QUESTIONS FOR GROUP DISCUSSION

1. Compare the types of infections that this patient has with those of the patient in Case 1.

2. What is the significance of the fact that this patient has a history of viral and fungal infections but not bacterial infections? What types of cell defects in (a) innate immunity or (b) adaptive immunity would present in this manner?

3. Why would a history of viral infections not necessarily accompany a history of infection with Candida albicans?

4. The chest radiograph revealed that the thymic shadow was missing. What is the function of the thymus? Review the steps that precursor lymphoid cells undergo to differentiate to CD4+ T cells or CD8+ T cells. Be sure to include the following terms in your discussion: (a) somatic recombination, (b) interactive avidity, (c) positive selection, (d) negative selection, (e) death by neglect, (f) T cell receptor excision circles (TRECs), (g) lineage determination/gene silencing, and (h) T cell repertoire. As well, be prepared to describe the phenotype of developing cells with respect to the following molecules: CD2, CD3, CD4, and CD8.

5. Given that the thymic shadow is lacking in the radiograph, it seems reasonable to investigate T cell function and T cell numbers. A commonly used test of T cell function (providing the child is not a newborn) is the skin test. What does this test measure? What common antigen is used in this test? Explain why this particular antigen is selected.

6. Another test of T cell function is T cell proliferation using mitogenic lectins. Which lectins are used to stimulate T cells? Describe the assay in general terms.

7. Explain how a congenital thymic defect could affect antibody titers to immunization antigens (e.g., tetanus).

8. What does the acronym “CATCH22” signify?

9. For many immunodeficiency disorders, bone marrow transplantation (BMT) is the treatment of choice when an appropriate donor can be identified. Why would BMT not be considered for this patient? What would you recommend?

10. Explain why a “successful” thymic graft could allow a patient’s T cells to respond to mitogenic lectins but not necessarily to foreign antigens.

RECOMMENDED APPROACH

Implications/Analysis of Family History

A family history is not possible because the child has been adopted.

Implications/Analysis of Clinical History

Viral and fungal infections point us immediately to a defect in T cells (Fig. 2-1). Although a defect in phagocytes may result in increased susceptibility to fungal infections, T cells are the key players in both viral and fungal infections. (Recall that the patient in Case 1 had a clinical history of fungal, viral, and bacterial infections.)

FIGURE 2-1 Cutaneous Kaposi’s sarcoma lesions in HIV-infected individual. Patients with T cell deficiencies are susceptible to opportunistic infections.

(From Skoner D, Urbach A, Fireman P: Slide Atlas of Pediatric Allergy and Immunology. St. Louis, Mosby, 1992.)

Implications/Analysis of Laboratory Investigation

The absence of a thymic shadow indicates a defect during embryogenesis resulting in partial or complete absence of a thymus. When the defect is very severe, T cell maturation cannot occur in the thymus. A deficiency of T cells would explain RD’s clinical history. Additionally, RD’s physical appearance is consistent with that of children diagnosed with this defect.

Additional Laboratory Tests

To do a complete workup, RD’s physician ordered (1) a complete blood cell count (CBC) with differential, (2) a skin test for T cell function, (3) antibody titers for immunization antigens, and (4) flow cytometric analysis of T and B cells. The skin test for T cell function consists of an intracutaneous injection of two or three standardized test antigens, one of which is C. albicans. Antigens to which most members of the population have been exposed are selected for this test. Prior exposure to any one of the antigens activates memory T cells and results in the formation of an induration at the site of injection that is maximal 48 to 72 hours later. This is also referred to as a “delayed-type hypersensitivity reaction.”

RD’s blood cell count was low (see Appendix for reference value), whereas the differential showed a profound decrease in lymphocytes (normal pediatric value = 48%). Skin tests to all three antigens were negative, despite the documented infection with Candida albicans. A negative skin test to all three antigens is interpreted as a defect in T cell function. (When the skin test is not definitive, a test that measures polyclonal activation of peripheral blood cells with T cell mitogens [concanavalin A and phytohemagglutinin] is used to assess T cell function.) In this assay, peripheral lymphocytes are incubated with a mitogenic lectin and proliferation (DNA synthesis) is assessed by measuring the amount of ³H-thymidine that is incorporated into the cells. Results of RD’s tests to detect the presence of serum antibodies specific for antigens used in pediatric immunization, DPT (diphtheria toxoid, pertussis, and tetanus toxoid), were all below normal. This is not surprising in that antibody responses to protein antigens require both cognate interaction with T cells as well as T cell–derived cytokines.

Flow cytometry can be used to assess the ratio and absolute cell number of cells (e.g., T and B cells). When this technique is used, fluorophore-labeled monoclonal antibodies, specific for proteins on the various cell types, are required. To analyze the lymphocyte subsets, antibodies to T cell proteins (anti-CD3, anti-CD4, anti-CD8) and to B cell proteins (CD19) are used. In RD’s case the differential had indicated a significant decrease in the percentage of lymphocytes. Flow cytometric analysis used to determine the ratio of circulating B cells to T cells indicated a profound decrease of circulating T cells (normal ratio of B cells/T cells is 5%-24%/57%-84%). The absolute number of T cells was less than 100 cells/mm³, suggesting that RD’s problems were due to an inherited defect that led to a deficiency in the number of T cells. The prototypical T cell immunodeficiency disorder is DiGeorge syndrome.

A diagnosis of DiGeorge syndrome was confirmed after fluorescence in situ hybridization (FISH) analysis of blood smears. For this test, a fluorochrome-labeled single-stranded DNA probe is hybridized to denatured metaphase chromosome (single-stranded) smears on a glass slide. Hybridization with the labeled DNA probe is detected by fluorescence microscopy. The absence of hybridization indicates a deletion. For this syndrome, the deletion was on chromosome 22.

DIAGNOSIS

Based on the molecular cytogenetic analysis (FISH), RD was diagnosed with congenital thymic aplasia, also referred to as DiGeorge syndrome.

THERAPY

Depending on the severity of the defect, pharmacologic intervention may suffice during infections. However, more severe cases require transplantation of functional components of fetal/postnatal allogeneic thymic tissue slices placed into both quadriceps muscles or under a renal capsule. Because thymic aplasia is the result of abnormal development of the third and fourth pharyngeal pouches during embryogenesis, patients also have hypoplasia (or aplasia) of the parathyroids.

Allogeneic Thymus

In thymic fragment transplantation, the major histocompatibility complex (MHC) proteins that are expressed on the allogeneic thymic epithelium are a major factor in the selection of thymocytes (i.e., developing T cells) that will be positively selected for export to the periphery (or death). Consequently, antigen recognition in the periphery will occur only when antigenic peptides are presented to T cells within the groove of the MHC proteins that are the same as those that were present on the thymic epithelium (self-MHC/self peptide) during T cell maturation/ education.

Therefore, the question arises, “How can T cells educated on an allogeneic thymus (allo-MHC) recognize self MHC-antigenic peptide in the periphery?” What is surprising is that 1 year after transplantation, even non–MHC-matched patients have been reported to have developed antigen-specific proliferative responses. Recognition of antigenic peptide/MHC complexes on host cells by T cells that have been educated in a thymus expressing a different MHC (allo-MHC) is inconsistent with current dogma.

The presence of T cells that have matured in the allogeneic thymus can be assessed by detecting T cell receptor rearrangement excision circles (TRECs, see later) in circulating T cells. T cell proliferative responses after stimulation with a T cell mitogen do not involve MHC recognition, and so one would predict that T cells isolated from these patients would proliferate when so stimulated. This is, in fact, what has been observed in several of the patients receiving a thymic transplant.

In addition to the thymic aplasia, patients with DiGeorge syndrome may lack parathyroid glands. These patients require regular therapy with an active form of vitamin D. In some cases patients are prescribed calcitriol, which is the active form of vitamin D required to increase calcium and phosphorus in the bloodstream. In the normal individual, precursor vitamin D is ingested with food and absorbed throughout the body, including the skin, where it is modified by sunlight radiation. Subsequent modifications in the liver and the kidney generate calcitriol. Calcitriol enhances absorption of calcium and phosphate from the gastrointestinal tract.

ETIOLOGY: DIGEORGE SYNDROME (CONGENITAL THYMIC APLASIA)

DiGeorge syndrome results from defective embryogenesis leading to a reduced and defective (or absent) thymus and other organs derived from the third and fourth pharyngeal pouches. These patients are hypocalcemic and exhibit distinctive physical features, including widely separated eyes, low ears, and a cleft palate. The acronym CATCH22 (cardiac defect, abnormal facial features, thymic hypoplasia, cleft palate, hypocalcemia, and chromosome 22) is often used to refer to the collection of symptoms that characterize this disorder. Not all children have congenital heart disease, although this disorder is the second most common cause of congenital heart disease. In virtually all cases of congenital thymic aplasia there is a microdeletion in one copy of chromosome 22.

Because the thymus is the organ of differentiation for all T cells (see later) these individuals have few or no circulating T cells and have increased susceptibility to viral, fungal, and intracellular bacterial infections. Patients with reduced or partial thymus function have low T cell numbers and improve with age, perhaps owing to an extrathymic maturation site.

T CELL DEVELOPMENT IN THE THYMUS

Role of the Thymus

The rationale for transplanting a thymic tissue fragment into patients with DiGeorge syndrome is to provide an organ of differentiation for precursor T cells generated in the bone marrow (Fig. 2-2). To understand the complexities associated with this therapeutic intervention, it is important to know something about how T cells recognize antigenic peptides and the role of the thymus in selecting antigen-specific T cell receptors (TCRs). Each TCR chain consists of a variable and a constant region. Although most T cell clones express T cell receptors constructed with the same constant regions, each clone expresses receptors with a unique variable region. The key event in the construction of a unique TCR variable region is somatic recombination.

FIGURE 2-2 Defects in thymus development lead to DiGeorge syndrome. The absence of a thymus leads to a deficiency in T cells and associated immune dysfunction in T cell–mediated responses.

Somatic Recombination Determines the Variable Regions of TCRs

Variable regions are constructed during somatic recombination from segments termed variable (V), diversity (D), and joining (J). More specifically, the TCRα variable regions are composed of a “V” and a “J” segment (VJ), whereas the TCRβ regions are composed of “V,” “D,” and “J” segments (VDJ). During the somatic recombination process, which is initiated by the action of recombinase activation genes 1 and 2 (RAG1 and RAG2), intervening DNA segments that existed between the selected V, D, or J segments are excised, forming circular episomes called TRECs. In any given individual, somatic recombination and diversification can create a repertoire of 10¹² to 10¹⁵ different TCRs, each potentially giving rise to a unique T cell clone.

Selection of TCRs in the Thymus

The random selection of V, D, and J gene segments again leads to the generation of T cells expressing TCRs that may be autoreactive or TCRs that are unable to activate T cells. Consequently, those T cells must be deleted during the screening process in the thymus. The fate of the thymocyte (life or death) depends on the interactive avidity between the developing T cell (thymocyte) and the thymic epithelium. Interactive avidity includes the (1) intrinsic affinity of the TCR for self antigen/MHC complex, (2) density of TCRs, (3) density of self antigen/MHC complexes on the thymic epithelium, and (4) density of antagonistic peptide complexes (i.e., cell/cell molecular interactions that repel one another).

In addition to these factors there is a contribution resulting from the interaction of a variety of accessory molecules and adhesion molecules as they interact with their counterpart on the thymic epithelium (cognate ligand). Insignificant interactive avidity with the thymic epithelial cell results in death from neglect. If recognition exceeds a predetermined threshold, thymocytes are clonally eliminated or functionally inactivated by an active process termed negative selection. Finally, positive selection and preferential expansion of T cells occurs when the interactive avidity of recognition is intermediate (i.e., between the two extremes noted). Following the screening process in the thymic cortex, lineage determination/gene silencing occurs, a phenomenon in which gene silencing of either the CD4 or the CD8 gene occurs. As a result, each T cell expresses either CD4 or CD8, which determines both function and antigen recognition by class I or class II MHC. A more finely tuned process of negative selection occurs in the medulla.