Dissecting the mechanistic basis of effective anti-leukemic immunityagainst chronic lymphocytic leukemia
Final Report Abstract
Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a highly effective treatment modality for many hematologic malignancies, a major treatmentassociated toxicity is the induction of a prolonged state of T cell immunodeficiency in the transplant recipient, which in turn contributes to critical clinical outcomes such as infectious complications, and the risk of relapse. The development of strategies to enhance immunocompetence and the induction of more robust graft-versus-leukemia (GvL) responses, the crucial anti-leukemic principle of allo-HSCT, in these patients remains a high priority. The aim of this project was to dissect the mechanistic basis for effective tumor immunity in patients with advanced chronic lymphocytic leukemia (CLL) who developed durable GvL effects without damage of normal tissues by graft-versus-host disease (GvHD) following early post-transplant tumor cell vaccination. For a detailed analysis of T cell recovery in vaccinated and control patients I proposed to integrate the assessment of minimal residual disease, immunophenotyping and T cell receptor excision circle (TREC) analysis with targeted deep sequencing of the T cell receptor beta-chain (TCRβ). Although TCRβ sequencing has emerged as a promising technology for enabling the qualitative and quantitative monitoring of T cell recovery following transplant with unprecedented resolution, major challenges remained in the establishment of informative analysis tools for the characterization of TCRβ repertoire dynamics. Together with bioinformatics and biostatistics experts, I have developed a novel analysis approach that can now be applied to gain detailed biological insight into T cell reconstitution following early post-transplant vaccination. To this end, T cell subpopulations from 14 patients with advanced CLL who received tumor cell vaccination, developed GvHD or did not experience a major immunologic event within the first 100 days following allo-HSCT were immunophenotypically characterized. In addition, naïve/effector (CD45RO-) and memory (CD45RO+) CD4+ and CD8+ T cells were isolated at three informative time points (post-transplant days 30, 120 and 365). From these T cell subpopulations, genomic DNA was extracted and our 168 samples were applied to TCRβ sequencing. As a comparison, we further studied repertoire data from naïve/effector (CD45RO-) and memory (CD45RO+) CD4+ and CD8+ T cells collected from 9 healthy adult volunteers and 4 transplant donors. To account for the variable input cell numbers of our samples, we surmised the need to develop an approach to reconstruct the full range of repertoire diversity irregardless of starting sample size. Our novel algorithm is an optimized version of the ‘unseen species’ question, which allowed us to make an inference about the clone size distribution of the total TCRβ repertoire. Using the Hill framework of species ecology and control sample sets, we identified Richness (0D, i.e. number of unique TCRβ clones within the repertoire), Entropy (1D, i.e. effective number of smaller clones present in the sample) and iBPI (infD, i.e. effective number of clones that would fit in the repertoire, if all clones were as large as the largest clone) as meaningful measures for the comprehensive characterization of T cell repertoire reconstitution in our transplant patients. Ongoing analyses focus on integrating the TCRβ repertoire analysis with the other experimental measures of immune reconstitution. The results are anticipated to provide valuable information for the design and optimization of future leukemia cancer vaccine trials and for improving allo-HSCT procedures.
Publications
- Boosting leukemia-specific T cell responses in patients following stem cell transplantation. Oncoimmunology 2013; 2(11): e26587
Burkhardt UE and Wu CJ
- Towards the next waves of cancer immunotherapy: 11th Annual Meeting of the Association for Cancer Immunotherapy (CIMT), Mainz, Germany, May 14-16, 2013. Cancer Immunol Immunother. 2014; 63(7): 749-55
Kloke BP, Rae R, Mahr A, Burkhardt UE, Kvistborg P, Britten CM
(See online at https://doi.org/10.1007/s00262-013-1495-6) - Hematological malignancies: at the forefront of immunotherapeutic innovation. Nat Rev Cancer. 2015; 15(4): 201-5
Bachireddy P, Burkhardt UE, Rajasagi M, Wu CJ
(See online at https://doi.org/10.1038/nrc3907)
