Crossing major immune barriers in bone marrow transplantation and in cell therapy
Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative approach for the treatment of a broad range of malignancies, as well as acquired and genetic defects. It remains a high-risk form of treatment because of the problem of reactivity of donor immune cells against the recipient, graft-vs.-host disease (GVHD), and delayed reconstitution of immunity.
Our group has made many contributions over the past three decades to the advancement of mis-matched HSCT, and developed successful strategies to extend the use of HSCT to “half matched”, haploidentical donors, which are available for almost all patients. Two major milestones have already been translated into clinical achievements, including the:
- first successful T cell depleted haploidentical HSCT in SCID patients (late 70s)
- use of ‘mega dose’ transplants in leukemia patients (1994-98)
More recently, our mechanistic research on veto cells has further suggested ways to improve the safety and effectiveness of haploidentical transplantation. These same principles apply to the burgeoning field of cellular immunotherapy. Chimeric antigen receptor T-cells targeting malignancies can be produced from veto T-cells, thus protecting them from rejection.
Lung injury repair by fetal and adult lung stem cells
Our long term interest in the use of committed embryonic progenitor tissues or cells for organ regeneration (Nature Medicine 2003, PLOS Medicine 2006; PNAS 2009) coupled with our insights from HSCT has led us to hypothesize recently that lung progenitors could be transplanted effectively and heal injured lung through approaches similar to those employed in HSCT. Using this strategy, we demonstrated that intravenous infusion of lung progenitors obtained from fetal or recently deceased donors (after vacating endogenous host progenitors) can induce robust lung chimeras and heal major lung injury (Nature Medicine 2015).
Mechanism of action and the physiological role of Veto CD8 T cells in steady state homeostasis
Veto activity of CD34 cells and their myeloid immature derivatives plays a major role in maintaining the peripheral tolerance responsible for the successful engraftment of high dose TDBM in leukemic patients. The intrinsic ability of veto cells to selectively eliminate cognate CD8 T cells bearing the specific TCR which can productively bind to their MHC-I-peptide complex is extensively studied in our lab.
Phase 1-2 clinical trial using veto CD8 T cells
We've developed new generation human veto central memory CD8 T cells with anti-viral activity. A clinical trial will commence this year investigating the efficacy of these cells in promoting engraftment of T depleted megadose haploid SCT in elderly patients with hematological malignancies following non-myeloablative conditioning (NMAC).
Significance: If successful, the new cell composition overcoming the challenge of graft rejection following NMAC could enable safer T cell depleted haploid SCT in patients with hematological malignancies. This could be important not only for elderly patients who cannot tolerate the toxicity of myeloablative conditioning, but could also fully exploit anti-tumor immunity by avoiding any need for immune suppressive therapy post- transplant in this GVHD free protocol. Furthermore, such novel and safer transplants could serve as a platform for cell therapy and organ transplantation from the same HSCT donor.
Tolerance induction by veto cells for induction of tolerance towards off-the-shelf donor T cells such as CAR or TCR transgenic T cells (without hematopoietic transplantation)
Chimeric antigen receptor T-cells (CAR T-Cells) have been a major advance for treatment of hematologic malignancies and solid tumors. Rejection of CAR T-cells from allogeneic donors greatly limits the efficacy of this approach. Recently, we found that ex-vivo expanded veto cells enable prolonged engraftment of genetically modified T cells such as CAR or TCR transgenic T cells. Furthermore, preliminary results suggest that it might be possible to transduce the veto cells themselves to express the CAR or TCR transgene. This approach could be especially attractive for the use of ‘off-the-shelf’ allogeneic CAR T cells in cancer patients. In essence, veto cells will lyse host T cells with specificity against anti-tumor CAR cells.
Significance: CAR T cell therapy using autologous cells has demonstrated remarkable results, achieving durable remissions in end stage patients with otherwise refractory leukemia’s. If successful, our approach could pave the way for clinical use of ‘off-the-shelf ’ CAR or TCR transgenic T cells.
Preclinical models to assess the feasibility of veto-based protocols for enabling successful allogeneic HSCT in sickle cell anemia and in type 1 diabetes
Learn how the Reisner Lab shaped this field
- Optimizing conditions for induction of immune tolerance in preclinical models towards transplantation of allogeneic lung progenitors (based on preliminary results published in ASH abstract 2016). In particular, we're aiming to replace naphtalene with clinically approved agents.
- Developing protocols for harvesting human adult lung progenitors using GMP-grade reagents, and prepare IND.
- Investigating safety and efficacy of lung stem cell transplantation in HSCT patients suffering from severe lung injury as a consequence of radio/chemotherapy or comorbid disease.
- Investigating the potential curative role of lung stem cell transplantation in animal models of cystic fibrosis.
- Characterizing the putative lung progenitor able to form lung patches in our in-vivo mouse assay.
This novel approach for transplantation of committed lung progenitors can spearhead efforts at jumpstarting a lung regeneration program not only for COPD patients, but also for a substantial number of cancer patients developing lethal lung injuries as a consequence of radio-chemotherapy and/or GVHD.
Previous studies demonstrated the role of different death molecules in different veto cells, including the TNF-α, Fas-FasL and perforin. Most of the mechanistic studies were carried out in-vitro. However, for Tcm which exhibit marked activity in-vivo and very poor veto activity ex-vivo, this mechanistic interrogation required a more challenging evaluation in-vivo. To that end, Perforin-KO mice were used to generate veto Tcm and LPR mice, which are FAS-KO, were used as recipients. We found that Tcm that originated from Perforin-KO (Balb\c X C57BL/6) F1 donors were potent facilitators of engraftment in C57BL/6 WT hosts, suggesting that the perforin mediated mechanism isn’t responsible for the Tcm veto activity. In contrast, PRF-KO F1 Tcm or WT F1 Tcm, which were potent facilitators of engraftment in WT C57BL/6 recipients, couldn’t induce engraftment in LPR mice (FASKO), suggesting that the Fas-FasL pathway is likely mediating the toleragenic effect of anti-3 -party Tcm (manuscript in preparation).
Considering that these veto Tcm are generated artificially ex-vivo and are tested for their activity under artificial transplantation setting, a major question remaining is whether such veto CD8 T cells play any role in maintenance of immune tolerance in steady state. To address this question, we're using gene editing to generate knockout mice in which the CD8 T cells will not express MHC-I which is essential for veto activity of CD8 T cells.
Once established, we intend to investigate in this model potential development of particular autoimmune phenotype as opposed to wild-type control mice treated by the same anti-NK antibodies.
Haploidentical transplantation has been a formidable challenge due to alloreactivity producing rejection or graft-vs.-host disease. In a series of successive steps, we have provided the medical community with tools enabling patients to be cured by haploidentical HSCT. These advances started in the late 1970’s, with the determination that soybean lectin could be utilized to remove alloreactive T lymphocytes from bone marrow preparations used for HSCT, effectively preventing graft versus host disease (GVHD). Following in vitro studies, and tests in preclinical animal models, we successfully used this approach together with colleagues at Memorial Sloan Kettering Cancer Center in the early 1980’s, to treat children with severe combined immunodeficiency (SCID), thereby providing the first cure of a patient by haploidentical hematopoietic stem cell transplantation.
This is now an international standard of care, and hundreds of SCID patients have been treated successfully in many centers using this approach around the world. This method is still in use in the most active transplantation center in the field, at Duke University (Prof RH Buckley). Our studies were published in seminal papers in PNAS (1978), Lancet, and Blood (1980-1983), with recent updates in N.Eng.J.Med (2014), (for review, see Or-Geva and Reisner J.Brit. Hematol 2016).
Next, together with our medical colleagues, we attempted to extend the use of T cell depleted haploidentical HSCT to the treatment of leukemia patients. Our initial studies clearly showed that despite the pre-treatment of these patients with supra-lethal radiation, graft rejection still presented a major barrier. Subsequently, in a second critical step, we found that graft rejection could be overcome by greatly increasing the dose of hematopoietic progenitors transplanted; an approach termed the “mega dose concept”. Experimental evidence was obtained in a murine model (Nature Medicine 1995) and successfully applied to leukemic patients (N.Eng.J.Med 1998), (extension and confirmation J Clin Oncol 2005) (Figure 1, below).
We defined the mechanism facilitating donor hematopoietic stem cell engraftment; the donor progenitor cells exert a “veto” effect on the host immune system, preventing their destruction (Blood 2002, 2005) through a TNF-based process (Figure 2, below). We also recently showed in mouse models that this veto activity can be further utilized to attain engraftment following reduced intensity, nonmyeloablative conditioning protocols. Thus, in another series of publications we demonstrated that anti -3rd party CD8 T cells which also exert veto activity can be harnessed to overcome rejection of mis-matched HSCT (Figure 3, below).
The veto mechanism mediating tolerance induction by veto CD8 T cells was extensively studies by our group (Immunity 2000, J Immunol 2004, 2007, Transplantation 2010 , Blood 2011, Blood 2013) (Figure 4, below).
As highlighted by an Editorial to our Blood plenary paper (Blood 2013), this approach offers new exciting possibilities to support HSCT with minimal toxicity and it is currently ready for clinical trials.
Repair of injured lungs represents a longstanding therapeutic challenge. We've shown that human and mouse embryonic lung tissue from the canalicular stage of development (20–22 weeks of gestation for humans, and embryonic day 15–16 (E15–E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sub-lethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism (Figures 1-3, below). Donor type structures or ‘patches’ contained epithelial, mesenchymal and endothelial cells (Figures 4 and 5, below), and express functional markers. Transplantation of differentially labeled E16 mouse lung cells indicated that these donor derived patches were probably of clonal origin (Figure 6, below).
Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. This study (Nature Medicine, 2015) provided proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche.
Considering the ethical limitations associated with the use of fetal cells, more recently we investigated whether adult lungs could offer an alternative source of lung progenitors for transplantation. We found that intravenous infusion of a single cell suspension of adult mouse lungs from GFP+ donors following conditioning of recipient mice with naphthalene and subsequent sub-lethal irradiation, led to marked colonization of the recipient lungs, at 6-8 weeks post-transplant, with donor derived structures including epithelial, endothelial and mesenchymal cells. Epithelial cells within these donor-derived colonies expressed markers of functionally distinct lung cell types, and lung function, which is significantly compromised in mice treated with naphthalene and radiation, was found to be corrected following transplantation. Dose response analysis suggests that the frequency of patch forming cells in adult lungs was about three fold lower compared to that found in E16 fetal lungs. However, as adult lungs are much larger, the total number of patch forming cells that can be collected from this source is significantly greater. Thus, as for fetal lung cells lung regeneration can be attained by adult lung cells after preconditioning to vacate the pulmonary niche.
Figure 1: GFP+ foci in chimeric lung.
Figure 2: Morphometric analysis of chimeric lungs.
Figure 3: Incorporation of donor cells in epithelial, endothelial and mesenchymal compartments.
Figure 4: Colocalization of functional markers with donor cells.
Figure 5: Engrafted donor cells exhibit the ion transport channel CFTR.
Figure 6: Clonal origin of chimeric patches in vitro and in vivo.