hepatosplenomegaly • Pancytopenias • Fevers • Hepatic, pulmonary and CNS dysfunction • Usually fatal if not treated • Liver function tests elevated, serum C-reactive protein elevated • Bone marrow biopsy shows histiocytosis
What are the 5 genetic subtypes of FHL?
5 subtypes of FHL, each associated with different mutations:
• FHL1 → HPLH1
• FHL2 → PRF1 (Perforin)
• FHL3 → Munc13.4 (Granules cant release perforin, they don’t open)
• FHL4 → Syntaxin 11 (SNARE protein involved in exocytosis)
• FHL5 → Syntaxin binding protein
→ Nearly half the cases are due to bi-allelic PRF1 mutations
Name two differential diseases of FHL
Griscelli Syndrome:
• Rare (less than 100 reported cases) autosomal recessive disorder
• Characterised by partial albininism, hepatosplenomegaly, pancytopenia, hepatitis, immunologic abnormalities and lymphohistiocytosis
• Associated with albinism because the mechanism of perforin granule secretion is the same way that melanocytes secrete melanin
• 3 types of Griscelli:
− Type 1 → Neurologic symptoms, mutations in MYO5A
− Type 2 → Haemophagocytic syndrome with abnormal T cell and macrophage activation. Mutations in RAB27A. Grave prognosis if untreated
− Type 3 → Mutations in melanopilin. Associated with albinism. Non-harmful
Chediak-Higashi Syndrome:
• Autosomal recessive
• Arises from mutation of lysosomal trafficking regulator protein LYST → the granules cant dock
• Results in a decrease in phagocytosis due to failure of phagolysosome formation, so bacteria are not killed
• Characterised by:
− Recurrent pyrogenic infections
− Large lysosome vesicles in neutrophils
− Abnormalities in nuclear structures in leukocytes
− Anemia
− Hepatomegaly
FHL is characterised by defective perforin secretion. However, overactive perforin is also problematic. What diseases does it cause?
• FHL, GS and CHS associated with reduced perforin activity, but overactivity can also be detrimental
• SLE →
− Chronic autoimmune disease caused by autoantibodies to nuclear antigens
− Affects skin, kidneys, joints and CNS
− Patients with disease flares high higher proportions of perforin and/or granzyme positive lymphocytes, and the frequency of these cells in the peripheral blood correlates with disease severity
• Polymyositis →
− Chronic systemic autoimmune inflammatory disorder
− Inflammation and weakness of muscles
− Infiltrating cells contribute to inflammation by release of TNF and perforin
• Rheumatoid arthritis →
− CD8 deficient mice had lower incidence
− Therefore proposed that perforin contributes to pathology by promotion of autoimmunity and destruction of target tissues.
How do we create humanised mouse models to study IDs?
• Humanised mouse models based on cell transfers
− A humanized mouse is a mouse carrying functioning human genes, cells, tissues and/or organs.
− Can transplant human HSCs into an immunodeficient mouse, and therefore study human immune responses
− All based on work by Irv Weisman – identified HSCs
Creating humanised mouse models (Commercial Jackson Lab):
• Myeloablate mouse so it becomes ID
• Inject human HSCs
Can be used to study: • Human specific pathogens, eg) HIV • Test bone marrow toxicity • Test gene defects • Try out different drugs in human cells • Study haematopoesis • Analyse GvHD
Timeline of ID Mouse Generation:
- Nude → 1962. From a strain with a genetic mutation that causes a deteriorated thymus = reduced T cells. Main outward appearance is lack of body hair, giving ‘nude’ name.
- SCID → 1983. From a recessive mutation on chromosome 16 giving faulty DNA repair. Don’t undergo VDJ recombination, so SCID mice have no T or B lymphocytes.
- NOD → 1995. Non-obese diabetic mice have susceptibility to spontaneous autoimmune insulin dependent diabetes.
- NSG → 2005. NOD SCID Gamma (+IL-2Ry KO). Among the most immunodeficient described to date. Lack T, B and NK cells. Also deficient in many cytokine signaling pathways and have defects in innate immunity. Permits the engraftment of primary human immune cells.
Purification of human stem cells:
• Use MACS → Allows cells to be sorted by incubating with magnetic nanoparticles coated with antibodies against a surface antigen. The cells attacked to the nanoparticle (expressing the antigen) stay in the colum, while others flow through.
• Positive selection → against the antigen of interest. Initial flow through is discarded, and cells attached to the column flushed into separate vessel.
• Negative selection → against an antigen known to be expressed on cells not of interest. The initial flow through is collected.
• Used to obtain a highly enriched population of CD34+ HSCs
Note:
• A problem with these mouse models is they don’t have human HLA
• So if you are putting human cells into these mice, they are restricted to human HLA, not mouse – so they wouldn’t work
• Need to put the human HLA genes into the mice
What is the NSG mouse?
NOD SCID Gamma
• To get good engraftment of HSCs, need to create a niche
− NOD → autoimmune mouse model
− SCID → No B or T cells
− Common gamma chain deficiency → No NK
• NSG mouse
− Has reduced complement activity
− Dysfunctional macrophages and DCs
− Very low incidence of lymphoma (unlike NOD/SCID model)
− Doesn’t develop diabetes
− Applications in research for cancer, infectious disease, immunology and regenerative medicine
− Flow cytometry shows us that when you engraft human or mouse cells, you get good reconstitution of both cell types.
How have ID mice been used to study HIV, hepatitis and cancer?
Studying HIV:
• Kumar et al, → T cell specific siRNA delivery suppresses HIV 1 infection in humanized mice
− CD7 specific antibody conjugated to oligo-9-arginine peptide for T-cell specific siRNA delivery in NSG mice reconstituted with human HSCs.
− In HIV infected mice, treatment with anti-viral siRNAs conjugated to the oligo-9-arginine peptide controlled viral replication and prevented the disease-associated CD4+ T cell loss
Studying hepatitis:
• Hepatitis only infects human cells, not mouse, so need to make a humanized mouse in order to study it in the lab
• Take a human liver explant, and put it in a mouse
• A the same time, take human HSCs of the same person into the same mouse → end up with a mouse with a human liver and human immune system
• Can now infect them with hepatitis and you have a perfect animal model
Studying cancer:
• Used to be hard to get human tumour engraftment as mouse NK cells would attack
• Now works well in NOD/SCID mice
How might iPSC and humanised mouse technologies be combined?
(N.B: this doesn’t exist yet for IDs)
Example of what is currently available:
• Correcting alpha 1 anti-trypsin deficiencies
− Enzyme secreted by the liver
− Can lead to liver problems in childhood
− Severe complications in the smoking population
• Methods:
− Isolate iPS cells form alpha 1 anti-trypsin deficient patient
− Correct genetic defect
− In vitro differentiate to hepatocytes
− Reimplant into ID mouse
− Proof the genetic modification worked
The big challenge:
• We cannot currently generate HSCs from iPSCs
• If we could – would give us an almost unlimited source of HSCs for transplant
Maybe 10 years from now: • Generate iPS cells from SCID patients • Correct the defect • Generate and expand HSCs • Replace the immune system from SCID patients by autologous HSCs derived from the corrected iPS cells.
