ITGB1-Expressing Effector Memory CD4 ⁺ T-Cell Response Associates with Clinical Recovery in Childhood Immune Thrombocytopenia

Background: The autoimmune bleeding disorder childhood immune thrombocytopenia (ITP) features antibody and T-cell immune responses against platelet self-antigens. This involves activation of B-cells, CD4 ⁺ T-cell help, and CD8 ⁺ T-cells. Novel prognostic biomarkers are needed to predict recovery and treatment responses. Compared to healthy children, changes in total lymphocytes, CD4 ⁺, regulatory CD4 ⁺, CD8 ⁺T-cells, CD19 ⁺ B-cells, and NK cells were described in childhood ITP. Children with transient ITP were also found to have a fewer CD25 ^hi CD4 ⁺ T-cells than those who develop chronic ITP. In contrast, the platelet-stimulated peripheral blood mononuclear cells (PBMC) of children with chronic ITP produced more IL-2 than those of transient ITP. At present, none of these biomarkers have been validated for ITP prognosis, and consequently they are not in clinical use. This emphasizes the need for clinical-grade biomarkers to predict clinical responses in childhood ITP.

Aims: To predict spontaneous recovery and IVIg response in newly diagnosed ITP by (1) validating previously suggested changes in immune cell frequencies, and (2) identifying novel immune cell subsets as predictors.

Methods: Children with newly diagnosed ITP were randomized 1:1 to observation or IVIg treatment (TIKI trial). Recovery was determined by platelet counts according to IWG criteria 1, 4, 13, 26 and 52 weeks after the diagnosis. For validation, the CD4 ⁺, regulatory CD4 ⁺ (CD25 ⁺/CD127 ^lo), CD8 ⁺, CD19 ⁺, and NK cells were quantified at the time of diagnosis in a centralized laboratory by flow cytometry. For identification of novel predictors, CD4 ⁺ central (CD45RO ⁺ CD27 ⁺) and effector memory (CD45RO ⁺ CD27 ^-) frequencies were determined. The primary clinical outcome was longitudinal recovery, either spontaneous or after IVIg (adjusted Cox-proportional hazard model). Secondary outcomes included the mean age-adjusted difference between transient/persistent and chronic ITP by multivariate regression, and comparison to age-appropriate healthy control data. Additionally, PBMC (N=6) were analyzed at diagnosis by single-cell RNA sequencing (scRNA-seq) combined with T- and B- cell receptor sequencing (scTCR-seq and scBCR-seq).

Results: For validation of previously suggested predictors, the absolute CD3 ⁺, CD4 ⁺, CD8 ⁺, CD19 ⁺, and NK cell counts of newly diagnosed ITP patients were within the age-appropriate healthy reference range (N=158); they were not associated with recovery, and there were no age-adjusted differences between transient/persistent (N=139) and chronic ITP (N=19). The regulatory CD4 ⁺ T cell frequency was not reduced compared to age-appropriate reference data; not associated with recovery, and not different between transient/persistent and chronic ITP patients. For identification of novel predictors, we found that high CD4 ⁺effector memory cells numbers were associated with a reduced recovery rate, with an adjusted hazard ratio for complete recovery over a one-year follow-up to be 0.55 (95% CI, 0.35 - 0.85; ≥ median; adjusted for age and treatment; N=150). Chronic ITP patients displayed a mean age-adjusted increase in effector memory CD4 ⁺cells of 1.4 % (95% CI, 0.4 - 2.4; P= 0.005). The association with recovery was independent of a preceding infection, total leukocyte and lymphocyte counts, and the presence of anti-platelet IgG or IgM autoantibodies. ScRNA-seq analysis of 7965 PBMC also showed an effector memory CD4 ⁺ T-cell cluster that was expanded in chronic ITP, present in at a frequency of 8.1 ± 1.4 % (mean ± sem) in transient vs 14.8 ± 1.2 % in chronic ITP. This cluster also expressed high levels of fibronectin receptor integrin b1 ( ITGB1) interleukin 32 (IL32) interleukin 7 receptor (IL7R) andlymphotoxin beta (LTB). The effector memory phenotype of CD4 ⁺ ITGB1 ⁺ T-cells was confirmed by flow cytometry analysis in healthy individuals and ITP patients.

Conclusions: Previously suggested changes in T-, B-, or NK cell frequencies could not be validated as predictors of spontaneous recovery or IVIg response. However, we identified the frequency of ITGB1-expressing effector memory CD4 ⁺ T cells as an independent predictor of spontaneous recovery and IVIg response. Thus, T cell phenotyping at the time of diagnosis may be suitable to determine prognosis and personalize treatment decisions in newly diagnosed childhood ITP.