Patient Vignette
A Confusing Case of Class Switching?
Two brothers were seen in our Immunodeficiency Clinic for an evaluation of possible X-linked Hyper-IgM (XHIGM; a.k.a. HIGM1) syndrome.
The oldest brother (12 at the time of evaluation) was suspected of having HIGM syndrome based on his clinical phenotype – recurrent sinus and ear infections – and the presence of high IgM levels and reduced to absent IgG, IgA, and IgE. A previously performed CD40L assay (a screening assay for HIGM1) was reportedly abnormal. The younger brother (2 yrs old) was diagnosed shortly after birth, based on a similar pattern of increased IgM reduced IgG, IgA and IgE. Both patients receive IVIG and bactrim for PCP prophylaxis. While the CD40L assay was in progress, we reviewed their B-cell panels. The figures, shown below, represent the percentage of CD27+ B cells (on gated B-cells). In the older brother, 14% of B cells expressed CD27, while the younger brother showed a percentage of 12% CD27+ B cells.
As discussed in our Spring-2006 Newsletter, CD27+ B cells are measured as part of the comprehensive B-cell panel, to determine the percentage of memory B cells in peripheral blood. As shown in the figure below, the percentage of memory B cells in peripheral blood is age-dependent, and typically constitutes >10% of B cells. It can be as high as 25-35% of B cells, even at a young age (and independent of prior vaccinations).
The presence of CD27+ B cells in both patients was unexpected, since HIGM1 patients typically lack memory B cells in peripheral blood (compare with figures below, obtained from a 8 year old HIGM1 patient, and four controls with increasing age [CD27 on Y-axis]).
The results of the CD40L assay provided the context for these results, as both patients showed normal baseline CD40L expression, as well as normal upregulation of CD40L expression following T-cell activation. The CD40L gene was sequenced in both patients to verify the discrepancy between these and previously obtained results. No mutations in the gene encoding CD40L were identified. In light of the clinical phenotype and immunological phenotype – our workup showed normal to slightly increased IgM levels and near-absent IgA and IgE levels, other forms of HIGM were considered. Given the apparent X-linked inheritance pattern, mutations in the gene encoding NEMO were ruled out, while normal CD40 expression (also part of the B-cell panel) ruled out HIGM3. Subsequent genetic testing of the gene encoding Activation- Induced Cytidine Deaminase (AICDA) revealed bi-allelic mutations, consistent with a diagnosis of HIGM2, while the gene encoding uracil-N-glycosylase (UNG, HIGM5) was normal.
Hyper-IgM syndromes are due to so-called B-cell intrinsic immunoglobulin class switch recombination (Ig-CSR) deficiencies (see reference 1 in side-bar). CSR is important during the germinal center reaction to improve the response to infections, through isotype-switching from IgM to IgG, IgA, or IgE (with specialized functions, e.g. complement activation or presence in mucosal secretions). The germinal center reaction also includes somatic hypermutation (SHM), which incorporates mutations in the immunoglobulin molecule in a stochastic high-frequency manner, and increases the affinity of the molecule for its antigens. Both CSR and SHM occur in the germinal center, but are not dependent on each other. Although it has been considered that both CSR and SHM require CD40L interaction on activated T cells with CD40 on B cells, it has been shown that CD40Lindependent pathways exist. In the case of CSR, the BAFF system (see Fall-2007 Newsletter) can substitute for CD40L/CD40 (with appropriate cytokines and B-cell antigen receptor engagement), while SHM has been found in IgM-expressing B cells, residing in the splenic marginal zone (representing a defense mechanism against blood-borne encapsulated bacteria).
Going back to the issue of CD27+ B cells in HIGM syndromes; it has been proposed that peripheral blood memory B cells are derived from B cells that have (successfully) undergone SHM during the germinal center reaction. Immunophenotypically, they express CD27. While in many cases, the lack of CD27+ B cells is indicative of a defective generation of memory B cells (see figure on page 4 of the Spring-2006 Newsletter), the reverse – presence of CD27+ B cells indicative of the presence of memory B cells – is not the case, as demonstrated by HIGM2 patients. In the case of Ig-CSR deficiency on the basis of autosomal recessive AICDA defects, many patients show a normal percentage of CD27+ B cells; yet these cells lack acquisition of somatic hypermutations.
The situation, however, is more complex as AICDA mutations located in the Cterminal part of AICDA have been found in which only CSR is defective, but SHM is preserved. In these cases, the presence of CD27+ B cells may thus appropriately reflect the presence of memory B cells. Lastly, autosomal dominant transmission of AICDA mutations have been found in several patients with variable immunodeficiency phenotypes, and preserved SHM in some, but not all, patients. The mutations affect the nuclear export signal (NES) domain of AICDA. CD27+ B cells are present in these patients.
An important clinical clue in the evaluation of HIGM patients is the presence of lymphoid hyperplasia in certain patients with HIGM2. Pathological examination of biopsied lymph nodes revealed the presence of giant germinal centers, filled with highly proliferating B cells (also referred to as progressive transformation of germinal centers). This histopathological entity is characteristically found in ALPS and in forms of Hodgkin’s lymphoma. Of note, the older brother had shown lymphadenopathy, as well as tonsillar hypertrophy in the past. Obtaining an accurate (genetic) diagnosis has practical and prognostic implications. These include the association with autoimmunity, and liver disease (associated with Cryptosporidium infection), the risk of PCP pneumonia (and need for PCP prophylaxis), and the risk of lymphoma that are relatively well characterized in HIGM1, but – so far -do not appear to be present in HIGM2. On the other hand, HIGM2 (as wells the other HIGM forms) are somewhat “new” disorders. And thus, more needs to be learned about these, and other conditions of defective CSR and/or SHM. From a practical standpoint, CD27 measurement on B cells should be regarded with caution, and in the context of other information. Other (flow-based) methods are needed as well in order to measure memory B cells (see future Newsletter).
