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Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.

The Recombination Activating Genes, RAG1 and RAG2, are essential for V(D)J recombination and adaptive immunity. Mutations in these genes often cause immunodeficiency, the severity of which reflects the importance of the altered residue or residues during recombination. Here, we describe a novel RAG1 mutation that causes immunodeficiency in an unexpected way: The mutated protein severely disrupts binding of the accessory protein, HMGB1. Although HMGB1 enhances RAG cutting in vitro, its role in vivo was controversial. We show here that reduced HMGB1 binding by the mutant protein dramatically reduces RAG cutting in vitro and almost completely eliminates recombination in vivo. The RAG1 mutation, R401W, places a bulky tryptophan opposite the binding site for HMG Box A at both 12- and 23-spacer recombination signal sequences, disrupting stable binding of HMGB1. Replacement of R401W with leucine and then lysine progressively restores HMGB1 binding, correlating with increased RAG cutting and recombination in vivo. We show further that knockdown of HMGB1 significantly reduces recombination by wild-type RAG1, whereas its re-addition restores recombination with wild-type, but not the mutant, RAG1 protein. Together, these data provide compelling evidence that HMGB1 plays a critical role during V(D)J recombination in vivo.

Background: Immune reconstitution after AlloSCT is a highly complex process influenced by both graft & recipient-related factors including post-graft therapies. Delayed T-cell reconstitution remains a source of morbidity & mortality and may contribute to limited anti-tumour effect. We sought to evaluate the immune reconstitution kinetics in the setting of post-graft Lenalidomide (Revlimid®) to augment the graft-versus-myeloma effect, by defining key immune biomarkers, in the NCRI LenaRIC phase II trial. Material & Methods: All patients recruited into the study underwent a reduced intensity conditioned (RIC) AlloSCT using Fludarabine & 2Gy TBI with ATG (Fresenius) as in vivo T-cell depletion, following a high dose melphalan ASCT (planned tandem SCT programme). Patients were scheduled to receive Lenalidomide (5mg-10mg/day) on day+35 if stable donor engraftment in the absence of GvHD. Lenalidomide discontinuation was planned at 12 months post-transplant, with donor lymphocyte infusions (DLI) given to patients with evidence of residual disease, relapse or mixed chimerism. Sequential peripheral blood samples were drawn at predetermined time frames before (baseline) and after graft infusion (D+30,+60,+90,+180,+270 & D+360) in all trial registrants. Using multi-parameter flow cytometry (MFC), a comprehensive panel of immune subset & functional/activation markers were used to define characteristics of T-cell, B-cell, regulatory T-cell & NK cells. The inflammatory proteome (IL-1b, IL-4, IL-5, IL-6, IL-10, IFNg, TNFa, CXCL8 & GM-CSF) and soluble immune checkpoints (PD-1 & ICOS) were analyzed using LUMINEX assays on serial serum samples at parallel points. PB cell subset-specific Complete Donor Chimerism (CDC) was analysed by single tandem repeat PCR at day (D)+100,+180,+270 and D+365. Results: From January 2011 to May 2015, 40 patients with myeloma ≥VGPR after 1st or 2nd ASCT, were recruited into the LenaRIC protocol. The median age was 49 years (range 35-65) with PB as the graft source (Matched Related Donor n=16, 10/10 Matched Unrelated Donor n=23; 1 patient not transplanted). Viable samples were received from 37/40 patients (92.5%), with no cycles of Lenalidomide received by 3/37 (8.1%) patients, 1-6 cycles received by 13/37 (35.1%) patients & >6 cycles received by 21/37 (56.8%) patients. 5 patients completed 12 cycles as per protocol treatment; of those completing >= 10 cycles, 5/16 received DLI. Only 10/37 (27%) of patients achieved a CD4+ cell count >200/µl in a median of 180 days compared with 30/37 (81%) of patients who achieved a CD8+ cell count >200/µl in a median of 60 days. In patients who did not receive any doses of Lenalidomide, higher levels of CD8+ cells (p=0.013) but lower NK cell levels (p=0.071) were evident at D+30 compared with those eligible to proceed with Lenalidomide. No difference was noted between Lenalidomide exposure subgroups in relation to CD8+ or CD4+ cell recovery. Treg cells were increased at D=270 & D+365 in those who received >6 cycles of Lenalidomide, though this corresponded with a higher level of HLA-DR expression on CD8+ T cells representing global immune activation. Significant differences between Lenalidomide exposure subgroups were evident in the inflammatory proteome readout at D+90. CXCL8 levels peaked at D+270, especially in those who received >6 cycles of Lenalidomide. Of 30 evaluable patients, T-cell CDC was seen in 63.3% at D100, with 84% of evaluable patients having CDC at D365. No difference between subgroups was evident in relation to rapidity of CDC and stability of CDC. Conclusions: The sequential monitoring of immune biomarkers in the setting of post-graft Lenalidomide following a T-deplete RIC-AlloSCT highlights the rapid and sustained quantitative and functional reconstitution of donor immune homeostasis. The clinical significance of these findings requires correlation with both the anti-tumour immune effect and theimmunopathy ofAlloSCT (GvHD). Unrestricted educational grants were provided by Cancer Research UK and byCelgene.Lenalidomideprovided free of charge byCelgene. The support and time of participating patients and their families is gratefully acknowledged Figure 1 a) Changes in circulating CD8+ T-cells according to Lenalidomide exposure following RIC-Allo SCT for myeloma b) Changes in circulating CD4+CD25+FoxP3+ T-Reg cells according to Lenalidomide exposure following RIC-Allo SCT for Myeloma Figure 1. a) Changes in circulating CD8+ T-cells according to Lenalidomide exposure following RIC-Allo SCT for myeloma. / b) Changes in circulating CD4+CD25+FoxP3+ T-Reg cells according to Lenalidomide exposure following RIC-Allo SCT for Myeloma Disclosures Cook: Glycomimetics: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Brock:AstraZeneca: Equity Ownership; GlaxoSmithKline: Equity Ownership. Cavenagh:Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Cook:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria; Cancer Research UK: Research Funding; Takeda: Honoraria.

Abstract 2228 Poster Board II-205 B lymphocyte homeostasis may have a role in regulating the immune response, both stimulatory & regulatory/tolerogenic, in Allogeneic Stem Cell Transplantation (AlloSCT). The predictive relationship of donor B-cell reconstitution with relapse risk, chronic Graft versus Host Disease (cGvHD) & overall survival (OS) was investigated. We performed prospective comprehensive immune reconstitution studies in 71 patients undergoing AlloSCT (Matched Related Donor=33, Matched Unrelated Donor =34, Umbilical Cord Blood =3, Haplo-identical Related Donor =1) for haematological malignancy & marrow failure syndromes who were available for follow-up beyond Day+100. 27 patients received full intensity conditioning & 44 reduced intensity conditioning with 34 patients receiving Alemtuzumab & 8 patients receiving ATG as in vivo T-cell depletion with a median PAM score of 22 (range 8-36). The grafts were bone marrow in 23, peripheral blood stem cells in 40 and umbilical cord blood in 3 with a median CD34+cell dose of 4.3×106/kg (range 0.8-10.5) & median duration of immune-suppression of 7.1 months (range 2, 49). Samples were analysed for lymphocyte subsets by multi-parameter flow cytometry & cell subset-specific Complete Donor Chimerism (CDC) by single tandem repeat PCR at day (D)+100, D+180, D+270 and D+365. The proportion of patients with a CD4+, CD8+, CD19+ & NK cell count within the normal range at D+100 & D+180 were: 10% & 27% for CD4+, 28% & 57% for CD8+, 24% & 57% for B-cells, 68% & 83% for NK cells, respectively. The proportion of patients with a CD4+, CD8+, CD19+ & NK cell CDC at D+100 & D+180 were: 80% & 78% for CD4+, 86% & 80% for CD8+, 89% & 91% for B-cells, respectively. 28 patients (38%) experienced cGvHD (22/28 extensive) with a median time to onset of 4 months (range 3-12) & median duration of immune suppression of 14.5 months (range 6.9-48.3). In univariate analysis, only the B-cell CDC at D+100 was associated with a higher incidence of cGvHD (mean 99.8%±0.2% vs. 90.4%±4.4 CDC, respectively; p=0.044) though a trend was seen in regard to D+180 B-cell CDC. No influence of mature/immature B-cell reconstitution at D+100 or D+180 was detected. 24 patients (34%) have relapsed at a median of 5.8 months (range 3-34). A trend to lower absolute B-cell counts at D+100 & D+180 was seen in those who relapsed with significantly lower B-cell CDC at D+100 and D+180 in those who relapsed compared to those who did not (D+100: mean 87.9%±2.4% vs. 98.3%±1.5 CDC, respectively; p=0.038; D+180: mean 80±5.6 vs. 100%±0 CDC, respectively; p=0.013). With a median follow-up of 16.7 months (range 5.1-51.8), 27 (38%) have died (disease progression n=12, infection n=4, GvHD-related n=6). In univariate analysis, no effect on OS was demonstrated by the absolute B-cell count or B-cell CDC at D+100 or D+180, though a trend was seen to lower levels in those who did not survive. In conclusion, delayed donor B-cell reconstitution was associated with a higher relapse risk. On the other hand, cGvHD was associated with higher early donor B-cell reconstitution, which suggests that residual recipient B-cells after AlloSCT may protect from cGvHD. This data provides further clinical evidence for a role of B-cells in the immune-modulation associated with the graft-versus-tumour/graft-versus-host disease paradigm of AlloSCT. Disclosures: No relevant conflicts of interest to declare.