Shelby M. Hutcherson

Background: Polymerized immunoglobulins can be difficult to interpret when observed on serum immunofixation electrophoresis (IFE). The use of reducing agents, such as beta-mercaptoethanol (BME), has been instrumental in resolving cases in which the monoclonal protein is unidentifiable due to polymerization. However, this procedure can be inconvenient to laboratories. We therefore investigated the use of immunotyping (IT) to resolve immunoglobulin aggregates as a potential replacement for BME pretreatment. Methods: Patient samples with paraprotein polymerization were selected from our routine testing and interpretation workflow in the protein immunology laboratory. Identified specimens were subjected to both repeat IFE testing after BME treatment and IT without BME treatment. Results from both types of repeat testing were compared to each other and to patient history if available. IFE was performed on a Hydrasys 2 (Sebia), and IT was performed on a Capillarys 3 Tera (Sebia). Results: Five patient samples were identified with an aggregate band visible in all IFE lanes. Results of BME treatment with repeat IFE or IT using the original sample were 100% concordant. One patient was determined to have no monoclonal protein, 3 had immunoglobulin M (IgM) lambda monoclonal proteins, and one had an IgM kappa monoclonal protein. Conclusions: IT was able to successfully identify paraproteins without the use of BME in samples that were difficult to interpret using IFE due to polymerization. This finding suggests that routine protocols for IT can be used in lieu of a secondary protocol and additional resources for BME treatment prior to IFE analysis.

Antigen receptor signaling pathways that control lymphocyte activation depend on signaling hubs and negative regulatory proteins to fine-tune signaling outputs to ensure host defense and avoid pathogenic responses. Caspase recruitment domain–containing protein 11 (CARD11) is a critical signaling scaffold that translates T cell receptor (TCR) triggering into the activation of nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK), mechanistic target of rapamycin (mTOR), and Akt. Here, we identify glutamine-rich protein 1 (QRICH1) as a regulator of CARD11 signaling that mediates an intracellular checkpoint for CD8 ⁺ T cell activation. QRICH1 associates with CARD11 after TCR engagement and negatively regulates CARD11 signaling to NF-κB. QRICH1 binding to CARD11 is controlled by an autoregulatory intramolecular interaction between QRICH1 domains of previously uncharacterized function. QRICH1 controls the antigen-induced activation, proliferation, and effector status of CD8 ⁺ T cells by regulating numerous genes critical for CD8 ⁺ T cell function. Our results define a component of antigen receptor signaling circuitry that fine-tunes effector output in response to antigen recognition.

CARD11 is a multidomain scaffold protein required for normal activation of NF-κB, JNK, and mTOR during Ag receptor signaling. Germline CARD11 mutations cause at least three types of primary immunodeficiency including CARD11 deficiency, B cell expansion with NF-κB and T cell anergy (BENTA), and CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS). CADINS is uniquely caused by heterozygous loss-of-function CARD11 alleles that act as dominant negatives. CADINS patients present with frequent respiratory and skin infections, asthma, allergies, and atopic dermatitis. However, precisely how a heterozygous dominant negative CARD11 allele leads to the development of this CADINS-specific cluster of symptoms remains poorly understood. To address this, we generated mice expressing the CARD11 R30W allele originally identified in patients. We find that CARD11^R30W/+ mice exhibit impaired signaling downstream of CARD11 that leads to defects in T, B, and NK cell function and immunodeficiency. CARD11^R30W/+ mice develop elevated serum IgE levels with 50% penetrance that becomes more pronounced with age, but do not develop spontaneous atopic dermatitis. CARD11^R30W/+ mice display reduced regulatory T cell numbers, but not the Th2 expansion observed in other mice with diminished CARD11 activity. Interestingly, the presence of mixed CARD11 oligomers in CARD11^R30W/+ mice causes more severe signaling defects in T cells than in B cells, and specifically impacts IFN-γ production by NK cells, but not NK cell cytotoxicity. Our findings help explain the high susceptibility of CADINS patients to infection and suggest that the development of high serum IgE is not sufficient to induce overt atopic symptoms. Key Points CARD11^R30W/+ mice exhibit pathway-specific defects in T, B, and NK cells. CARD11^R30W/+ mice develop high IgE in an age-dependent manner with 50% penetrance. High IgE in CARD11^R30W/+ mice is uncoupled from Th2 expansion and atopic dermatitis.

CARD11 functions as a key signaling scaffold that controls antigen-induced lymphocyte activation during the adaptive immune response. Somatic mutations in CARD11 are frequently found in Non-Hodgkin lymphoma, and at least three classes of germline CARD11 mutations have been described as the basis for primary immunodeficiency. In this review, we summarize our current understanding of how CARD11 signals, how its activity is regulated, and how mutations bypass normal regulation to cause disease.