The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.

Epstein–Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) with central nervous system (CNS) involvement is a severe complication after solid organ transplantation. Standard treatment with reduction of immunosuppression and anti-CD20 antibody application often fails leading to poor outcome. Here, we report the case of an 11-year-old boy with multilocular EBV-positive CNS PTLD 10 years after liver transplantation. Complete remission was achieved by repeated intravenous and intrathecal anti-CD20 antibody rituximab administration combined with intrathecal chemotherapy (methotrexate, cytarabine, prednisone) over a time period of 3 months. Due to the poor prognosis of CNS PTLD and lack of EBV-specific T-cells (EBV-CTLs) in patient’s blood, we decided to perform EBV-directed T-cell immunotherapy as a consolidating treatment. The patient received five infusions of allogeneic EBV-CTLs from a 5/10 HLA-matched unrelated third-party donor. No relevant acute toxicity was observed. EBV-CTLs became detectable after first injection and increased during the treatment course. Next-generation sequencing (NGS) TCR-profiling verified the persistence and expansion of donor-derived EBV-specific clones. After two transfers, epitope spreading to unrelated EBV antigens occurred suggesting onset of endogenous T-cell production, which was supported by detection of recipient-derived clones in NGS TCR-profiling. Continuous complete remission was confirmed 27 months after initial diagnosis.

Donor lymphocyte infusion (DLI) is a standard of care for relapse of AML after allogeneic hematopoietic stem cell transplantation (aHSCT). Currently it is poorly understood how and when CD8+ αβ T cells exert graft-versus-leukemia (GvL) activity after DLI. Also, there is no reliable biomarker to monitor GvL activity of the infused CD8+ T cells. Therefore, we analyzed the dynamics of CD8+ αβ T cell clones in DLI-patients. In this prospective clinical study of 29 patients, we performed deep T cell receptor β (TRB) sequencing of sorted CD8+ αβ T cells to track patients' repertoire changes in response to DLI. Upon first occurrence of GvL, longitudinal analyses revealed a preferential expansion of distinct CD8+ TRB clones (n=14). This did not occur in samples of patients without signs of GvL (n=11). Importantly, early repertoire changes 15 days after DLI predicted durable remission for the 36 months study follow-up. Furthermore, absence of clonal outgrowth of the CD8+ TRB repertoire after DLI was an early biomarker that predicted relapse at a median time of 11.2 months ahead of actual diagnosis. Additionally, unbiased sample analysis regardless of the clinical outcome revealed that patients with decreasing CD8+ TRB diversity at day 15 after DLI (n=13) had a lower relapse incidence (P=0.0040) compared to patients without clonal expansion (n=6). In conclusion, CD8+ TRB analysis may provide a reliable tool for predicting the efficacy of DLI and holds the potential to identify patients at risk for progression and relapse after DLI.

Donor lymphocyte infusion (DLI) can (re-)induce durable remission in relapsing patients after allogeneic hematopoietic stem-cell transplantation (alloHSCT). However, DLI harbors the risk of increased non-relapse mortality due to the co-occurrence of graft-versus-host disease (GVHD). GVHD onset may be caused or accompanied by changes in the clonal T-cell receptor (TCR) repertoire. To investigate this, we analyzed T cells in a cohort of 21 patients receiving DLI after alloHSCT. We performed deep T-cell receptor β (TRB) sequencing of sorted CD4+CD25+CD127low regulatory T cells (Treg cells) and CD4+ conventional T cells (Tcon cells) in order to track longitudinal changes in the TCR repertoire. GVHD following DLI was associated with less diverse but clonally expanded CD4+CD25+CD127low Treg and CD4+ Tcon TCR repertoires, while patients without GVHD exhibited healthy-like repertoire properties. Moreover, the diversification of the repertoires upon GVHD treatment was linked to steroid-sensitive GVHD, whereas decreased diversity was observed in steroid-refractory GVHD. Finally, the unbiased sample analysis revealed that the healthy-like attributes of the CD4+CD25+CD127low Treg TCR repertoire were associated with reduced GVHD incidence. In conclusion, CD4+CD25+CD127low Treg and CD4+ Tcon TRB repertoire dynamics may provide a helpful real-time tool to improve the diagnosis and monitoring of treatment in GVHD following DLI.

Background: Relapse of disease remains a frequent cause of mortality after allogeneic hematopoietic stem cell transplantation (HSCT). For patients with imminent relapse, therapeutic donor lymphocyte infusions (DLI) offer a treatment option for re-induction of complete remission of the underlying disease by employing the graft-versus-leukemia (GvL) effect. Moreover, DLI is used pre-emptively to prevent relapse in patients with high risk disease. DLI is given in increasing doses, adapted to clinical signs of graft-versus-host disease (GvHD) or molecular markers of disease, i.e. donor chimerism or minimal residual disease. There is a need for reliable monitoring of the immunologic response preceding the clinical outcome, enabling a precise administration of DLI dose escalation and thus possibly preventing overshooting immune reactions. With the advent of deep sequencing technology direct measure of high resolution T cell receptor (TCR) diversity can be used as a read-out of the immunologic response in the patient at the molecular level. Aims: In this study we aim to elucidate whether TCR-diversity can serve as a biomarker for clinical outcome of DLI treatment. Patients and Methods: We assessed 19 patients, who received DLI after HSCT. Therapeutic DLI was given in 14 patients and prophylactic DLI in 5 patients. The majority of patients (n=14) was treated for AML or MDS, 2 for ALL, and 3 for MPN. Peripheral blood samples for TCR-sequencing were obtained from the patient pre-DLI, at 2 and 4 weeks post DLI, and subsequently when available. Also, donor samples were collected. Donor's and patient's lymphocytes were FACS-sorted into CD8+ and CD4+ conventional (Tconv) and CD4+CD127-CD25+ regulatory T cells (Treg). Sorted subpopulations were subject to cDNA-based CDR3-region amplification by RACE-PCR allowing assessment of the entire TCR-β repertoire. Reliable generation of CDR3 amplicons was possible from as few as 4000 cells. These were then sequenced using the Illumina MiSeq platform. Reads were annotated by the IMGT.org database; further bioinformatics analyses included VDJtools and the tcR R-package. Results: GvL response (remission or stable disease) could be achieved in 14/19 patients; 8 of these patients developed acute GvHD ≥III°. Flow cytometric analysis showed that the ratio between CD8+ and CD4+ Tconv in the DLI is predictive of response to DLI: DLIs containing a majority of CD4+ Tconv were associated with development of GvHD (n=8, CD8:CD4 = 35.3%:54.9%) , whereas patients responding to DLI with GvL only had received a higher proportion of CD8+ T cells (n=6, CD8:CD4 = 59.1%:29.5%); comparison of ratios met statistical significance (Mann Whitney test, p=0.0027). TCR sequencing revealed that the diversity of the TCR-repertoire seems to be predictive of the clinical course of the patient after DLI. GvHD development within 2 weeks of blood sampling time (n=4) was preceded by an increase of Treg repertoire diversity (assessed with inverse Simpson's diversity index, 1/Ds). Compared to the pre-DLI repertoire 1/Ds, mean increase was 143.67% vs. a 36.04% decrease in patients not developing GvHD (n=7; Mann Whitney test, p=0.02). Steroid treatment of GvHD (n=2) led to a mean decrease of 49.71% of Treg TCR 1/Ds compared to the previous time point. Analysis of GvL response revealed an association of remission induction with a trend towards decreased 1/Ds of the CD8+ TCR-repertoire (mean decrease of 27.66% at d28 after DLI compared to pre-DLI vs. 0.31% decrease in patients showing no GvL effect). Assessment of TCR CDR3 region clonotype expansion over time is shown for a patient responding with GvHD (Fig. 1A) and GvL (Fig. 1C) to DLI and a patient progressing without response to DLI (no GvHD Fig. 1B, no GvL Fig. 1D). Conclusion: Our data indicate that TCR sequencing allows the assessment of TCR-diversity change as a surrogate parameter of DLI response. While an increase of Treg diversity seems to indicate the development of GvHD, repertoire compression of the CD8 compartment may be predictive of GvL response. Taken together, monitoring TCR repertoires may become a valuable predictive tool to improve DLI therapy and furthermore, analysis of expanding clonotypes after DLI enables identification of individual CDR3 sequences associated with DLI response. Disclosures Heuser: Pfizer: Research Funding; Novartis: Consultancy, Research Funding; BerGenBio: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Tetralogic: Research Funding; Celgene: Honoraria; Bayer Pharma AG: Research Funding.