Laboratory Testing: Innate and Adaptive Immune Systems

Thomas A. Fleisher, M.D., FAAAAI, FACAAINational Institutes of Health

Bethesda, MD, USA

Dr. Fleisher has no conflicts of interest related to this presentation

Principles of Immune Testing

STARTING POINT INVOLVES A PATIENT:• Medical history of recurrent infections

including microorganism(s), frequency, site(s), therapy required

• Family history with attention to early deaths and recurrent infections

• Physical examination

Evaluation of Adaptive Immunity: B Cell Function Screening Tests

• History of recurrent sinopulmonary infections with encapsulated bacteria

• Screening Tests• Immunoglobulin (IgG, IgA, IgM, IgE) levels• Specific antibody response

• Protein antigens• CHO antigens

• HIV testing

Immunoglobulin Levels• Age related differences are significant and must

be considered for each evaluation (i.e. use age specific reference intervals to evaluate values)

• Normal range = 95% confidence interval

• Total immunoglobulin level (for each class) is the net of production, consumption, and loss

2.5% controls below 2.5% controls above

IgG Subclass Levels• Test is relatively expensive• Frequency of isolated IgG subclass deficiency

remains controversial• May be useful in evaluating IgA deficient

patients with recurrent infections• Generally still need to establish a failure to

produce specific antibody before initiating IgG replacement therapy

Specific Antibody Response• Represents the “ standard” regarding the

capacity to mount an antibody response• “Natural antibody” screen: testing for

isohemagglutinins (<1-2 yrs unreliable), antibody level to prior immunization

• Provocative testing: immunize with protein and CHO vaccines, obtain pre- and post- antibody levels

Evaluation of Adaptive Immunity: Screening of T Cell Function

• History recurrent opportunistic infections often with failure to thrive

• Screening Tests– HIV test– Lymphocyte count (T cells = ~75% of lymphs)– DTH testing (used less frequently in USA)

• Specific response to recall antigens in vivo: antigen specific T cell activation, cytokine production, inflammatory cell migration

• Lack of response: anergy versus no prior exposure

Secondary Adaptive Immune Testing: Primary Focus on T cellsIn vitro assays that help identify the cellular level or degree of T cell dysfunction• Immune cell and specific protein identification:

flow cytometry• Immune cell function (ordered based on strong

history of a cellular immune defect)• T cell proliferation/cytokine assay (mitogens,

recall antigens)• T cell cytotoxicity assays: used infrequently in

clinical evaluation

Flow Cytometry in the Evaluation of Adaptive Immunity:

Cell Directed Evaluation• Evaluation for absence of a lymphocyte population/

subpopulation (e.g. B cells - XLA, B & NK cells - XSCID)• Evaluation for a specific cell surface protein (e.g. CD40

ligand/CD154 [activated CD4+], IFNR [monocytes])• Test for an intracellular protein: specific disease screen (e.g. BTK

– XLA [monocytes], FOXP3 – IPEX [Tregs])

Assessment for Biologic Effect• Memory/naive T cells (CD45RO/RA, CD62L)• Memory B cells (CD27)• B cell isotype switch

Flow Cytometric Evaluation of a Child with Agammaglobulinemia

Low or absent B cells = XLA or AR agammaglobulinemia;

B cells =0.2%

T cells =95%

Flow Cytometric Evaluation of an Infant with Opportunistic Infections

No NK cells

Normal B cells

Very low T cells

T-/B+/NK- XSCID; flow allows four cell based phenotypes of SCID: T-/B-/NK-; T-/B-/NK+; T-/B+/NK-; T-/B+/NK+

Lymphocyte Function Testing• Response to mitogens, recall antigens, allogeneic

cells– Proliferation assessed by 3 H-thymidine incorporation– Cytokine release into culture supernatant– Activation antigen upregulation (e.g. CD69 by flow) – Cell division(e.g. CFSE) or cell cycle (e.g. BrDU)

• Cytotoxicity:– Antigen specific: requires presensitization, initiated thru

TcR recognition of viral (or other) peptide on MHC• 51Cr release from target cells• Flow cytometry test for CD107a expression by cytotoxic T cell

Evaluating Innate Immunity

Evaluate NK cells

• Very few patients identified with isolated NK cell absence

• HLH and XLP are associated with functional NK cell deficiency

• Enumerate NK cells using flow cytometry• Evaluate NK cell functional activity using in vitro

cytotoxicity assay with K562 targets– 51Cr release from target cells– Flow cytometry test for CD107a expression by NK cells

Evaluating TLR Function

• Clinical phenotype of TLR defects– Recurrent pyogenic infections with limited

systemic symptoms (minimal fever, normal or low CRP, etc)

– Herpes simplex encephalitis• Screening tests of TLRs utilize stimulation by

ligands for specific TLRs• Currently this testing has relatively limited

availability in clinical laboratories

37ºC5% CO2

TLR specific ligand

PBMC

I- Activation in vitro

II- Quantify Response

TNF ELISA

MC MC MC

2.0 x 105

cells/well

Von Bernuth, et al, Pediatrics, 118:2498-2503, 2006; Deering and Orange, Clin Vaccine Immunol, 12:68-76, 2006

Multiplex assay

Supernatant

MC

Cells

CD62L shedding (Flow)

Evaluating TLR Response

MC

Neutrophil Immunodeficiency• History of recurrent/chronic infections with

bacteria and fungi involving the skin, lungs, bone, liver and oral cavity

• Most often is 2o to neutropenia• Defined genetic defects primarily impact

microbial killing (CGD) or neutrophil migration (LAD)

Screening of Neutrophil Function• Absolute neutrophil count (ANC)• Evaluation of oxidative burst

– CGD results from defective oxidative burst– DHR test, NBT test, chemiluminescence

• Evaluation of adhesion molecules– Leukocyte adhesion deficiency (LAD) I results

from defective CD18 (2 integrin) expression– Test for CD18 (+ CD11a,b,c) expression

DHR Assay to Diagnose CGD

Normal

Phox47 deficient CGD (AR)

Phox91 deficient CGD (X-linked)

X-linked CGD carrier

Flow Cytometric Analysis of CD18 Surface Expression in Unstimulated CellsLy

mph

ocyt

es

PM

Ns

Normal LAD1 patient

Absent/markedly decreased CD18 expression = LAD type I

Complement Deficiency• Clinical presentation

• Recurrent encapsulated bacterial infections: early component deficiency (C1q, C2, C4, C3)

• Recurrent meningococcal infections: deficiency of terminal complement components (C5-C9)

• Screening test: CH50 (functional assay of total hemolytic complement activity)

• Additional testing: AP50 and complement component assays

Summary• Laboratory testing is crucial in evaluating patients for

possible immune defects• The choice of tests should be determined based on the

specific clinical history of recurrent and/or chronic infections

• There remain patients with a clinical history strongly suggesting an immune defect that using current testing remain undiagnosed

• Genetic testing is emerging as an important diagnostic test in resolving possible PID

References• Notarangelo LD, et al. Primary immunodeficiencies: 2009

update. J Allergy Clin Immunol. 2009, 124:1161.• Oliveira JB, Fleisher TA. Laboratory evaluation of primary

immunodeficiencies. J Allergy Clin Immunol. 2010, 125:S297.• Oliveira JB, Fleisher TA. Molecular and flow cytometry-based

diagnosis of primary immunodeficiency disorders. Curr Allergy Asthma Rep. 2010, 10:460.

• Rosenzweig SD, Holland SM. Recent insights into the patho-biology of innate immune deficiencies. Curr Allergy Asthma Rep. 2011, 11:369.