HighlightsClinical outcomes are excellent using an alemtuzumab-containing hematopoietic stem cell transplantation regimen for aplastic anemia.Outcomes are excellent despite prolonged abnormality of the T cell profile.Recipient-derived CD8 T cells shape persistent mixed chimerism.

Antibodies to the voltage-gated potassium channel (VGKC) complex and glutamic acid decarboxylase (GAD) have been reported in some cases of psychosis. We conducted the first systematic review and meta-analysis to investigate their prevalence in people with psychosis and report a case series of VGKC-complex antibodies in refractory psychosis. Only five studies presenting prevalence rates of VGKC seropositivity in psychosis were identified, in addition to our case series, with an overall prevalence of 1.5% (25/1720) compared to 0.7% in healthy controls (12/1753). Meta-analysis established that the pooled prevalence of GAD65 autoantibodies was 5.8% (95% confidence interval [CI]: 2.0-15.6%; I = 91%; nine studies) in psychotic disorders, with a prevalence of 4.6% (95%CI: 1.2-15.9%; nine studies; I = 89%) and 6.2% (95%CI: 1.2-27.0%; two studies; I = 69%) in schizophrenia and bipolar disorder, respectively. People with psychosis were more likely to have GAD65 antibodies than controls (odds ratio [OR], 2.24; 95%CI: 1.28-3.92%; P = 0.005; eight studies; I = 0%). Among 21 participants with treatment-resistant psychosis, none had VGKC antibodies. The prevalence of VGKC antibodies is low in psychosis. Our preliminary meta-analysis suggests that GAD autoantibodies are more common in people with psychosis than in controls, although few studies accounted for the possibility of co-existing type 1 diabetes mellitus and the clinical significance of reported GAD titers remains unclear. The paucity of studies reporting thresholds for defining GAD abnormality and rates of comorbid type 1 diabetes mellitus precludes interpretations regarding the influence of GAD antibodies on the development of psychotic disorders and may have led to an overestimate of the prevalence of GAD. Our case series fails to support the hypothesis that VGKC antibodies are linked to treatment resistance in psychosis, but the literature to date is remarkably sparse.

We have previously shown that HSCT conditioning regimen for severe aplastic anemia (SAA) using fludarabine, cyclophosphamide and alemtuzumab (FCC), is associated with very low rate of GvHD (especially chronic, cGVHD), and excellent overall survival (OS) of 83% and 95% for matched unrelated (MUD) and matched sibling donor (MSD) HSCT, respectively. Sustained full donor myeloid chimerism occurred most frequently with mixed T-cell chimerism, and this persisted despite discontinuation of post graft immunosuppression with ciclosporin (CSA). We proposed that prolonged mixed T-cell chimerism with alemtuzumab conditioning may facilitate tolerance and hence a low risk of GVHD. We have investigated the basis for the mixed T-cell chimerism in a cohort of 35 patients, median age 32y (range 15-63), transplanted for acquired idiopathic SAA from 2007 - 2014 at King’s College Hospital. FCC conditioning comprised fludarabine 30 mg/m2 iv (day -7 to -4), cyclophosphamide 300 mg/m2 iv (day -7 to -4) and alemtuzumab (median dose 70mg, range 45-100). CSA was used as post graft immune suppression for 9 months, followed by dose tapering to zero over the next 3 months depending on PB chimerism. Stem cell source was BM in 7(20%) patients and 28(80%) PBSC, with a median cell dose of 6.8 (1.9-12.4) CD34+ cells x 106/Kg. Donor type was MSD in 9(26%) pts and UD in 26(74%), of whom 20 were matched (MUD) and 6 were 9/10 match ( received FCC with 2 Gy TBI). Median time to neutrophils >0.5x109/l was 12(10-20) days and to platelets >50x109/l was 10(9-61) days. Primary graft failure occurred in only one patient (2%). Acute GvHD was seen in 6(17%) pts (all grade I-II), and chronic GvHD in 5(14%) pts (3 mild, 1 moderate, 1 severe, all with only skin involvement). On univariate analysis with Cox regression model, factors significantly predicting cGVHD were previously diagnosed acute GVHD (p=0.035) and CD3+ chimerism at day+100 ≥90% (p=0.006). Four patients developed autoimmune anemia post HSCT (2 hemolytic anemia, 2 PRCA). Median follow-up was 30 months (4.3-86.8), OS 94% and event free survival 90%. One year TRM was 6%. Of 32(91%) patients evaluable for chimerism, 8 (23%) were fully donor chimeric for unfractionated BM, PB CD15+ and PB CD3+, while 24 (77%) patients had persistent mixed CD3+ chimerism. Median CD3+ chimerism values were 77% at day+30, 60% at day+60, 41% at day +100,57% at +day180, 70,5% at 1 year, 77% at 2 years and 80% at 3 years. The proportion of evaluable patients no longer taking CSA was 0/23 at 1yr, 9/19 at 1.5yr, 12/16 at 2yr, 12/12 at 2.5yr and 4/4 at 3yr. PB immunophenotyping was performed in 19 patients. During the first year post HSCT, there was profound and prolonged lymphopenia, NK cells recovered first but numbers remained below normal. T cells comprised only 2.6% of lymphocytes at day 30, rising slowly to 41% at day 360 but still significantly below normal (66% in 11 aged-matched healthy individuals), with deficiency greatest for CD4+ T cells. Despite slow recovery of CD4+ T cells, percentage of T-regulatory cells (CD4+ CD25high CD27+ FoxP3+) within this population was normal (range 4.2-7.4% for all time points compared to 5.3% for healthy individuals). Patients aged <50y showed recovery of naïve T cells that expressed markers of recent thymic emigrants (CD31+ CD62L+ CD45RA+), indicating renewed thympoiesis and implementation of central tolerance B cell recovery was notably robust, with numbers comparable to healthy individuals for most patients by day 100. Subset analysis showed absence of memory B-cells but significantly increased proportion of immature transitional (T1 and T2) B cells (CD24+ CD38+, CD27-, IgM high IgD high) for 6 months after FCC HSCT. Cells within this subset are known to possess immune suppressive activity. The mechanism for prolonged mixed T-cell chimerism and associated low incidence of chronic GVHD following FCC conditioning for SAA HSCT may be multifactorial, but we here report novel findings: apart from a markedly low proportion of T cells, we show for the first time in SAA post HSCT the presence of T and B cell subsets with potential regulatory properties and establishment of central tolerance of naïve T-cells in younger patients. Our results also confirm the excellent outcomes of UD HSCT using alemtuzumab with reduced intensity conditioning in SAA, and we propose it is now time to re-think the up-front MUD transplant at all ages in eligible patients. Disclosures No relevant conflicts of interest to declare.

Mixed T-cell chimerism is frequent and persists despite discontinuation of immunosuppression after hematopoietic stem cell transplantation (HSCT) for severe aplastic anemia (SAA) using the fludarabine, cyclophosphamide and alemtuzumab/campath (FCC) conditioning regimen. To investigate the basis for long-term co-existence of donor and patient T cells and remarkably low rates of graft versus host disease (GVHD), the lymphocyte composition and lymphocyte subset chimerism of adult patients transplanted for SAA using FCC was assessed. Patients We have transplanted 42 patients (pts) for acquired idiopathic SAA using FCC conditioning from 2007- March 2015 at King's College Hospital. Median age was 33 years (range 15-63). BM was stem cell source in 7(17%) pts and PBSC in 30 (83%). Median cell dose was 6.85 (1.9-12.4) CD34+ cells x106/Kg. Donor was matched sibling in 11(26%) and unrelated in 31 (74%) pts, 6 of whom also received 2 Gy TBI for 9/10 match. Post graft immunosuppression comprised ciclosporin (CSA) alone. Primary graft failure occurred in 1 pt (2%). Acute GVHD was seen in 6 (14%) pts (all grade I-II), and chronic GVHD in 5 (12%) patients (all skin, only 1 severe in a 9/10 match patient). Factors associated with cGVHD were previous aGVHD (p=0.035) and CD3+ chimerism at day+100 ³ 90% (p=0.006). Median follow-up of survivors was 41 months (4.3-96), with 93% overall survival and 90% event-free survival. At 1, 2 and 3 years post HSCT, 4/30, 20/23 and 16/16 patients, respectively, had discontinued CSA. Results Comparison to 11 healthy age-matched individuals showed prolonged lymphopenia and abnormal proportions of T, B and NK cells. T cells were profoundly deficient comprising only 1.76% of lymphocytes at day 30, rising to 43.8% by day 360 but still significantly below normal (66%, p=0.018). CD8 T cells recovered faster than CD4 T cells producing inversion of the normal CD4 to CD8 T cell ratio. Composition of na•ve, memory and effector subsets within the CD4 T cell population was near normal by day 360 and na•ve CD4 T cells expressed CD31 indicative of renewed thymopoiesis. In contrast, CD8 T-cell subset composition remained abnormal at day 360 due to a high proportion of effector T cells (57.2% of CD8 T cells compared to 9.3% for healthy individuals, p=<0.005). T-cell subsets from five patients were isolated by fluorescence-activated cell sorting, and chimerism determined by analysis of informative alleles from polymorphic short tandem repeat loci. Results shown in the figure below revealed that mixed T-cell chimerism at day 360 was principally due to persistence of patient CD8 T cells, with notable contribution of the effector subset. Follow-up analysis of three patients at >2 years after HSCT showed the mixed chimerism profile within T-cell subsets was stable. A significant correlation was observed between lower donor T-cell chimerism at day 360 and CMV reactivation or EBV viremia early after HSCT (p=0.036), suggesting that antigen-driven expansion of virus-specific patient CD8 effector T cells may substantially contribute to persistent mixed T-cell chimerism. Sustained co-existence of donor and patient T cells and low rates of GVHD indicates conditions favor mutual tolerance. Findings suggested a potential role for cells with immunomodulatory properties. B cell recovery was robust, with normal numbers by day 100. Notably, the B cell population present early after HSCT contained a significantly increased proportion of cells with an immature transitional phenotype (CD24+ CD38+, CD27- IgMhigh IgDhigh 17.1% compared to 2.7% for healthy individuals, p=<0.005) and significantly more B cells produced IL-10 after in vitro stimulation with CD40L (14.6% compared to 6.3% for healthy individuals, p=0.013), consistent with the known immunosuppressive activity of immature B cells. Furthermore, proportions of regulatory T cells (CD4+ CD25high CD27+ FoxP3+) within the CD4 T-cell population were normal (range 4.2-7.4% compared to 5.3% in healthy controls). Conclusions Patient-derived effector CD8 T cells shape mixed T-cell chimerism after FCC conditioning and presence of regulatory B and T cells may contribute to long-term tolerance of mixed T-cell chimerism with associated low incidence of GVHD in the absence of post graft immunosuppression. Note: Marsh JCWand Barber LD joint last authors Figure 1. Figure 1. Disclosures Kulasekararaj: Alexion: Consultancy.