Evaluation of primary immunodeficiency diseases by flow cytometry analysis

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Vol. 14, No. 2, 1994

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magiobulinemia produce C-mu without attached VH region. Nature 304:355-358, 1983. Siminovitch KA, Greer WL, Axeisson B, et al.: Selective impairment of CD43-mediated T-cell activation in the Wiskott-Aldrich syndrome. Immunedeficiency 4:99-108, 1993. Simon H-U, Higgius EA, Demetrion M, et al.: Defective expression of CD23 and autocrine growth-stimulation in Epstein-Barr virus (EBV)transformed B cells from patients with WiskottAldrich syndrome. Clin Exp Immuno191:43-49, 1993. Simon H-U, Mills GB, Hashimoto S, Siminovitch KA: Evidence for defective transmembrahe signaling in B cells from patients with Wiskntt-Aldrich syndrome. J Clin Invest 90:1396-1405, 1992. Skare JC, Grierson IlL, Sullivan JL, et aL: Link-

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age analysis of seven kindreds with the X-linked lymphoproliferntive syndrome (XLP) confirms that the XLP locus is near DXS42 and DXS37. Hum Genet 82:354-358, 1989. Skate J, Wu BL, Madan S, et al.: Characterization of three overlapping deletions causing X-linked lymphoproliferative disease. Genomics 16:254-255, 1993. StandenGR: Wiskott-Aldrich syndrome: New perspectives in pathogenesis and management. J Roy Coil Plays London 22:80-83, 1988. Teahan C, Rowe P, Parker P, et al.: The X-linked chronic granulomatuus disease gene codes for the ~chain of cytuchrome b-245. Nature 327:720-721, 1987. Yen PIL Patel P, Chinanlt AC, et al.: Differential methylation of hypoxanthine phosphoribesyltransferase genes in active and inactive X chro-

Pruc Nail Acad Sci USA 81:17591763, 1984. Tsnkada S, Saffran DC, Rawlings DJ, et al.: Deftcient expreasion of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell 72:279-290, 1993. Vetrie D, Vorechovsky I, Sideras P, et al.: The gene involved in X-linked agammaglobulinemia is a member of the SRC family of protein-tyrosine kinases. Nature 361:226-233, 1993. Wadelius G, P i u M, Sundvall M, et al.: Linkage analysis in proi~rdin deficiency families: Refined location in proximal Xp. Clin Genet 42:812, 1992. Weinberg K, Parkman R: Severe combined immunodeficiency due to a specific defect in the production of intedeadtin-2. N Engl J Med 322:1718-1723, 1990. mosomes.

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Evaluation of Primary l m m u n o d e f i c i e n c y Diseases by Flow Cytometry Analysis Steven J. M e l n i c k 1 a n d N a y n e s h R. K a m a n i 2 IDepartment of Pathology and 2Division of Clinical Immunology, Miami Children's Hospital, Miami, Florida

low cytometry (FCM) is a powerful tool for the evaluation of the human immune response and immunologic disease states. Its primary applications derive f~om its ability to characterize the cells of the immune system using an extensive repertoire of monoclonal antibodies (MAbs) reactive to leukocyte surface and cytoplasmic antigens. Although most applications of FCM are directed at quantifying lymphocytes in the peripheral blood or cells of hematopoietic neoplasms, the growing array of characterized leukocyte antigens permits the evaluation of cell lineage, differentiation, and maturation and the analysis of processes associated with the active immune response (activation and transformation). These features have clearly expanded the role of FCM in the study of hematologic and immunologic diseases, including primaryimmunodeficiency diseases. Primary immunodeficiency diseases, which were important areas of interest in the early years of flow cytometry, have been largely overlooked because of the growing AIDS (acquired immunodeficiency syndrome) pandemic, applications in transplant medicine, and the relative rar-

F

ity of primary immunodeficiency diseases. Also neglected have been many other secondary immunodeficiency states such as those associated with viral and bacterial infections, immunosuppressive drugs, and those seen in patients with malignancies treated with chemo- and/or radiotherapy. As a prelude to a discussion of the applications of FCM in the evaluation of primary immune deficiency diseases, a conceptual framework is described that correlates the leukocyte differentiation antigens with stages of hematopoiesis and cellular activation. Normal Hematopoiesis The extensive, though far from comprehensive, repertoire of determinants of immune cells described by the cluster designation (CD) nomenclature has permitted the mapping of their stages of differentiation and maturation. The best characterized leukocytes are the lymphocytes. Both B and T cell lymphopoiesis begins in the bone marrow. However, only B cell lymphopoiesis occurs there exclusively. One of the earliest markers that defines cells of lymphoid lineage (T and B cells) is inlranuclear terminal deoxynucleotidyl transferase (TdT). 1 © 1994 Elsevier Science Inc.

During B cell lymphopoiesis, other determinants that appear in order of maturation include CD19, CD10, CD20, cytoplasmic la and surface immunoglobulin. The end stage is a mature or naive B lymphocyte that may be destined for the peripheral blood or lymphoid organs. Markers expressed upon activation may include CD10 or CALLA, CD71 (transferrin receptoO, and CD25 (IL-2 receptor). 1A similar scheme exists for T cells that differentiate and mature in the thymus gland instead of in the bone marrow. 2 An understanding of this framework has aided in creating a perspective on many primary immunodeficiency diseases, particularly those associated with mamrational defects. By categorizing these diseases within this framework, it also becomes practical to use FCM techniques such as immunophenotyping and func. tional assays to precisely define the nature of the immunodeficiency as humoral (B cell), cell-mediated (T cell), or combined (B and T cells) (Table 1).

Immunophenotype Panels and Normal Ranges Flow cytometry immunophenotyping tech0197-1859/94/$0.00 + 7.00

26 C L I N I C A L I M M U N O L O G Y Newsletter

TABLE 1. FLOW CYTOMETRIC ANALYSIS IN PRIMARY IMMUNODEFICIENCY DISEASES Humor~ defects X-linked agammaglobulinemia Common variable inmaunodeficiency Selective IgA deficiency Hyper IgM immunodeficiency Cell mediated defects DiGeorge syndrome Humoral and cell-mediated effects Severe combined immtme deficiency Adenosine deaminase deficiency Bare lymphocyte syndrome Ataxia-telangiectasia Omenn's syndrome Wiskott-Aldrich syndrome Granulocytic cell defects Leukocyte adhesion deficiency Defecls of oxidative systems Hyper IgE syndrome Defects of phagocytosis

niques use MAbs directed against cellular antigenic determinants that characterize the lineages, stages of maturation, transformation and activation of immune cells. Since these different states are uniquely def'med by combinations of expressed antigens rather than by a single antigen, it is essential to use specific combinations of MAb's simultaneously. A screening panel (Table 2--basic monoclonal antibodies) is commonly used by most flow cytometry laboratories for the immunophenotypic evaluation of peripheral blood lymphocytes in patients suspected of having an immunodeficiency or in monitoring the immune status of immunocompromised patients. The panel is designed to provide basic quantitative information of the lymphocyte subsets that includes T, B, and natural killer (NK) cells. Follow-up with a secondary panel more precisely defines the nature of the disease state (Table 2----additional monoclonal antibodies). Diseases of host defense affecting myeloid cells may also be assessed by FCM analysis. With the basic immunophenotype panel, virtually 100% of lymphocytes in peripheral blood can be accounted for in the light scatter histogram by seven MAbs. Mature T cells are characterized by the expression of CD3, a component of the CD3/I" cell re-

Vol. 14, No. 2, 1994

ceptor (TCR) complex that is the main recognition site for antigens on the T cell. The CD3 molecule is composed of three noncovalently linked monomorphic peptides thai function in signal transduction following binding of antigen to the TCR. The TCR is a disulfide linked heterodimer composed of o;/~ or T/fi chains) The helper]inducer and cytotoxic/suppressor function in T cells is defined by the CD4+/CD3+ and CDg+/CD3+ phenotypes, respectively. Neither CD4 nor CD8 is unique to either subset. CD4 is expressed on monocytes that may contaminate the lymphocyte light scatter window, whereas CD8 is expressed on NK cells that cannot be distinguished from other lymphocytes by light scatter. Combining an anti-CD3 MAb with anti-CD4 and/or anti-CD8 uniquely def'mes the T-helper/inducer and T-cytotoxic/suppressor subsets. 3 In addition to CD8, NK cells also express the CD16 and CD56 antigens3 ,5 However, CD16 and CD56 are not specific for NK cells. The NK cell population is characterized by expression of CD16 and CD56 but lacks CD3 expression. Thus, using antiCD3 with anti-CD 16+CD56 distinguishes between NK cells and T cells with NK function (Fig. 1). B cells are usually characterized by expression of the CD19 or CD20 antigens. CD19 is expressed very early in pre-B maturation in the bone marrow and persists through maturity3'7 The CD10 antigen is expressed on B cells at a slightly later stage of maturation but is lost on mature B cells. The combination of antiCD19 with anti-CDl0 is thus used to differentiate mature B cells from those that are immature. There is a subset of mature B cells that may co-express CD10. In lymphoid tissue, activated or transformed B cells present within the germinal centers of lymphoid follicles may express CD10 along with other markers associated with activation or transformation,a To effectively utilize the information obtained, results should be compared to normal rat~ges of lymphocyte numbers and percentages in age-matched controls. Lymphocyte immunophenotype studies in children have shown that CD3, CD4, and CD8 counts are significantly higher in children, particularly in neonates and infants, than in

0197-1859/94/$0.00 + 7.00 I

adults. These studies also show that the percentage of T cells rises with increasing age with a concurrent decrease in the percentage of B cells and NK cells. Similarly, the percentage of C-qM cells declines and the percentage of CD8 cells rises, thereby resulting in a declining CD4/CD8 ratio.9-14 Subtle quantitative deficiencies may thus be overlooked if this phenomenon is not considered. This is particularly true when evaluating CD4 counts in infants and children infected with the haman immunodeficiency virus (HIV). Other factors that can affect the quantitative aspect o f F C M immunopbenotyping must be considered. Preparations of peripheral blood lymphocytes using a density gradient method of separation have been shown to yield fewer CD8+ cells and other subsets than those prepared using red cell lysis techniques. 15This may represent an important source of interlaboratory variation. In practice, however, this is not a significant problem as long as there is consistency of preparation methods with appropriate controls. Humoral Immune Defects

X-Linked Agammaglobulinemia This is an X-linked immunodeficiency disease that results from a defect somewhere in the pathway of B cell maturation) 6 Two groups have recently described mutations at critical sites in the gene coding for a tyrosine kinase [Bruton's tyrosine kinase (Btk), previously known as Atk for agammaglobulinemia tyrosine kinase or Bpk for B cell progenitor kinase in patients with X L A . 17.18 As a result, B cells are markedly decreased or absent in the peripheral blood and plasma cells are absent in lymphoid tissues. Pre-B cells express immunoglobulin (Ig) heavy chains but not light chains. Serum immunoglobulins are either significantly decreased (IgG <20 mg/dl) or absent (IgD, IgM, IgA, and IgE). The peripheral blood immunophenotype demonstrates a virtual absence of lymphocytes expressing CD19, CD20, and surface immunoglobulin. Although mature B cells are absent in the bone marrow, pre-B cells, characterized by the expression of HLADR, CDI9, CD10, CD20, CD38, and cytoplasmic g, are present) 9 T cells and T-cell subsets are quantitatively normal.

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Common Variable Immonodeficiency This disease represents an acquired form of hypogammaglobulinemia seen in children and adults and associated with recurrent pyogenic infections involving the upper and lower respiratory tracts and intestinal manifestations such as giardiasis. Common variable immunodeficiency (CVID) represents a clinically and immunologically heterogeneous group of disorders characterized by hypogammaglobulinemia and failure of ant i b o d y p¢oduction. 19~

Farrant et al)3 have classifiedthis disease into different groups based on the ability of patient B cells to secrete IgM and IgG in vitro following stimulation with anti-tt or IL-2. Those CVID patients whose B cells can secrete neither IgG or IgM (group A) have lymphopenia with a marked decrease in B cells and CD4+ cells (both CD45RA+ CD4+ and CD45RO+ CD4+) and an inversed CD4+/CD8+ ratio. Those patients whose B cells are normally functional in vilro (group C) have normal lymphocyte numbers and subsets. Patients whose B cells can secrete IgM but not IgG in vitro (group B) have moderate lymphopenia with a decrease in CIMSRA+CIM+ cells.23A small number of patients with clinical CVID lack circulating B cells. Some of these may, in facL have XLA. The etiology of CVID is clearly heterogeneous and includes an intrinsic B cell defect resulting in unresponsiveness following stimulation and abnormalities in T-cell signals for B-cell function.2*,25 Selective IgA Deficiency Selective IgA deficiency is the most common primary immunodeficiency disorder, with a prevalence of about I in 700 individuals. Like CVID, it is associated with a maturation arrest of B cells at a terminal stage of differentiation. The etiology of this disease, though unclear, has been associated with neonatal viral infectious and abnormalities in chromosome 18, which possibly contains a regulatory gene for IgA synthesis. 26 Immunophenotypically, T and B cells are quantitatively normal. B cells that express IgA are decreased in number, and the majority of IgA B cells in these patients co-

express IgD and IgM, indicating an arrest in B cell differentiation at an early stage. 27 Defective T-helper and suppressor function have also been impficated. 2s Serum IgA levels are typically less than 5 mg/dl while IgG and IgM levels are normal. Hyper IgM Immunodeficiency Hyper IgM immunodeficiency (HIgMI) is predominantly an X-linked recessive disease, although acquired forms have been described. The defect in this disorder is at the stage of isotype switching. B cells are able to synthesize IgM and IgD but not IgE, IgA, or IgG. Immunophenotyping reveals the presence of B cells that express IgM but not lgG or IgA. Serum IgM levels are normal or elevated (150-1,000 mg/dl) and IgG and IgA are decreased or absent.29 The gene defect in I-HgMI has been independently identified by several groups to be in the gene that codes for the CD40 ligand (CD40L) on the surface of T cells. CD40L binds to the CD40 molecule present on B cells to induce isotype switching. The CD40L molecule (also called TRAP for TNF-related activation protein) is expressed exclusively on activated T cells and not on B cells or monocytes. Activated T cells from patients with HIgMI do not express CD40L when evaluated by immunofluc~scence flow cytometry using anti-TRAP rabbit serum: ° CD40-Ig chimeric molecule,3~or soluble CD40.s2 Other T-cell antigens are normally expressed. Patients with HIgMI have normal numbers of T cells, B cells, and CD4+ and CD8+ T cells. Other Humoral Defects There are other defects of humoral immunity that are rare and not well characterized by FCM. These diseases include IgG subclass deficiencies, immunodeficienties with normal or hyperimmunoglobulinemia, fight chain deficiencies and ecto-5"-nncleotidase deficiency. A deficiency of ecto-5"-nucleotiduse activity, an enzyme found in the plasma membranes of subclasses of T and B cells, is seen in some patients with B-cell deficiencies." It is believed that this enzyme is associated with B-cell maturation because it is most highly expressed in mature B cells. In patients with hypogammagiobulinemia, this O 1994 Elsevier Science Inc.

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TABLE 2. MONOCLONALANTIBODIES USED TO EVALUATE PRIMARY IMMUNODEFICIENCY DISEASES Detenninant

Distribution

Basic monodommlantibodies CD2

T and N K subsets

CD3

Mature T cells(specific)

CD4/CD3

T helperfmducersubset

CD8/CD3

T cytotoxic/suppressorsubset

CDI9, CD20

Mature B cells

CDI9/CDI0

Immature B cells(bonemarrow)

CDI6/CD56

N K cells

Addifiomd monuclonal antibodies TCR ct/13

M o a T cells

TCR 3¢8

4-8% of T cells

CiM5RA

Naive, suppressor/inducer T cells

CD45RO

Memmy, helper/inducer T cells

CD43

T cellsand PMNs; absentin Wiskott-Aldrich syndrome

CD4/CD8

Thymic T cells; some SCID

CIM/CD71

Some SCID

CIM5/CDI8

All leukocytes. ,I, or absent in leukocyteadhesiondeficiency

HLA-ABC (Class I)

All nucleated cells; absent in Brae lymOtocyte syndrome

HLA-DR (Class II)

B cells, monocyte,, activated T cells; absent in MHC Class II deficiency

CDI la

Most leukocytes; binds to ICAM- I (CD54) and ICAM- I

CDI Ib

Gnmulocytes, monocytes, and NK cells

CDI Ic

Granulocytes, monocytes, and NK cells

enzyme is decreased on the B cells corre. latlng to a block in maturation) 3 Defects of Cell-Mediated Immunity DiGeorge Syndrome The DiGeorge anomaly (DGA) or developmental field defect is characterized by congenital aplasia or hypoplasia of the thymus gland, abnormal facies, hypocalcemia secondary to hypoparathyroidism, and cardiovascular malformations. It results from an abnormality in the development of the third and fourth pharyngeal pouches that normally give rise to the thymus and parathyroid glands, part of the thyroid gland, aortic arch, mandible, and ears. 0197-1859/94/$0.00 + 7.00

28 CLINICAL IMMUNOLOGY Newsletter

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Fig. 1. Five color (fluorochrome) flow cytometry immunophenotype of peripheral blood lymphocytes. The cluster designation (CD): fluorochrome combinations used are CD3- fluorescein isothioyanate (EITC), CDl6+ CD%- R-phycoerythrin (PE), CD19-ECD, CD4-Per CP, and CD8-allophycocyanin (AK). The lymphocyte population (gated region”A”) is characterized by the CD combinations shown. (CD16-KD56~~D3-natural killer cells, CD3+/CD4+ T helper/inducer cells, CD3+/CD8+ T cytotoxic/suppressor cells and CD19+ B cells.

The etiology of the DGA is heterogeneous. It is known to be associated with chromosomal abnormalities, single gene defects, and exposure to teratogens and has been associated with other anomalies and syndromes. Cytogenetic studies reveal abnormalities in chromosome 22 in the band 22qll in many patients. Driscoll et al. have been able to identify microdeletions in the 22qll region using molecular analysis in all patients studied and postulate that submicroscopic deletions of 22qll are etiologic for most patients with DGA.% The immunologic manifestations of this syndrome can range from normal immune function to a severe combined immunodeficiency. Most cases involve a variable degree of T-cell hypofunction depending on the degree of thymic hypoplasia.2 Flow cytometry evaluation of these patients demonstrates a variation in the number of mature (CD3+) T cells from normal to markedly reduced22*35(Fig. 2). In some patients, the number of immature thymocytes (CD5+, CD7+) may be higher than the number of CD3+ cells, suggesting inadequate thymic maturation. B cells and NK cells are quantitatively normal or may be relatively increased in number. 019%1859/94R0.00 + 7.00

Combined T- and B-Cell Immunodefieiencies Reticular Dysgenesis This is a rare, rapidly lethal, inherited disorder characterized by a failure of maturation of both myeloid and lymphoid progenitors. There is marked lymphopenia and granulocytopenia with a normal to slightly reduced hemoglobin and platelet count, resulting in overwhelming infections shortly after birth. The primary defect in this disorder remains undefined. Severe Combined Immune Deficiency Severe combined immune deficiency (SCID) represents a family of immunodeficiency disorders in which there is a profound deficiency of the cellular and humoral immune systems. These diseases result from different defects and are characterized by distinct immunophenotypic features.x*37SCID may be inherited as an autosomal recessive or X-linked disease. The classical form of SCID, inherited as an autosomal recessive trait, is characterized by severe lymphopenia with an absence of mature T and B lymphocytes. NK cells are present and functional.38.39The molecular basis of classical SCID remains undefined but the clinical syndrome resem@ 1994 Elsevier Science Inc.

bles the mouse model of SCID, in which there is defective recombination of the VD-J elements of the Ig and TCR genes. In X-linked SCID, which accounts for about 50% of SCID cases, mature T cells are absent but B cells are present in the blood. The B lymphocytes express CD5, CD1 lc, and CD23 and phenotypically and functionally resemble neonatal B cells.” Recently, the candidate gene for XSCID has been identified as the gene encoding the y chain of the IL-2 receptor (IL-2R y), located in a region within Xq13.41?12 Other forms of SCID have been associated with specific genetic defects.” In adenosine deaminase deficiency (ADA), the deficiency of this purine salvage pathway enzyme leads to a buildup of toxic metabolites that affect T- and B-cell maturation.43These patients rarely have lymphocytes of any kind, including immature T and B cells. The defect appears to occur at the stem cell stage of lymphopoiesis. Purine nucleosidase phosphorylase deficiency also yields toxic metabolites that affect T-cell maturation leading to a progressive decrease in T cells.n*43As in other forms of SCID, B cells are also affected, though in this case they appear quantitatively normal. The final form of SCID discussed is MI-E deficiency or Bare lymphocyte syndrome. In the immunodeficiency originally reported as the Bare lymphocyte syndrome (BLS), patient cells lack class I HLA molecules but normally express class II molecules on B cells and monocytes. These patients present with an early onset of hypogammaglobulinemia, CD4+ lymphopenia, and diarrhea.” A few nonimmunodeticient individuals with aberrant class I antigen expression have also been describedP5*~ Patients with MI-IC Class II deficiency (Type II) fail to express class II molecules on B cells, monacytes, and activated T cells. These molecules are not inducible by y interferon. A defect in a transacting regulatory factor leads to an absence of HLA class II gene transcription. The expression of class I molecules is decreased in most patients but can be corrected in vitro with interferon. The abnormal expression of MHC molecules leads to a profound combined humoral and cellular immunodefi-

CLINICAL IMMUNOLOGY Newsletter 29

Vol. 14, No. 2, 1994

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Omenn's Syndrome Omenn's syndrome is a combined immunodeficiency inherited in an autosomal re-

cessive fashion. It is characterized by early onset diffuse erythroderma, protracted diarrhea, failure to thrive, and hepatosplenomegaly. Immunologic evaluation reveals hypogammaglobulinemia, hy~ ~ :ii!:~i : [ " " ~ ..i i :iiiiiiii!!!iiii:.: I ' pereosinophilia, and progressive cellular immune dysfunction. Although there may be a T-cell lymphocytosis, there is a restricted heterogeneity of the T-lymphocyte repertoire. T cells in these patients are de" 3 c rived from a small number of T-cell clones and belong to one predominant subset with riiil l rlJMlll l lli11N1 I •1 I 10 100 1000 a restricted use of T-cell receptor genes, st CD3 Wirt et al. described a child with Omenn's Fig. 2. Flow cytometryhistogram from a patient syndrome whose T cells were TCR-tx/~with DiGeorge anomaly. The proportion of T positive but lacked both CD4 and CD8. s2 ceils (CD3+) is markedlydecrease~ (16%). This suggests that Omenn's syndrome represents a SCID variant with the emergence of a small number of leaky T-cell clones. ciency. B cells and CD3+ T cells are normal in number. CIM lymphocytes are deWiskott-Aldrich Syndrome creased with a relative increase in CD8 lymphocytes.4~,4s This rare X-linked recessive disease of A familial T-cell receptor immunodefiearly childhood is characterized by the ciency (TCR ID) characterized by defectriad of eczematous dermatitis, thrombocytive surface expression of the TCR/CD3 topenia, and recurrent viral and bacterial infections.53During infancy, T cells are complex and impaired mitogen- and antiquantitatively normal but decline with age. gen-induced activation of T cells via the Eventually, these children develop T-cell TCR/CD3 complex has been recently delymphopenia. scribed?9 Proliferative responses to antiCD2, IL-2, and PMA were normal. The The immunologic deficiency is related to an absence or decreased expression of patient with the phenotype of severe TCR CD43 (sialophorin), a surface glycoprotein ID had many classical features of SCID. found on most circulating blood cells. The patient's T lymphocytes had markedly CD43 is normally expressed on mature T deficient expression of CD3, TCR-od~l and cells, pre-B, and activated B cells but not TCR-),/~, with normal numbers of CD2 or on mature resting B cells.~ This antigen CD4 and CD8 expressing T cells. The appears to have a role in the activation of membrane defect has been ascribed to a deboth T and B cells, proliferation of T cells, fective association of CD3-~ chain with and maturation of B cells. Loss of these the other chains of the complex. Milder asfunctions may he linked to the quantitative ymptomatic phenotypes of this immunodedeficiencies of T and B cells and the poor ficiency may exist. functional response of B cells to polysacIndividLtalswith cellular immunodeficharide antigens. Serum immunoglobulin ciency and selective deficiencies of either levels are variable. IgG is usually normal CD8+ T cells or CD4+ T cells have also while IgA and IgE levels are increased and been reported? ? A male infant with SCID IgM levels are decreased. The other signifihas been described in whom there was a cant hematopoietic abnormality is thrombofailure of IL-2 production due to a pretrans- cytopenia with abnormal platelet morphology. It is less clear whether a lack lational defect in IL-2 synthesis, s° There of CD43 expression plays a role in this dewere normal numbers of CD2-bearing fect since the antigen is weakly expressed cells but moderately decreased numbers of on platelets if at all.S' CD3+, CD4+, and CD8+ cells. llill

© 1994 E l s e v i e r S c i e n c e Inc.

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Fig. 3. Flow cytomela'yhistogramfrom a patient with at~ia-tel~giect~ia. Expression of T cell receptor (~/ct) shown in quadrant 2 is elevated (15%). Evaluation of the CD3 proportion is of most value in following the clinical course of these patients since the CIM/CD8 ratio does not change. There is another T-cell subset associated with this disease that may also he clinically useful. The proportion of T cells expressing TCR-~/8 instead of T C R - t ~ may be increased, ss This observation may he made on an immunophenotype screen since the majority of CD3+/CD4-/CD8- cells express TCR-?/8 that normally account for about 5% of T cells in the peripheral blood. The proportion of T cells bearing the TCR ?/~iphenotype may be elevated in a number of disease states that include Wiskott-Aldrich syndrome, ataxia-telangiectasia, the Chediak-Higashi syndrome, and HIV infection.5~ Ataxia-Telangiectasia Ataxia-telangiectasia is an autosomal recessive disease associated with a defect in the chromosome region 1 lq 22-23. It is characterized clinically by early onset progressive cerebellar ataxia, oculo-cutaneous telangiectasia, recurrent sinopulmonary infections, gonadal dysgenesis, mental retardation, hematopoietic malignancies, and a combined immunodeficiency, s7 Although there is no single characteristic immunologic defect in this disorder, most patients have deficiencies of serum IgA, IgE, IgG2, and IgG4 and defective cellular ira0 1 9 7 - 1 8 5 9 / 9 4 / $ 0 . 0 0 + ZOO

30 C L I N I C A L I M M U N O L O G Y Newsletter

mune responses. Serum alpha-fetoprotein levels are elevated in most patients. Underlying the immunologic abnormalities are defects of DNA repair and gene rearrangement. These result in an increased susceptibility to chromosomal breakage and an increased vulnerability to neoplastic transformation. These are reflected in the diversity of immunophenotypic aberrancy involving T- and B-cell function. A defect in B-cell gene rearrangement during the isotype switch from IgM to other heavy chains is believed to cause the immunoglobulin abnormalities while a defect in T-cell gene rearrangement produces some of the T-cell abnormalities including increased expression of TCR-~//&58 decreased T-helper/inducer cells, and normal or increased T-cytotoxic/suppressor cells (Fig. 3). T cells expressing CD2 and CD3 are quantitatively normal or slightly decreased. Defects of Granulocyte Function The main method of FCM evaluation of lymphocyte-associated primary immunodeficiency diseases is immunophenotyping. Certain aspects of granulocyte analysis also utilize immunophenotyping. However, there are a number of other flow cytometric techniques that can assess specific aspects of neutrophil function. These methods aid in the determination of the level of the granulocyte or monocyte defect. Granulocyte functions that can be assessed include evaluation of the oxidative burst and phagocytosis. Leukocyte Adhesion Deficiency Leukocyte adhesion deficiency (LAD) is an autosomal recessive disease associated with recurrent infections and an inadequate inflammatory response. The disease is caused by defective synthesis of the 13chain (CDI8) of the adhesion molecule complex CDI 1/CD18. There is also failure of expression of the a-chain (CDI la, CD1 lb, and CD1 lc) subunitsfl '~ The result of this deficiency is that there is no upregulation of the cell adhesion molecules with the appropriate stimulation. All leukocytes are affected. However, the major effects are observed in granulocytes and monocytes and manifest with impaired phagocytosis. 0197-1859/94/$0.00 + 7.00

Vol. 14, No. 2, 1994

Flow cytometry immunophenotyping is very useful in defining the degree as well as the presence or absence of expression of CD18, CD1 la, CDIlb, and CD l lc. This is essential since the severity of the disease correlates with the degree of expression, which ranges from absent to low levels. Care must be made to focus on the approprime cell type when using FCM analysis in this case. Since most flow cytometry users are more accustomed to working with lymphocytes, it is important to be aware of immunophenotypic applications in other leukocytes. A novel type of leukocyte adhesion deficiency has recently been described in two unrelated boys with recurrent pyogenic infections. Neutrophils from both patients lacked the fucosylated Sialyl-Lewis X carbohydrate structure that serves as a ligand for E-and P-selectins. Patient neutrophils expressed CD18 normally but failed to express Sialyl-Lewis X using the anti-SialylLewis X antibody CSLEX1.61 Defects of Oxidative Systems The primary disease associated with a defect in oxidative metabolism in granulocytes is chronic granulomatous disease (CGD). The inheritance of this disease, which us~mlly begins in early childhood or infancy, is either X-linked (60%) or autosomal recessive (40%). Clinically, these patients experience recurrent bacterial infections with ca!ala~-positive microorganisms such as Staphylococcus aureus and Aspergillus species. The main defect is the inability of phagocytes to produce an oxidative burst response when stimulated.62 The gene defects that lead to the inability of patient phagocytes to undergo the respiratory burst have now been identified. Gene mutations affecting one of at least four different components of the NADPH oxidase enzyme have been identified in CGD patients. Mutations of the gene coding for gp 91-phox are responsible for Xlinked CGD whereas gene defects for the components p22-phox, p47-phox, or p67phox lead to autosomal recessive CGD. There are very sensitive FCM assays that evaluate the ability of phagocytic cells to undergo a respiratory burs! by assessing the amount of H202 produced, u The assay © 1994 Elsevier Science Inc.

involves the incorporation of 2"-7' dichlorofluorescein diacetate (DCF) within the hydrophobic region of the cells lipid bilayer.~ The acetate moieties are cleaved by hydrolytic enzymes to produce a nonfluorescent polar compound (DCF) that becomes trapped within the cell. The H:O2 is able to oxidize the trapped DCF following stimulation by PMA and produce a fluorescent compound that fluoresces at 530 nm (green fluorescence). The green fluorescence is proportional to the H202 generated. In patients with CGD there is no increase in fluorescence intensity. In some carriers, a dual population may be seen indicative of normal and defective cells.~ Hyper IgE Syndrome The hyper IgE syndrome is a rare disorder of early childhood characterized clinically by elevated serum IgE levels, an inconsistent defect in neutrophil chemotaxis, and eosinophilia associated with recurrent pyogenic sinopulmonary and skin infections.~ Although T and B call numbers are normal, many patients have deficient numbers of CD8+ T cells. Defects of Phagocytosis A number of FCM methods have been developed to assess phagocytic function.67,~ The methods involve measurement of fluorescent beads or fluorescently labeled heatkilled bacteria or fungi that have been ingested by neutrophils. Use of opsonized and nonopsonized microorganisms are of value in distinguishing between a defect in phagocytosis or opsonization. There are a number of clinical conditions that are associated with defective phagocytosis. Since phagocytosis is a complex, multistage process, a defect at any stage can affect the end result. The primary cell receptors for phagocytosis are C3b (complement receptor) and FcR (receptor for the Fc portion of IgG). Additionally, normal actin polymerization and microtubule function are required for phagocytosis. Thus, complement C3b deficiency or defects in actin polymerization may interfere with phagocytosis. Defective phagocytosis is also inherent in immature neutrophils. Therefore, situations associated with increased immature neutrophils, as in neonates, and conditions associated with stress, such as thermal in-

CLINICAL IMMUNOLOGY Newsletter 31

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jury, trauma, and sepsis, may also result in decreased phagocytosis. A deficiency in Tuftsin, a tetrapeptide produced in the spleen that normally enhances neuax)phil phagocytic function, can also produce defective phagocytic function.69'~°Tuftsin deficiency may be inherited or acquired following splenectomy. A male infant with an autosomal recessive disorder of neutrophil chemotaxis and phagocytosis has been described whose neutrophils demonslrated a defect in neutrophil actin assembly. A defect in the polymerization of F-actin, a component of the cell cytoskeleton, has been documented in this child with an FCM assay that uses the fluorescent compound nitrobenzoxadiazole-phallacidin, which binds to F - a c t i n . 7t Summary This review has attempted to highlight the applications of flow cytometry assays in the evaluation of primary immunodeficiency diseases. While immunophenotyping is presently the most widely used FCM method, a variety of functional assays are being developed and becoming available clinically. With the CD-antlgen repertoire expected to double in the next few years and with the development of methods to probe cell structure and function, FCM is likely to become one of the most important methodologies for evaluating the human immune system.

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61(2):$94--$99, 1991. 41. Puck JM, Deschenes SM, Porter JC, et al.: The intedeukin-2 receptor gamma chain maps to Xq 13.1 and is mutated in X-linked severe combined inununodeficiency (SCIDxl). Hum Mol Genet 2:1099-1104, 1993. 42. Vou SD, Hong R, Sondel PM: Severe combined immunodeficiency, intedeukin-2 (IL2), and the IL-2 receptonExperiments of nature continue to point the way. Blood 83:626-635, 1994. 43. Morsan G, Levintky RJ0 Hugh JK, et al.: Heterogeneity of biochemical, clinical and immumoiogical parameters in severe combined innn~ficiency due to adenosine deaminase deficiency.Clin Exp Immunol 7~.491--499, 1987. 44. Tonraine JL, Betuel H: Immonodeficiency diseases and expression of HLA antigem. Hum lmmunol 2:147-153, 1981. 45. Payne R, Brodeky FM, Petedin BM, Young LM: "Bare lymphocytes" without immunodeficiency. Hum lmmtmol 6:219-227, 1983. 46. Maeva H, Hirata R, Chert RF, et al.: Defective expression of HLA class I antigens: A case of the Bare lymphocyte without inammndeficiency. Immunogenetics 21:549-558, 1985. 47. Clement LT, Plaeger-Marshall S, Haas A, et al.: Bare lymphocyte syndrome: Consequences of absent class II major histocompotihility antigen expression for B lymphocyte differentiation and function. J Ciin Invest 81:669--675, 1988. 48. Griscelli C, Limwaska-Grospierre B, Mach B: Combined immonodefielency with defective expression in MHC d u s II 8enes. lmmunodef Rev 1:135-154, 1989. 49. Alarcon B, Resueiro JR, Amaiz-Yillena A, Terhont C: Familial defect in the surface expression of the T-cell receptor-CD3 complex. N En81J Med 319:1205-1208, 1988. 50. Weinherg K, Parkman R: Severe combined immunodeficiency due to a specific defect in the

Editor-in.Chief: M.R.G. O ' G o r m a n , M.Sc., Ph.D.,

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Editertal Beard Kenneth Beaman,WaD,North Chicago, IL; lnme J. Check, PaD, Evanston, IL; Peter Domer, MD, Chicago, IL; Mary Ann Fletcher,Phi), Miami, FL; James D. Folds, PhD, Chapel Hill, NC; Marc Golightly, Phl), Stony Brook, NY; Ann Jackson, PhD, Ridgefield,WA; Alan L. Landay, Phl), Chicago, IL; Joel Oger, MD, Vancouver,BC, Canada; Launm Pachman, MD, Chicago, IL Please address editorial correspondenceto M.R.G. O'Gommn, MSc, Phi), A~istaut Pmfemor ef Pediatrics, Northwestern University, Director, Diagnostic ]mmuuelngy end Plow Cytomett~ Laboratories,Children's Memodal Hospital, 2300 Children's Plaza, Chicago,IL 60614. Genorsl Information Clinical ImmwwtogyNewsletter is published monthly by Elsevier Science Inc., 655 Avenue of the Amedcm, Now Yodc,NY 10010. See inside front cover for subscription infornmtico.

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