Rabbit anti-thymocyte globulin (ATG (Thymoglobulin)) kills T cells in vitro and probably also in vivo as it prevents graft-vs-host disease (GvHD) in patients. Recently we demonstrated that ATG at a clinically relevant concentration (10–50 mg/L) kills in vitro not only T cells but also leukemic blasts. In the present study, we investigated whether ATG kills not only leukemic blasts but also leukemic stem cells (LSCs). We used a flow cytometric assay of complement-mediated cytotoxicity (CDC). ATG-induced death of acute myeloid leukemia (AML) cells from patients newly diagnosed with AML was measured among blasts as well as LSCs. At 10 mg/L ATG, blasts but not LSCs were killed. At 50 mg/L ATG, both blasts and LSCs were killed. We also measured ATG-mediated killing of healthy individuals’ hematopoietic stem cells (HSCs). Median 2% HSCs from blood and 15% HSCs from filgrastim-mobilized grafts were killed with 50 mg/L ATG, compared to 30% LSCs from the blood of AML patients (p = 0.001 and 0.022, respectively). In conclusion, LSCs are sensitive to ATG, however, only at a relatively high ATG concentration. At that concentration, LSCs are killed to a higher degree than HSCs.
Allogeneic hematopoietic cell transplantation (HCT) is a potential cure for patients with acute myeloid leukemia (AML). However, leukemia relapse and graft-versus-host disease (GvHD) are two major limitations to the success of HCT . Small molecule immunosuppressive drugs such as cyclosporine and methotrexate, although effective for decreasing the incidence of GvHD, increase the incidence of relapse [2, 3]. Interestingly, polyclonal rabbit anti-human thymocyte globulin (ATG, Thymoglobulin) added to myeloablative conditioning has been shown to reduce GvHD without affecting relapse [4,5,6]. The reason why ATG decreases GvHD but does not increase relapse is unclear. A potential explanation could be that ATG kills not only donor-anti-host T cells, responsible for mediating GvHD and graft-versus-leukemia effect (GVL), but also leukemic cells [7,8,9]. Recently, we showed that ATG induces complement-dependent cytotoxicity (CDC) and complement-independent cytotoxicity (CIC) of leukemic blasts . The killing of the blasts was relatively minor at 10 mg/L, which is the median maximum ATG serum concentration achieved in patients (range 1–25 mg/L) who received 4.5 mg/kg ATG . Major killing of the blasts was achieved with higher concentrations , including 50 mg/L, which is the median maximum ATG serum concentration achieved in patients who received 6 mg/kg ATG . There is a high patient-to-patient variability in the sensitivity of blasts to ATG—most are sensitive, but some appear resistant to the ATG-mediated killing .
It is unclear whether the “no increase in relapse” applies only to Thymoglobulin or also to Grafalon (rabbit anti-Jurkat T cell globulin). The “no increase in relapse” has been documented for Thymoglobulin in 3 of 3 randomized studies [4,5,6]. For Grafalon, there have been four randomized studies; two of them showed no increase in relapse [11, 12], but the remaining two studies showed a trend toward increased incidence of relapse [13, 14]. Our present study is focused on Thymoglobulin. Throughout this manuscript, the abbreviation “ATG” refers to anti-thymocyte globulin (Thymoglobulin) unless otherwise stated.
Relapse is the primary cause of mortality after chemotherapy without as well as with HCT [15,16,17,18]. The reason for relapse remains unclear. However, it has been long proposed that relapse occurs due to the persistence of a rare subset of relatively quiescent malignant cells that are not eradicated efficiently with current treatments, the so-called leukemic stem cells (LSCs) [19,20,21]. LSCs are defined as cells that (1) are capable of initiating the disease in an immunodeficient animal as well as sustaining the disease through multiple serial transplants (self-renewal) [22, 23], and (2) give rise to the bulk of leukemic cells. LSCs are difficult to eradicate with standard chemotherapy. This leads to disease resistance and relapse. It is thought that active anti-LSC therapies would prevent relapse. It is not known whether ATG kills LSCs.
AML LSCs are known to be more similar to healthy hematopoietic stem cells (HSCs) than to their differentiated progeny, including CD34+ CD38− phenotype and capacity to self-renew [22,23,24]. Therefore, ideal anti-LSC therapy must spare normal HSCs to preserve long-term hematopoiesis or prevent graft failure after HCT.
Here we studied the effect of ATG on leukemic blasts, LSCs, and HSCs.
Patients and methods
Patients and donors
To study LSCs, we obtained blood samples from 27 patients newly diagnosed with AML (before induction chemotherapy). Patient characteristics are shown in Table 1. To study healthy HSCs, we obtained blood from 10 healthy volunteers and filgrastim-mobilized blood stem cells (graft specimens) from 17 allogeneic graft donors. Characteristics of the healthy volunteers and the graft donors are shown in Table 1. Informed consents were obtained from both the patients and the donors. The study was approved by the Health Research Ethics Board of Alberta.
Determination of ATG cytotoxicity against stem cells by flow cytometry
Despite some controversy, both LSCs and HSCs are believed to be enriched in the CD34+ CD38– compartments . Here we assumed that the CD34+ CD38– cells from AML patients at diagnosis contain virtually only LSCs (the number of healthy HSCs being negligible) and that the CD34+ CD38– cells from healthy volunteers contain only healthy HSCs (and no LSCs). Thus, both LSCs and HSCs were defined as CD45dim/–, side scatter low (SSclow), lineage– (not expressing CD3, CD19, CD14, CD56, CD16, CD41a, or CD235a), CD34+, CD38– cells (Fig. 1). Blasts were defined as CD45dim/–, SSclow, and lineage– cells (Fig. 1). The borders between CD38 positive and negative cells and CD34 positive and negative cells were determined using fluorescence minus one (FMO) controls (Supplementary Fig. 1).
Percent dead cells (7-aminoactinomycin D (7AAD) positive) after exposure to ATG was measured in CDC assay. Percent apoptotic cells (Annexin V positive) after exposure to ATG was measured in CIC assay. The details of these assays, as well as the statistical analysis are described in Supplementary Appendix.
ATG at 50 mg/L, but not 10 mg/L, kills leukemic stem cells
CDC of LSCs was induced by 50 mg/L but not at 10 mg/L ATG (Fig. 2). At the 50 mg/L concentration, the median-adjusted (background subtracted) percent 7AAD+ LSCs was 38%. The sensitivity of LSCs to ATG at the 50 mg/L concentration was highly variable—the adjusted percent of 7AAD+ LSCs ranged from 0.2 to 98% (Supplementary Fig. 2).
Similar to CDC, CIC of LSCs was induced by 50 mg/L but not 10 mg/L ATG (Fig. 3). Contrary to CDC, even at the 50 mg/L concentration, the ATG-mediated CIC appeared weak—the median-adjusted percent Annexin V+ LSCs was only 13%. Similar to CDC, the sensitivity of LSCs to 50 mg/L ATG was variable—the adjusted percent Annexin V+ LSCs ranged from 0 to 37% (Supplementary Fig. 3).
For comparison, the killing of T cells in the patients was as follows: For CDC, the median-adjusted percent 7AAD+ T cells was 15% at 10 mg/L ATG (vs 0.5% for LSCs) and 59% at 50 mg/L ATG (vs 38% for LSCs) (Supplementary Fig. 4). For CIC, the median-adjusted percent Annexin V+ T cells was 17% at 10 mg/L (vs 7% for LSCs) and 27% at 50 mg/L (vs 13% for LSCs) (Supplementary Fig. 5).
Leukemic blasts are more sensitive to ATG than LSCs via CDC, but equally sensitive via CIC
Due to phenotypic and functional differences between leukemic blasts and LSCs [26, 27], ATG might have a different effect on leukemic blasts versus LSCs. Therefore, we compared leukemic blasts and LSCs in ATG-mediated CDC and CIC. We did this in 20 AML patients.
In the CDC assay, leukemic blasts were killed to a greater degree than LSCs (Fig. 4). At 10 mg/L ATG, the median-adjusted percent 7AAD+ cells was 3% among blasts vs 0.5% among LSCs (p = 0.006). At 50 mg/L ATG, it was 64% among blasts vs 38% among LSCs (p = 0.009).
In the CIC assay, at both 10 and 50 mg/L ATG, the killing of both blasts and LSCs was minor. In contrast to CDC, at both 10 and 50 mg/L concentration, there was no difference in killing of leukemic blasts vs LSCs (Supplementary Fig. 6).
Complement is not consumed in patients receiving standard dose of ATG
Given that LSCs were observed to be killed by ATG in the CDC assay at 50 mg/L but not 10 mg/L ATG, it might be theoretically desirable to use a high ATG dose (resulting in serum levels of ≥50 mg/L, achieved after 6–8 mg/kg ATG) , as this could minimize both relapse and GvHD. However, it is not known whether complement is depleted in patients after they have received a lower ATG dose. To address this question, we performed CDC assay against human lymphocytes (obtained from a healthy volunteer). We used serum of patients who received our conventional dose of ATG (0.5 mg/kg on day −2, 2.0 mg/kg on day −1, and 2.0 mg/kg on day 0, expected to lead to pre-graft infusion ATG concentration of ~10 mg/L)  as the source of complement. The sera were obtained from 10 allogeneic HCT patients on day 0, between finishing ATG infusion and starting graft infusion. The allogeneic HCT patient sera were compared to sera of autologous HCT patients, who did not receive ATG, in their ability to lyse lymphocytes with ATG at 50 mg/L and 300 mg/L. Pooled serum from healthy volunteers was also used for comparison. As negative controls, we used cells alone (without serum and without ATG), cells incubated with serum but no ATG, and cells incubated with ATG in the presence of heat-inactivated sera (56 °C for 1 h to destroy complement). As shown in Fig. 5, percent dead (7AAD+) lymphocytes after 15 min incubation with 50 mg/L ATG was higher when using sera from patients who received ATG (allogeneic HCT patients) vs patients who did not receive ATG (autologous HCT patients) (median 79% vs 72%, p = 0.03). Similar result was obtained for ATG at 300 mg/L. This suggests that complement is not depleted after administration of 4.5 mg/kg ATG. On the contrary, there appears to be more complement in the ATG-treated patients than the non-ATG-treated patients or healthy volunteers. The reason is unclear; it is conceivable that the partial consumption of complement on day −2 and −1 stimulated the liver to release or produce more complement. We also confirmed the “no depletion of complement” in an alternative experiment where we performed the CDC assay against HPB-ALL cells (T-cell line) instead of primary lymphocytes. As shown in Supplementary Fig. 7, percent dead (7AAD+) HPB-ALL cells after 15 min incubation with 300 mg/L ATG was similar when using sera from patients vs healthy volunteers (median 98% vs 96%, respectively). Thus, complement is not depleted after the standard ATG dose of 4.5 mg/kg.
ATG kills LSCs to a greater degree than healthy HSCs
Given similarities between LSCs and healthy HSCs [22, 28], the effect of ATG on healthy HSCs should be considered. We compared ATG-mediated killing of LSCs from the blood of 10 AML patients vs HSCs from the blood of 10 healthy volunteers. As HSCs in healthy blood are sparse (Table 1), we immunomagnetically enriched CD34+ cells from the blood of both the patients and the volunteers. Given limited number of CD34+ cells obtained from the volunteers, we performed only the CDC (and not CIC) assay, since CDC appears to be the more important mechanism of ATG-induced killing (compare Figs. 2 and 3), and used only the 50 mg/L ATG concentration, which is the relevant concentration for killing LSCs (Fig. 2). As shown in Fig. 6, the median percents dead (7AAD+) LSCs and HSCs after treatment with 50 mg/L ATG were 40% (range 1 to 90%) and 1% (range 0 to 30%), respectively (p = 0.001). This suggests that LSCs are more sensitive to ATG-induced killing than HSCs.
During HCT, patients receive apheresed MNCs from donors treated with filgrastim (to mobilize HSCs from marrow to blood). Thus, comparing LSCs to HSCs from the grafts (post-filgrastim) may be clinically more relevant than to HSCs from the blood of healthy volunteers (who have not received filgrastim). Therefore, we compared LSCs from the blood of 17 AML patients to HSCs from 17 graft samples. No immunomagnetic enrichment for CD34+ cells was done (as percentages of CD34+ cells in both patient blood and donor grafts were sufficient). As shown in Fig. 7, median percents 7AAD+ LSCs and graft HSCs after treatment with 50 mg/L ATG were 39% (range 0 to 98%) and 16% (range 0 to 39%), respectively (p = 0.022). This suggests that LSCs are also more sensitive to ATG-induced complement-mediated lysis than graft HSCs.
Unlike the case of healthy HSCs from blood, in the graft specimens, we were able to perform not only CDC but also CIC assays due to sufficient number of HSCs. Thus, we evaluated CIC (using 50 mg/L ATG) against LSCs and graft-HSCs. Similar to CDC, the LSCs were significantly more sensitive to ATG in the CIC assay than the graft HSCs (p = 0.036) (Supplementary Fig. 8).
Anti-leukemic activity of Thymoglobulin versus Grafalon
We wished to compare Thymoglobulin and Grafalon in their ability to kill (by CDC) LSCs, including at concentrations above 50 mg/L as Grafalon is known to kill immune cells at higher concentrations than Thymoglobulin [29, 30]. However, our CDC assay did not provide meaningful results when >50 mg/L ATG (Thymoglobulin or Grafalon) was used. At those concentrations ATG outcompeted fluorochrome-labeled antibodies in binding to the antigens used to define LSCs. Therefore, we decided to instead compare Grafalon and Thymoglobulin in their ability to kill leukemic cells (blasts + LSCs). We used blood mononuclear cells (MNCs) from 10 AML patients with very high blast percentage (median 90% CD45dim/– CD34+ cells among the MNCs, range 79–96%) and assumed that virtually all the MNCs were leukemic cells. This avoided the need to identify cells by surface antigens. As shown in Supplementary Fig. 9, both Grafalon and Thymoglobulin killed leukemic cells, however, a higher concentration of Grafalon was needed to achieve the same effect—approximately 40% leukemic cells were killed by 100 mg/L Grafalon or 50 mg/L Thymoglobulin. Thus, we conclude that both Grafalon and Thymoglobulin kill (by CDC) leukemic cells. However, as previously shown for immune cells [29, 30], a higher concentration of Grafalon is needed to achieve the same degree of killing of leukemic cells (by CDC). This is reminiscent of CIC, which was induced in leukemic cells from 4 of 8 AML patients using a relatively high concentration of Grafalon (250 mg/L) .
Previously, we demonstrated that ATG (Thymoglobulin) at clinically relevant concentration induces cytotoxicity against leukemic blasts . In the present study, we show that ATG induces cytotoxicity also against LSCs. However, the cytotoxicity occurred with 50 mg/L and not 10 mg/L, the latter being the approximate median maximum concentration achieved in patients at our center using 4.5 mg/kg ATG. Nonetheless, 50 mg/L is clinically achievable—Remberger et al. reported median maximum concentration ≥50 mg/L using 6.0 mg/kg ATG and ≥80 mg/L using 8.0 mg/kg ATG . As even higher ATG doses (10–40 mg/kg) have been used in clinic [32,33,34], it is likely that even higher ATG concentrations (>50 mg/L) can be achieved in patients.
Chemotherapeutic agents clinically used today eradicate blasts/rapidly dividing cells. However, the same agents demonstrate little to no effect on the blast progenitor cells, i.e., the LSCs [35, 36]. For example, cytarabine has virtually no activity against LSCs . However when tested on leukemic blasts from the same patients, cytarabine shows potent cytotoxicity against blasts. Here we showed that Thymoglobulin kills both blasts and LSCs, albeit LSCs only at a relatively high concentration.
Even though the majority of the studies indicate that phenotypically LSCs resemble more HSCs than their mature progeny, recent studies on AML suggest that some differences between LSCs and HSC exist [38,39,40]. Therefore, it is possible that LSCs might be more or less sensitive to therapy than HSCs. From a therapeutic perspective, it is crucial, when studying the anti-leukemic activity of drugs against LSCs, to also consider their effect against HSCs to account for non-specific toxicities such as graft failure or delayed engraftment. Both Thymoglobulin and Grafalon are known to delay engraftment [4, 41]. Whereas Thymoglobulin has not been conclusively shown to induce graft failure, one study has shown that the incidence of graft failure was higher in patients treated with Grafalon compared to placebo . Our results suggest that via CDC, neither LSCs nor HSCs are sensitive to ATG at 10 mg/L. With a fivefold higher concentration, both LSCs and HSCs are sensitive to ATG. However, LSCs are more sensitive. The reason for the different sensitivity of LSCs vs HSCs is unclear. Perhaps, LSCs express antigens targetted by ATG not expressed or less expressed by HSCs. For example, ATG is known to contain antibodies against CD44, and the expression of CD44 is higher on LSCs than HSCs [9, 42, 43]. Moreover, treatment with H90, an anti-CD44 antibody was shown to eradicate AML LSCs in vivo in immunodeficient mice while not altering normal hematopoiesis, making it an attractive target for LSCs therapy while sparing HSCs . In addition to CD44, ATG also contains antibodies against CD25, CXCR4, CD32 which are either exclusively expressed on LSCs (and not HSCs) or more expressed on LSCs than on HSCs [9, 44].
What is the relevance of our in vitro finding of ATG killing LSCs to patients, given that so far no study has shown a lower incidence of relapse with ATG compared to no ATG and that ATG is expected to kill not only LSCs but also donor T cells mediating graft-vs-leukemia effect (GVL)? We speculate that the “no decrease in relapse” could be due to different effects of pre-HCT vs post-HCT ATG area under the curve (AUC) on relapse. Only the post-HCT AUC (but not the pre-HCT AUC) is expected to interfere with the GVL. Consistent with that, a high post-HCT AUC has been associated with a trend toward increased incidence of relapse . Theoretically (if our in vitro observation applies to in vivo), a high pre-HCT AUC should be associated with a decreased incidence of relapse. Consistent with that, we have preliminary data showing a trend towards decreased incidence of relapse in patients with a high pre-HCT AUC and a trend towards increased incidence of relapse in patients with a high post-HCT AUC. If this is confirmed, it will suggest that administration of ATG designed to result in high pre-HCT AUC and relatively low post-HCT AUC (high-dose ATG given early in conditioning) could be beneficial for minimizing relapse.
There are several limitations in our study: (1) We defined both LSCs and HSCs as CD34+ and CD38–, as is common practice in other studies . However, some studies show that LSCs and HSCs are not limited to the CD34+ CD38– compartment but are also present in the CD34+ CD38+ or the CD34– compartment [47, 48]. Nonetheless, both in vitro and in vivo studies have provided compelling evidence that the CD34+ CD38– LSCs are probably the most relevant LSCs. For example, CD34+ CD38– frequency, but not CD34+ CD38+, total CD34+, and CD34– frequencies at diagnosis correlates with engraftment in immunocompromised mice or with relapse rates in patients [25, 49]. We could have theoretically overcome this limitation by using additional markers of LSCs, but in practice, generally accepted additional LSC markers are lacking. Following the initial publications that supported the LSC activity to be limited to the CD34+ CD38– population [22, 23], additional identification markers for LSCs have been proposed including CD123 [38, 50], CD32 , CD33 , CD45RA , CD47 , CD96 , CD99 , IL1RAP , and TIM-3 . However, to date, there is no standard phenotype for defining LSCs in AML. Therefore, and because of the high intra-patient and inter-patient heterogeneity of AML LSC populations [47, 58,59,60], we defined LSCs as per the conventional phenotype, i.e., CD45dim/–, SSclow, Lin–, CD34+, and CD38–. (2) Due to the lack of a sophisticated antigen panel, we were unable to differentiate between LSCs and HSCs in our study phenotypically. Therefore, it is possible that the LSCs defined in our study could also contain healthy HSCs. However, the fraction of healthy HSCs (defined as CD34+ cells) is meager (0.01% of the total nucleated cells in blood) . Given the high percentage of CD34+ CD38– cells among the leukemic blasts in the majority of the specimens (Table 1), we assume that the majority of cells having the CD45dim/–, SSclow, Lineage–, CD34+, CD38– phenotype are LSCs. (3) Our experiments were performed using blood, whereas the marrow is the “home” tissue of both LSCs and HSCs. Therefore, comparison of bone marrow-derived LSCs with bone marrow-derived HSCs would have been more relevant. However, the feasibility of a comparison of marrow LSCs versus marrow HSCs is limited by practical and ethical constraints of obtaining marrow from healthy volunteers. (4) The CD34+CD38− population may contain not only LSCs or HSCs but also their progeny. We cannot exclude the possibility that the killed (7AAD+ or Annexin V+) cells among the CD34+CD38− cells belonged to the progeny rather than the LSCs/HSCs. (5) We measured the anti-leukemic activity only in vitro. Studying the anti-leukemic activity in vivo using immunocompromised mice harboring human leukemia could be more informative. However, it may also have the disadvantage of using mouse complement, which may be triggered with the rabbit ATGs to a different degree than human complement.
Despite the limitations mentioned above, our study is the first to demonstrate the effect of ATG against LSCs and, to a lesser degree, HSCs. The anti-LSC effect is, however, significant only at a relatively high ATG concentration, achievable probably with ATG doses of ≥6 mg/kg. Targeting high pre-transplant ATG AUC might lead to further reduction in GvHD  and possibly also relapse, if what we have described in vitro applies in vivo.
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We thank the patients for participating in research that could not benefit them but only future patients. We thank the healthy volunteers involved in the study. This study could not happen without the dedication of Mamta Kantharia, Jennifer LeBlanc, Lori Rackel, Laura Spilchen, many inpatients nurses, pharmacists, particularly Michelle Dowhan, and physicians, notably Dr. Michelle Geddes, Dr. Mona Shafey, Dr. Peter Duggan, and Dr. Lynne Savoie. We also thank the staff of Calgary Laboratory Services, including Glenis Doiron. Finally, we thank Douglas Mahoney for invaluable feedback during this study.
R.D. developed the cytotoxicity assays, ran the assays, performed statistical analysis, and analyzed the results. P.D.K. and J.L. provided input into assay development and interpretation. M.M., T.v.S., and J.B. collected or arranged for the collection of specimens. A.D., D.M., F.M.K., and J.S. provided critical feedback. R.D. and J.S. designed the study, and J.S. supervised its conduct. R.D. wrote the manuscript.
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The authors declare that they have no conflict of interest.
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Dabas, R., Dharmani-Khan, P., Modi, M. et al. Anti-thymocyte globulin’s activity against acute myeloid leukemia stem cells. Bone Marrow Transplant 54, 549–559 (2019). https://doi.org/10.1038/s41409-018-0296-0