A longitudinal analysis of TCRβ repertoire diversity in immune tolerance of pediatric patients post-UCBT: T cell clonal deletion over clonal anergy

Abstract

Background & Aim The mechanisms of long-term immune tolerance after HLA mismatched unrelated cord blood transplant (UCBT) is not understood. Both central (clonal deletion) and peripheral mechanisms (anergy, Treg, or Tr1 mediated suppression) are suspected. In this study, we set up serial experiments to test for each of these mechanisms in the GvH direction. Next-generation TCR sequencing was applied for tracking and quantitating alloreactive T cell clones within the TCRVβ repertoire. Methods, Results & Conclusion Methods: 12 patients (median age 1y/o) with non-malignant diseases were enrolled on a reduced-intensity conditioning trial (NCT01852370) with Alemtuzumab 2 weeks pre-UCBT. Patients were tolerant if completed immunocompetent, off immunosuppression for months without GvHD or rejection. T cell responses to host antigen-presenting cells (APC) were measured utilizing EBV transformed B cells in seronegative children by mixed lymphocyte reaction (MLR) and cytotoxic T lymphocyte (CTL) reaction, along with cytokine profiling and ImmunoSEQ® monitoring the fate of host-reactive T cell clones. To identify the key mechanisms of tolerance, we depleted Tregs with Denileukin diftitox prior to MLR and CTL. The involvement of Tr1 or anergy was examined either by blocking IL10R or by adding low dose IL2, respectively. Result and discussion In tolerant patients, there was no significant proliferative or CTL response towards host APC by donor-derived T cells while they responded vigorously to 3rd party APC (Fig1A). Similarly, cytokine measurements by Bioplex revealed host-specific non-responsiveness (Fig1B) similar to anti-self-responses of healthy volunteers. TCRβ clonotype tracking with ImmunoSEQ® revealed the disappearance of host-specific clones identifiable from an aliquot of the infused CB graft (pre-UCBT) (Fig1C). These alloreactive T cell clones became undetectable even in the presence of exogenous IL2 (Fig1D). There was no indication of Treg or Tr1 involvement in sustaining tolerance since neither deletion of Tregs, nor IL-10R blockade could reverse T cell hypo-reactivity in MLR, CTL, and Bioplex assays. In summary, the rapid acquisition of immune tolerance in the GvH direction post-UCBT is characterized by hypo-reactivity towards host APC in proliferation, cytotoxicity, cytokine secretion. While a small degree of anergy may also contribute, ImmunoSEQ® data confirmed that clonal deletion is a dominant mechanism towards long term immune tolerance.