A Comparative Gene Expression Study of CD3+ T-Cells In Bone Marrow From Patients with Chronic ITP and Healthy Controls by DNA Microarray.
Authors: Jernås Margareta , Olsson Bob , Wadenvik Hans 
Published: October 2019

Abstract
Abstract 1445

Background:
The pathophysiology of ITP (immune thrombocytopenia) is more complex than initially believed. The importance of regulatory T-cells, platelet specific cytotoxic T-cells, suppression of megakaryocyte proliferation and maturation, functional thrombopoietin deficiency and inappropriately low bone marrow production of platelets has been unraveled over the last decade. T-cells are important in all forms of autoimmunity including ITP. However, very little is known about the T-cell activity in organs where platelets and their precursors are targeted by effectors of the immune system. To provide new insights into the pathophysiology of ITP, we performed a DNA microarray analysis on isolated bone marrow derived T-cells from chronic ITP patients and healthy controls.

Patients and Method:
Bone marrow was collected from 6 chronic ITP-patients (3 males and 3 females, mean age 39.5 years) and 6 healthy controls (3 males and3 females, mean age 33.8 years) through iliac crest bone marrow aspiration. T-cells were isolated by immunomagnetic cell sorting (Miltenyi Biotec, Surrey, UK). RNA was prepared using the Chomczynski method (Chomczynski and Sacchi, 1987), followed by RNeasy minielute clean-up (Qiagen, Hilden, Germany). RNA was amplified from 20 ng of starting total RNA with the Ovation RNA Amplification System V2 (NuGEN Technologies, Inc.), and cDNA was synthesized using the Encore Biotin Module kit (NuGEN Technologies, Inc.). After standard labeling, each sample was hybridized to an Affymetrix U133Plus 2.0 Human Genome array (Santa Clara, CA).

Results:
We identified 397 differently regulated genes in the bone marrow derived T-cells between ITP patients and controls by the DNA microarray analysis (P-value <0.05 and fold change ± 1.3). Some of the regulated genes have previously been implicated in ITP, such as integrin alpha 4 (ITGA4 or CD49D or VLA-4), Fas ligand (FASLG) and cytotoxic lymphocyte-associated 4 (CTLA-4). Furthermore, several apoptotic genes were lower expressed in ITP patients, such as Fas apoptotic inhibitory molecule 2 (FAIM2), BCL2-like 11 (BCL2L11), BCL2-like 13 (BCL2L13), peptidyl-tRNA hydrolase 2 (PTRH2) and the enzyme inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB).
Integrin alpha 6, (ITGA6) and the adhesion molecule endothelial cell adhesion molecule (ESAM) were higher expressed in ITP patients compared to controls and are important for T-cell adhesion and migration or “homing” of lymphocytes to different lymphoid organs and inflammatory sites.

Conclusion:
We have identified a number of regulated genes in T-cells from bone marrow that differed between patients with ITP and healthy controls. The importance of several of the identified genes has already been implicated in ITP, proving the usefulness of this strategy. The identification of novel regulated genes may provide new insights into the physiology and pathophysiology of ITP and for autoimmunity in general.

Disclosures:
No relevant conflicts of interest to declare.