Murine Hematopoietic Cell Transplantation Model Research Articles

Posttransplantation cyclophosphamide expands functional myeloid-derived suppressor cells and indirectly influences Tregs.

Posttransplantation cyclophosphamide (PTCy), given on days+3 and+4, reduces graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT), but its immunologic underpinnings are not fully understood. In a T-cell-replete, major histocompatibility complex-haploidentical murine HCT model (B6C3F1→B6D2F1), we previously showed that PTCy rapidly induces suppressive mechanisms sufficient to prevent GVHD induction by non-PTCy-exposed donor splenocytes infused on day+5. Here, in PTCy-treated mice, we found that depleting Foxp3+ regulatory T cells (Tregs) in the initial graft but not the day+5 splenocytes did not worsen GVHD, yet depleting Tregs in both cellular compartments led to fatal GVHD induced by the day+5 splenocytes. Hence, Tregs were necessary to control GVHD induced by new donor cells, but PTCy's impact on Tregs appeared to be indirect. Therefore, we hypothesized that myeloid-derived suppressor cells (MDSCs) play a complementary role. Functionally suppressive granulocytic and monocytic MDSCs were increased in percentages in PTCy-treated mice, and MDSC percentages were increased after administering PTCy to patients undergoing HLA-haploidentical HCT. PTCy increased colony-stimulating factors critical for MDSC development and rapidly promoted the generation of MDSCs from bone marrow precursors. MDSC reduction via anti-Gr1 treatment in murine HCT did not worsen histopathologic GVHD but resulted in decreased Tregs and inferior survival. The clinical implications of these findings, including the potential impact of expanded MDSCs after PTCy on engraftment and cytokine release syndrome, remain to be elucidated. Moreover, the indirect effect that PTCy has on Tregs, which in turn play a necessary role in GVHD prevention by initially transplanted or subsequently infused T cells, requires further investigation.

Post-Transplantation Cyclophosphamide Uniquely Restrains Alloreactive CD4+ T-Cell Proliferation and Differentiation After Murine MHC-Haploidentical Hematopoietic Cell Transplantation.

Post-transplantation cyclophosphamide (PTCy) reduces the incidence and severity of graft-versus-host disease (GVHD), thereby improving the safety and accessibility of allogeneic hematopoietic cell transplantation (HCT). We have shown that PTCy works by inducing functional impairment and suppression of alloreactive T cells. We also have identified that reduced proliferation of alloreactive CD4+ T cells at day +7 and preferential recovery of CD4+CD25+Foxp3+ regulatory T cells (Tregs) at day +21 are potential biomarkers associated with optimal PTCy dosing and timing in our B6C3F1→B6D2F1 MHC-haploidentical murine HCT model. To understand whether the effects of PTCy are unique and also to understand better the biology of GVHD prevention by PTCy, here we tested the relative impact of cyclophosphamide compared with five other optimally dosed chemotherapeutics (methotrexate, bendamustine, paclitaxel, vincristine, and cytarabine) that vary in mechanisms of action and drug resistance. Only cyclophosphamide, methotrexate, and cytarabine were effective in preventing fatal GVHD, but cyclophosphamide was superior in ameliorating both clinical and histopathological GVHD. Flow cytometric analyses of blood and spleens revealed that these three chemotherapeutics were distinct in constraining conventional T-cell numerical recovery and facilitating preferential Treg recovery at day +21. However, cyclophosphamide was unique in consistently reducing proliferation and expression of the activation marker CD25 by alloreactive CD4+Foxp3- conventional T cells at day +7. Furthermore, cyclophosphamide restrained the differentiation of alloreactive CD4+Foxp3- conventional T cells at both days +7 and +21, whereas methotrexate and cytarabine only restrained differentiation at day +7. No chemotherapeutic selectively eliminated alloreactive T cells. These data suggest that constrained alloreactive CD4+Foxp3- conventional T-cell numerical recovery and associated preferential CD4+CD25+Foxp3+ Treg reconstitution at day +21 may be potential biomarkers of effective GVHD prevention. Additionally, these results reveal that PTCy uniquely restrains alloreactive CD4+Foxp3- conventional T-cell proliferation and differentiation, which may explain the superior effects of PTCy in preventing GVHD. Further study is needed to determine whether these findings also hold true in clinical HCT.

Phase I/II Study of Reduced Dosing of Post-Transplantation Cyclophosphamide (PTCy) after HLA-Haploidentical Bone Marrow Transplantation

Background: Post-transplantation cyclophosphamide (PTCy) is widely used for graft-versus-host-disease (GVHD) prophylaxis after hematopoietic cell transplantation (HCT). However, the standard PTCy dose (50 mg/kg/day) and timing (given on days +3 and +4) were largely extrapolated from murine skin allografting models, and these have never been rigorously tested to determine if they are optimal. We have shown in murine HCT models that an intermediate PTCy dose of 25 mg/kg/day on days +3/+4 is superior at preventing severe GVHD compared with 50 mg/kg/day on days +3/+4 (Wachsmuth et al., JCI. 2019). Furthermore, PTCy 25 mg/kg/day given on day +4 alone is equivalent to 25 mg/kg/day given on days +3/+4 at preventing severe GVHD (Wachsmuth et al. BBMT. 2019). Thus, the standard clinical dosing may be higher than necessary and potentially come at the cost of increased toxicity, delayed engraftment, and impaired immune reconstitution.Methods: This is a single institutional prospective phase I/II study (NCT03983850) to de-escalate PTCy exposure for adult patients with hematologic malignancies. All patients received myeloablative conditioning with IV daily busulfan/fludarabine, HLA-haploidentical bone marrow, and GVHD prophylaxis with PTCy (dose and timing based on Dose Level (DL)), mycophenolate mofetil (days +5 to +35), and sirolimus (days +5 to +80).The first 5 patients received PTCy 50 mg/kg/day on days +3/+4 (standard dosing, Dose Level 1) for comparative data. This was followed by a 3+3 dose de-escalation design testing 25 mg/kg/day on days +3/+4 (experimental, Dose Level 2) and 25 mg/kg on day +4 only (experimental, Dose Level 3), followed by a phase II expansion cohort at the better experimental dose level. The primary endpoint and dose limiting toxicity for the dose de-escalation was grade III-IV acute GVHD.Results: Phase I enrolled 19 patients, and the phase II expansion has enrolled 13 of 14 patients, 9 of whom have sufficient follow-up (60 days) to be considered evaluable for the primary endpoint; median follow-up of survivors evaluable for the primary endpoint is 288 (range 60-676) days. Patient and disease characteristics and outcomes for these patients are summarized in Table 1.No grade III-IV acute GVHD was seen at either experimental DL, and there have been no cases of grade II-IV acute GVHD at DL2 (Figure 1). Based on more reliable early engraftment (see below) but less intense and shorter duration of engraftment fevers, DL2 was taken to phase II.Engraftment was faster at the experimental DLs but was most consistent with DL2. Median neutrophil engraftment was at day 14 at DL2 compared with day 19 at the standard dosing (p=0.0004) (Figure 2A). Median platelet engraftment was at day 23 at DL2 compared with day 33 at the standard dosing (p=0.026) (Figure 2B). Correspondingly, transfusional requirements were lessened with DL2 (Figure 2C-D). Engraftment syndrome, manifesting as fever, rash, and mild transaminitis, occurred in 1 patient at DL2 and 2 patients at DL3, but resolved rapidly without intervention in all cases. Primary graft failure was seen in 1 patient at DL2, and relapse prior to engraftment was seen in 1 patient at DL3.Mucositis was less severe and shorter in duration for both experimental DLs when compared with standard PTCy dosing (Figure 2E). CMV reactivation requiring pre-emptive therapy was less frequently seen after lower PTCy dosing (Figure 3A). Symptomatic BK virus-associated cystitis in at-risk patients was shorter in duration for patients receiving lower PTCy; median duration was 74 days for the standard DL versus 26 days at DL2 and 7 days at DL3 (Figure 3B). At DL2, there have been 3 cases to date of chronic GVHD requiring systemic immunosuppression among the 13 engrafting patients with at least 100 days of follow-up.Conclusions: De-escalating PTCy exposure is feasible and effective in maintaining protection against severe acute GVHD while promoting more rapid engraftment and less early post-transplant toxicity. Two-day dosing of 25 mg/kg/day PTCy appears to allow for more consistent early engraftment and protection against protracted engraftment fevers compared with day +4 only. Longer follow-up and comparative studies are needed to understand if de-escalated PTCy is superior to the standard dosing schedule. [Display omitted] DisclosuresNo relevant conflicts of interest to declare. OffLabel Disclosure:The use of cyclophosphamide as graft-versus-host disease prophylaxis

Post-Transplantation Cyclophosphamide Induces Alloreactive T-Cell Functional Impairment and Depends on Foxp3+ Regulatory T Cells Not Selective Elimination or Intrathymic Clonal Deletion of Alloreactive T Cells

Our understanding of how post-transplantation cyclophosphamide (PTCy) prevents graft-versus-host disease (GVHD) in hematopoietic cell transplantation (HCT) is largely extrapolated from major histocompatibility complex (MHC)-matched murine skin allografting models. This current paradigm of understanding posits that PTCy preferentially eliminates alloreactive T cells and is also dependent on intrathymic clonal deletion of alloreactive T cells. Skin allografting data used to generate this model leveraged strain-specific differential expression of certain T-cell receptor (TCR) Vβs as markers of alloreactive cells.To test the hypothesis that PTCy works via selective elimination of alloreactive T cells and investigate the biology of PTCy, we developed a murine MHC-haploidentical HCT model that directly parallels human sibling haploidentical HCT. In this model (B6C3F1→B6D2F1), the administration of 40 x 106 splenocytes and 10 x 106 T-cell-depleted bone marrow cells following 10.5 Gy irradiation induced universally fatal GVHD, with the Vβ6 TCR serving as a marker of alloreactive T cells. Administration of PTCy on days +3 and +4 at doses ranging from 10 to 50 mg/kg effectively prevented lethal GVHD.In assessments of the blood, lymph nodes, spleen, and liver, PTCy 25 mg/kg did reduce proliferation of CD4+ T cells but not CD8+ T cells at day +7 post-transplant. Vβ6+ T cells in both the CD4+ and CD8+ subsets in all four tissue compartments persisted at or above normal donor frequencies at days +7, +21, and +200 post-transplant. Persistence of Vβ6+ T cells in all four tissue compartments also was demonstrated in three other murine HCT models: B6→B6D2F1 (MHC-haploidentical; day +7), C3H→B6D2F1 (MHC-disparate; days +6 and +200), and C3H→AKR (MHC-matched; day +7). Using our B6C3F1→B6D2F1 model, we also created grafts in which 8% of donor CD8+ T cells expressed the 2C TCR, which is specific for H2-Ld (a major histocompatibility antigen in our model). 2C TCR+ T cells continued to proliferate highly and markedly expand from days +3 to +7 despite administration of PTCy 25 mg/kg on days +3 and +4, disproving selective alloreactive T-cell elimination after PTCy treatment in HCT. Additionally, PTCy was equally effective in our wild-type B6C3F1→B6D2F1 model when using thymectomized or non-thymectomized recipients, negating a necessary role for the thymus in GVHD prevention by PTCy.Compartmentalization could not explain GVHD amelioration despite persistence of alloreactive T cells as similarly normal Vβ6+ frequencies were found infiltrating the liver, a GVHD target organ, as were found in the blood, lymph nodes, or spleen. Indeed, 2C TCR+ T cells composed 70-80% of liver-infiltrating CD8+ T cells at day +7 in mice treated with or without PTCy. Yet, compared with vehicle-treated mice, liver-infiltrating wild-type T cells obtained at day +21 from PTCy-treated mice had slightly reduced proliferation but dramatically lower cytokine production when restimulated in vitro with DBA/2 (host parental) irradiated splenocytes. Similar results were seen when repeating these experiments using liver-infiltrating T cells that were flow cytometrically depleted of CD4+CD25+ T cells, suggesting impaired functionality of alloreactive T cells after PTCy.CD4+CD25+Foxp3+ T cells did preferentially expand in all four tissue compartments by day +21 post-transplant. A one-log reduction of these cells in the donor graft using an AutoMACS had minimal impact on transplant outcomes in our model. However, a two-log reduction of Foxp3+ cells immediately post-transplant in vivo using Foxp3-DTR-expressing donor cells resulted in marked worsening of the clinical scores and weights, which began to diverge starting approximately 30 days post-transplant. PTCy-treated mice also were resistant to reinfusion of 40 or 120 x 106 new donor splenocytes at +120-150 days, consistent with suppression being an active mechanism underlying PTCy's efficacy. Overall, our findings disprove the existing paradigm of understanding of how PTCy prevents GVHD, support our previously demonstrated role for regulatory T cells, and may have direct translational relevance towards improving outcomes for patients. DisclosuresNo relevant conflicts of interest to declare.

Recipient-specific T-cell repertoire reconstitution in the gut following murine hematopoietic cell transplant

AbstractGraft-versus-host disease (GVHD) is a complication of hematopoietic cell transplantation (HCT) caused by alloreactive T cells. Murine models of HCT are used to understand GVHD and T-cell reconstitution in GVHD target organs, most notably the gastrointestinal (GI) tract where the disease contributes most to patient mortality. T-cell receptor (TCR) repertoire sequencing was used to measure T-cell reconstitution from the same donor graft (C57BL/6 H-2b) in the GI tract of different recipients across a spectrum of matching, from syngeneic (C57BL/6), to minor histocompatibility (MHC) antigen mismatch BALB.B (H-2b), to major MHC mismatched B10.BR (H-2k) and BALB/c (H-2d). Although the donor T-cell pools had highly similar TCR, the TCR repertoire after HCT was very specific to recipients in each experiment independent of geography. A single invariant natural killer T clone was identifiable in every recipient group and was enriched in syngeneic recipients according to clonal count and confirmatory flow cytometry. Using a novel cluster analysis of the TCR repertoire, we could classify recipient groups based only on their CDR3 size distribution or TCR repertoire relatedness. Using a method for evaluating the contribution of common TCR motifs to relatedness, we found that reproducible sets of clones were associated with specific recipient groups within each experiment and that relatedness did not necessarily depend on the most common clones in allogeneic recipients. This finding suggests that TCR reconstitution is highly stochastic and likely does not depend on the evaluation of the most expanded TCR clones in any individual recipient but instead depends on a complex polyclonal architecture.

Phenotypic Myeloid-Derived Suppressor Cells (MDSCs) are expanded after Post-Transplantation Cyclophosphamide (PTCy), but are not necessary for Graft-Versus-Host Disease Prevention by PTCy

The use of PTCy substantially lowers the rates of severe acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT), but the mechanisms of its action are incompletely understood. Recently, we showed in a T-cell–replete, MHC-haploidentical, murine HCT model (B6C3F1→B6D2F1) that PTCy prevents GVHD not by eliminating alloreactive T cells but instead by inducing alloreactive T-cell dysfunction and suppression. Depletion of CD4+regulatory T cells (Tregs) early post-transplant did not result in substantially worsening GVHD for ∼4 weeks. Surprisingly, we found that alloreactive T cells could be infused in extremely high numbers early after PTCy but not cause GVHD. Here, we used this same MHC-haploidentical murine HCT model to test the role of Tregs in preventing GVHD induced by infusion of new splenocytes after PTCy; depleting Tregs in the initial graft but not the reinfused splenocytes did not result in worsening GVHD in these mice. However, when Tregs were depleted both in the initial graft and in the reinfused splenocytes, universally fatal GVHD ensued. These results suggested that Tregs were necessary to prevent GVHD but a direct effect of PTCy on Tregs was not required. Thus, we hypothesized that other suppressive cell populations may be playing a critical role early after PTCy. We examined the frequency of MDSCs after PTCy in our model and found that both granulocytic and monocytic MDSCs were increased at post-transplant days +7 and +21 compared with vehicle-treated mice. Next we examined the role of MDSCs in GVHD prevention after PTCy by using a Gr-1-depleting antibody. Gr-1 depletion immediately post-transplant led to decreased percentages of Tregs and inferior survival, clinical scores, and weights in PTCy-treated mice. Conversely, Gr-1 depletion at days +28 or +150 had a transient effect only on weights but no impact on clinical scores or survival. Yet, histopathologic evaluation revealed that mice depleted of MDSCs early post-transplant did not have worse GVHD; instead the worse survival in those mice appeared related to infection presumably as a result of neutrophil depletion by the anti-Gr-1 antibody in mice with mucosal disruption by radiation and GVHD. We also tested the suppressive functionality of MDSCs on alloreactive T cells in in vitro mixed lymphocyte cultures. We found neither population of MDSCs from either vehicle-treated or PTCy-treated mice suppressed the proliferation of vehicle-treated T cells nor was inflammatory cytokine secretion reduced. Overall, these findings confirm that Tregs are required for GVHD prevention by PTCy but a direct effect of PTCy on Tregs is not required; although phenotypic MDSCs are expanded after PTCy and may contribute to Treg expansion, these MDSCs do not appear functionally capable of suppressing alloreactive T-cell response to alloantigen in vitro or decreasing GVHD in vivo.

ST2/MyD88 Deficiency Protects Mice against Acute Graft-versus-Host Disease and Spares Regulatory T Cells.

Acute graft-versus-host disease (aGVHD) hinders the efficacy of allogeneic hematopoietic cell transplantation (HCT). Plasma levels of soluble membrane-bound ST2 (ST2) are elevated in human and murine aGVHD and correlated to type 1 T cells response. ST2 signals through the adapter protein MyD88. The role of MyD88 in T cells during aGVHD has yet to be elucidated. We found that knocking out MyD88 in the donor T cells protected against aGVHD independent of IL-1R and TLR4 signaling in two murine HCT models. This protection was entirely driven by MyD88-/- CD4 T cells. Transplanting donor MyD88-/- conventional T cells (Tcons) with wild-type (WT) or MyD88-/- regulatory T cells (Tregs) lowered aGVHD severity and mortality. Transcriptome analysis of sorted MyD88-/- CD4 T cells from the intestine 10 d post-HCT showed lower levels of Il1rl1 (gene of ST2), Ifng, Csf2, Stat5, Batf, and Jak2 Transplanting donor ST2-/- Tcons with WT or ST2-/- Tregs showed a similar phenotype with what we observed when using donor MyD88-/- Tcons. Decreased ST2 was confirmed at the protein level with less secretion of soluble ST2 and more expression of ST2 compared with WT T cells. Our data suggest that Treg suppression from lack of MyD88 signaling in donor Tcons during alloreactivity uses the ST2 but not the IL-1R or TLR4 pathways, and ST2 represents a potential aGVHD therapeutic target sparing Tregs.

Post-transplantation cyclophosphamide prevents graft-versus-host disease by inducing alloreactive T cell dysfunction and suppression.

Post-transplantation cyclophosphamide (PTCy) recently has had a marked impact on human allogeneic hematopoietic cell transplantation (HCT). Yet, our understanding of how PTCy prevents graft-versus-host disease (GVHD) largely has been extrapolated from major histocompatibility complex (MHC)-matched murine skin allografting models that were highly contextual in their efficacy. Herein, we developed a T-cell-replete, MHC-haploidentical, murine HCT model (B6C3F1→B6D2F1) to test the putative underlying mechanisms: alloreactive T-cell elimination, alloreactive T-cell intrathymic clonal deletion, and suppressor T-cell induction. In this model and confirmed in four others, PTCy did not eliminate alloreactive T cells identified using either specific Vβs or the 2C or 4C T-cell receptors. Furthermore, the thymus was not necessary for PTCy's efficacy. Rather, PTCy induced alloreactive T-cell functional impairment which was supported by highly active suppressive mechanisms established within one day after PTCy that were sufficient to prevent new donor T cells from causing GVHD. These suppressive mechanisms included the rapid, preferential recovery of CD4+CD25+Foxp3+ regulatory T cells, including those that were alloantigen-specific, which served an increasingly critical function over time. Our results prompt a paradigm-shift in our mechanistic understanding of PTCy. These results have direct clinical implications for understanding tolerance induction and for rationally developing novel strategies to improve patient outcomes.

PD-L1/CD80 and PD-L1/PD-1 Signaling Reciprocally Regulate Alloreactive CD8+ T Cell Glycolysis, Proliferation, Apoptosis and Gvhd-Inducing Capacity

Graft versus host disease (GVHD) remains the major obstacle for widespread application of allogeneic hematopoietic cell transplantation (HCT), a curative therapy for hematological malignancies. We and others have reported that while sorted CD4+ T cells induce severe acute GVHD, the same numbers of CD8+ T cells induce no acute GVHD. CD8+ T cells can facilitate donor cell engraftment and mediate graft versus leukemia (GVL) effect in the absence of GVHD, but the mechanisms remain largely unknown. Programmed death-ligand 1 (PD-L1) interacts with both CD80 and PD-1. We have recently reported that simultaneous PD-L1/CD80 and PD-L1/PD-1 signaling augments activated alloreactive CD4+ T cell proliferation and apoptosis and ameliorates acute GVHD. However, the impact of this PD-L1-mediated signaling on alloreactive CD8+ T cells has not been investigated. In addition, T cell glycolytic metabolism has been recently reported to be able to regulate acute GVHD, and PD-L1/PD-1 signaling inhibits CD4+ T cell glycolytic flux, but how PD-L1/CD80 modulates T cell metabolism remains unstudied. In the current studies, we evaluated the role of PD-L1/CD80 and PD-L1/PD-1 signaling on alloreactive CD8+ T cell glycolysis, proliferation, apoptosis and GVHD-inducing capacity, using a MHC-mismatched murine HCT model of C57BL/6 (H-2b) donor to BABL/c recipient (H-2d). We have observed the following early (3-5 days) after HCT: 1) In wild-type (WT) recipients, PD-1-/- CD8+ T cells significantly upregulate expression of Glucose Transporter I (Glut1) and exhibit much higher rates of glycolytic flux; PD-1-/- CD8+ T cells also show markedly enhanced proliferation and reduced apoptosis as well as up-regulation of gut tissue homing and chemokine receptors (i.e. α4β7 and CCR9) and induce lethal GVHD, as compared to WT CD8+ T cells that induce little signs of GVHD. 2) In PD-L1-/- recipients, PD-1-/- CD8+ T cells reduce expression of Glut1 and no longer exhibit enhanced glycolytic flux or reduced apoptosis; PD-1-/- CD8+ T cells transplanted into PD-L1-/- recipients also induce little GVHD, similar to WT CD8+ T cells. 3) After injection of anti-PD-L1 mAb (clone 43H12) that specifically blocks PD-L1/CD80 interaction and preserves PD-L1/PD-1 interaction, WT CD8+ T cells in WT recipients show drastically reduced expansion, as compared to control recipients treated with rat-IgG. Blockade of PD-L1/CD80 interaction reduces phosphorylation of key elements in the TCR signaling cascade and CD28 co-stimulatory pathway including ZAP-70, AKT, mTOR and rpS6. Taken together, these studies indicate that 1) PD-L1/CD80 signaling alone augments alloreactive CD8+ T cell expansion and GVHD-inducing capacity; 2) PD-L1/CD80 and PD-L1/PD-1 signaling reciprocally regulate CD8+ T cell glycolysis, proliferation, and apoptosis as well as their GVHD-inducing capacity, and a balance of the two signaling pathways would be required to allow donor CD8+ T cells to facilitate engraftment and mediate GVL effect without causing GVHD. This work was supported by Institutional Funds of The Beckman Research Institute of City of Hope. DisclosuresNo relevant conflicts of interest to declare.

Murine Cytomegalovirus Immediate-Early 1 Gene Expression Correlates with Increased GVHD after Allogeneic Hematopoietic Cell Transplantation in Recipients Reactivating from Latent Infection

The success of allogeneic (allo) hematopoietic cell transplantation (HCT) is limited by its treatment related complications, mostly graft versus host disease (GVHD) and fungal and viral infections. CMV reactivation after HCT has been associated with increased morbidity and mortality, and a causal relation between GVHD, immunosuppressive therapy and vice versa has been postulated. Using a low GVHD severity murine HCT model, we assessed the role of MCMV reactivation and GVHD development. BALB/c mice were infected with either murine CMV (MCMV) or mock and monitored for 25 weeks to establish latency, followed by sublethal irradiation conditioning and infusion of bone marrow plus splenocytes from either syngeneic (syn) BALB/c or allo B10.D2 donors. Engraftment of allo donor cells was confirmed by PCR for D2Mit265 gene product size. Day+100 mortality and overall GVHD severity in allo MCMV pre-infected recipients was higher than in allo mock controls. Pathologic changes of lung and liver GVHD in immediate-early gene 1 (IE1) positive recipients were significantly increased compared to mock controls, and were only slightly increased in IE1 negative. No significant gut injury was seen in any group. Aggravated lung injury in IE1 positive recipients correlated with higher BAL cell counts both for total cells and for CD4+ T cells when compared with mock controls, and also with protein expression of lung IFN-gamma and liver TNF. No evidence for CMV specific morphologic changes was seen on histopathology in any organ of IE1 positive recipients, suggesting that CMV reactivation is related to increased GVHD severity but does not require active CMV disease, strengthening the concept of a reciprocal relationship between CMV and GVHD.

Innate immune activation by the viral PAMP poly I:C potentiates pulmonary graft-versus-host disease after allogeneic hematopoietic cell transplant

Respiratory viral infections cause significant morbidity and increase the risk for chronic pulmonary graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT). Our overall hypothesis is that local innate immune activation potentiates adaptive alloimmunity. In this study, we hypothesized that a viral pathogen-associated molecular pattern (PAMP) alone can potentiate pulmonary GVHD after allogeneic HCT. We, therefore, examined the effect of pulmonary exposure to polyinosinic:polycytidylic acid (poly I:C), a viral mimetic that activates innate immunity, in an established murine HCT model. Poly I:C-induced a marked pulmonary T cell response in allogeneic HCT mice as compared to syngeneic HCT, with increased CD4+ cells in the lung fluid and tissue. This lymphocytic inflammation persisted at 2weeks post poly I:C exposure in allogeneic mice and was associated with CD3+ cell infiltration into the bronchiolar epithelium and features of epithelial injury. In vitro, poly I:C enhanced allospecific proliferation in a mixed lymphocyte reaction. In vivo, poly I:C exposure was associated with an early increase in pulmonary monocyte recruitment and activation as well as a decrease in CD4+FOXP3+ regulatory T cells in allogeneic mice as compared to syngeneic. In contrast, intrapulmonary poly I:C did not alter the extent of systemic GVHD in either syngeneic or allogeneic mice. Collectively, our results suggest that local activation of pulmonary innate immunity by a viral molecular pattern represents a novel pathway that contributes to pulmonary GVHD after allogeneic HCT, through a mechanism that includes increased recruitment and maturation of intrapulmonary monocytes.

GvHD Prophylaxis with Everolimus and Mycophenolate in Allogeneic Hematopoietic Cell Transplantation - A Phase I/II Study.

Abstract 4642Next to the control of malignant disease, tolerance induction of the grafted cells remain to be a critical issue for longterm survivors after allogeneic hematopoietic cell transplantation (HCT). Regulatory T cells (Tregs) are believed to be involved in the process of tolerance induction to solid organ grafts and in the regulation of alloreactivity, e.g. graft versus host disease (GvHD) and graft versus leukemia effect (GvL), after allogeneic HCT. Since GvHD causes substantial morbidity, medication with calcineurin inhibitors (CI) like cyclosporine are established prophylactic measures for the prevention of GvHD after HCT. Next to the substantial renal toxicity of CI, tolerance induction might be hampered in HCT patients due to deteriorated Treg function. In contrast, data from in vitro and animal experiments suggest that inhibition of the mammalian target of rapamycin (mTOR) has not only an antiproliferative effect on many malignant cell lines but also results in an inhibition of proliferation of alloreactive T cells with sustained Treg function in a murine HCT model. Therefore we initiated a phase I/II, monocenter trial using everolimus and mycophenolate sodium (MMF-Na) as GvHD prophylaxis in patients undergoing allogeneic HCT with peripheral stem cell (PBSC) grafts after conditioning with fludarabine, melphalan, and BCNU (FBM). No additional T cell depleting agents were used for conditioning/GvHD prophylaxis. Enrolment was started in april 2008, and up to august 2009 10 patients were included (median age: 50.7 years, range: 26-64). The diagnoses included de novo AML (n=3), sAML (n=4), RAEB II (n=1), CML (n=1), T-PLL (n=1). 6/10 patients were regarded as high risk (not in CR1) for early relapse. PBSC grafts were obtained from unrelated (n=5) and related (n=5) HLA-matched donors. With no graft failures, engraftment kinetics for myeloid cells were normal, and reconstitution of the T cell compartment reached median cell counts of 251 CD4+ cells/μl and 163 CD8+ cells/μl at day +30. No grade IV/V toxicities (according to CTC criteria) were observed due to the study medication. After a median follow-up of 6 month two patients have died. The causes were acute GvHD, refractory to several lines of treatment, in a patient with CML, and severe pulmonary toxicity/BOOP in a patient with sAML. Out of 9 patients reaching CR after HCT, only one high risk patient relapsed after 6 month. In total 6 patients are alive and show complete donor chimerism for time periods of 1-14 months post transplant. The observed early recovery of T cell immunity correlated in 8/10 patients with an early brief period of acute GvHD, with 4 patients experiencing grade III/IV severities. Most of the cases could be controlled with steroids alone. Chronic GvHD could be observed in 6/7 patients, with mild to moderate forms in 5 cases, mainly involving skin, mucosa and liver. Interestingly, while early tapering of MMF-Na did not cause any problems, reduction of everolimus earlier as 6 month after HCT resulted in an induction of GvHD symptoms. Although viral reactivation (CMV, HHV6) did occur in patients receiving additional immunosuppression with steroids, no severe bacterial or fungal infections were observed even in cases with prolonged everolimus treatment. In conclusion, GvHD prophylaxis with everolimus and MMF-Na is feasible but results in an increased frequency of mild to moderate chronic GvHD. Since this sustained mild alloreactivity might reduce the risk of relapse, this GvHD prophylaxis could well be suited for patients undergoing HCT with advanced or uncontrolled malignant disease. Disclosures:Marks:Novartis: Research Funding. Off Label Use: Everolimus for prophylaxis of GvHD. Finke:Novartis: Research Funding.

Clinical Outcomes Following Allogeneic Hematopoietic Cell Transplantation (HCT) Using Nonmyeloablative Host Conditioning with Total Lymphoid Irradiation and Anti-Thymocyte Globulin Confirm a Low Incidence of Graft Versus Host Disease (GVHD) and Retained Graft Anti-Tumor Activity.

Murine models of HCT established that nonablative conditioning using low doses of irradiation targeted to lymphoid tissues (TLI) and depletive anti-T cell antibodies protects against GVHD by skewing residual host T cell subsets to favor regulatory natural killer T cells that suppress GVHD by polarizing donor T cells toward secretion of non-inflammatory cytokines such as IL-4. We recently translated the murine protocol to a clinical study of 37 patients (pts) using nonmyeloablative TLI and ATG host conditioning with HLA matched related (MRD) and unrelated (URD) donors, and showed a marked reduction in the incidence of acute GVHD (aGVHD) while retaining graft anti-tumor activity (Lowsky et al., NEJM 2005; 353). We now report the clinical outcomes following HCT of a larger set of pts using the same TLI and ATG regimen. Seventy consecutive pts with hemato-lymphoid malignancies (46 with lymphoid malignancies and 24 with acute leukemias) received MRD (38) or URD (32) G-CSF mobilized HCT. Of the 46 pts with lymphoid malignancies, 40 (87%) had advanced stage disease, 25 (54%) had relapsed after a prior autologous transplant, 31 (67%) were in partial remission (PR) at the start of the allogeneic transplant regimen, 6 (13%) had progressive disease (PD) and 9 (20%) were in a complete remission (CR). Of the 24 pts with acute leukemia 13 (54%) were in a first CR, 8 (33%) in second CR or beyond, and 3 (13%) were not in remission at the start of the transplant regimen. Sixty-eight of the 70 (97%) pts achieved multilineage donor cell engraftment within 56 days post transplantation. All pts were monitored for manifestations of aGVHD using standard scoring scales during the first 100 days after transplantation. Among the 38 recipients of an MRD graft aGVHD was grade 0 in 36 (95%) pts and grade I in 2 (5%) pts. Among the 32 pts that received grafts from an URD aGVHD was scored as grade 0 in 24 (75%) pts, grade I in 5 (16%) pts, grade II in 2 (6%) pts, and grade III in 1 (3%). All cases of aGVHD were treated to resolution. Thirty-seven of 38 (97%) pts with grafts from a MRD survived more than 100 days and were at risk for chronic GVHD (cGVHD). Among these, 26 (70%) had no cGVHD, 4 (11%) had limited cGVHD and 7 (19%) extensive cGVHD. Twenty-eight pts with grafts from URDs were at risk for cGVHD; 19 (68%) had no cGVHD, 3 (11%) had limited cGVHD and 6 (21%) had extensive cGVHD. Among the 46 transplant recipients with lymphoid malignancies, the follow-up ranged from 1674 to 197 days; 12 of these patients died and six were from disease relapse. The Kaplan-Meier (K-M) actuarial event free survival (EFS) at 3 years is 43%. EFS for patients that relapsed with lymphoma after failing a prior autologous transplant (n=25) was not different compared to patients that did not have a prior autologous transplant (n=21) as 6 events occured in both groups. The period of observation for the 24 pts with acute leukemia ranged from 190 to 1631 days; 10 of 11 pts died from disease relapse. The K-M actuarial EFS at 3 years is 50%. These data confirm the low incidence of acute and chronic GVHD using the TLI and ATG regimen in patients receiving grafts from MRD and URD. The low incidence of disease relapse in this group of patients with high-risk disease demonstrates the retention of graft anti-tumor activity.

A single-platform approach using flow cytometry and microbeads to evaluate immune reconstitution in mice after bone marrow transplantation

The monitoring of immune reconstitution in murine models of HC transplantation, using accurate and automated methods, is necessary in view of the recent developments of hematopoietic cell (HC) transplantation (including reduced intensity conditioning regimens) as well as emerging immunological concepts (such as the involvement of dendritic cells or regulatory T cells). Here, we describe the use of a single-platform approach based on flow cytometry and tubes that contain a defined number of microbeads to evaluate absolute blood cell counts in mice. This method, previously used in humans to quantify CD34+ stem cells or CD4+ T cells in HIV infected patients, was adapted for mouse blood samples. A CD45 gating strategy in this “lyse no wash” protocol makes it possible to discriminate erythroblasts or red blood cell debris from CD45+ leukocytes, thus avoiding cell loss. Tubes contain a lyophilized brightly fluorescent microbead pellet permitting the acquisition of absolute counts of leukocytes after flow cytometric analysis. We compared this method to determine absolute counts of circulating cells with another method combining Unopette® reservoir diluted blood samples, hemocytometer, microscopic examination and flow cytometry. The sensitivity of this single-platform approach was evaluated in different situations encountered in allogeneic HC transplantation, including immune cell depletion after different conditionning regimens, activation status of circulating cells after transplantation, evaluation of in vivo cell depletion and hematopoietic progenitor mobilization in the periphery. This single-platform flow cytometric assay can also be proposed to standardize murine (or other mammalian species) leukocyte count determination for physiological, pharmacological/toxicological and diagnostic applications in veterinary practice.