SummarySystemic sclerosis (SSc) is a complex and heterogeneous autoimmune disorder with a multi-factorial pathogenesis. Like other autoimmune disorders, the possible role of specific cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms in predisposing to SSc has been hypothesized, but it remains controversial. CTLA-4 promoter (-318C/T) and exon 1 (+49 A/G) polymorphisms have been analysed in 43 Italian females with SSc and in 93 unrelated matched healthy controls by a newly designed tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method. No significant association has been found with either polymorphisms. Nevertheless, SSc patients without concomitant Hashimoto's thyroiditis (HT) were carrying both the -318T allele (P = 0·031) and the +49 G allele (P = 0·076) more frequently than SSc patients with HT [defined by positivity for anti-thyroperoxidase (TPO) and anti-thyroglobulin (TGA) autoantibodies] than controls. Haplotype analysis confirms this association (P = 0·028), and suggests the predominant role of the -318T, whereas that of the +49 G, if any, seems weak. Thus, in Italian SSc patients the CTLA-4 -318C/T promoter polymorphism appears to be associated with the susceptibility to develop SSc without thyroid involvement. Larger studies are needed to confirm these findings and to clarify whether the -318C/T polymorphism is the functional responsible or whether it reflects the presence of another linked genetic element in the same chromosomal region.

Summary We have previously reported that about 80% of acute myeloid leukaemia (AML) samples tested at diagnosis constitutively expressed cytotoxic T‐lymphocyte‐associated antigen‐4 (CTLA‐4). The present study compared CTLA‐4 expression and function of leukaemic cells from AML patients at diagnosis with those from AML patients resistant to conventional chemotherapy. We also explored the possibility of targeting CTLA‐4 for apoptosis induction in chemoresistant AML cells. AML cells either from untreated patients (n = 15) or in chemoresistant phase (n = 10) were analysed for CTLA‐4 protein and transcript expression by flow cytometry and reverse transcription‐polymerase chain reaction respectively. CTLA‐4 expression was similar in untreated and in chemoresistant samples and was not associated with patients’ clinical features. In chemoresistant AML cells, CTLA‐4 transduced an apoptotic signal on engagement with its recombinant ligands r‐CD80 and r‐CD86, which induced an average of 71% and 62% apoptotic cells, respectively, at highest concentration. Apoptosis was equally induced in untreated leukaemic cells accompanied by cleavage of procaspase‐8 and ‐3. Thus, this study provides the first evidence that killing of leukaemic cells from AML patients may be obtained by the engagement of CTLA‐4 with its ligands, opening the way to a novel potential therapeutic approach based on triggering the CTLA‐4 molecule to circumvent chemoresistance in AML.

Title: CTLA-4 +49A>G polymorphism of recipients of HLA-matched sibling allogeneic stem cell transplantation is associated with survival and relapse incidence polymorphisms. At variance, we found a significant association of donor +49A>G GG genotype with longer overall survival (OS) (log rank P=0.04), and the number of +49A>G G-alleles in the recipient with longer OS (P for trend=0.027), longer disease-free survival (P for trend=0.036) and reduced relapse rate (P for trend=0.042). However, only recipient +49A>G polymorphism was retained as independent prognostic factor in a multivariate analysis, suggesting that the expression of CTLA-4 on the cells of recipient may be relevant for the clinical outcome of HSCT. According to the Reviewer's request, we have indicated in the figure that the p values are from the log rank test. In the figure legend, we have also specified that the log rank test estimates differences in terms of OS or DFS among the 3 genotypes of Recipient considered as separate discrete categories (+49GG, AG, AA). As requested we have included "*log rank p=0.027" and "*log rank p=0.036" at the bottom left of the OS and DFS graphics, respectively, in Figure 1 and the following new sentence: "* the P values, obtained by log rank tests, refer to the differences in terms of OS and DFS across the 3 subgroupss of the discrete variable (recipient genotype)" in the Legend to Figure 1. Point 2In the last paragraph of Results, page 8, line 1, the word "response" has been deleted with the new sentence becoming: "In view of these results and of the known unfavorable effect of acute GvHD on both OS and DFS, we further investigated a possible correlation between acute, as well as chronic GvHD, and the +49A/G or G/G recipient genotype". Point 3Since this statistical analysis was not performed, we agree with the Reviewer that our sentence was too speculative. We thus have deleted the following sentence: "According to this hypothesis, the GvL effect in our series of patients was independent from the severity of GVHD" in the Discussion Page 10, lines 8-9. We believe that we have addressed the minor concerns raised by the Reviewer #1 and hope that the revised manuscript will meet the requirements for publication in Annals of Hematology. We thank you very much for your attention and look forward to hearing from you. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 However, only recipient +49A>G polymorphism was retained as independent prognostic factor in a multivariate analysis, suggesting that the expression of CTLA-4 on the cells of recipient may be relevant for the clinical outcome of HSCT.

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