Synergistic effect of KIR ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation from HLA-matched sibling donor for treatment of myeloid malignancies

Human Immunology xxx (2016) xxx–xxx

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Synergistic effect of KIR ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation from HLA-matched sibling donor for treatment of myeloid malignancies Daniela Maira Cardozo a, Amanda Vansan Marangon a, Rodrigo Fernandes da Silva a, Francisco José Penteado Aranha a, Jeane Eliete Laguila Visentainer b, Sandra Helena Alves Bonon a, Sandra Cecília Botelho Costa a, Eliana Cristina Martins Miranda a, Carmino Antonio de Souza a, Fernando Guimarães c,⇑ a b c

Faculdade de Ciências Médicas – FCM, University of Campinas, Campinas, Brazil Laboratório de Imunogenética, Universidade Estadual de Maringá, Maringá, Brazil Hospital da Mulher Prof. Dr. José Aristodemo Pinotti – Centro de Atenção Integral à Saúde da Mulher, University of Campinas, Campinas, Brazil

a r t i c l e

i n f o

Article history: Received 18 September 2015 Revised 5 July 2016 Accepted 6 July 2016 Available online xxxx Keywords: Natural killer cells GVHD Leukaemia Myeloid leukaemia

a b s t r a c t The goal of this study was to evaluate the influence of KIR-HLA genotypes on the outcome of patients undergoing treatment for haematological malignancies by non-T-depleted lymphocyte haematopoietic stem cell transplantation (HSCT) from HLA-matched sibling donors. The prospective study was conducted at the Center of Hematology, University of Campinas, and 50 patients and their donors were followed up from 2008 to 2014. KIR and HLA class I genes were genotyped and patients grouped based on the presence of KIR ligands combined with KIR genotype of their respective donors. Patients with all KIR ligands present (n = 13) had a significantly higher (p = 0.04) incidence of acute graft-versus-host-disease (GVHD) than patients with one or more KIR ligands missing (n = 37). The overall survival following transplantation of patients with myeloid malignancies (n = 27) was significantly higher (p = 0.035) in the group with one or more KIR ligands missing (n = 18) than in the group with all ligands present (n = 9). Presence of KIR2DS2 was associated with a worsening of HSCT outcome while reactivation of cytomegalovirus (CMV) infection improved the outcome of patients with one or more KIR ligands missing. Our results indicate that KIR-HLA interactions affect the outcome of the HLA-matched transplantation, particularly in patients with myeloid malignancies. Ó 2016 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

1. Introduction The lack of one or more HLA class I alleles, whose protein products are the ligands for KIR receptors, has been exploited as a prognostic factor for the outcome of patients with haematological malignancies treated by haematopoietic stem cell transplantation (HSCT). The genes encoding KIR and HLA class I molecules are located on different chromosomes (19q13.4 and 6p21, respectively) and therefore they are segregated independently, enabling

⇑ Corresponding author at: Rua Alexander Fleming 101, 13083-881 Campinas, SP, Brazil. E-mail address: [email protected] (F. Guimarães).

asynchrony among the KIR and HLA class I repertoires of the patients [1,2]. The inhibitory KIR receptors recognise allele groups of the HLA class I molecules. Thus, KIR2DL1 inhibitory receptor recognises the HLA-Cw molecules from group 2 (HLA-C2), which is characterised by the presence of a lysine residue at position 80, while KIR2DL2 and KIR2DL3 recognise the HLA-Cw molecules from group 1 (HLA-C1), which is characterised by the presence of an asparagine residue at position 80 [1,2]. Additionally, polymorphic alterations in the KIR2DL2/3 gene can affect its receptors, producing low affinity to HLA ligands from group 2 [3]. As a result of an HSCT from a donor who has the KIR2DL1 gene to a patient homozygous for HLA alleles of group C1 (C1C1), the resulting NK cells can express the KIR2DL1 inhibitory receptor, which will not find their specific

http://dx.doi.org/10.1016/j.humimm.2016.07.003 0198-8859/Ó 2016 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

Please cite this article in press as: D.M. Cardozo et al., Synergistic effect of KIR ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation from HLA-matched sibling donor for treatment of myeloid malignancies, Hum. Immunol. (2016), http://dx.doi. org/10.1016/j.humimm.2016.07.003

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D.M. Cardozo et al. / Human Immunology xxx (2016) xxx–xxx

ligand, even in the setting of HLA-matched transplantation. Similarly, the HSCT from a donor with the KIR2DL2/3 gene to a patient with HLA alleles of group C2 (C2C2) can give rise to NK cells expressing KIR2DL2/3 molecules, which will not find their specific ligand. Additionally, HLA alleles from the group Bw4 are specific ligands for the KIR3DL1 receptors, and HLA-A⁄03 or A⁄11 are specific ligands for KIR3DL2. The absence of the HLA-Bw4 in patients receiving HSCT from donors with the KIR3DL1 gene can also result in NK cells that will not find their specific ligand [4]. The clinical relevance of HLA-ligand absence in the outcome of HSCT was first observed in the setting of haploidentical transplantation for treatment of patients with acute myeloid leukaemia (AML), which resulted in lowered risk of relapse, improved survival and decreased post-transplant complications such as the development of graft-versus-host disease (GVHD) [5–7]. These results were explained in terms of the generation of potentially alloreactive NK cells following HSCT, i.e., NK cells expressing a given inhibitory KIR receptor for which the respective HLA class I ligand is absent on the patient cells [5,6]. However, due to the process by which NK cells gain functional competence, known as education, it is unlikely that NK cells with the potential to mediate alloreactivity could emerge after HLA-matched HSCT from sibling donors. The education process relies on the expression of KIR receptors on NK cells and their interaction with self-HLA [8]. As a result, possible self-responsive NK cells, i.e., those missing inhibitory KIR receptors, or those which do not meet the respective HLA-ligand, are hypothesised to remain hyporesponsive (anergic) after transplantation, due to the similarities in the HLA genes of the donor and patient [9–11]. Although it has been accepted that KIR-HLA interactions may influence the outcome of the HLA-mismatched HSCT, there is no consensus regarding the settings of HLA-matched transplantation. There are studies that have reported either benefits, or no effects, under the influence of inhibitory KIR-HLA interactions [12–16]. Additionally, certain activating KIRs and/or reactivation of cytomegalovirus (CMV) infection have been reported to affect the outcome of HLA-matched transplantation [17–20]. The goal of this study was to contribute to this discussion by providing data relating to the outcome of a group of Brazilian patients undergoing treatment for haematological malignancies by non-T-depleted lymphocyte HSCT from HLA-matched sibling donors. 2. Subjects and methods 2.1. Subjects Fifty patients undergoing HSCT without depletion of T lymphocytes, together with their HLA-matched (10/10 HLA-A, B, C, DRB1 and DRQ1) sibling donors, were included in this study. The characteristics of patients and therapeutic procedures are presented in Table 1. Patients were assisted at the Center of Hematology, University of Campinas, during the period 2008–2014, and the study was conducted in accordance with ethical guidelines (466/2012 CNS-MS) and with approval (1107/2008) from the Research Ethics Committee of Unicamp. Informed consent was obtained from all subjects in accordance with the Declaration of Helsinki. 2.2. Samples and DNA extraction Blood samples were collected through venipuncture into vacuum tubes containing EDTA (Vacuette, Campinas, Brazil). Genomic DNA was isolated from 200 ll of whole blood using the QIAamp extraction kit (Qiagen, Missisauga, Canada) according to the manufacturer’s instructions.

Table 1 Patient-donors and transplantation characteristics (n = 50). Variable Gender (male/female) – n Patient age (years) – mean/median (range) Donor age (years) – mean/median (range) Transplantation type – n Myeloablative/RIC No Myeloablative Pre-transplantation risk category – n Low risk High risk Conditioning regimen type – n Bu + CY Bu + CY + VP Bu + Fludara Fludara + Melfalan Fludara + TBI TBI + CY + VP Stem cell source – n Bone Marrow Periferal blood stem cell GVHD prophylaxix – n CsA + MTX CsA + MMF Cell CD34+ n/kg, median (range) (106) Months from diagnosis to HSCT – median (range) Platelet days until 20 k/ll mean (range) 45 patients

6 45.5 (20–68) 44 (12–72) 44 6 24 26 22 05 08 08 06 01 13 37 44 06 5.67 (1.14–18.9) 17 (0–61) 19 (10–35)

Low risk: complete remission or chronic phase CML; High risk: partial remission, relapse, progression, accelerate phase for CML; Bu: busulfan; CY: cyclophosphamide; VP: etoposide; GVHD: graft versus host disease; MTX: methotrexate; CsA: cyclosporine; MMF: mycophenolate mofetil; RIC: reduced intensity conditioning; TBI: total body irradiation.

2.3. KIR and HLA class I genotyping The following KIR genes KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, KIR2DP1 and KIR3DP1, and HLA class I alleles HLA-A, -B and -C, were genotyped using polymerase chain reaction with sequence specific oligonucleotide probe (PCR-SSOP) protocols using commercial KIR and HLA genotyping test kits (One Lambda Inc., Canoga Park, CA, USA) according to the manufacturer’s instructions. The amplified DNA was used for hybridisation with beads linked to oligonucleotides specific to the alleles. The hybridisation was evaluated by flow cytometry using a LABScanTM 100 flow analyser (One Lambda Inc.). The data was analysed using the HLA VisualTM software, version2.2.0 (One Lambda Inc.). The sequences of alleles of the HLA genes were analysed one by one in the HLA database (http://www.ebi.ac.uk/imgt/hla/) for the presence of certain amino acids at specific positions and classified into C1, C2, and Bw4 groups.

2.4. KIR and HLA class I profiles Patients were grouped based on the presence of HLA ligands combined with the KIR genotype of their respective donors. Four possible combinations of Bw4 and/or Cw ligands for inhibitory KIRs of the donors were initially considered for comparison: a) Cw heterozygous (C1C2) with Bw4 (all ligands present); b) Cw heterozygous (C1C2) without Bw4 (one ligand missing); c) Cw homozygous (C1C1 or C2C2) with Bw4 (one ligand missing); d) Cw homozygous (C1C1 or C2C2) without Bw4 (two ligands missing). Subsequently, patients were compared based on the patient groups presenting all evaluated KIR ligands or with one or more KIR ligands missing. For the HLA-A ligand, patients were grouped based on the presence of A⁄03 or A⁄11 combined with KIR3DL2 of their respective donors. The impact of certain activating KIR genes

Please cite this article in press as: D.M. Cardozo et al., Synergistic effect of KIR ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation from HLA-matched sibling donor for treatment of myeloid malignancies, Hum. Immunol. (2016), http://dx.doi. org/10.1016/j.humimm.2016.07.003

D.M. Cardozo et al. / Human Immunology xxx (2016) xxx–xxx

was also evaluated, since their presence has been implicated in the transplantation outcome. 2.5. CMV reactivation The antigenaemia (AGM) assay was performed at least once a week from the time of engraftment onwards, as previously described by Van Der Bij et al. [21] and modified by Halwachs et al. [22] and Pannuti et al. [23]. Nested PCR (N-PCR) was used to detect CMV DNA in blood specimens using primers described by Demmler et al. [24] and Shibata et al. [25]. 2.6. Endpoint definitions Patients were considered to have relapsed when the disease reappeared in patients more than 30 days following transplantation. GVHD was diagnosed and graded according to the National Institutes of Health criteria, which includes acute and chronic main categories and their respective subcategories [26]. Overall survival represents the time elapsed from the HSCT until patient death from any cause. Risk categories were defined according to the Center for International and Blood Marrow Transplant Research (CIBMTR) criteria [27]. Active CMV infection was defined based on one or both of the following criteria: one or more positive cells in the AGM assay, and two or more consecutive positive N-PCR results. For the diagnosis of CMV disease, the active infection had to be accompanied by clinical symptoms and histopathological identification of CMV [28]. 2.7. Statistics Survival rates were estimated using the Kaplan-Meier method and compared using the log-rank test. Univariate Cox regression was performed to test the association between variables and survival rates. Cumulative incidence analysis was performed to calculate acute and chronic GVHD probabilities, considering primary disease relapse and early death as competing risks. To compare their curves, the Gray’s test was applied [29]. Chi-square or Fisher’s exact tests were used to establish differences among distributions of categorical variables. The level of significance was p < 0.05. The major statistical analysis was performed using the Statistical Package for the Social Sciences, version 15.0 (IBM-Corp, Armonk, NY, USA) and Stata version 8.3 (Stata Corp LP, College Station, TX, USA) was applied in the calculation of cumulative incidence. 3. Results We performed a prospective analysis of 50 patients with haematological malignancies (Chronic Myeloid Leukaemia, CML n = 7; Acute Myeloid Leukaemia, AML n = 17; Acute Lymphoid Leukaemia, ALL n = 7; Chronic Lymphoid Leukaemia, CLL n = 2; Myelodysplastic Syndrome, MDS n = 3; Hodgkin Lymphoma, HL n = 1; Non-Hodgkin Lymphoma, NHL n = 7; Myelofibrosis n = 3; Hyperplasia, HP n = 3) who had undergone non-depleted T lymphocyte HSCT from HLA-matched sibling donors. Table 1 shows the main characteristics of patients and donors, and transplantation procedures. The overall incidence of GVHD was 58% (29/50 patients), being 41% acute (12/29 patients) and 82% chronic (24/29 patients). Relapses were observed in 30% of the patients (15/50 patients) and 52% of the patients (26/50 patients) were alive at the end of the observation period (80 months).

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3.1. HLA class I distribution and HSCT outcome The analysis of HLA class I profiles showed that HLA-Bw4 was present in 56% (28/50) of the patients and absent in 44% (22/50), and the HLA-A alleles A⁄03 or A⁄11 were present in 28% (14/50) and absent in 72% (36/50). Regarding HLA-Cw, 46% (23/50) of the patients were heterozygous (C1C2) and 54% (27/50) were homozygous (C1C1 or C2C2). No significant differences were observed between Cw homozygous and Cw heterozygous patients in relation to overall survival, GVHD or relapse incidences. Similarly, no significant differences were observed in the frequencies of overall survival, GVHD or relapse, when comparing the presence or absence of HLA-Bw4, or the presence or absence of HLA alleles A⁄03 or A⁄011 (Table 2). A significant association (p = 0.03) was found between the presence of HLA-Bw4 ligands and the occurrence of acute GVHD, however, this result was not confirmed by the cumulative incidence analysis. In addition, HLA class I profiles were characterised by the combination of Cw and Bw4, showing that 26% of the patients expressed all the evaluated ligands (group Cw heterozygous with Bw4, n = 13), 50% had one ligand missing (groups Cw heterozygous without Bw4, n = 10 or Cw homozygous with Bw4, n = 15) and 24% of the patients had two ligands missing (groups Cw homozygous without Bw4, n = 12). All the donors had inhibitory KIR genes (KIR2DL1, KIR2DL2/3 and KIR3DL1) for the evaluated ligands. No significant differences were observed among the four groups in relation to the overall survival at the end of the observation period (80 months, n = 50, p = 0.170). A similar pattern of response, was observed for the overall survival when the same analysis was performed on patients with leukaemia (n = 33, p = 0.246) or on patients with myeloid leukaemia and MDS (n = 27, p = 0.207), extracted from the whole haematological malignancies group (data not shown). Overall survival was further analysed by grouping patients according to those with all KIR ligands present (Cw heterozygous with Bw4, n = 13) and those with one or more ligands missing (n = 37). No significant differences were observed between the two groups when all diagnostic types were analysed together (n = 50, p = 0.151, Fig. 1A) or when patients with leukaemias were extracted from the whole group (n = 33, p = 0.246). However, when patients with myeloid malignancies (AML and MDS, n = 27) were analysed independently, the group with one or more KIR ligands missing (n = 18) had a significantly higher (p = 0.035) overall survival than patients with all ligands present (n = 9) (Fig. 1B). Patients with all KIR ligands present (n = 13) had a significantly higher (p = 0.040) cumulative incidence of acute GVHD than patients with one or more KIR ligands missing (n = 37) (Fig. 2A). The occurrence of acute GVHD was further evaluated by stratifying the patients in relation to the source of stem cell grafts. As a result, patients with all KIR ligands present and receiving grafts from blood (n = 8) had a significantly higher (p = 0.006) cumulative incidence of acute GVHD than those with one or more KIR ligands missing (n = 29) (Fig. 2B). Furthermore, it was observed in patients with chronic GVHD that the time to relapse or death was significantly shorter (p = 0.020) among those with all KIR ligands present (n = 3) than in those with one or more KIR ligands missing (n = 21) (Fig. 3A), whilst in patients without chronic GVHD, such a difference was not observed (Fig. 3B). 3.2. Impact of donor activating KIR and haplotype on HSCT outcome The analysis of KIR profiles showed that 22% (11/50) of the donors were haplotype A and 78% (39/50) were haplotype B. Activating KIRs were distributed as follows: 2DS1 was present in 32% (16/50) and absent in 68% (34/50); 2DS2 was present in 44% (22/50) and absent in 56% (28/50); 2DS3 was present in 34%

Please cite this article in press as: D.M. Cardozo et al., Synergistic effect of KIR ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation from HLA-matched sibling donor for treatment of myeloid malignancies, Hum. Immunol. (2016), http://dx.doi. org/10.1016/j.humimm.2016.07.003

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Table 2 Chi-square or Fisher’s exact test were employed to establish differences among the distributions of categorical variables in different groups based on HLA class I (⁄significant p < 0.05). Alive

Dead

p

With GVHD

Without GVHD

p

With relapse

Without relapse

p

HLA-Cw Homozygous (n = 27) Heterozygous (n = 23)

15 11

12 12

0.39

15 15

12 8

0.34

6 9

21 14

0.16

HLA-Bw4 Present (n = 28) Absent (n = 22)

15 11

13 11

0.51

18 12

10 10

0.16

9 6

19 16

0.47

HLA-A⁄03 or A⁄11 Present (n = 14) Absent (n = 36)

7 19

7 17

0.55

10 20

4 16

0.24

6 9

8 27

0.18

≥ one KIR ligands missing, n=37

p=0.151 KIR ligands present, n=13

B. Myeloid leukemias or MDS (n=27)

≥ one KIR ligands missing, n=18

Overall Survival (%)

Overall Survival (%)

A. Hematological malignances (n=50)

p=0.035

KIR ligands present, n=9

Months

Months

Fig. 1. Overall survival of patients who underwent haematological stem cell transplantation (HSCT) from HLA-matched sibling donors, without depletion of T lymphocytes, for treatment of haematological malignancies. (A) Comparison of patients with all KIR ligands present (n = 13) with patients with one or more KIR ligands missing (n = 37). No significant differences (p = 0.151) were observed between the two groups in relation to the overall survival at the end of the observation period (80 months); (B) Comparison of between patients with all KIR ligands present (n = 9) with and patients with one or more KIR ligands missing (n = 18). A significant difference (p = 0.035) was observed when patients with myeloid malignancies (n = 27) were extracted from the whole set of haematological malignancies. Kaplan-Meier plots with p values obtained from logrank tests for difference.

B. Gra source: blood

A. Gra source: bone marrow and blood

p=0.04

≥ one KIR ligands missing, n=37

Days

acute GVHD

KIR ligands present, n=13

Cumulave incidence of

acute GVHD

Cumulave incidence of

p=0.04

KIR ligands present, n=8

p=0.006

≥ one KIR ligands missing, n=29

Days

Fig. 2. Cumulative incidence of acute graft-versus-host-disease (GVHD) in patients who underwent haematological stem cell transplantation (HSCT) from HLA-matched sibling donors, without depletion of T lymphocytes, for treatment of haematological malignancies. (A) Comparison of patients with all KIR ligands present (n = 13) with patients with one or more KIR ligands missing (n = 37). Patients with all KIR ligands present had a significantly higher (p = 0.04) cumulative incidence of acute GVHD than patients with one or more KIR ligands missing. (B) Comparison of patients with all KIR ligands present (n = 8) with patients with one or more KIR ligands missing (n = 29). Patients with all KIR ligands present had a significantly higher (p = 0.006) cumulative incidence of acute GVHD than patients with one or more KIR ligands missing when patients treated with stem cell grafts from blood (n = 37) were extracted from the whole group. Curves compared using the Gray’s test.

(17/50) and absent in 66% (33/50); 2DS4 was present in 96% (48/50) and absent in 4% (2/50); 2DS5 was present in 28% (14/50) and absent in 72% (36/50); and 3DS1 was present in 30% (15/50) and absent in 70% (35/50). The donor’s haplotype (A or B) did not significantly affect the HSCT outcome in relation to over-

all survival, GVHD or relapse incidence. Similarly, no significant differences were observed between patients who received a graft from donors with, or without, activating KIRs 2DS1, 2DS3, 2DS4, 2DS5 or 3DS1. However, the presence of KIR2DS2 in the donor was significantly associated with the worsening of overall survival

Please cite this article in press as: D.M. Cardozo et al., Synergistic effect of KIR ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation from HLA-matched sibling donor for treatment of myeloid malignancies, Hum. Immunol. (2016), http://dx.doi. org/10.1016/j.humimm.2016.07.003

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B. Without cGVHD (n=26)

KIR ligands present, n=3

p=0.02 ≥ one KIR ligands missing, n=21

Probability of relapse or death caused by disease progression

Probability of relapse or death caused by disease progression

A. With cGVHD (n=24)

KIR ligands present, n=10

p=0.97 ≥ one KIR ligands missing, n=16

Months

Months

Fig. 3. Probability of relapse or death caused by disease progression of patients who underwent haematological stem cell transplantation (HSCT) from HLA-matched sibling donors, without depletion of T lymphocytes, for treatment of haematological malignancies. (A) Patients without chronic graft-versus-host-disease (cGVHD) (n = 26) were compared with those with all KIR ligands present (n = 10) and those with one or more KIR ligands missing (n = 16). (B) Patients with cGVHD (n = 24) were compared with those with all KIR ligands present (n = 3) and those with one or more KIR ligands missing (n = 21). A significantly (p = 0.02) higher probability of relapse or death caused by disease progression was observed in patients with cGVHD and presenting all KIR ligands. Curves compared using Gray’s test.

(p = 0.026) (Table 3) and event-free survival (p = 0.017) after HSCT (Fig. 4). The association of 2DS2 gene with risk of death was confirmed using univariate Cox regression (Hazard Risk = 2.45, 95% CI: 1.08–5.56 and p = 0.03). 3.3. CMV reactivation and HSCT outcome The pre-transplant analysis for immunity against CMV showed that 47 donor/patient pairs were IgG positive/positive, respectively, 2 were negative/positive, respectively, and 1 was negative/ negative, respectively. After transplantation, reactivation of CMV occurred in 66% (33/50) of the patients, which was significantly beneficial for the overall survival (p = 0.015) and event free survival (p = 0.001, Fig. 5A). The impact of CMV on HSCT outcome was further evaluated by extracting the patients with myeloid malignancies (AML, CML and MDS) from the reactivation group (n = 18). Interestingly, among patients with reactivation of CMV infection, those with one or more KIR ligands missing (n = 12) had a higher overall survival (p = 0.085) and event free survival (p = 0.013, Fig. 5B) compared with patients with all ligands present (n = 6).

4. Discussion Several studies have investigated the influence of KIR-HLA class I genotypes on the outcome of HLA-matched stem cell transplantation for the treatment of haematological malignancies. The data currently available seem controversial, possibly a consequence of the complexity of the biological interactions of the KIR-HLA genes combined with the variety of diagnoses and protocols for transplantation. Here we provided data obtained over six years of observation of patients undergoing non-T-depleted lymphocyte HSCT from HLA-matched sibling donors, which support the influence of missing KIR ligand on the outcome of transplantation. Seventy-four percent of the patient-donor pairs included in this study had one or more KIR ligands missing, an amount that is within the known range, of 60–85% for matched transplantations [13,14,30,31]. The lack in the patients of HLA-B or -C, which are ligands for the inhibitory KIR receptor present in their donors, was initially associated with an improved outcome of bone marrow HSCT from matched sibling donors in the treatment of AML [30]. Similar results were found by Sobecks et al. [32], who also showed that HLA-C heterozygous patients had worse survival than

Table 3 Chi-square or Fisher’s exact test were employed to establish differences among the distributions of categorical variables in different groups based on the presence or absence of activating KIR genes (⁄significant p < 0.05). aKIR

Alive (n = 26)

Dead (n = 24)

p

With GVHD (n = 30)

Without GVHD (n = 20)

p

2DS1 Present Absent

8 18

8 16

0.543

7 23

9 11

0.09

2DS2 Present Absent

8 18

14 10

0.046⁄

11 19

11 9

2DS3 Present Absent

7 19

10 14

0.21

9 21

2DS4 Present Absent

26 0

22 2

0.22

2DS5 Present Absent

6 20

8 16

3DS1 Present Absent

7 19

8 16

With relapse (n = 15)

Without relapse (n = 35)

p

7 8

9 26

0.13

0.16

9 6

13 22

0.11

8 12

0.33

6 9

11 24

0.39

29 1

19 1

0.64

15 0

33 2

0.48

0.31

7 23

7 13

0.28

4 11

10 25

0.59

0.42

6 24

9 11

0.06

7 8

8 27

0.09

Please cite this article in press as: D.M. Cardozo et al., Synergistic effect of KIR ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation from HLA-matched sibling donor for treatment of myeloid malignancies, Hum. Immunol. (2016), http://dx.doi. org/10.1016/j.humimm.2016.07.003

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KIR2DS2 / 2DL2 absence, n= 28

p=0.026 KIR2DS2 / 2DL2 presence, n= 22

B. Hematological malignances (n=50)

Event-free Survival (%)

Overall Survival (%)

A. Hematological malignances (n=50)

Months

KIR2DS2 / 2DL2 absence, n= 28

p=0.017

KIR2DS2 / 2DL2 presence, n= 22

Months

Fig. 4. Overall survival and event-free-survival of patients who underwent haematological stem cell transplantation (HSCT) from HLA-matched sibling donors, without depletion of T lymphocytes, for treatment of haematological malignancies. Significant differences were observed in (A) Overall survival (p = 0.026) and (B) Event-free survival (p = 0.017) between patients who received grafts from donors with (n = 22) or without (n = 28) KIR2DS2/2DL2 genes. Kaplan-Meier plots with p values obtained from log-rank tests for difference.

yes, n=33

p=0.001

B. Myeloid leukemia or MDS reacvated CMV (n=18)

Event-free Survival (%)

Event-free Survival (%)

A. Hematological malignances (n=50)

≥ one KIR ligands missing, n=12

p=0.013

no, n=17 KIR ligands present, n=6

Months

Months

Fig. 5. Event-free survival of patients who underwent haematological stem cell transplantation (HSCT) from HLA-matched sibling donors, without depletion of T lymphocytes, for treatment of haematological malignancies. (A) Significant differences (p = 0.001) were observed between patients with (n = 33) and without (n = 17) reactivation of CMV infection at the end of the observation period (80 months). (B) Significant differences (p = 0.013) were observed between patients with one or more KIR ligands missing (n = 12) and patients with all KIR ligands present (n = 6), when subjects with myeloid malignancies and MDS (n = 18) were extracted from the group of patients with reactivation of CMV infection. Kaplan-Meier plots with p values obtained from log-rank tests for difference.

those homozygous for C1 or C2. Subsequently, the benefits of missing KIR ligands on the outcome of matched transplantations were extended to transplant settings using T-cell depleted stem cell grafts from peripheral blood [12], and additionally, this same study showed that greater amounts of NK cells in the grafts could produce better outcomes. Thus, the benefits of missing KIR ligands on the outcome of matched transplantation seemed to be restricted to the T-cell depleted grafts, as pointed out by Clausen et al. [33]. Correspondingly, three other studies on non-manipulated grafts (with Tcells) have failed to reproduce the overall survival-increasing effect of missing KIR ligands [13–15] previously shown in T-depleted grafts. However, two of them have established an association between the missing KIR ligand and other post-transplant complications, such as the development of GVHD [13,15]. In contrast, Wang et al. [16] observed a significantly lower incidence of chronic GVHD, and higher disease-free and overall survival, as an effect of the missing KIR ligand, in a two-year follow-up study of patients receiving non-manipulated HSCT from matched-sibling donors for treatment of myeloid malignancies. Similarly to other studies [16,30,34], our results showed that missing KIR ligand can distinctively affect the outcome of HSCT

in patients with myeloid malignancies, since overall survival was significantly higher (p = 0.035) in patients lacking one or more KIR ligands than in those presenting all ligands. Notwithstanding, our result was observed in the setting of non-manipulated HSCT, in contrast to the aforementioned studies [13–15,33]. Additionally, the absence or presence of KIR ligands was also associated with differences in development of post-transplant complications. Firstly, the incidence of acute GVHD was significantly lower in patients with one or more KIR ligands missing than in those with all ligands present. A previous study involving a specific Chinese population that underwent HSCT from matched sibling donors also associated the decreased incidence of acute GVHD with the lack of KIR ligands [13]. However, two other studies involving HSCT from unrelated matched donors observed the opposite, i.e., they associated the increased incidence of acute GVHD with the lack of Bw4 [35] or C2 [36] ligands. Secondly, our results showed a beneficial association between the absence of KIR ligands and chronic GVHD, since patients with this combination had a significant reduction in the probability of relapse or death caused by disease progression. The benefits attributed to the missing KIR ligand, favouring the outcome of HSCT, are frequently explained in terms of the alloreactivity of NK cells [4,37,38]. NK cell alloreactivity has been well

Please cite this article in press as: D.M. Cardozo et al., Synergistic effect of KIR ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation from HLA-matched sibling donor for treatment of myeloid malignancies, Hum. Immunol. (2016), http://dx.doi. org/10.1016/j.humimm.2016.07.003

D.M. Cardozo et al. / Human Immunology xxx (2016) xxx–xxx

demonstrated in the settings of haploidentical transplantation, particularly when T cell depleted grafts with high numbers of stem cells were used for the treatment of AML in adults, and ALL in children [39]. However, the possibility of alloreactive NK cells emerging in the setting of non-T-depleted lymphocyte HSCT from HLAmatched sibling donors is uncertain. Studies investigating this issue have reported either a temporary break of tolerance [34], or maintenance of NK hyporesponsiveness [31] throughout NK cell repopulation after transplantation. Nevertheless, it has been suggested that NK cell responsiveness could be modulated based on the number and type of HLA ligands expressed on patient cells, as well as by the expression of certain activating receptors, which could benefit the outcome in matched transplantation [39–41]. In this regard, grafts with activating KIRs 2DS1, 3DS1 and 2DS5 were associated with improvement in disease-free survival of HLA-Cw heterozygous patients with CML [17]. Additionally, the higher number of activating KIRs, provided by donors with haplotype B was associated with a better (graftversus-leukaemia) GvL response and a reduction in relapse rates when compared with grafts from donors with haplotype A [4,42]. Finally, Impola et al. [43] associated the presence of KIRs 2DL2/2DS2 in grafts from haplotype B donors with improvement in disease free-survival of patients with AML. In contrast, in our study, the outcome of HSCT was not affected by the origin of the grafts, either coming from donors with haplotype A or B. In addition, we found that the absence of the activating KIR2DS2 favours overall and disease-free survival of patients. It is noteworthy that, in our cohort, the absence or presence, respectively, of KIR2DS2 was associated with the absence or presence, respectively, of KIR2DL2. Another factor that has been hypothesised to modulate NK cell responsiveness in the post-transplant period is the reactivation of CMV infection. Recent studies have implicated the reactivation of CMV infection in the outcome of HSCT, mainly decreasing relapse rates of patients with AML [18–20]. Similarly to the aforementioned studies, our results showed that reactivation of CMV infection had a favourable effect, reducing relapse rates in patients treated for haematological malignancies by non-T-depleted lymphocyte HSCT from HLA-matched sibling donors. Additionally, they also support the breaking of tolerance of self-responsive NK cells as a possible mechanism implicated in the improvement of disease-free survival of patients with myeloid malignancies (Fig. 5A and B). In this group, only patients with one or more KIR ligands missing benefited from reactivation of CMV infection, while patients with all KIR ligands present did not. Since the study was conducted in a single center, the number of subjects was limited. The cases were included sequentially based on the employment of non-depleted T lymphocyte grafts taken from HLA-matched sibling donors. This procedure resulted in heterogeneity of diagnoses, a characteristic frequently observed in similar studies. To overcome this, we employed appropriate statistical analysis, and whenever possible, specific data were extracted from the whole group of patients. On the one hand, the use of non-depleted T lymphocyte grafts in our transplantation protocol may favour an undesirable effect, such as the occurrence of GVHD [34]. However, on the other hand, the presence of T lymphocytes may elicit a GvL effect, which is desirable for the elimination of malignant cells [44]. Taken together, our results suggest a possible contribution of NK cells to the modulation of chronic GVHD and the promotion of GvL, resulting in a reduced probability of relapse or death in the patients with missing KIR ligands. In conclusion, our results indicate that KIR-HLA interactions can affect the outcome of HLA-matched transplantation, particularly in patients with myeloid malignancies.

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Please cite this article in press as: D.M. Cardozo et al., Synergistic effect of KIR ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation from HLA-matched sibling donor for treatment of myeloid malignancies, Hum. Immunol. (2016), http://dx.doi. org/10.1016/j.humimm.2016.07.003