Combination treatment of rituximab and donor platelets infusion to reduce donor‐specific anti‐HLA antibodies for stem cells engraftment in haploidentical transplantation

Abstract Background Donor‐specific anti‐human leukocyte antigen (HLA) antibodies (DSAs) in recipients is a risk factor for donor stem cell graft failure in haploidentical hematopoietic stem cell transplantation (haplo‐HSCT), and the treatment to reduce the levels of DSAs is not unanimous. This study was to analysis the role of DSAs for stem cell engraftment and to discuss the effective treatment to reduce DSAs in haplo‐HSCT. Methods We retrospectively evaluated the levels of DSAs and the effect of the combination treatment of rituximab and donor platelets (PLTs) for donor stem cell engraftment in haplo‐HSCT patients from June 2016 to March 2018 at our center. Results Nine patients (11.5%) out of the total 78 patients were DSAs‐positive and multivariate analysis revealed DSAs was the only factor that affected engraftment. Seven out of the 9 DSAs (+) patients received therapy: Four had antibodies against donor HLA class I (HLA‐I) antigens and were administered two therapeutic amounts of donor apheresis platelets (platelet count approximately 3‐5 × 1011) before donor stem cell infusion and the other three patients received a combination therapy of donor apheresis platelets and rituximab due to the antibodies against both donor HLA‐I antigens and HLA class II (HLA‐II) antigens. All the seven patients achieved donor stem cell engraftment successfully, and the DSAs levels decreased rapidly after transplantation. Conclusions DSAs is an important factor affecting engraftment in haplo‐HSCT. Donor platelet transfusion is one simple and effective treatment for HLA‐I DSAs, and a combination therapy should be administered if patients have both HLA‐I and HLA‐II antibodies.


| INTRODUC TI ON
Allogeneic hematopoietic stem cell transplantation is an effective treatment for hematologic malignancies and bone marrow (BM) failure diseases. However, the probability of finding human leukocyte antigen (HLA) matched sibling donors is <30%, and it is even lower in unrelated donors. 1 Haploidentical donors have gradually become substitutes for traditional donors in recent years, whereas primary graft failure (PGF) remains a life-threatening complication after transplantation either due to increased transplant-related mortality following infectious and hemorrhagic complications or due to early relapse in the absence of a functional graft. 2,3 HLA mismatch and donor-specific anti-HLA antibodies(DSAs) in recipients are critical causes of graft failure and malfunction in solid organ transplantation. 4,5 Recent researches revealed that DSAs played an equally important role in haploidentical hematopoietic stem cell transplant (haplo-HSCT). [6][7][8][9] In haplo-HSCT, the engraftment rate was much lower in DSAs (+) recipients than that in DSAs (−) recipients (61.9% vs 94.4%), and a higher fluorescence intensity of the DSAs was associated with a higher risk of graft failure and transplant-related mortality. [10][11][12] Therefore, DSAs must be considered when choosing a haploidentical donor and if a patient had no other alternative donors, he or she should been administered appropriate treatment to reduce the levels of DSAs for donor stem cell engraftment. The pre-transplanting treatment of DSAs is not unanimous. Plasmapheresis was often used before conditioning, but the antibody levels usually rebounded when donor stem cells were infused. 10 Rituximab or proteasome inhibitor could inhibit B lymphocytes from producing new antibodies, but it could not reduce the levels of already existing DSAs.
Since HLA class I (HLA-I) antigens are highly expressed in platelet(PLT), donor PLT infusion might neutralize the recipient's anti-HLA-I DSAs and rapidly reduce the DSAs levels without increasing the risk of developing acute graft-versus-host disease(aGVHD) or other adverse effect. 13 In the present study, we had analyzed the impact of DSAs on engraftment and the therapy to reduce the levels of DSAs in haplo-HSCT patients.

| Patients
Consecutive patients (N = 78) who received a haplo-HSCT at our All patients provided informed consent before participating in the study.

| Measurement of anti-HLA Ab levels
Serum samples were collected from the patients for screening anti-HLA antibodies with a LABScreen panel reactive antibody (PRA) Kit (One Lambda). Positive samples were further tested for the specificity of the antibodies against HLA-I (ie, HLA-A/B/C) and HLA-II (ie, HLA-DR/DQ) antigens using a LABScreen Single Antigen Kit (One Lambda). Fluorescence was measured using a Luminex100 flow analyzer (Luminex), and the data were analyzed using the LABScan 100 software (One Lambda). The median fluorescence intensity (MFI) of the PRA beads' reactions was obtained from the output file generated by the flow analyzer, adjusted for the background signal using the formula: sample beads −negative control beads. The fluorescence intensity of the negative and positive control beads was <100 and >9000, respectively. If the sample data did not fit these conditions, the serum or plasma was treated with ADSORB OUT beads (One Lambda) to reduce background fluorescence. All samples with fluorescence intensity >500 were tested with single Ag beads to confirm and identify sample specificity. MFI was adjusted for the back-

| Hematopoietic reconstitution and chimerism analysis
Neutrophil recovery was defined as an absolute neutrophil count (ANC) >0.5 × 10 9 /L for 3 consecutive days, and PLT recovery was defined as PLT count >20 × 10 9 /L for 7 consecutive days without platelet infusion. Graft failure was defined as no appearance or the complete loss of donor-derived neutrophils by day 28 using short tandem repeat (STR) chimerism analysis. BM puncture was performed on 14, 28, 42, and 60 days after donor stem cell infusion.
Chimerism analysis involved the quantitative polymerase chain reaction (PCR) of informative STRs in the recipient and donor.
DNA was amplified with fluorescent PCR primers for markers that would distinguish the donor and recipient alleles. Fluorescent PCR products were separated using an Applied Biosystems 3730 Genetic Analyzer (Applied Biosystems), and the Genemapper software (Applied Biosystems) was used to correlate allele peak areas with the percentage of donor or recipient DNA. It was defined as complete donor chimeras when the result of STR was ≥97%.
The ratio of X/Y chromosomes was tested using fluorescence in situ hybridization if the gender of donor and recipient was not matched.

| Statistical analysis
SPSS 21.0 was used for data processing and analysis. Fisher's exact test was used to compare the differences of engraftment rate among groups, whereas multivariate analysis was performed with logistic regression analysis to identify risk factors that affect donor cell engraftment. Statistical significance was considered when P < .05.

| Patient characteristics and donor stem cell engraftment
Overall, 78 patients who underwent haplo-HSCT from June 2016 to March 2018 were included in this study, and 69 patients were DSAs (−) and 9 (11.5%) were DSAs (+). The characteristic of the patients was showed in Table 1

| Characteristics of transplant in DSAs (+) patients and the measures for reducing DSAs
There were nine patients with DSAs (+) in this study including the first four patients (Patients 1-4) mentioned above and the following

| DSAs levels of patients during transplantation
The median fluorescence intensity (MFI) was used semiquantitatively for estimation of levels of DSAs and for predicting crossmatch results and assessing immunological risk. Crossmatch assays, the patient's serum was incubated with the donor's T and B lymphocytes to assess antibody reactivity directly, were not done in this study.
The DSAs levels of Patient 1 and Patient 2 in their secondary transplants and Patients 5-9 in their primary transplants were shown in antigens and the levels of DSAs (MFI) were more than 10 000 in four patents (Patients 1, 5,7, and 9), more than 5000  (Table 5). PLT infusion might be a simple, effective, and safe treatment for anti-HLA-I DSAs. But it is important to note that PLTs do not express HLA-II antigens, and DSAs against donor HLA-II antigens will not be consumed.

| D ISCUSS I ON
In our study, plasmapheresis was not used due to: (a) most of our patients were diagnosed as high-risk MDS without remission and the peripheral blood platelet counts were very low before conditioning, the risk of serious bleeding would be increased during or after plas-