Impact of Anti-T-lymphocyte globulin dosing on GVHD and Immune reconstitution in matched unrelated myeloablative peripheral blood stem cell transplantation

Data on the influence of different Anti-lymphocyte globulin (ATLG) doses on graft versus host disease (GVHD) incidence and immune reconstitution in matched unrelated (MUD) allogeneic Stem

cell transplantation (allo-SCT) is limited. This retrospective study conducted at the University Medical-Center Hamburg compares GVHD and Immune reconstitution after myeloablative MUD (HLA

10/10) PBSC allogeneic stem cell transplant between 30 mg/Kg (n = 73) and 60 mg/Kg (n = 216) ATLG. Detailed phenotypes of T, B natural killer (NK), natural killer T (NKT) cells were analyzed

by multicolor flow at day 30, 100, and 180 posttransplant. Neutrophil and platelet engraftments were significantly delayed in the 60 mg/kg group with a higher Cumulative incidence of

Infections (67% vs 75% p = 0.049) and EBV (21% vs 41% p = 0.049) reactivation at day 100 in this group. In the 30 mg/kg group, we observed a faster reconstitution of naïve-B cells (p 0.5 × 

109/L. Platelet engraftment was defined as the first consecutive days with a platelet count >20 × 109/L without transfusion support. Acute GVHD was graded according to standard criteria

[10]. Chronic GVHD was graded according to National Institute of Health (NIH) criteria routinely at every visit after transplantation [11]. Infections were defined as any microbial testing

with a positive result and requiring therapy at any time-point after allo-SCT.

As per institution guidelines, blood samples were collected for each patient on days +30, +100, and +180 post-allo-SCT. Routine analyses for absolute concentrations of CD3+, CD4+, CD8+, NK,

and γδ T cells were performed by flow cytometry according to an internal protocol: (1) CD4-APC, CD8-PE, Multitest (CD3 FITC, CD16 + 56 PE, CD45 PerCP, CD19 APC); (2) CD4-APC, CD45-V450,

Multitest (CD45RA FITC, CD45RO PE, CD3 PerCP, CD8 APC); (3) CD45-V450, CD3-PerCPl, anti-TCR-PE, anti-HLA DR-APC in peripheral blood samples. All antibodies were obtained from Becton

Dickinson (BD Biosciences, New Jersey, USA). Up to 5000 events (25,000 per sample) were acquired per tube. Sample acquisition was performed using a BDTM FACS-Canto flow cytometer with the

BDTM FACSDiva software which was also used for data analyses.Immunophenotypes were assessed using four color cytometry using mouse anti-human antibodies for the following cells:

T-lymphocytes (CD3+), activated-T-lymphocytes (CD3+ HLADR+), T-helper (CD3+/CD4+), T-cytotoxic (CD3+/CD8+), B-lymphocytes (CD19+), B-lymphocytes subpopulations (CD19+CD5+ CD1d+)(CD19+ 

CD27+), naïve-B-cells (CD19+ CD27-CD10+), NK-cells (CD56+ CD3-), NKT-cells (CD56+ CD3+), naïve-T-helper (CD4+ CD45RA+), memory-T-helper (CD4+ CD45R0+), naïve-T-cytotoxic (CD8+ CD45RA+),

memory-T-cytotoxic (CD8+ CD45R0+ ), γδT-cells (γδTCR+, CD3+), regulatory-T-cells (CD4+ CD25+ CD127low-neg).

All data was retrospectively collected and was summarized by standard descriptive statistical methods. χ2 test was used to compare categorical variables, whereas continuous variables were

compared using student’s t-test. We defined PFS as survival without relapse or progression of hematological disease; we censored patients without disease or progression at the time of last

follow up. We defined OS and NRM as death from any cause, and without evidence of relapse, respectively. We used the Kaplan-Meier method to calculate the probabilities of DFS and OS; and the

cumulative incidence functions were used to estimate incidence of GVHD, Infections, viral reactivations, RI and NRM. All analysis was performed using SPSS version 26.0 and R version 4.0.5.

A total of 289 consecutive patients were included in the study. Seventy-three patients (25%) received ATLG-30 and 216 Patients (75%) received ATLG-60with a tendency to give lower doses in

more recent years. The median age at transplant was 57 years (range, 18–71) and 50 years (range, 18–74) in the ATLG-30 and ATLG-60 (p = NS), respectively. All patients, donor and transplant

characteristics are listed in Table 1.

Platelet and neutrophil engraftment were significantly delayed in ATLG-60 group when compared to the ATLG-30 group with a median of 11 days (range, 8–23) to neutrophil in the ATLG-30 vs 12

days (range, 8–27) in ATLG-60 group (P = 0.009) (Fig. 1a); and a median of 14 days (range, 9–53) to platelet engraftment in ATLG-30 group vs. 16 days (range, 8–237) in the ATLG-60 group (p =

 0.011).

a Comparison of neutrophil engraftment between 30 mg/kg and 60 mg/kg ATLG. b Incidence of infections after 30 mg/kg and 60 mg/kg ATLG.

We observed no significant differences in incidence of CMV reactivation before day 100 (ATLG-30 43%, ATLG-60 45%). The overall incidence of infection before day 100 was significantly higher

in the ATLG-60 (78%) when compared to the ATLG-30 (67%), p = 0.04 (Fig. 1b). The incidence of EBV reactivation before days 100 in the ATLG-30 group was lower than the ATLG-60 group (21% vs.

41% p = 0.049). IRM at 1-years was 10% in the ATLG-30 vs 11% in the ATLG-60 group (p = 0.7)

The cumulative incidence of aGVHD grade II-IV (47% vs 37%, P = 0.09) and III-IV (19% vs 14%, P = 0.2) were comparable in the ATLG-30 vs ATLG-60 groups, respectively. We observed a higher

incidence of aGVHD grade IV in patients receiving ATLG-30 when compared to the ATLG-60 group (8% vs 0.5% p = 0.0002).

On univariate analysis, we observed no difference in the cumulative incidence of cGVHD all grade was (32% vs 37%, p = 0.47), moderate/severe (14% in both groups, p = 0.48) and grade severe

(5% vs 4%, p = 0.64) were similar between the 30 mg/Kg ATLG and the 60 mg/Kg ATLG, respectively.

The estimated 3-year OS was 55% for patients in the ATLG-30 group and 51% in the ATLG-60 group (p = 0.16) (Fig. 2a). On univariate analyses patients’ age, patient and donor CMV serology,

donor gender and disease status at transplant significantly affected OS. However, on multivariate analyses only Status at Transplant, Recipient CMV serology and Donor Gender were significant

(Table 3).

a comparison of overall survival (OS) between 30 mg/kg and 60mg/kg ATLG. b comparison of disese-free survival (DFS between 30 mg/kg and 60 mg/kg ATLG and c comparison of cumulative incidence

of non-relapse mortality (NRM) between 30 mg/kg and 60 mg/kg ATLG.

The estimated 3-year PFS was 45% for patients in the ATLG-30 group and 54% in the ATLG-60 group (p = 0.18) (Fig. 2b). On univariate analyses, older patients, negative recipient CMV serology,

female donor, SCT chronology>1 and active disease at time of transplant were associated with decreased DFS. All the variables except patient age retained their negative impact on DFS in the

multivariate analysis (Table 3).

The 2-years cumulative incidence of NRM was comparable between the two groups, with 14% vs 12% in the ATLG-30 and ATLG-60 (p = 0.89), respectively (Fig. 3). On Univariate analysis: older

patients, female donor, and negative recipient CMV serology negatively affected NRM. These variables remained significant on multivariate analyses (Table 4).

Comparison of imnune reconstitution between 30 mg/kg and 60 mg/kg ATLG after transplantation regarding (a) gamma-delta T cells (b) naive T cells c Natural Killer (NK) cells and d naive

B-cells.

At day +30, we observed a faster γδTcells reconstitution in the ATLG-30 group (p = 0.045) (Fig. 4a), however the values at day +100 and +180 were comparable between the two groups.

Furthermore, helper naïve T-cells (CD4 + /CD45RA + ) (Fig. 4b) and NK cells reconstitution was faster at day +180 in the ATLG-30 group (Fig. 4c) and reconstitution of naïve B-cells

(CD19+/CD27−/CD10+) was faster at days +30 and +180 in the ATLG-30 group (p