Despite advances in gene therapy allogeneic hematopoietic stem cell transplants (HSCT) remains the most effective way to cure sickle cell disease (SCD). However, there are substantial challenges including lack of suitable donors, therapy-related toxicity (TRM) and risk of graft-versus-host disease (GvHD). Perhaps the most critical question is when to do a transplant for SCD. Safer transplant protocols for HLA-disparate HSCT is needed before transplants are widely accepted for SCD. Although risk of GvHD and TRM are less with T-cell-deplete HSCT and reduced-intensity conditioning (RIC), transplant rejection is a challenge. We have reported graft rejection of T cell-depleted non-myeloablative HSCT can be overcome in wild type fully mis-matched recipient mice, using donor-derived anti-3rd party central memory CD8-positive veto cells combined with short-term low-dose rapamycin. Here, we report safety and efficacy of this approach in a murine model for SCD. Durable donor-derived chimerism was achieved using this strategy with reversal of pathological parameters of SCD, including complete conversion to normal donor-derived red cells, and correction of splenomegaly and the levels of circulating reticulocytes, hematocrit, and hemoglobin.
Sickle cell disease (SCD) is one of the most common genetic diseases. In the US 1 out of 365 African Americans is reported to have SCD with a prevalence of about 100,000 Americans [1,2,3,4]. Hemoglobin-S polymerizes under deoxygenated conditions causing intra-vascular hemolysis with acute and chronic clinical features, such as vaso-occlusive and spleen sequestration crises, osteo-necrosis, acute chest syndrome and stroke [5,6,7,8]. The clinical course of SCD is highly variable but in general people with SCD have a reduced life expectancy.
HSCT can successfully treat SCD patients when a human leukocyte antigen (HLA)-matched sibling donor is available; however, the scarcity of such donors remains a major challenge. This problem has been addressed to some extent by new approaches to reduce the toxicities associated with unrelated matched donors or haploidentical related donors, with special emphasis on new means to reduce the incidence and severity of GvHD and graft failure [9,10,11,12]. Likewise, new insights from the development of less toxic reduced intensity conditioning (RIC) protocols in the treatment of patients with hematological malignancies are being translated to the treatment of SCD and thalassemia patients [9, 13,14,15]. In principle, the problem of GvHD could be substantially addressed by the use of T cell-depleted (TD)-HSCT, but it was shown that such transplants are more prone to graft failure in the context of RIC.
In acute leukemia patients receiving haplo-identical TD-HSCT and treated with myeloablative protocols, this barrier can be overcome by the use of ‘megadose’ transplants . It has been suggested that the mechanism underlying the ability to resist residual CD8 anti-donor host T cells is mediated by “veto” activity exhibited by CD34 progenitors and their myeloid derivatives . Veto activity, first defined by Miller , is based on the ability of certain cells to attack host CTL-precursors (CTLp), which are directed against the antigens of the veto cells themselves, sparing other T cell clones including pathogen-specific T cells that are not targeted against donor-derived veto cells. Notably, while the megadose CD34 cell inoculum could neutralize the residual anti-donor CD8 T cells surviving myeloablative protocols, the number of CD34 cells attained by G-CSF mobilization of the donor, is not sufficient to enable engraftment in patients conditioned with non-myeloablative protocols.
To address this challenge, new sources of veto cells that could be expanded ex-vivo to large numbers and combined safely with haploidentical TD-HSCT, have been investigated in different murine models . One potential source of veto cells is central memory CD8-postive T-cells (Tcm) depleted of graft-versus-host (GvH) reactivity by selective loss of anti-host T cell clones. To that end, naive CD8-postive T-cells are cultured with 3rd party stimulator cells without cytokines for 6 days followed by expansion for 10 days with IL15 [20, 21]. Notably, such Tcm were shown to effectively induce donor-specific immune tolerance and enhance T cell depleted Bone Marrow (TDBM) allografts in fully H2-mis-matched recipients following RIC (5.5 Gy TBI) without GvHD. Furthermore, combining Tcm therapy with 5 days of rapamycin, enabled to reduce the conditioning radiation dose to 4.5 Gy TBI . Here, we describe using this strategy to correct SCD in the Berkeley SCD mouse model.
Materials and methods
Experimental model and animal subjects
Animal studies followed Institutional Animal Care and Use Committee (IACUC) approval and guidelines at the faculty of veterinary resources, MD Anderson Cancer Center. Sickle mice (B6; Hba < tm1Paz > Hbb < tm1Tow > Tg(HBA-HBBs)41Paz/J) were purchased from Jackson Laboratories (Bar Harbor, Maine; Sacramento, California). Breeding colonies of sickle mice were maintained in the animal house facility at MD Anderson Cancer Center. Age-matched and sex-matched, 8-week-old males and females were randomly allocated to the experimental arms. Balb/c (H-2d), FVB (H-2q), C57BL/6 (H2-b), and Balb/c nude (H-2d) mice were purchased from the Jackson Laboratory. Pre-established exclusion criteria were based on IACUC guidelines including systemic disease, toxicity, respiratory distress, refusal to eat and drink, and substantial (>15%) weight loss. Most mice were in good health and were included in the appropriate analyses.
RIC model for bone marrow transplantation
Long bones were harvested from Balb/c-Nude mice (H-2d, 10–12 weeks of age) and bone marrow was extracted by grinding the bones to achieve a single cell suspension. Each sickle recipient mouse received sub-lethal total body irradiation (TBI) on day −1 in a cesium-137 irradiator (JL Shephard & Associates, Glendale, CA). On day 0 mice received 25 × 106 Balb/c-Nude bone marrow cells with or without 5 × 106 anti 3rd-party Tcm given on day +7 with or without rapamycin, 0.5 mg/kg body weight (Rapamune®, Pfizer Inc., New York) on days −1 to +4. Mice were evaluated twice weekly for overall appearance and weight. Chimerism analyses were conducted as indicated.
Mice were maintained under specific pathogen-free conditions with antibiotics (Baytril), 4 ml per 350 ml drinking water days 0 to +14 (Bayer Healthcare LLC, Whippany, NJ).
Preparation of host non-reactive donor anti-3rd-party cells
Anti-3rd-party Tcm were prepared as described . Briefly, spleen cells from donor mice were cultured with irradiated 3rd-party spleen cells in RPMI-1640 for 60 h. CD8 cells were positively selected using magnetic-activated cell sorting [MACS® Cell Separation, Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in an Ag-free environment. rhIL-15 (20 ng/mL; R&D Systems, Inc., Minneapolis, MN, USA) was added on every 2nd day. Next, Tcms were positively selected for CD62L expression and retrieved for FACS analysis.
WBC chimerism analysis
Chimerism was determined by flow cytometry. Blood was collected by retro-orbital bleeding, cells fractionated on Ficoll-Paque Plus (Amersham Pharmacia Biotech, AB), and mononuclear cells of each mouse were stained by direct immuno-fluorescence against donor and host surface markers.
Flow cytometry analysis
Blood or selected cell populations were analyzed by fluorescence-activated cell sorting (FACS) by using following fluorochrome-labeled antibodies against murine antigens, anti-mouse CD3-PE/Cy7/FITC, anti-mouse CD4-APC, anti-mouse CD8-PB/PerCP, anti-mouse CD45-PE/Cy7/APC/Cy7, anti-mouse CD44-PE, anti-mouse CD62L-FITC/PB, anti-H2Kd-FITC/PE/APC, anti-H2Kb-PE/FITC, and Ter119-APC (Biolegend, San Diego, CA, USA), or IgG isotype controls (Biolegend) corresponding to each antibody in a specific panel. The stained cells were acquired on an LSRFortessa X-20 cytometer (Beckton, Dickinson Franklin Lakes, NJ) with BD FACSDiva software 8.0.1. Data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
Analysis of hematologic parameters
A complete blood count was performed using a Siemens ADVIA 2120i hematology analyzer (Siemens Healthcare Diagnostics, Erlangen, Germany). Reticulocyte level was determined by FACS as described . Briefly, whole heparinized blood was stained with fluorochrome-labeled antibodies specific for RBCs (anti-Ter-119 APC, Biolegend), WBCs (anti-CD45 PECy7 or APC/Cy7, Biolegend) and the nucleic acid-binding fluorescent dye, thiazole orange (TO; Sigma, St Louis, MO). Percentage of blood cells that were Ter-119+, TO+, and CD45− were defined as reticulocytes.
Analysis of RBC chimerism
Differential hemoglobin electrophoresis was performed to determine the RBC chimerism in experimental mice, as reported earlier . The assay was performed on the Helena Titan III electrophoresis system (Helena Laboratories, Beaumont, TX). The pattern of hemoglobin in recipient, donor, sickle and wild-type mice was determined based on differences in electrophoretic mobility of hemoglobins of each strain.
Kidneys, spleen, liver, and lungs were excised and fixed in 4% paraformaldehyde solution and processed for paraffin sections. Sections were stained with H&E and examined microscopically.
Blood slides were prepared under oxygenated conditions. Wright-stained and examined microscopically.
Survival analyses were performed using Kaplan–Meier curves (log-rank test). Comparison of means co-variates were performed using the Student’s t-test and the means of >2 co-variates were compared by one-way ANOVA using SPSS Statistics 24.0 software. P-values < 0.05 were considered significant.
Chimerism induction in SCD mice with T-cell-depleted (TCD) allogenic bone marrow, veto cells, and short-term rapamycin after sub-lethal irradiation
We previously demonstrated that a combination of mega-dose TCD allogeneic bone marrow from C57BL/6 donors combined with veto CD8+ T cells and short-term post-transplant treatment with a low dose of rapamycin, could successfully induce chimerism in Balb/c recipients conditioned with sublethal 4.5 GY TBI .
Considering that the SCD mouse model (Berkeley model, H-2Kb) is based on the genetic background of C57BL/6 mice, known to be more resistant to TBI , we initially attempted to define the optimal dose of TBI in SCD recipients. As shown schematically in Fig. 1A, we compared conditioning with 4.5 Gy TBI and 5 Gy TBI prior to transplantation of megadose Balb/c ‘nude’ bone marrow (Nu/BM) cells (H-2Kd; 25 × 106), in conjunction with 5 × 106 anti-3rd party veto Tcm. In both groups, rapamycin was administered from days −1 to +4. Notably, higher levels of engraftment were found in the group receiving 5 Gy TBI (7/7) compared to the group receiving 4.5 Gy TBI (5/9) (Fig. 1B) at day +35, and this high level of chimerism was found to be durable when examined at day +140 post-transplant (Fig. 1C). All mice of both treatment groups survived, indicating very low risk for transplant-related mortality (Fig. 1D) with no evidence of GvHD as measured by loss of body weight (Fig. 1E). Notably, in the long term, non-chimeric mice exhibited a tendency to gain more weight, typical of SCD mice, while all the chimeric mice in the 4.5 and 5 Gy TBI groups exhibited donor type RBC chimerism as detected by hemoglobin electrophoresis (Fig. 1F) although clearly WBC and red cell chimerism was significantly lower in the group receiving 4.5 Gy TBI.
Further long-term follow-up over 381 days, revealed continued stable chimerism in the group conditioned with 5 Gy TBI (Fig. 2A) with complete conversion to normal hemoglobin, as measured by electrophoresis (Fig. 2B). Only a single death occurred in this group at day 215 likely due to ocular bleeding (all mice were repeatedly bled for chimerism analysis during the long-term follow-up period and this death occurred immediately after the last bleeding day+213) (Fig. 2C). In contrast, we found continued gradual loss of chimerism in the group receiving 4.5 Gy TBI. Thus, by day +213, the three remaining mice were not chimeric. Furthermore, six out of nine mice in this group died during the 381 day follow-up period (Fig. 2C) with marked pathology of SCD. The histopathology reveals that these mice had abnormal renal and splenic pathology. The splenic sequestration which is characterized by the enlarged spleen, drop in hemoglobin, thrombocytopenia and reticulocytosis are likely the reason of death of these mice (data not shown).
Based on these data we used 5 Gy TBI in a subsequent experiment. At day +44 six out of seven mice receiving Nu/BM + Tcm + rapamycin had donor chimerism levels of 77–94% (Fig. 3A–C). In contrast, there was no donor chimerism in mice not receiving veto cells and in recipients of veto cells + Nu/BM cells without rapamycin (Fig. 3B). Durable donor chimerism was also found at day +318 in lymph nodes (83.7 ± 7.7), bone marrow (78.8 ± 13.0), spleen (75.4 ± 10.5), and thymus (59.6 ± 26.7; Fig. 3D, E)
Normalization of pathology in chimeric mice
To evaluate the extent of SCD correction by donor-derived hematopoietic chimerism we initially interrogated blood parameters of chimeric mice (n = 8) compared to SCD mice (n = 14). None of the 14 mice transplanted in two independent experiments died by day +120 (Table 1). Thereafter two mice died of ocular bleeding on day +215 and day +231 and 1 mouse had graft-failure on day +44. Ten of eleven survivors had a normal hemoglobin electrophoresis at >day +300; 1 mouse had a mixed hemoglobin pattern (Table 1; Fig. 4A). Chimeric mice had a normal percentage of reticulocytes (Fig. 4B, C), normal concentrations of WBCs (Fig. 4D) and hemoglobin (Fig. 4E) and normal hematocrit (Fig. 4F) and mean corpuscular hemoglobin concentration (Fig. 4G). Blood slides of chimeric mice had no sickle cells (Fig. 5C, D).
Upon termination of the experiments mice were euthanized and necropsies performed. Average spleen weight of chimeric mice was 118.3 ± 18.5 mg (n = 9) compared with 893.2 ± 162.0 mg in SCD control mice (n = 6; p < 0.001; Fig. 5A). Typical spleen sizes in sickle and chimeric mice are shown in Fig. 5B. Chimeric mice had normal histology in spleen, kidney, liver and lung with no sickle cells in sinusoids of these organs (Fig. 5E–H).
Although SCD can be effectively cured by HSCT [22, 24,25,26,27] this curative approach has been limited to patients with matched donors and who are fit enough to receive myeloablative conditioning. More recently, studies by Brodsky et al. 2008 using non-myeloablative conditioning coupled with high dose cyclophosphamide, indicated that this approach could be potentially extended to haploidentical donors, and thereby markedly extend its use to almost every patient eligible for transplantation . Notably, in one study in which 2 Gy TBI was administered for conditioning, a high rate of graft rejection was observed , while more recently this problem was significantly resolved by increasing the radiation dose to 4 Gy . However, while the latter study represents a significant improvement, this approach is still associated with some risk for GvHD and for infections due to the long-term immune suppression required post-transplant for GvHD prophylaxis when using T cell-replete HSCT. Considering that other potential curative options for SCD, namely, gene therapy and genome editing, are increasingly feasible [29, 30], further improvement of safety and efficacy of haploidentical HSCT in SCD patients is needed to make this approach competitive.
Clearly, a major alternative strategy to enhance the safety of haploidentical HSCT is based on the use of rigorously TD-HSCT. To overcome the enhanced graft rejection associated with such transplants when applied following non-myeloablative conditioning, Archer’s group suggested the use of co-stimulation blockade (CTLA4-Ig and anti-CD40L) ; however, the use of anti-CD40L was found in a clinical trial to be complicated by cardiovascular thromboembolic events, which may have been caused by immune complexes consisting of soluble CD40L and anti-CD40L antibodies [31,32,33]. More recent development of anti-CD40 antibodies suggests that this strategy could potentially be safer .
Our data offer a strategy to potentially cure SCD with T-cell-depleted non-myeloablative HLA disparate transplants using central memory CD8 veto T-cells depleted of GvH reactivity by exposure to 3rd party stimulator cells with cytokines deprivation. The efficacy of these veto cells was initially confirmed in the context of wild type recipient mice . Furthermore, we found that upon transplantation of TDBM from C57BL/6 mice into Balb/c mice conditioned with sublethal 4.5GY TBI, veto central memory CD8 T cells were more effective when combined with a short treatment with low-dose rapamycin .
We have now found similar synergism between anti-3rd party central memory veto CD8 T cells and rapamycin in SCD mice, but sustained success required a slightly higher radiation dose of 5 Gy TBI. Neither veto Tcm nor rapamycin alone were able to prevent graft-failure but adding both resulted in long-term chimerism with no GvHD, despite no posttransplant immune suppression after day +4. Notably, while administration of 5 million CD8 T cells is associated with marked GvHD following such conditioning, we confirmed in the present study in the Berkeley mouse model for SCD that indeed as shown by Ophir et al. , CD8 T cells cultured against third party stimulators are markedly depleted of GvH reactivity while exhibiting strong veto activity. This synergism between the veto CD8 T-cells and rapamycin can be explained by the different mechanisms underlying their activities. The activity of veto CD8 T-cells is based on TCR activation in cognate anti-donor host T-cells and subsequent Fas upregulation resulting in apoptosis via triggering of FasL on the veto cells . In contrast, rapamycin interferes with a down-stream pathway of T-cell activation by inhibiting mammalian target of rapamycin (mTOR) complex activation, without blocking TCR-induced signaling. Although, inhibition of mTOR signaling leads to inhibition of Th1, Th2, and Th17  effector T-cells, it also promotes T-cell differentiation to a Foxp3+T-reg phenotype [37, 38]. Consequently, rapamycin induces tolerance and chimerism in H2-mis-matched mice, through veto T-cell-independent mechanisms [39,40,41,42] which can synergize with the Fas-FasL-based veto mechanism.
As expected, the long-term hematopoietic chimerism we observed resulted in complete conversion to normal donor-derived RBC and resolution of hematological and histo-pathological features of SCD in previously affected mice.
In conclusion, our results provide proof of concept for a curative approach that could potentially enable translation with low risk of transplant-related mortality, into an ethically justified clinical protocol for SCD patients. A clinical trial testing the safety and efficacy of central memory CD8 veto cells in recipients of non-myeloablative T-cell-depleted HLA-haplotype-matched transplants in patients with hematological cancers is in progress.
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Singh, A.K., Schetzen, E., Yadav, S.K. et al. Correction of murine sickle cell disease by allogeneic haematopoietic cell transplantation with anti-3rd party veto cells. Bone Marrow Transplant 56, 1818–1827 (2021). https://doi.org/10.1038/s41409-021-01237-6