Impact of anti-t-lymphocyte globulin dosing on gvhd and immune reconstitution in matched unrelated myeloablative peripheral blood stem cell transplantation

ABSTRACT 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_ < 0.0001) and γδ T cells (_p_ = 0.045) at day+30 and a faster naïve helper T-cell (_p_ = 0.046), NK-cells (_p_ = 0.035), and naïve B-cell reconstitution (_p_ = 

0.009) at day+180. There were no significant differences in aGVHD, cGVHD, NRM, RI, PFS, and OS between the groups. The choice of ATLG dose has significant impact on IR but not on GVHD after

MUD-allo-SCT. Higher doses are associated with delayed engraftment and increased infections. SIMILAR CONTENT BEING VIEWED BY OTHERS IMMUNOLOGICAL RECONSTITUTION AND INFECTIONS AFTER ALLOHCT

- A COMPARISON BETWEEN POST-TRANSPLANTATION CYCLOPHOSPHAMIDE, ATLG AND NON-ATLG BASED GVHD PROPHYLAXIS Article Open access 19 November 2024 LONG-TERM RESULTS AND GVHD AFTER PROPHYLACTIC AND

PREEMPTIVE DONOR LYMPHOCYTE INFUSION AFTER ALLOGENEIC STEM CELL TRANSPLANTATION FOR ACUTE LEUKEMIA Article Open access 08 November 2021 THE ROLE OF ANTI-THYMOCYTE GLOBULIN IN ALLOGENEIC STEM

CELL TRANSPLANTATION (HSCT) FROM HLA-MATCHED UNRELATED DONORS (MUD) FOR SECONDARY AML IN REMISSION: A STUDY FROM THE ALWP /EBMT Article 02 September 2023 INTRODUCTION Due to the graft

versus tumor effect, allogeneic stem cell transplantation (allo-SCT) is a potentially curative treatment strategy for hematological diseases [1, 2]. However its benefits may be offset by

increased non related mortality (NRM), mainly due graft-versus-host-disease (GVHD) and infections [3]. The use of an unrelated donor and peripheral blood stem cells (PBSC) is associated with

an increased risk of GVHD. Randomized studies have shown that pretransplant anti-T-Lymphocyte globulin (ATLG) can prevent severe acute and chronic GVHD [4,5,6]. Although lower ATLG doses

may compromise the immunosuppression effects, higher ATLG doses may offset its benefit by decreasing antiviral and graft-versus-malignancy effect by depletion of donor effector T cells [7].

Even though the use of ATLG in allo-SCT is well established, however, data on optimal ATLG dosing in the setting of MUD-PBSC is scarce, only one study has compared different ATLG doses in

this setting [8]. In the present study, we aim to compare the IR kinetics and transplant outcomes between recipients of 30 mg/Kg vs 60 mg/Kg ATLG as TCD strategies undergoing

MUD-PBSC-allo-SCT with myeloablative conditioning (MAC). MATERIALS AND METHODS This retrospective study conducted at University Medical Center Hamburg-Eppendorf (UKE) with a primary goal to

compare IR between 30 mg/Kg (ATLG-30) vs. 60 mg/kg (ATLG-60) ATLG in recipients of MAC PBSC allo-SCT. The choice of ATLG dose was according to treating physician preference. Secondary

outcomes included incidence of viral reactivations, engraftment, rate of infections and Infection related mortality (IRM), aGVHD, cGVHD, non relapse mortality (NRM), progression free

survival (PFS), and overall survival (OS). All patients signed written informed consents for treatment and the study was approved by the institutional review board at UKE. To have comparable

groups, we selected only patients receiving MAC with PBSC as stem cell source, and MUD (HLA 10/10). MAC regimens were defined according to published working group definition [9]. ATLG

(Grafalon®, Neovii, Switzerland) was given at a dose of 30 mg/kg or 60 mg/kg. A test dose of 200 mg was given at day −4 and the remaining ATLG doses were fractionated between days −3 to −1.

Similar supportive care was used for all patients per institutional guidelines including antimicrobial prophylaxis consisting of fluoroquinolone for bacterial infections,

trimethoprim-sulfamethoxazole or pentamidine for _Pnemocystis jiroveci_, micafungin for fungal infections and acyclovir for viral infections. Patients were screened weekly for CMV and EBV by

blood PCR. Neutrophil engraftment was defined as the first 3 consecutive days with a measure of absolute neutrophil count >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). STATISTICAL METHODS 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. RESULTS PATIENTS AND

TRANSPLANT CHARACTERISTICS 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. TRANSPLANT OUTCOMES All transplant outcomes are summarized in Table 2. ENGRAFTMENT 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).

INFECTIONS CMV AND EBV REACTIVATION 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) GRAFT-VERSUS-HOST DISEASE 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. OVERALL SURVIVAL 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). PROGRESSION FREE SURVIVAL 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). NON-RELAPSE MORTALITY 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). IMMUNE RECONSTITUTION 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_ < 0.0001) (Fig. 2d). All our data is summarized in Supplementary Table 1. DISCUSSION Although ATLG is recommended for GVHD prevention in allo-SCT [12], data on optimal ATLG dosing in

the setting of MUD-PBSC is still lacking. Recently a consensus-based recommendation by an international expert panel recommended the use of 30 mg/Kg and 60 mg/Kg of ATLG for sibling and

unrelated MAC allo-SCT, respectively [13]. A retrospective study conducted in 2003 compared two doses of ATLG (<60 mg/Kg vs 60 mg/Kg) in CML Patients undergoing MUD allo-SCT reports

improved OS and DFS in patients receiving >60 mg/Kg ATLG. These differences were attributed to the higher incidence of severe aGVHD in the lower ATLG dose group. A number of 31 patients

received a lower ATLG Dose with 58% receiving 20 mg/Kg ATLG and 36% receiving 40 mg/Kg ATLG [14]. Our findings fall in line with the study, as we reported a higher incidence of severe aGVHD

on univariate and multivariate analysis in the lower ATLG dosing, however this did not impact any of the other outcomes. The discrepancy can be explained by the lower ATLG Doses in this

study and improved supportive care in our population. A study published from our center compared 30 mg/Kg ATLG with 60 mg/Kg ATLG in the MUD-allo-SCT setting between 1997–2005. ATLG was

administered at a dose of 30 mg/kg on day –1 in the 30 mg/Kg group or 20 mg/kg/day on days –3 to –1 in the 60 mg/Kg group. This study reported a higher NRM in the ATLG-60 group, mainly due

to increased incidence of infections in this group with no differences in incidences of aGVHD, cGVHD and Relapse [8]. Our results fall in line with the present study. We observed a higher

incidence of infections in the ATLG-60 group, however, due to improved supportive care this had no impact on NRM. In a multicenter, randomized, open label phase 3 Trial Locatelli et al.

assessed the impact of two doses of ATLG (15 mg/Kg vs 30 mg/Kg over 3 days, from day −4 to −2) in children undergoing MUD-allo-SCT with MAC. They reported lower NRM, higher event free

survival and OS in the lower ATLG dose group. In addition, higher doses of ATLG were associated with increased EBV and Adenovirus reactivation and increased IRM. They observed no significant

differences in incidences of aGVHD and cGVHD between the two groups. Our results fall in line with Locatelli et al. In our we observed a higher incidence of EBV reactivation in the lower

ATLG dose group, however, no differences in any of the long-term outcomes [15]. Butera et al. retrospectively evaluated the impact of two different anti-thymocyte globulin dose

(Thymoglobulin, 5 mg/Kg vs 6.5 mg/Kg) in adults undergoing MUD-allo-SCT at three Italian centers [16]. They report an increased Infection related mortality in the higher ATG dose group, and

no significant differences in any of the other outcomes. In our study, we observed a higher incidence of Infection, however no differences in Infection related mortality between the two

groups. The differences in our outcomes can be explained in terms of the discrepancies in transplant characteristics. Butera et al. included both MAC and RIC, Bone Marrow and PBSC grafts and

they reported a higher proportion of patients with 1HLA mismatch in the higher ATG dose group; while in our population Contrary in the present study, patients were more homogeneous, they

all received MAC, PBSC with 10/10 HLA matched grafts. In addition, in contrast to ATLG Thymoglobulin may contain not only antibodies specific against mature T cell antigens but also antigens

on the thymus-specific cells, thus directly impairing Thymic T cell regeneration and deepening the immunosuppression [17], thus explaining the increased IRM in patients receiving ATG in

contrast to ATLG. Gooptu et al. investigated the effect of ATLG on IR in MUD-PBSCT (_n_ = 44 ATLG, _n_ = 47 placebo) [18]. They reported improved CD3+, CD4+, CD8+, and Treg Lymphocytes in

the Placebo group. They additionally reported delayed CD3+ and CD4+ Lymphocytes reconstitution up to 6 months. Our study falls in line with these findings, the CD3+ and CD4+ Lymphocytes did

not normalize at last follow up. Bacigalupo et al. compared in two randomized trials two different doses of thymoglobuline (15 mg/kg vs 7.5 mg/kg) in patients undergoing MUD-allo-SCT with

MAC conditioning and BM grafts [19]. They reported decreased aGVHD in patients receiving higher doses (37% vs 69%). Two additional studies have compared different ATLG dosing, in

haplo-identical and cord-blood setting [20, 21], both studies have reported increased infectious complications. Taking into consideration the basic differences in transplant types, our

results fall in line with these findings, higher doses of ATLG have reduced incidence of grade IV aGVHD however at the cost of increasing infections in our patient’s population, without any

impact on the long-term outcomes. It has been recently suggested that ATG/ATLG dose should be calculated not only based on Body weight but also according to absolute Lymphocyte count on the

first day of infusion [22, 23]. In two studies this approach has maximized the benefit by decreasing GVHD and reduced the risks of increased infections and relapse. This was also validated

in a post hoc analysis of a RCT where lower ALC counts on first ATLG infusion day was associated with lower PFS and OS [24]. In our study we have not evaluated the impact of ALC on any of

the outcomes. γδTcells and NK-cells protect against viral and bacterial infections [25,26,27,28]. In addition administration of ATLG is associated with a faster γδTcell recovery [29].

Moreover, a recent study on the impact of γδTcells recovery on transplant outcomes in pediatric patients with acute leukemia reported improved DFS and OS in Patients with higher γδTcells

[30], which was also confirmed in the adult populations [29, 31, 32], in addition they reported a decreased incidence of infections in patients with higher γδTcell count. The decreased

γδTcells and longer period of aplasia in the ATLG-60 group can explain the higher incidence of infections in this group. After allo-SCT the numbers of B cells normalize within a year

[33,34,35]. In addition, ATLG induces apoptosis in CD20 + B cells and has shown to induce complement dependent cell lysis in myeloma cells [36, 37]. In our study the B cell have increased

but did not normalize at day 180. In addition, the ATLG-60 group had a significantly lower naïve B-cell Population which supports the hypothesis that CD19 + B cells reduction by ATLG is dose

dependent [38,39,40,41]. Early NK-cells recovery after allo-SCT has been previously reported and it has been postulated that ATLG spares NK cells when compared with other in vivo TCD

strategies [42, 43]. In our study NK-cells recovered at day 30 in both groups and we observed no differences in cell count at all time points after transplant. However, we observed a higher

percentage of NK cells in the ATLG 30 group. This can be clarified by the improved overall lymphopoiesis observed in the ATLG-30 group. ATLG mediates in vivo T cell depletion by complement

mediated cytolysis, Fas-receptor dependent apoptosis and antibody dependent cell mediated cytotoxicity [44]. In addition, Servais et al. analyzed the impact of ATG on IR post MAC PBSC

allo-SCT [45]. It has been established that Tregs suppress GVHD without decreasing GVL [46], and that they accelerate T-cell IR in murine models [47]. Tregs in our study persisted after

allo-SCT and we observed no significant differences in Tregs IR between both groups. This can be explained by previous reports of ATG selectively sparing Tregs [45]. Two studies have

associated higher NKT-cell count with increased GVHD and relapse [48,49,50]. In our study, we observed early recovery of NKT cells without significant differences in IR between the two

groups. In both groups the CD8 + T cell compartment recovered at day +100, while the CD4 + Tcell compartment recovery was not achieved at day +180. This has been previously reported by Fehse

et al. where CD8 + Tcells recovered within the first year after allo-SCT however CD4 + T cells failed to reconstitute within 2 years after allo-SCT [51]. This can be explained by the fact

that: The reconstitution of the Tcell compartment after allo-SCT arises from both homeostatic peripheral expansion (HPE) of donor T-cells transferred with the graft and from novel production

of naïve T-cells in the thymus [52, 53]. In patients receiving MAC most of the T-cells originate from HPE [54, 55]. In addition, HPE occurs more asymmetrically between T-cells, with CD8 + 

T-cells having higher proliferating potential by HPE when compared to CD4 + T-cells [45]. Moreover, It has been hypothesized that ATLG targets naïve T cells while sparing memory T cells

[45]. Our study falls in line with previous findings, ATLG selectively compromised the recovery of naïve CD4+, memory CD4+, and naïve CD8+ cells, while it spared memory CD8 + T cells.

However, in our study higher doses of ATLG had more pronounced effects on naïve T CD4 + Tcells, which supports the theory that ATLG exerts a dose dependent T cell effect [38, 56]. In

addition, we observed a higher incidence of EBV reactivation in the ATLG-60 group. This can be attributed to the more pronounced severe depletion of naïve CD4 + Tcells and NK cells in this

group. More patients in the ATLG-30 group were not in CR at time of transplant, however we observed no differences in relapse incidence between the two groups which suggests a higher Graft

versus Malignancy effect in the ATLG-30 group. However, it should be noted that more Patients in the ATLG-30 group had active disease at time of transplant, therefore calcineurin inhibitor

may have differed between the two groups and may have influenced immune reconstitution. Acknowledging the retrospective nature of our study, the choice of ATLG dose has significant impact on

IR post MUD-allo-SCT, higher doses reduce aGVHD however they delayed engraftment, impair B-cell, γδTcells, NK and CD4 + T cell reconstitution and increase the risk for infection and EBV

reactivation. However, this did not affect any of the long-term outcomes. DATA AVAILABILITY The datasets generated during and/or analysed during the current study are available from the

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FUNDING Open Access funding enabled and organized by Projekt DEAL. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Stem Cell Transplantation, University Medical Center

Hamburg-Eppendorf, Hamburg, Germany Radwan Massoud, Evgeny Klyuchnikov, Nico Gagelmann, Tatiana Zabelina, Christine Wolschke, Francis Ayuk, Ulrike Fritzsche-Friedland, Axel Zander & 

Nicolaus Kröger Authors * Radwan Massoud View author publications You can also search for this author inPubMed Google Scholar * Evgeny Klyuchnikov View author publications You can also

search for this author inPubMed Google Scholar * Nico Gagelmann View author publications You can also search for this author inPubMed Google Scholar * Tatiana Zabelina View author

publications You can also search for this author inPubMed Google Scholar * Christine Wolschke View author publications You can also search for this author inPubMed Google Scholar * Francis

Ayuk View author publications You can also search for this author inPubMed Google Scholar * Ulrike Fritzsche-Friedland View author publications You can also search for this author inPubMed 

Google Scholar * Axel Zander View author publications You can also search for this author inPubMed Google Scholar * Nicolaus Kröger View author publications You can also search for this

author inPubMed Google Scholar CONTRIBUTIONS RM and NK designed the study, analyzed data, interpreted results, and wrote the manuscript. EK, NG, TZ, CW, FA, UF and AZ collected and analyzed

data. All authors approved the final version of the manuscript. CORRESPONDING AUTHOR Correspondence to Nicolaus Kröger. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Massoud, R., Klyuchnikov, E., Gagelmann, N. _et al._ Impact of Anti-T-lymphocyte globulin dosing on GVHD and Immune reconstitution in matched

unrelated myeloablative peripheral blood stem cell transplantation. _Bone Marrow Transplant_ 57, 1548–1555 (2022). https://doi.org/10.1038/s41409-022-01666-x Download citation * Received:

27 September 2021 * Revised: 23 March 2022 * Accepted: 29 March 2022 * Published: 13 July 2022 * Issue Date: October 2022 * DOI: https://doi.org/10.1038/s41409-022-01666-x SHARE THIS ARTICLE

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