Haploidentical hematopoietic stem cell transplant (haplo-HSCT) has been applied with promising results for patients diagnosed with hematological malignancies lacking an appropriately matched sibling or unrelated donor and urgently requiring transplantation (1–5). Although considerable progress has been made to overcome the challenging human leukocyte antigen (HLA)-barriers in the setting of haplo-HSCT through various graft-versus-host disease (GVHD) prevention approaches, GVHD remains a major factor contributing to variable degrees of transplantation-related morbidity and mortality, as well as quality of life compromise. Currently, T-replete strategies using unmanipulated allografts have been the dominant procedures for haplo-HSCT, in which the GVHD prophylaxis consists of an immunosuppressive therapy mainly based on antithymocyte globulin (ATG) or post-transplant cyclophosphamide (PTCy) (5–12). Both ATG and PTCy have consistently demonstrated efficacies in GVHD prophylaxis, but each strategy uniquely affects post-transplant immune reconstitution resulting in concordant increased incidences of opportunistic infections and malignancy relapse (9, 11, 13–15). The optimal approach to manipulate the delicate balance between controlling GVHD and timely T-cell immune reconstitution remains uncertain and has become a main focus of attention.

Basiliximab is a chimeric murine-human monoclonal antibody that is directed against the IL-2Rα chain (CD25) to inhibit T lymphocyte activation. Compared with ATG, basiliximab has similar immunosuppressive efficacies but leads to a lower incidence of infection and infection-related mortality in renal transplantation and heart transplantation (16–18). In terms of HSCT, basiliximab has been used to treat acute steroid-refractory GVHD with satisfactory responses (19–23). Considering that basiliximab only selectively eliminates the donor-specific alloreactive T cells without affecting the resting T cells present in the graft (24), basiliximab may prevent GVHD without compromising immune function. In fact, the protective effect of CD25 blockade on GVHD is controversial. It has been reported that prophylactic administration of daclizumab (another CD25 blockade) does not prevent acute GVHD (aGVHD) but may increase the risk of chronic GVHD (cGVHD) (25). On the contrary, we and others have indicated that basiliximab alone or with the combination of ATG contributes to GVHD prophylaxis in HSCT without increasing infections (26–32). Nevertheless, these studies contained a relatively small number of patients; and a larger study with more patients and longer follow-up is warranted. Therefore, we modified the ATG-based GVHD prophylaxis with the addition of basiliximab in the setting of haplo-HSCT. Furthermore, in order to reduce the risk of GVHD without increasing the incidence of infection events or compromising overall survival, and simultaneously decrease expense related to ATG, we attempted to explore the appropriate dose of ATG combined with basiliximab.

In this study, we retrospectively analyzed a large series of patients with hematological malignancies who underwent haplo-HSCT using GVHD immunosuppressive prophylaxis with different dosages of ATG incorporated with basiliximab. On the one hand, we investigated the efficacy and safety of this combined GVHD prophylaxis option. On the other hand, we performed a preliminary assessment of the appropriate lower dose of ATG coupled with basiliximab in our haplo-HSCT system.

In the present retrospective study, basiliximab was administrated in addition to the ATG-based GVHD prophylaxis regimen in 239 patients who were diagnosed with intermediate- or high-risk hematologic malignancies and had received haplo-HSCT with a median follow-up of 2.4 years (range, 0.8-6.6). The efficacy of this similar protocol in high-risk hematologic malignancies has been explored (28–30). However, with respect to the experience of these groups, the higher number of patients, the longer follow-up of our study, the use of more intensive conditioning regimens, the different graft sources as well as the different dosages and types of ATG provide relevant novelty to the present report. Herein, this combined GVHD prophylaxis strategy could result in very favorable rates of acute and chronic GVHD and achieve satisfactory results of 3-year OS 60.9% with acceptable relapse rates and TRM. Remarkably, lower dose of ATG (≤6mg/kg) combined with basiliximab was associated with less CMV viremia but comparable GVHD preventive effect and survival rates.

Haploidentical donors provide the benefits of rapid and near universal donor availability. However, immunological barriers resulting from the high degree of HLA-mismatch in the haplo-HSCT settings were formidable (39, 40). In vivo T-cell depletion or modulation with ATG (the Beijing protocol) or PTCy (the Baltimore protocol) has been the basis for the development of multiple novel GVHD prophylaxis approaches used in haplo-HSCT. The clinical benefit of incorporating ATG or PTCy as GVHD prophylaxis in haplo-HSCT varied among studies, which could be related to the dose and formulation of ATG or PTCy administration, the conditioning regimen, the donor type, the stem cell source, the concomitant immunosuppressive medications and the background hematological malignancy (15, 41–50). We summarized several studies on the effects of ATG-based, PTCy-based, and two schemes combined GVHD prophylaxis (4–7, 12, 42, 43, 45, 47, 48, 51–54) ( Supplementary Table 3 ). Comparable incidences of grade II-IV aGVHD (18%-42%) and severe cGVHD (10%-23%) were shown in ATG-based and PTCy-based prophylaxis. Meanwhile, PTCy-based regimen appears to be more effective in the incidence of grade III-IV aGVHD (5%-14%). Additionally, a novel regimen of combining ATG and PTCy after haplo-HSCT for hematological malignancies showed promising activity with grade II–IV aGVHD of 17%-26% and grade III–IV aGVHD of 3.2%-6.9%. It is worth noting that in our study, we observed that GVHD prophylaxis using the described ATG plus basiliximab combination results in very low rates of both acute and chronic GVHD, with estimated incidences of grade II-IV aGVHD, grade III-IV aGVHD and 3-year extensive cGVHD of 15.8%, 5% and 5.2%, respectively. The rates of clinically significant grade III–IV aGVHD and extensive cGVHD were significantly decreased, a benefit that has not been achieved in most of the studies using standard ATG regimen and PBSC. Accordingly, the effect of the use of ATG and basiliximab on the incidence of GVHD converted into the reduced risk of TRM and improved GRFS. Compared with PTCy-based studies, although our study showed lower severe aGVHD, lower TRM, and higher GRFS, this could be due to the younger baseline characteristics of the patients (47, 48, 51, 55). Older patients who receive reduced conditioning are often associated with more severe infections, GVHD, and recurrence, which results in lower GRFS. Therefore, clinical studies with larger sample sizes and older patients are needed to confirm the effectiveness.

Furthermore, our results observed that the female to male combination results in a higher incidence of severe acute GVHD than other donor-recipient sex combinations, which coincides with the results of others (5, 56, 57), but it did not translate into any higher TRM. Nevertheless, the incidence of GVHD was not influenced by the stem cell source, the types of hematological malignancy or intensity of conditioning regimens in multivariate analysis. Importantly, the incidence of GVHD in our study using unmanipulated PBSC combined or not with BM with intensified myeloablative or myeloablative conditioning was comparable to that in previous studies reported for similar GVHD prophylaxis, using unmanipulated BM haplo-HSCT with myeloablative or reduced intensity conditioning (29, 30). Thus, we concluded that the effect of this enhancing immunosuppression strategy may be independent of stem cell source and the intensity of conditioning regimen.

Infectious complications remain the major factors affecting overall survival and are central to advances seeking to improve haplo-HSCT. As far as we know, in vivo T-cell depletion with ATG results in delayed immune reconstitution, leaving patients vulnerable to severe infections, including viral reactivations with CMV or Epstein-Barr virus (EBV), bacterial infection, as well as infection-related deaths (13, 15, 58). The incidence of CMV viremia was 62.76% in our study, which was comparable with ATG-based regimens (60-78.6%) (5, 28, 30, 43, 59–61). Despite pre-emptive therapy of ganciclovir or foscarnet, 3% patients still progress to CMV disease. Antiviral drug resistance and treatment-limiting side effects remain major challenges. Letermovir was approved for prophylaxis of CMV-infection and -disease in adult CMV-seropositive recipients after allogeneic HSCT and demonstrated lower risk of CMV-infection than placebo without apparent safety concerns (62). Letermovir has a novel mechanism of action that inhibits CMV DNA synthesis at a late step by targeting the pUL56 subunit of the terminase enzyme complex (63–66). Because of a unique mechanism of action that is distinct from ganciclovir and other CMV DNA polymerase inhibitors, letermovir has the potential for treating multidrug-resistant CMV. We believe that with additional clinical efficacy data, this medication could emerge as a primary option for the prevention and treatment of CMV in the immunocompromised patient population.

As for the use of PTCy in haplo-HSCT for hematologic malignancies, a major concern is the high relapse rate, which is up to 50% (9, 11, 67–69). While the relapse rate at 1-year and 3-year were 20.5% and 28.1%, respectively, which was comparable to those in previous studies, ranging from 15% to 34.5% depending on the diagnosis and the disease risk (4, 5, 55, 70). In line with the published data, patients suffering from ALL showed a higher risk of relapse (51, 55). Different from ATG, basiliximab binds specifically to the IL-2R of activated T cells and only selectively eliminates the alloreactive donor lymphocytes, without affecting the resting T cells (24), which is important for preventing infectious complications. It was also reported that antibody mediated CD25 blockade may be useful to promote anti-tumor immunity (25). This may account for the comparable incidences of CMV viremia and relapse between our haplo-HSCT setting using ATG plus basiliximab and other regimens using ATG alone to prevent GVHD. Thus, we could conclude that ATG combined with basiliximab was complementary integration to reinforce GVHD prophylaxis without increasing infections and significantly compromising disease control.

The limitations associated with the use of ATG as a protocol for GVHD prophylaxis include the profound immune deficiency and higher risk of infectious complications, depending on the dose of ATG administered (15, 61, 71–73). To date, the optimal dose of ATG balancing the efficacy of GVHD prophylaxis and the risk of severe infections has not been established for haplo-HSCT. Hang et al. has documented that 6 mg/kg ATG applied in haplo-HSCT was related to a faster recovery of T cell reconstitution and lower incidence of EBV infection but a higher rate of severe GVHD and GVHD related death than 10 mg/kg ATG (15, 61, 74). Recently, a multicenter randomized study comparing two different doses of ATG (7.5 and 10 mg/kg) as GVHD prophylaxis for haplo-HSCT, showed that patients receiving 7.5 mg/kg ATG had a lower incidence of EBV and CMV DNAemia and a similar incidence of aGVHD and cGVHD compared with those receiving 10.0 mg/kg (75). In our haplo-HSCT system, we also made an initial attempt to investigate whether low doses of ATG, 6mg/kg and below, were more beneficial in the outcome of haplo-HSCT. Consistently with the mentioned results, the reduced incidence of CMV DNAemia was found in the lower dose arm(≤6mg/kg), compared to the higher dose arm (>6mg/kg). Surprisingly, there was no difference noted with regard to the occurrence of acute and chronic GVHD between the lower dose and higher dose groups. In addition, in the context of haplo-HSCT with similar GVHD prophylaxis, the incidence of GVHD in the lower dose arm seems to be better or equivalent to that in previous studies using higher dose of ATG, ranging from 6 to 9 mg/kg, or using different source of ATG (ATG-Fresenius, 20 mg/kg) (28–30). These findings suggest that lower dose ATG has sufficient efficacy for GVHD prophylaxis and minimizes the risk of infection when used in combination with basiliximab. Furthermore, long-term transplant outcomes did not show any significant differences between the two groups concerning disease recurrence, TRM, OS and DFS. It’s worth noting that a beneficial effect of TRM was not seen in the lower dose arm despite the lower incidences of CMV viremia and the comparable morbidity and mortality associated with GVHD. This may attribute to the effective preemptive treatment for CMV DNAemia at our center. Although survival benefit has not been achieved, it might bring a potential economic benefit as well as less side effects of preemptive therapies in the patients receiving lower dose ATG. In general, in our study, a lower dose ATG(≤6mg/kg) in combination with basiliximab was as effective as higher doses for GVHD prevention after haplo-HSCT. A higher dose did not confer any additional benefit; conversely it appeared to be associated with increased CMV DNAemia. Based on these results, a lower dose ATG(≤6mg/kg) seems to be more appropriate for balancing infection control and GVHD prophylaxis when used in combination with basiliximab. A prospective randomized trial would be required to reach a definite conclusion on the superior efficacy of lower dose ATG plus basiliximab regimen in haplo-HSCT.

Indeed, our study was limited by its retrospective nature and the heterogeneity of the sample size. To some extent, the dose of ATG was related to HLA mismatch and the impact of ATG dose on the incidence of GVHD may be confounded by HLA mismatch. Although several studies have shown that HLA mismatch has little effect on GVHD in haplo-HSCT, this issue cannot be ignored (5, 28, 76, 77). Thus, further prospective and randomized control studies are needed. In addition, more detailed analyses are needed to get information on other virus infections including EBV and immune reconstitution in this haplo-HSCT circumstance. However, we included a relatively large patient number with a rather long follow-up, which allowed us to reliably estimate the impact of ATG in conjunction with basiliximab on long-term clinical outcomes at our center.

In conclusion, the evidence reported in this paper indicates that the combination of ATG with basiliximab is a feasible and effective protocol to promote protection against GVHD and improve haploidentical transplant outcomes in the context of both intensified myeloablative and myeloablative conditioning regimens. Moreover, the combination with a lower dose of ATG has shown to be safer and equally effective than the original one with a higher dose, in preventing GVHD. We conclude that the optimal scheme of using a combined regimen of ATG and basiliximab is that administration of lower dose ATG(≤6mg/kg), which could exert a synergistic activity to reduce the risk of GVHD without increasing severe infection, particularly CMV viremia.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Ethical Committee of Huazhong University of Science and Technology. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

LX and WS designed the study and enrolled patients. HY and ZH analyzed the data and wrote the paper. ZH and YT were responsible for the data collection. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (No.81974003, No. 81500168, No. 81974007), the National Key R&D Program of China (Grant No 2019YFC1316204), and Collaborative Innovation Center of Hematology of China.

We are very grateful to all the participants at the institute who provided information in this study.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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