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4584 Clonal Deletion Plays a Major Role to Achieve Immune Tolerance after Reduced Intensity Unrelated Donor Cord Blood Transplantation (UCBT)

Clinical Allogeneic Transplantation: Acute and Chronic GVHD, Immune Reconstitution
Program: Oral and Poster Abstracts
Session: 722. Clinical Allogeneic Transplantation: Acute and Chronic GVHD, Immune Reconstitution Poster III
Monday, December 5, 2016, 6:00 PM-8:00 PM
Hall GH (San Diego Convention Center)

Paul Szabolcs, MD1, Xiaohua Chen, MD PhD1*, Mark Vander Lugt, MD2* and Memphis J Hill, BS1*

1Department of Pediatrics, Division of Blood and Marrow Transplantation and Cellular Therapies, Children's Hospital of Pittsburgh, Pittsburgh, PA
2Department of Pediatrics, Division of Blood and Marrow Transplantation and Cellular Therapies, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA

Generation of tolerance to transplanted hematopoietic cells or organs is essential if elimination of immunosuppressive therapy (IST) is desired. Immune tolerance is the expected outcome after successful hematopoietic stem cell transplantation (HSCT). It is characterized by immunocompetence without any alloreactivity (GVHD) in the absence of IST. However, the mechanism(s) required to prevent alloreactivity are not fully understood. Both central (clonal deletion) and peripheral (anergy, suppression by Treg, Tr1) mechanisms are suspected. In this study, we set up serial experiments to test for these mechanisms after unrelated donor cord blood transplant (CBT) in the GvH direction. We also studied these mechanisms at the time of GVHD.


Seven patients (age range from 1m to 9y) with non-malignant diseases were transplanted and studied for host-specific alloreactivity following reduced intensity conditioning (RIC) regimen (NCT01852370) that also utilized Alemtuzumab (1.2mg/kg) ~ 2 weeks prior to UCBT. Five of the patients were off immunosuppression therapy when blood was drawn (6 -12 months after UCBT) while two were not and had either limited chronic GVHD or recently resolved acute GVHD. Donor T cell chimerism at the time of blood draw was a median 97% (range 87-100%). Purified T cell responses to host APC (EBV-LCL for EBV seronegative patients, or monocyte derived DC) were measured by mixed lymphocyte reaction (MLR) and cytotoxic lymphocyte (CTL) reaction after 5 or 7 days in culture, respectively. Th1/Th2/Th17 cytokines along with IL-10 secreted during MLR were quantified from day 5 supernatant by bio-plex assay. To track host-reactive T cell clones, TCRb repertoire was monitored and quantitated using targeted amplification of rearranged TCR genes followed by high-throughput sequencing (Adaptive Biotechnologies®). The data was analyzed with ImmunoSEQ® Analyzer Software. To further identify the key mechanism(s) of tolerance, we attempted to break tolerance by inhibiting or amplifying putative pathways using the following methods: Regulatory T cell (Treg) deletion was achieved by Denileukin diftitox (Ontak) treatment prior to MLR and CTL reactions. Involvement of Tr1 or anergy was examined either by blocking IL10R or by adding low dose IL2, respectively.

Results and Discussion:

Patients with clinical tolerance (n=5): There was no significant proliferative or cytotoxic T cell response towards recipient APC while T cells responded vigorously to 3rd party APC (Fig 1, 2). Similarly, Th1/Th2/Th17 cytokine profiles revealed recipient-specific non-responsiveness by both proliferation and cytokine secretion (Fig 1A,B) fulfilling a critical tenet of tolerance in three independent assays (MLR, CTL, cytokine secretion).  In addition, tracking TCRb clonal profiles with ImmunoSEQ® revealed the disappearance of recipient-specific T cell clones that were amplified from the cord blood graft itself by 7 days of stimulation with purified host APC (labeled pre-UCBT in Fig 1.C). There was no indication of Treg or Tr1 involvement in sustaining tolerance since neither deletion of Tregs by IL-2 IT, nor IL-10R blockade had any impact on T cell hypo-reactivity in MLR, CTL, and bioplex assays. Interestingly, addition of IL-2 in MLR did enhance T cell proliferative responses in 1 out of 5 tolerant patients tested, but did not activate host-specific CTL responses. Nevertheless, here we can not exclude the possibility of anergy playing some role in preventing host-specific alloreactivity.

Patients with GvHD at time of examination: T cells isolated from patients with recently resolved or still active limited chrGVHD receiving some IST (n=2) did proliferate against recipient APC in MLR, but were not cytotoxic against host APC (Fig 3).   IL-2 and IL-13 secretion towards host APC was detectable in both (data not shown).

In summary, the rapid acquisition of immune tolerance in the GvH direction post-UCBT on our prospective RIC clinical trial is characterized by hypo-reactivity in all assays employed towards host APC. The proliferation, cytotoxicity, and TH1/TH2, TH17 cytokine secretion data suggest that host-specific clonal deletion is a significant mechanism of tolerance in most if not all patients.  In one, there is possibility for anergy as exogenous low dose IL-2 was able to overcome absent proliferation by MLR, while having no impact on host-reactive CTL activity or TH1/TH2/Th17 cytokine secretion.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH