Recovery of immunity is delayed in recipients of T-depleted grafts. Adoptive transfer of memory T-cells may improve immune response to common pathogens. A cohort of 53 patients with malignant (n = 36) and non-malignant conditions (n = 17) received TCR alpha/beta depleted grafts from haploidentical (n = 25) or MUD (n = 28) donors. Donor lymphocytes were depleted of CD45RA-positive cells. At a median of 48 days after transplantation, patients received DLI at 25 × 103/kg CD3 cells from haploidentical or 100 × 103/kg CD3 from MUD donors. Up to 3 doses of donor lymphocytes were administered at monthly intervals, escalating to 100 × 103/kg in haploidentical transplants and 300 × 103/kg in MUD transplants. At a median follow-up of 23 months, the cumulative incidence of de novo acute GVHD after DLI is 2% (1 of 43), while the rate of reactivation of preexisting aGVHD was 50% (5 of 10). The transplant-related mortality is 6%. The overall survival rates are 80% and 88% in malignant and non-malignant conditions, respectively. Among patients with absent CMV-specific immune reactivity at baseline (n = 31) expansion of CMV-specific T-cells was demonstrated in 20 (64.5%) within 100 days. Infusions of low dose donor memory T-lymphocytes are safe and constitute a simple measure to prevent infections in the setting of alpha/beta T cell-depleted transplantation.
Depletion of alpha/beta T-cells from the graft was developed as a method of graft engineering to improve the control of graft-vs.-host disease (GVHD) in the recipients of haploidentical grafts [1–3]. Early clinical results suggest that grafts depleted of alpha/beta T cells ensure fast and stable engraftment, a low rate of GVHD and improved immune recovery compared to the previous generation of T-cell depletion methods [4–6]. Although the quantitative recovery of peripheral T-cells seems to be improved, the generation of a broad TCR repertoire, which is required for normal immunity, is delayed . This is reflected in the significant rate of viral infections and associated morbidity and mortality [6, 8]. Antigen-specific adoptive T-cell therapy of viral infections after transplantation is an effective modality, which is based on either ex-vivo stimulation and expansion or selection methods [9–12]. Currently, the majority of methods depend on extraction of virus-specific memory T-cells from an antigen-experienced donor. Depletion of naïve (CD45RA-positive) T cells is a new method of selective T-cell depletion in HSCT and was successfully implemented in the setting of both matched and mismatched transplantation [13–16]. Depletion of CD45RA-positive cells results in grafts with decreased alloreactivity and retained pathogen-specific memory T-cell repertoire [15, 17]. Based on these results and an instructive case report, we hypothesized that infusion of the low doses of CD45RA-depleted donor lymphocytes can be used in the context of TCR-alpha/beta depleted grafts to improve pathogen-specific immune reconstitution and protect recipients from common viral infections . We report herein the results of a pilot trial evaluating the safety and potential efficacy of this approach.
The inclusion criteria for the study were as follows: any HSCT indication, allogeneic HSCT from haploidentical or matched unrelated donor, TCR alpha/beta and CD19 depletion of the graft, confirmed engraftment, day >+30 post-transplant, and CMV serological status donor+/recipient+. Patients with signs of active GVHD, systemic corticosteroid therapy >0.5 mg/kg, or unresolved sepsis were excluded. Fifty-three patients after their first (n = 50) or second (n = 3) allogeneic HSCT were recruited between 15.04.2014 and 15.07.2015.
Patient and donor cohort
The median age at transplantation was 8 (1–21) years, and indications included both malignant (n = 36) and non-malignant (n = 17) conditions. Donors were unrelated, HLA-matched in 28 cases and haploidentical in 25 cases. The baseline patient and donor characteristics and transplantation procedure details are presented in Table 1.
Conditioning regimens and GVHD prophylaxis
The conditioning regimens for malignant disorders included fludarabine at 150 mg/m2, treosulfan at 42 g/m2 (n = 27) and either melphalan at 140 mg/m2 (n = 22) or thiophosphamide at 10 mg/kg (n = 3). Six patients with acute lymphoblastic leukemia received fractionated total body irradiation at a total dose of 12 Gy, etoposide at 60 mg/kg and fludarabine at 150 mg/m2. Three patients received oral busulfan at 16 mg/kg, cyclophosphamide at 120 mg/m2 and fludarabine at 150 mg/m2. Patients with primary immune deficiency (PID) received fludarabine at 150 mg/m2 and treosulfan at 36–42 g/m2 (n = 6) with the addition of either melphalan 140 mg/m2 (n = 4) or thiophosphamide at 10 mg/kg (n = 2). One patient received thiophosphamide, cyclophosphamide and fludarabine. Patients with severe aplastic anemia or congenital bone marrow failure syndromes (n = 10) received fludarabine at 150 mg/m2, cyclophosphamide at 50–100 mg/kg and total lymphoid irradiation (TLI) irradiation at a dose of 2 to 6 Gy. Serotherapy included antithymocyte globulin (ATG) from either rabbit (Thymoglobulin, Genzyme) at 5 mg/kg (n = 40) or horse (ATGAM, Pfizer) at 100 mg/kg (n = 11). One patient received alemtuzumab (Campath, Genzyme) at 1 mg/kg, and one patient did not receive serotherapy. The GVHD prophylaxis regimen included tacrolimus from day −1 through day +60 either as a monotherapy (n = 12) or with the addition of methotrexate 5 mg/m2 days +1, +3, +6 (n = 9) or mycophenolate (n = 1). Since January 2014, patients with acute leukemia received immunosuppressive therapy with bortezomib 1.3 mg/m2 on days −5, −2, +2, and +5 without any additional agents (n = 25). Six patients did not receive any pharmacologic GVHD prophylaxis after transplantation. Forty-three patients received rituximab (100 mg/m2) on day -1 as an additional measure to deplete both donor and recipient B cells and to reduce the incidence of PTLD and GVHD.
Grafts were granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells. All grafts were depleted from alpha/beta T cells and B (CD19-positive) cells by CliniMACS Plus Instrument, Miltenyi Biotec, Bergish Gladbach, Germany. Depletion was effective with 4,6 log depletion for alpha/beta T cells and 3,5 log for B cells and the median recovery of CD34+ cells at 87% (supplementary Fig. 1). The median (range) dose of alpha/beta T cells within the graft was 12.8 × 103/kg (0.4–331), B cells—122 × 103/kg (1.4–3886), and CD34+ cells—8.6 × 106/kg (3.2–23).
Donor lymphocyte infusion preparation
Apheresis product from G-CSF-stimulated donors was used for donor lymphocyte infusion (DLI) preparation in 36 patients, unstimulated peripheral blood apheresis in 14 patients and whole blood in 3 patients. Briefly, (2–5) × 109 mononuclear cells (MNC) were depleted from CD45RA+ cells by either CliniMACS Plus, Depletion 2.0 program or CliniMACS Prodigy custom program, the immunomagnetic reagent dose was adjusted proportionally to the MNC input. The final product was evaluated for cell viability, residual content of CD45RA+ cells and microbial contamination. The released product was either infused fresh and/or aliquoted for cryopreservation in doses adjusted to the patient weight and planned dose range. The median log depletion achieved for CD3+CD45RA+ was 3.6 (2.5–5.5) and median recovery of CD3+CD45RO+ 37% (10–76). Thawed aliquots were retrospectively tested for reactivity to CMV, EBV and adenovirus with the gamma-IFN ELISPOT assay.
Schedule of donor lymphocyte infusions
The planned schema of CD45RA-depleted DLI included three infusions of escalating doses of T cells at monthly intervals. The planned dose range was 25, 50 and 100 × 103/kg for haploidentical and 100, 200 and 300 × 103/kg for MUD donors. Dose individualization was allowed to account for previous GVHD (step-down dose) or ongoing CMV viremia (step-up dose). The timing, composition and dosing of DLIs, including calculated doses of virus-specific T lymphocytes, are presented in Table 2 and supplementary Fig. 2.
Laboratory monitoring, supportive care and definitions
Either CMV or EBV viremia was defined as the presence of more than 500 copies of viral DNA per milliliter of whole blood. A positive PCR result in the blood after two consecutive weeks of negative results was regarded as reactivation. Either CMV or EBV disease was diagnosed if either virus was detected by PCR of virus or immunohistochemistry in the appropriate tissue sample with clinical signs of organ damage (independent of the presence of viral DNA in the blood). Neither foscarnet nor ganciclovir was administered prophylactically. CMV and EBV monitoring was routinely performed on a weekly basis by plasma quantitative PCR from the time of HSCT to day +30 after last memory DLI and then tailored based on continuing immunosuppression, a previous history of viral reactivations and immune reconstitution.
After the first detection of CMV viremia >500 genome equivalents per ml, ganciclovir or foscarnet (in patients with existing cytopenia) were administered. Preemptive administration of rituximab was triggered by either persistent EBV DNAemia above 10,000 genomes/ml.
Detection of CMV-specific T cells was performed with the gamma-IFN ELISPOT assay (see supplementary material) before first and each subsequent memory DLI, one month after the last DLI and at one year post-transplant. EBV and adenovirus immune responses were monitored in selected patients by the gamma-IFN ELISPOT assay. Among the donors with appropriate HLA alleles, n = 10 (HLA-A 0101, 0201, 0301, HLA-B 0702, 0801), the frequency of circulating CMV-specific T cells after DLI was additionally measured by flow cytometric CMV-dextramer staining (Immudex, Denmark). The GVHD grade was determined according to the Seattle criteria for acute GVHD .
Study design and statistical analysis
Primary end-point for the pilot study was the cumulative incidence of acute GVHD after DLI. Death from any cause without signs of GVHD, relapse of leukemia and graft rejection in non-malignant disease were considered competing risks. Secondary end-points included measures of the immune response to CMV (proportion of patients with recovery of CMV-specific reactivity) and relevant clinical outcomes, including overall survival, transplant-related mortality, cumulative incidence of chronic GVHD and cumulative incidence of relapse among patients with malignant conditions. The Mann–Whitney test and Fisher exact test were used for group comparison and contingency table analysis, respectively. The overall survival was calculated according to Kaplan–Meyer. Transplant-related mortality was calculated with relapse of malignant disease as a competing risk. The planned sample size was 30 patients. All patients and/or legal guardians provided informed consent to participate in this study. For survival and cumulative incidence analyses, alive patients were censored on 01.10.2016. The trial was registered under NCT02337595 at www.ClinicalTrials.gov.
Fifty-three patients received 134 infusions of CD45RA-depleted donor lymphocytes, and the median number of DLI was 3. The individual infusions contained variable levels of virus-specific T-cells and a negligible residual number of naïve CD45RA+ T cells (Table 2). The median follow-up time for survivors at the time of this report is 23 (14–31) months.
Patient status at first infusion
Regarding the preceding aGVHD, 10 patients developed first signs of grade 2 aGVHD before the first DLI, no case of grade 3–4 aGVHD was registered. At the time of the first DLI 11 patients had been receiving ongoing immune suppression for either prophylaxis (n = 5) or therapy (n = 6) for aGVHD. Twenty-nine patients (53.7%) had documented CMV viremia at a median of 19 days before the first DLI.
Safety (graft-versus-host disease)
None of the infusions was complicated by any type of immediate reaction or septic event. Six patients developed signs of aGVHD grade 2 (de novo n = 1 and reactivation n = 5) at a median of 14.5 (4–59) days after DLI, which included 3 after DLI#1, 2 after DLI#2 and 1 after DLI#3. Among 43 patients without preceding aGVHD, 1 patient developed grade 2 aGVHD post DLI with a cumulative incidence of de novo aGVHD of 2% (95% CI: 0–18) (Fig. 1a). Among 10 patients with preceding aGVHD, 5 developed aGVHD with a cumulative incidence of 50% (95% CI: 29–92) (Fig. 1b). Among the total cohort, the cumulative incidence of any aGVHD ≥ grade 2 (either after primary graft or after memory DLI) was 21% (95% CI: 12–35) (Fig. 1c). Among six patients who developed aGVHD post DLI, four responded to standard therapy, limited chronic GVHD (cGVHD) developed in 3 patients, and none of the patients developed de novo cGVHD. The cumulative incidence of cGVHD is 6% (95% CI: 2–17) (Fig. 1d). The detailed characteristics and outcomes of the patients who developed aGVHD after DLI are presented in Table 3.
Thirty-three (62%) patients had CMV viremia detected before or within 10 days after DLI#1. The median timing of CMV viremia was 19 days before DLI#1. Twenty-eight patients received preemptive pharmacologic therapy; the median duration of CMV-positivity was 3 (1–14) weeks and median number of reactivations was 1 (1–2). CMV disease developed in 2 patients, one case of CMV-chorioretinitis, who developed the symptoms before DLI, and one case of CMV-colitis. Both resolved with DLI and pharmacologic antiviral therapy. EBV viremia was registered in 4 (7.5%) patients at a median of 66 days after DLI. One patient received rituximab for lymphoproliferative disease. In two patients, severe lethal adenovirus infection developed. In one of the cases, the memory DLI did not contain any detectable reactivity towards adenovirus hexon antigen. In the second case, memory DLI contained 95 adenovirus-reactive lymphocytes per kg. Both patients received ongoing corticosteroids when diagnosed with fulminant adenovirus disease.
Monitoring of virus-specific immune response
In 13 patients, CMV-reactive T cells were detected before DLI#1 and 9 patients did not have valid ELISPOT results at one of the relevant time points; therefore, 31 patients could be evaluated for the recovery of the CMV-specific immune response in relation to the protocol intervention. Among 31 patients without CMV reactivity at the t 0 time point (immediately before DLI#1), 12 patients had expansion of CMV-reactive cells at time point t 1 (immediately before DLI#2). An additional 7 patients had CMV-reactive T cells detected at time point t 2 (before DLI#3) and in 1 patient at time point t 3 (30 days after DLI#3) (Table 3 and Fig. 2a). In total, recovery of CMV-specific immunity after DLI was documented in 20 patients (64.5%). Among these cases, the median increase in the CMV-reactive T lymphocytes between t 0 and t max1–3 was 698 (13–1175) (Fig. 2b). Several factors were tested for association with the recovery of CMV-specific T cells, including CMV viremia, the dose of CMV-specific T cells in the DLI, the dose of CMV-specific T cells in the graft, the dose of alpha / beta T cells in the graft, the naïve T cell count before each DLI, the total T cell count before DLI, T cell chimerism, the donor type (haplo versus MUD), the source of DLI (G-CSF-stimulated apheresis versus other), the donor and patient age and the ongoing immune suppression (Table 5). This analysis revealed that among 20 patients with recovered CMV-reactivity, 14 (70%) were documented to have CMV viremia, whereas none of the 11 patients without recovery had CMV viremia, Fisher exact p < 0.0001. Therefore, detection of CMV DNA was significantly associated with the recovery of CMV-specific immunity. The dose of CMV-reactive T lymphocytes in the DLI was paradoxically negatively associated with the recovery of CMV immunity, and median level of CMV-reactive lymphocytes was 170 cells/kg in patients with recovered CMV immunity versus 442 cells/kg in patients without recovery, Mann–Whitney p = 0.029. Only one of twenty patients with recovery of CMV-specific immunity had detectable naïve (CD3+CD45RA+CD197+) T cells in the peripheral blood at baseline.
Among 13 patients with detectable CMV-reactivity at time point t 0, 10 (70%) had documented CMV viremia at a median of 23 days before DLI. We did not detect any factor that was associated with the recovery of CMV-specific immunity before DLI. Thirty-four patients were followed at time point t 4 (1 year after transplantation). Among this cohort, 23 patients had detectable CMV-reactivity either before (n = 10) or after (n = 13) DLI, and all had persistent CMV-reactive T lymphocytes at time point t 4 (Fig. 2c). In selected patients with appropriate HLA alleles, the frequency of CMV-reactive CD8+ T cells was measured directly by CMV-dextramer staining. When measured concurrently (n = 10, 6 MUD and 4 haplo), the frequencies of CMV-reactive T lymphocytes detected by either Elispot or dextramer staining correlated significantly, Pearson r = 0,72, p = 0,019. Detailed data on the recovery of CMV immunity and ELISPOT results representative of different patterns of CMV immunity are illustrated in supplementary table 2 and Fig. 3. Immune responses to EBV and Adenovirus were tested in selected patients (Table 4).
Global immune recovery
Quantitative recovery of the major lymphocyte subpopulations is presented in the supplementary figure and also in Table 4. The tempo of immune reconstitution was similar to that observed in our previous studies [6, 7].
Three patients died of non-relapse causes at a median of 192 (190–295) days after HSCT and 129 (61–274) days after DLI#1. The cumulative incidence of TRM is 6% (95% CI:2–19) (Fig. 3a). In one case, the direct cause of death was sepsis / MOF; two patients succumbed to disseminated adenovirus infection (Table 5). In all three cases of transplant-related death, patients received ongoing corticosteroid therapy for GVHD control. The cumulative incidence of relapse in patients with leukemia was 18% (95% CI: 8–37) (Fig. 3b). The overall survival rates at 2 years for the whole group was 83% (95% CI: 68–94): 80% (95% CI: 72–93) in malignant conditions and 88% (95% CI: 73–100) in non-malignant conditions (Fig. 3c, d).
The aim of the current study was to evaluate the safety and potential efficacy of infusing low doses of donor T-cells, depleted of CD45RA+ naïve T lymphocytes. Although the potential for alloreactivity in the memory T-cell fraction was shown to be significantly reduced by several studies [15, 17], cross-reactivity of the memory T-cell repertoire with allogeneic HLA is a known phenomenon [20–23]. The true potential of memory T lymphocytes to induce GVHD in a particular clinical setting is unknown thus far. Published clinical experience suggests that depletion of naïve T-cells does not completely abrogate the risk of GVHD l [13, 14, 24]. Our data suggest that memory T cell DLI at doses of up to 100 × 103/kg in the setting of haploidentical HSCT and up to 300 × 103/kg in the MUD setting are associated with a low risk of de novo GVHD but might pose a risk of reactivation in patients with pre-existing GVHD.
Monitoring of CMV-reactive T-cells post DLI indicates that upon appropriate antigen exposure, CMV-reactive T cells undergo massive expansion. Detected CMV-specific clones could be derived from one of the sources (1) residual CMV-specific memory T lymphocytes in the primary graft; (2) CMV-specific memory T lymphocytes in the DLI; (3) naïve donor-derived T cells produced de novo and (4) recipient T lymphocytes in patients with mixed T-cell chimerism. Our analysis suggests that naïve T cells and recipient-derived T cells contribute little to the regeneration of the CMV immune response. Although it is not possible to fully dissect the CMV-directed immune responses derived from the primary graft and those transferred with the DLI, circumstantial evidence suggests that DLI significantly contributes.
It is of note that clinically relevant responses can be derived from less than ten CMV-reactive cells per kg. Transfer of the donor memory repertoire could be useful in the setting of haploidentical transplantation in which common pathogen exposures within the family would allow for the transfer of immune responses for particular strains of persisting and reactivating pathogens. We suggest that memory T cell infusion may represent a robust, direct and easy to implement measure to provide the protection from common infections until recovery of broad repertoire of T lymphocytes. Current automatic production may obviate the need for GMP facility, making this approach a widely available blood bank procedure.
Overall, our data suggest that donor lymphocytes depleted of CD45RA+ T cells can be safely studied at doses in the range of 25–100 × 103/kg in the haploidentical setting and 100–300 × 103/kg in the unrelated setting after T-depleted transplantation. To be effective as a prophylactic measure memory DLI would have to be used early, preferably as part of the primary graft, in the setting of minimal post-transplant immune suppression, including all types of polyclonal serotherapy or other pan-T-cell depleting antibodies. If the prophylactic administration of memory DLI is demonstrated as effective in comparative trials, including NCT02942173 randomized trial initiated by our group, it could become a significant cost-saving approach considering the cost of pharmacologic control of viral infections after HSCT .
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The authors thank the physicians and nursing staff of the Hematopoietic stem cell transplantation Units 1 and 2 for outstanding patient care, the staff of Molecular biology, Microbiology and Transplant biology laboratories for excellent service, Marina Persiantseva and Susanne Morsch for unrelated donor search, Dmitriy Pershin and Victoria Kiseleva for technical assistance. We are grateful to the “Podari Zhizn” foundation for continued support of the care of the patients and research in the field of hematopoietic stem cell transplantation.
MM planned the study, performed the analysis and wrote the manuscript. SB collected the data and wrote the manuscript. LS, AM, DB, and GN planned the study, analyzed the data and reviewed the manuscript. JS established the database. EK, YM, AK and PT performed graft and cell therapy product processing and formulation. EB, EO, NK and VZ performed immunomonitoring and cell therapy product quality control and contributed to the Methods section.
Conflict of interest
MM delivered lectures at Miltenyi Biotec satellite symposia at EBMT 2013 and EBMT 2016 annual meetings. Rest of the authors declare to have no conflict of interest.
The study was approved by Ethics committee of the Dmitriy Rogachev Federal Center of pediatric hematology, oncology and immunology.
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