Cell sorting in clinical immunology research

Attendees will see some common applications of cell sorting in a department of immunology. Beginning with an introduction of lymphoproliferative disorders in inborn errors of immunity.

1. How cell sorting can help in the molecular characterization of somatic autoimmune lymphoproliferative syndrome (sALPS).

2. How we performed a functional validation of a TET2 candidate variant by using cell sorting for epigenetic testing.

3. Learn how cell sorting was used to aid in the study of molecular variants in large granular lymphocyte leukemia (LGLL).

ABSTRACT

Human inborn errors of immunity (IEI) comprise a variety of diseases caused by molecular variants that impair both innate and adaptive immune responses. Patients with IEI often exhibit a susceptibility to a wide range of infectious diseases, along with autoimmune, autoinflammatory, allergic, and malignant conditions. These symptoms typically manifest in childhood, which is also when IEI is most frequently diagnosed.

In recent years, it has become increasingly recognized that somatic variants acquired in a subset of cells can also result in immune disorders or 'phenocopies' of IEI. The identification of somatic mosaicism as a cause of IEI has mainly come from studying sporadic cases where symptoms of autoinflammation and/or autoimmunity are present, but germline variants are not detected. Somatic mosaicism responsible for disease has been discovered in genes that also cause germline IEI, such as FAS or genes linked to autoinflammatory diseases. Cell sorting and DNA sequencing is critical for the diagnosis of somatic autoimmune lymphoproliferative syndrome (ALPS) patients. Cell sorting and functional validation may also be important for molecular variants with an unknown significance (VUS).

Large granular lymphocyte leukemia (LGLL) is an indolent lymphoproliferative disorder categorized under mature T and natural killer (NK) cell neoplasms. According to the 2016 World Health Organization classification, it is classified as cytotoxic T and NK cell lymphomas and leukemia. There are two main types of chronic LGL proliferation: T-LGL and NK-LGL, which represent over 85% and 10% of cases, respectively. Recent evidence highlights the clinical and phenotypic relevance of specific mutations in T-large granular lymphocytic leukemia (T-LGLL). The STAT3 mutation, primarily found in CD8+ T-LGLL and gamma/delta T-LGLL, is linked to neutropenia and reduced overall survival. The STAT5B mutation, which is more common in the rare CD4+ T-LGLLs (up to 30% of cases), is associated with a poor prognosis in CD8+ T-LGLL but does not affect the prognosis in CD4+ T-LGLL and gamma/delta T-LGLL. NK large granular lymphocytic leukemia (NK-LGLL) is increasingly understood to be a monoclonal or oligoclonal expansion of NK cells, sharing many characteristics with T-LGLL. Cell sorting and the study of molecular variants in the genes such as STAT3, STAT5B, TET2, and other in LGL leukemia is interesting for a better understanding of the pathophysiology of these diseases and also could correlate with the personalization of immunosuppression.