T Cell Receptor Repertoire Landscape Mapping: A Novel Paradigm for T Cell Mediated Autoimmune Disorders Based on Deep Vβ Chain Sequencing
Authors: Clemente Michael J , Przychodzen Bartlomiej , Makishima Hideki , Afable Manuel G , Lichtin Alan E. , Maciejewski Jaroslaw P. 
Published: October 2019

Abstract 1341

T cell large granular lymphocytosis (T-LGL), a chronic lymphoproliferation of cytotoxic T cells (CTL) is often associated with lineage-restricted cytopenias. CTL-mediated inhibition of hematopoiesis in T-LGL is, in principle, similar to other T cell-mediated bone marrow failure syndromes. Variation in the CTL target spectrum could explain the lineage-restricted cytopenias in T-LGL and pancytopenia in aplastic anemia (AA). Regardless of effector mechanisms, T-LGL can serve as a simplified, monoclonal model of otherwise polyclonal CTL-mediated AA. The study of TCR Vβ clonotypes has the potential to reveal the nature/derivation of antigenic targets. It is possible that clonal specificity is private, inherently unique, and thus suggestive of the existence of neo-antigens or unique exogenous antigenic drivers. In an alternative scenario, one could expect a high degree of clonal sharing between T-LGL patients who have a similar hematologic presentation and immunogenetic background. Finally, finding of expanded T-LGL clonotypes in controls could point toward autoantigens and breach of tolerance in T-LGL or possibly an inability to maintain viral defense. Consequently, the study of the TCR repertoire may reveal unique patterns allowing for classification of immune-mediated disease.

In the past, molecular analysis of the T cell repertoire allowed only a quantitatively limited insight into the clonotypic spectrum. With the advent of next generation sequencing (NGS), the clonotypic repertoire can now be analyzed at an incredible depth. Beginning with a cohort of 143 patients with T-LGL leukemia, we observed a marked increase in clone size as assessed by Vβ flow cytometry in patients with HLA-B7 (p<0.001). Based on this finding, we performed NGS of the TCR CDR3 in 10 patients with CD8 T-LGL leukemia and 3 controls. We selected 7 patients who shared HLA-B7 and 9 with anemia as presentation; 3 had Vβ17 (TRBV19) as the immunodominant clone by flow. Several levels of bioinformatic analysis have been performed; the average depth allowed for characterization of 507574±7818 (338847–666567) sequences. First, we assessed the clonal size, degree of diversity, and determined the frequency of codominant clones. The dominant clone spanned 56.1±34.7 of the TCR repertoire. The diversity, i.e. the number of unique clonotypes per patient, was 3571±3520 (212–11185). Aside from the dominant clonotypes, there were, on average, 107±94 expanded/redundant clonotypes (frequency >5/10K) per patient. In controls, the diversity was 20283±8525 (12428–29349), the most expanded clonotype was 8.3%, and there were an average of 103±36 expanded/redundant sequences.

To assess antigenic specificity we first assessed the homology of the CDR3 regions and found that immunodominant clonotype-related clones (>75% homology) were present in other patients, albeit with a lower degree of expansion. In general, accounting for this level of homology, the diversity of patient repertoires was much lower (82% lower) as compared to controls. Interestingly, in 6 patients an additional clone with an amino acid sequence similar to the immunodominant clone (codominant clone) was detected. Similarly, while there was clonotype sharing between the patients, the immunodominant clonotypes were unique among the 10 cases subjected to Vβ NGS. In particular, clustering based on homology revealed cluster sharing with an average of 8 clones structurally related to the immunodominant clone. Most importantly, the immunodominant clones and those with >75% homology to immunodominant clones were generally found at extremely low (<10) copy numbers in controls. However, dominant and codominant clonotypes were not found in a database of over 6K clonotypes associated with various autoimmune and infectious conditions.

While these results are preliminary, clonotype sharing at the deepest level of molecular resolution between patients and controls suggests that antigens driving the immune response in T-LGL are unique to patients or may be related to other immunogenetic factors and not related to auto-antigens which would be expected to elicit responses at a lower level. Our study of T-LGL patients demonstrates that analogous analyses, currently ongoing in AA, may reveal distinct and possibly diagnostic landscapes which would allow for sub-classification of disease and identification of patterns predicting response to immunosuppression.

Disclosures:

No relevant conflicts of interest to declare.