SEMINAR 5005: WHAT’S NEW IN TESTING FOR PRIMARY IMMUNODEFICIENCIES (PIDs)?

ROSHINI SARAH ABRAHAM, PhD MAYO CLINIC

FEBRUARY 26th, 2013

TOPICS COVERED IN THIS SESSION

1. Assessment of functional antibody capacity (antibody potency) for pneumococcal antibodies (to S. pneumoniae vaccination) using a flow cytometric opsono- phagocytic activity assay (OPA) 2. Next-generation (Next-Gen) sequencing for identification of molecular defects in in PIDs 3. NK cell and CTL functional assays

1. MEASUREMENT OF OPSONOPHAGOCYTIC ACTIVITY OF PNEUMOCOCCAL ANTIBODIES BY FLOW CYTOMETRY

OPSONOPHAGOCYTIC ASSAY FOR FUNCTIONAL ASSESSMENT OF PNEUMOCOCCAL ANTIBODIES

● Quality of an antibody response includes both quantitative (antibody concentration/titer) and functional capacity of the antibodies induced in response to vaccination ● Serologic correlates of protection for pneumococcal vaccination not well established ● Opsonophagocytosis is the primary mechanism for clearance of pneumococcal bacteria from host ● Therefore, measurement of opsonohagocytic antibodies produced after vaccination appear to correlate with vaccine-induced protection ● Correlation of opsonophagocytosis and IgG antibody concentration varies according to serotype ● Antibody concentration measuring assays (ELISA, bead-based) assess total binding of antibodies with various avidities (strength of antigen-binding); opsonophagocytosis measures total binding of antibodies with higher avidities ● In vitro determination of opsonophagocytic activity (OPA) of antibodies is likely to represent a better surrogate marker for in vivo protection because it measures functional activity of the antibodies ● We have developed and are validating a multiplexed flow cytometric assay for measuring OPA for 23 serotypes of S. pneumoniae for clinical use

Fluorescently labeled bead (“Bacteria”)

Polysaccharide antigen

Pneumococcal antibody in patient serum recognizing pneumococcal polysaccharide

Complement

Complement activation resulting in opsonization of bead (“bacteria”)

Uptake (phagocytosis) by phagocytic cell line (HL-60)

PRINCIPLE OF OPSONOPHAGOCYTIC ACTIVITY MEASUREMENT

Measurement of uptake of opsonized fluorescently labeled beads by flow cytometry

Calculation of phagocytic titer (reciprocal dilution of the dilution determined to have 50% maximal uptake of labeled beads (opsonized “bacteria”)

MULTIPLEX FLOW CYTOMETRIC ASSAY TO MEASURE OPA OF PNEUMOCOCCAL ANTIBODIES

2. NEXT GENERATION SEQUENCING FOR IDENTIFICATION OF GENETIC DEFECTS FOR PRIMARY IMMUNODEFICIENCIES (PIDs)

RELEVANCE OF GENETIC TESTING IN PIDs

● The majority of PIDs are monogenic disorders affecting the immune system and are either inherited or sporadic gene defects ● Some PIDs, may be oliogenic or polygenic with a few to multiple genes involved in the clinical and immunological phenotype ● Approximately 200 gene defects described to date with significant clinical and immunological heterogeneity with 3-5 new genes being described each year ● Genetic analysis is essential for identification of the specific disease-causing mutation, which facilitates accurate diagnosis and classification of disease, allows genotype-phenotype correlations, carrier testing and counseling of family members ● While treatment cannot be delayed, when needed, for a genetic diagnosis, identification of specific defect may be helpful in refining treatment and management approaches ● Newborn screening for SCID and T cell lymphopenia has resulted in the identification of unexpected genetic defects associated with T cell lymphopenia (RAC2 deficiency, Barth syndrome, TAR syndrome etc. besides the more classic SCID genes) ● Several SCID and CID genes have been identified and it is onerous and expensive to sequence each gene individually when attempting to achieve a genetic diagnosis ● High throughput approaches that are cost and time-effective are required

INCREASING COMPLEXITY

EVOLUTION OF GENETIC TESTING

SINGLE GENE SEQUENCING FOR MULTIPLE GENES

WHOLE EXOME

WHOLE GENOME

SINGLE GENE SEQUENCING-BASED DIAGNOSTICS FOR MULTIPLE GENES

CLINICAL PHENOTYPE

MULTIPLE GENES MUTATIONAL SPECTRUM

LOCUS HETEROGENEITY ALLELIC HETEROGENEITY

ELEMENTS OF GENETIC DIAGNOSIS

GENETIC TESTING

ELEMENTS OF GENETIC DIAGNOSIS: A MORE RATIONAL AND COMPREHENSIVE APPROACH

DIAGNOSIS PROGNOSIS TREATMENT COUNSELING

GENETIC TESTING FOR MULTIPLE GENES

ANALYTICAL OPTIONS:

FULL-GENE (SANGER) SEQUENCING OF INDIVIDUAL GENES

MULTIPLE GENE RE-SEQUENCING MICROARRAYS

SCANNING AND SEQUENCING

NEXTGENERATION (NEXT-GEN) SEQUENCING

HOW DOES THE CURRENT APPROACH (SANGER SEQUENCING) WORK?

● The Sanger (full-gene) sequencing method involves PCR amplification and sequencing of all coding exons (and sometimes non-coding exons) along with intron-exon boundaries ● This is followed by electrophoretic separation of fluorescent dye chain termination products using capillary electrophoresis ● Sequences are analyzed using various mutation analysis software and compared to reference (wild-type) sequences

A T C G

HOW DOES NEXT-GEN SEQUENCING WORK? ● ANALYTICAL PROCESS IS MULTI-STEP ● LIBRARY OF DNA FRAGMENTS IS CREATED ● DNA LIBRARY SEQUENCED IN SITU ● MULTIPLE PARALLEL SEQUENCING TO PROVIDE ADEQUATE DEPTH OF COVERAGE ● PROVIDES BOTH QUALITATIVE AND QUANTITATIVE INFORMATION ● BIOINFORMATIC ALGORITHMS ESSENTIAL TO ACCURATE DATA INTERPRETATION ● SPECIFIC MUTATION WILL HAVE TO BE VERIFIED BY SANGER SEQUENCING

Reference sequence

Coverage

NEXT-GEN SEQUENCING FOR PIDS SCID/CID GENES: RAG1 SCID/OMENN SYNDROME (OS) RAG2 SCID/OMENN SYNDROME (OS) ADA SCID/OMENN SYNDROME (OS) IL2RG SCID/OMENN SYNDROME (OS) JAK3 SCID/OMENN SYNDROME (OS) IL7RA SCID/OMENN SYNDROME (OS CD45 SCID CD3D SCID CD3E SCID CD3Z SCID DLCRE1C SCID/OMENN SYNDROME (OS PRKDC SCID LIG4 SCID/OMENN SYNDROME (OS PNP PURINE NULEOSIDE PHOSPHORYLASE DEFICIENCY CD3G MILD SCID NHEJI1 CERNNUNOS AK2 RETICULAR DYSGENESIS FOXN1 FOXN1 DEFICIENCY CORO1A CORONIN-1A DEFICIENCY MTHFD1 FOLATE METABOLISM GENE/SCID CD8A T CELL DEFICIENCY/POSSIBLE- SCID-LIKE ZAP70 T CELL DEFICIENCY/POSSIBLE- SCID-LIKE RMRP CARTILAGE HAIR HYPOPLASIA/OMENN SYNDROME. RMBA TAR SYNDROME TP63 EEC SYNDROME STAT5B COMBINED IMMUNODEFICIENCY (CID) DOCK8 COMBINED IMMUNODEFICIENCY (CID ATM ATAXIA TELANGIECTASIA ITK EBV-ASSOCIATED T CELL DEFICIENCY MAGT1 T CELL DEFICIENCY RHOH T CELL DEFICIENCY (HPV) MST1 T CELL DEFICIENCY (HPV)

SCID/CID GENES: TAP1 BARE LYPHOCYTE SYNDROME- I (BLS-I) TAP2 BARE LYPHOCYTE SYNDROME- I (BLS-I) TAPBP BARE LYPHOCYTE SYNDROME- I (BLS-I) CTIIA BARE LYPHOCYTE SYNDROME- II (BLS-II) RFX5 BARE LYPHOCYTE SYNDROME- II (BLS-II) RFXAP BARE LYPHOCYTE SYNDROME- II (BLS-II) RFXANK BARE LYPHOCYTE SYNDROME- II (BLS-II) RAC2 CID WITH NEUTROPHIL DEFECTS CHD7 CHARGE SYNDROME TAZ BARTH SYNDROME TBX1 DIGEROGE SYNDROME ORA1 CALCIIUM CHANNELOPATHY STIM1 CALCIIUM CHANNELOPATHY

● Genes associated with SCID, T cell lymphopenia or combined immunodeficiencies or previously identified in infants from newborn screening for SCID ● Next-Gen sequencing convenient for gene panel testing ● Cost and time-effective ● Additional 72 genes included for other non-SCID/CID primary immunodeficiencies (e.g. genes for ALPS, FHL/HLH, neutropenia, complement defects, CMC, APECED, IPEX, CVID, XLA, TLR defects, Hyper IgE syndrome, CGD, WAS, Hyper IgM syndrome

3. EVALUATION OF NK CELL AND CTL FUNCTION BY FLOW CYTOMETRY

WHEN TO ASSESS NK CELL AND CTL FUNCTION: 1. NK cell deficiencies (intrinsic or 2o to another PID, e.g. MCM4 deficiency, ANKD, FNKD,CNKD, Wiskott-Aldrich Syndrome, Papillon-Lefevre syndrome, NEMO deficiency, SCID with NK defect, GATA2 deficiency), recurrent herpes virus infections 2. T cell defects, especially in the context of viral infections or part of detailed T cell functional studies in cellular or combined immunodeficiencies 3. Familial hemophagocytic lymphohistiocytosis (FHL) (STX11, UNC13D, PRF1…) 4. Secondary hemophagocytic lymphohistiocytosis (HLH), including VAHS (virus- associated hemophagocytic syndrome) 5. Other PIDs with granule defects, e.g. Chediak-Higashi syndrome, Griscelli syndrome type 2, Hermansky-Pudlak syndrome type II 6. Emerging evidence for role of NK cells in immune reconstitution post-hematopoietic cell transplantation and survival/outcomes (not routinely used clinically at present but NK cell subset quantitation and function may be used in this context in the future)

ASSAYS FOR NK CELL AND CYTOTOXIC T CELL (CTL) FUNCTION IN PIDs

WHAT TESTS ARE AVAILABLE TO MEASURE NK AND CTL FUNCTION

1. Degranulation assay: measures CD107a/b (LAMP1 and 2) expression on NK and CD8 T cells after stimulation (allows distinction between PRF1 deficiency-FHL2 and UNC13D- FHL3 / STX11 -FHL4/STXBP2 – FHL5. PRF1 deficiency – normal degranulation, abnormal in FHL3, 4 and 5 as well as other PIDs with earlier granule defects, e.g. LYST, RAB27A)

2. Cytotoxicity assays: 1. Natural cytotoxicity (using K562 target cells) at varying concentrations of effector (NK cells) to target cells

1. 4h stimulation with target cells 2. 24h stimulation with target cells

2. Lymphokine-activated killing (LAK) (using K562 target cells) at varying concentrations of effector (NK cells) to target cells NK cells can be stimulated with either IL-2 or IFN-γ or IL-15 +IL-18 3. Antibody-dependent cellular cytotoxicity (ADCC) Measures NK cell cytotoxicity of antibody-coated target cells; mediated through the FcγRI receptor, CD16 (uses a mouse mastocytoma cell line, P815, not killed by NK cells expressing high affinity Fc receptor and anti-CD16 antibody (3G8 clone) used to “coat” target P815 cells, which activate NK cells through CD16 and trigger killing)

All assays listed can be performed by flow cytometry (allows visualization of effector and target populations)

3. Polyfunctional T cell assay: measures cytokine (IL-2, IFN-γ, TNF-α) production of CD4 and CD8 T cells after stimulation with mitogens, PHA and/or PMA+ionomycin (Assess T cell activation (a functional measurement) and capability to produce different cytokines on activation)

Diagnosis NK function NKT cell count Total NK cell count CD3+56+ cells CD3-56+16+ (may also include CD16- NK cells ANKD Absent Absent Absent CKND Absent Present Absent FNKD Low Present Present

Absolute NK cell deficiency Classic NK cell deficiency Functional NK cell deficiency

Orange JS et al, Curr Opin Allergy Clin Immunol, 2006, 6: 399-409

POINTS TO REMEMBER FOR INTERPRETATION: ● If sample is being sent to a non-local lab, then it would be reasonable to include a shipping control to account for transportation variations ● NK cell functional assays usually refer to cytotoxicity assays, though any assay that measures NK function can be included ● For degranulation assays, NK cell and CD8 T cell-specific markers are used to differentiate degranulation capability in specific cell populations ● An absent or low NK cell cytotoxicity result should be repeated at least 3 times over a period of time (~ 3 months) to definitively establish NK cell dysfunction ● Perforin and Granzymes A and B protein expression can be assessed by flow cytometry in various NK cell subsets ● CD16 deficiency requires CD16 flow cytometric analysis with the B73.1 clone (3G8 clone cannot identify the specific mutation reported)

INTERPRETATION OF RESULTS AND CAVEATS

● If attempting to differentiate between the most common form of FHL (perforin deficiency) and other forms of FHL, a degranulation test would be very useful ● Standard measurement of NK cytotoxic function (natural NK cytotoxicity) should be the first level of testing, if NK cell function (actual target cell killing) has to be assessed ● If abnormal, then additional tests of NK cell function could be ordered (such as LAK/cytokine- activated killing and ADCC) and this algorithm would be reasonable for most patients where NK cell function is being evaluated as a rule-out or for HLH-type contexts ● However, in some patients where there is a higher degree of suspicion of NK cell dysfunction, or a more comprehensive analysis of NK cell activity is desired, it may be more economical from a time perspective to order a complete NK cell functional work-up at the beginning ● Sample stability (conditions under which sample is collected and transported) and cellular viability will strongly influence results ● 25% of healthy adults can have low NK cytotoxic function when tested randomly, which is why additional testing over time is required to definitively establish a functional NK cell defect ● It is helpful to do NK cell quantitation along with NK cell functional analysis to differentiate ANKD, CNKD and FNKD ● In secondary HLH, NK cell function is typically only transiently abnormal and when the under- lying cause is resolved, NK cell cytotoxicity normalizes, in contrast to FHL or other PID patients where there is an intrinsic NK cell deficiency

INTERPRETATION OF RESULTS AND CAVEATS (contd.)

● CD8 T cell (CTL) function is usually measured after some form of global activation, i.e. mitogen stimulation, since it is very difficult to develop and validate clinical assays for specific antigens ● The degranulation test, used for NK cell function, can also be used to measure CTL degranulation (a surrogate marker for CTL cytotoxic function) by adding the CD8 T cell marker in the flow assay ● Additionally, the ability of CTLs to produce cytokines on activation can be assessed by flow cytometry after mitogen stimulation (polyfunctional T cell assay) ● Usually, mitogen stimulation includes PHA (measures proximal T cell signaling pathways) and PMA+ ionomycin (measures distal T cell signaling pathways) ● Typically, there is a temporal regulation of CD8 T cell (and CD4 T cell) activation, and several populations of CD8 (and CD4) T cells exist producing various cytokines –e.g. IFN-γ, TNF-α and IL-2 ● Granzyme B activity (direct killing of target cells) can be measured in CTLs after T cell activation, however, such an assay requires T cell pre-sorting/enrichment to differentiate between T cell vs. NK cell activity (as opposed to using bulk PBMCs). This adds complexity to clinical test development and increases cost of the test ● Degranulation and polyfunctional T cell analysis serve the purpose of assessing global CTL function

Granule Membrane CD107a CD107b CD63

Granule Core Perforin Granzymes A and B Granulysin Proteoglycan

Cytotoxic granule

CD107a/b

Cell Membrane

Activation

Granules move to cell membrane

Membranes merge

Degranulation anti-CD107a/b

CYTOTXIC PROTEINS IN EFFECTOR LYMPHOCYTES (NK CELLS / CD8 T CELLS)

PHA1: 40% PHA2: 43% PHA3: 44% Unstimulated: 4%

PHA1: 87% PHA2: 92% PHA3: 95% Unstimulated: 12%

Bryceson Y et al, Blood, 2012, 119: 2754-2763

CD8 T cell degranulation NK cell degranulation

MEASUREMENT OF DEGRANULATION (GRANULE EXOCYTOSIS)

● Measures expression of CD107a/ b (cytotoxic granule membrane proteins) after stimulation as a marker for granule exocytosis

Smyth M et al, Mol Immunol, 2005, 42: 501-510

MECHANISMS OF NK CELL KILLING CYTOTOXICITY

CYTOKINE MEDIATED- ACTIVATION OF APOPTOSIS (LAK)

ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY (ADCC)

EXAMPLE OF POLYFUNCTIONAL T CELL ANALYSIS (MEASURES CYTOKINE PRODUCTION AFTER T CELL ACTIVATION- MITOGEN STIMULATION- IN CD4 AND

CD8 T CELLS)