Secondary Neoplasms After Hematopoietic Cell Transplant for Sickle Cell Disease.

PURPOSE: To report the incidence and risk factors for secondary neoplasm after transplantation for sickle cell disease. METHODS: Included are 1,096 transplants for sickle cell disease between 1991 and 2016. There were 22 secondary neoplasms. Types included leukemia/myelodysplastic syndrome (MDS; n = 15) and solid tumor (n = 7). Fine-Gray regression models examined for risk factors for leukemia/MDS and any secondary neoplasm. RESULTS: The 10-year incidence of leukemia/MDS was 1.7% (95% CI, 0.90 to 2.9) and of any secondary neoplasm was 2.4% (95% CI, 1.4 to 3.8). After adjusting for other risk factors, risks for leukemia/MDS (hazard ratio, 22.69; 95% CI, 4.34 to 118.66; P = .0002) or any secondary neoplasm (hazard ratio, 7.78; 95% CI, 2.20 to 27.53; P = .0015) were higher with low-intensity (nonmyeloablative) regimens compared with more intense regimens. All low-intensity regimens included total-body irradiation (TBI 300 or 400 cGy with alemtuzumab, TBI 300 or 400 cGy with cyclophosphamide, TBI 200, 300, or 400 cGy with cyclophosphamide and fludarabine, or TBI 200 cGy with fludarabine). None of the patients receiving myeloablative and only 23% of those receiving reduced-intensity regimens received TBI. CONCLUSION: Low-intensity regimens rely on tolerance induction and establishment of mixed-donor chimerism. Persistence of host cells exposed to low-dose radiation triggering myeloid malignancy is one plausible etiology. Pre-existing myeloid mutations and prior inflammation may also contribute but could not be studied using our data source. Choosing conditioning regimens likely to result in full-donor chimerism may in part mitigate the higher risk for leukemia/MDS.

Enabling allogeneic therapies: CIRM-funded strategies for immune tolerance and immune evasion.

A major goal for the field of regenerative medicine is to enable the safe and durable engraftment of allogeneic tissues and organs. In contrast to autologous therapies, allogeneic therapies can be produced for many patients, thus reducing costs and increasing availability. However, the need to overcome strong immune system barriers to engraftment poses a significant biological challenge to widespread adoption of allogeneic therapies. While the use of powerful immunosuppressant drugs has enabled the engraftment of lifesaving organ transplants, these drugs have serious side effects and often the organ is eventually rejected by the recipient immune system. Two conceptually different strategies have emerged to enable durable engraftment of allogeneic therapies in the absence of immune suppression. One strategy is to induce immune tolerance of the transplant, either by creating "mixed chimerism" in the hematopoietic system, or by retraining the immune system using modified thymic epithelial cells. The second strategy is to evade the immune system altogether, either by engineering the donor tissue to be "invisible" to the immune system, or by sequestering the donor tissue in an immune impermeable barrier. We give examples of research funded by the California Institute for Regenerative Medicine (CIRM) in each of these areas, ranging from early discovery-stage work through clinical trials. The advancements that are being made in this area hold promise that many more patients will be able to benefit from regenerative medicine therapies in the future.

Unleashing the cure: Overcoming persistent obstacles in the translation and expanded use of hematopoietic stem cell-based therapies.

Hematopoietic stem cell transplantation (HSCT) is broadly used for treating and curing hematological cancers and various disorders of the blood and immune system. However, its true therapeutic potential remains vastly constrained by significant scientific and technical hurdles that preclude expansion to new indications and limit the number of patients who could benefit from, gain access to, or financially afford the procedure. To define and overcome these challenges, the California Institute for Regenerative Medicine (CIRM) held multiple workshops related to HSCT and has subsequently invested in a new generation of approaches to address the most compelling needs of the field, including new sources of healthy and immunologically compatible hematopoietic stem cells for transplant; safe and efficient genome modification technologies for correction of inherited genetic defects and other forms of gene therapy; safer and more tractable transplantation procedures such as nongenotoxic conditioning regimens, methods to accelerate immune reconstitution and recovery of immune function, and innovations to minimize the risk of immune rejection; and other life-threatening complications from transplant. This Perspective serves to highlight these needs through examples from the recent CIRM-funded and other notable investigations, presents rationale for comprehensive, systematic, and focused strategies to unleash the full potential of HSCT, thereby enabling cures for a greatly expanded number of disorders and making HSCT feasible, accessible, and affordable to all who could benefit.

Proceedings: human leukocyte antigen haplo-homozygous induced pluripotent stem cell haplobank modeled after the california population: evaluating matching in a multiethnic and admixed population.

The development of a California-based induced pluripotent stem cell (iPSC) bank based on human leukocyte antigen (HLA) haplotype matching represents a significant challenge and a valuable opportunity for the advancement of regenerative medicine. However, previously published models of iPSC banks have neither addressed the admixed nature of populations like that of California nor evaluated the benefit to the population as a whole. We developed a new model for evaluating an iPSC haplobank based on demographic and immunogenetic characteristics reflecting California. The model evaluates haplolines or cell lines from donors homozygous for a single HLA-A, HLA-B, HLA-DRB1 haplotype. We generated estimates of the percentage of the population matched under various combinations of haplolines derived from six ancestries (black/African American, American Indian, Asian/Pacific Islander, Hispanic, and white/not Hispanic) and data available from the U.S. Census Bureau, the California Institute for Regenerative Medicine, and the National Marrow Donor Program. The model included both cis (haplotype-level) and trans (genotype-level) matching between a modeled iPSC haplobank and the recipient population following resampling simulations. We showed that serving a majority (>50%) of a simulated California population through cis matching would require the creation, redundant storage, and maintenance of almost 207 different haplolines representing the top 60 most frequent haplotypes from each ancestry group. Allowances for trans matching reduced the haplobank to fewer than 141 haplolines found among the top 40 most frequent haplotypes. Finally, we showed that a model optimized, custom haplobank was able to serve a majority of the California population with fewer than 80 haplolines.

Proceedings: Immune Tolerance and Stem Cell Transplantation: A CIRM Mini-Symposium and Workshop Report.

The mission of the California Institute for Regenerative Medicine (CIRM) is to accelerate stem cell treatments to patients with unmet medical needs. Immune rejection is one hurdle that stem cell therapies must overcome to achieve a durable and effective therapeutic benefit. In July 2014, CIRM convened a group of clinical investigators developing stem cell therapeutics, immunologists, and transplantation biologists to consider strategies to address this challenge. Workshop participants discussed current approaches for countering immune rejection in the context of organ transplant and cellular therapy and defined the risks, challenges, and opportunities for adapting them to the development of stem cell-based therapeutics. This effort led to the development of a Roadmap to Tolerance for allogeneic stem cell therapy, with four fundamental steps: (a) the need to identify "tolerance-permissive" immune-suppressive regimens to enable the eventual transition from current, drug-based approaches to a newer generation of technologies for inducing tolerance; (b) testing new biologics and small molecules for inducing tolerance in stem cell-based preclinical and clinical studies; (c) stimulation of efforts to develop novel therapeutic approaches to induce central and peripheral tolerance, including manipulation of the thymus, transplantation of purified stem cells, and cell therapy with T-regulatory cells; and (d) development of robust and sensitive immune monitoring technologies for identifying biomarkers of tolerance and rejection after allogeneic stem cell treatments in the clinical setting.

Bottlenecks in deriving definitive hematopoietic stem cells from human pluripotent stem cells: a CIRM mini-symposium and workshop report.

On August 29, 2013, the California Institute for Regenerative Medicine (CIRM) convened a small group of investigators in San Francisco, CA, to discuss a longstanding challenge in the stem cell field: the inability to derive fully functional, definitive hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs). To date, PSC-derived HSCs have been deficient in their developmental potential and their ability to self-renew and engraft upon transplantation. Tasked with identifying key challenges to overcoming this "HSC bottleneck", workshop participants identified critical knowledge gaps in two key areas: (a) understanding the ontogeny of human HSCs, and (b) understanding of the intrinsic and extrinsic factors that govern HSC behavior and function. They agreed that development of new methods and tools is critical for addressing these knowledge gaps. These include molecular profiling of key HSC properties, development of new model systems/assays for predicting and assessing HSC function, and novel technological advancements for manipulating cell culture conditions and genetic programs. The workshop produced tangible advances, including providing a current definition of the nature and challenge of the HSC bottleneck and identifying key mechanistic studies of HSC biology that should be prioritized for future funding initiatives (e.g., including higher risk approaches that have potential for high gain).

The potential for immunogenicity of autologous induced pluripotent stem cell-derived therapies.

Induced pluripotent stem cell (iPSC) technology offers the promise of immune-matched cell therapies for a wide range of diseases and injuries. It is generally assumed that cells derived from autologous iPSCs will be immune-privileged. However, there are reasons to question this assumption, including recent studies that have tested iPSC immunogenicity in various ways with conflicting results. Understanding the risk of an immune response and developing strategies to minimize it will be important steps before clinical testing. Here, we review the evidence for autologous iPSC immunogenicity, its potential causes, and approaches for assessment and mitigation.

Allogeneic T cells treated with amotosalen prevent lethal cytomegalovirus disease without producing graft-versus-host disease following bone marrow transplantation.

Infusion of donor antiviral T cells can provide protective immunity for recipients of hemopoietic progenitor cell transplants, but may cause graft-vs-host disease (GVHD). Current methods of separating antiviral T cells from the alloreactive T cells that produce GVHD are neither routine nor rapid. In a model of lethal murine CMV (MCMV) infection following MHC-mismatched bone marrow transplantation, infusion of MCMV-immune donor lymphocytes pretreated with the DNA cross-linking compound amotosalen prevented MCMV lethality without producing GVHD. Although 95% of mice receiving 30 x 10(6) pretreated donor lymphocytes survived beyond day +100 without MCMV disease or GVHD, all mice receiving equivalent numbers of untreated lymphocytes rapidly died of GVHD. In vitro, amotosalen blocked T cell proliferation without suppressing MCMV peptide-induced IFN-gamma production by MCMV-primed CD8(+) T cells. In vivo, pretreated lymphocytes reduced hepatic MCMV load by 4-log(10) and promoted full hemopoietic chimerism. Amotosalen-treated, MCMV tetramer-positive memory (CD44(high)) CD8(+) T cells persisted to day +100 following infusion, and expressed IFN-gamma when presented with viral peptide. Pretreated T cells were effective at preventing MCMV lethality over a wide range of concentrations. Thus, amotosalen treatment rapidly eliminates the GVHD activity of polyclonal T cells, while preserving long-term antiviral and graft facilitation effects, and may be clinically useful for routine adoptive immunotherapy.

Photochemical treatment of donor lymphocytes inhibited their ability to facilitate donor engraftment or increase donor chimerism after nonmyeloablative conditioning or establishment of mixed chimerism.

Donor T-cells can provide a graft-versus-leukemia effect and help to promote donor engraftment after allogeneic BMT; however, these benefits can be outweighed by the ability of the cells to induce life-threatening GVHD. Photochemical treatment (PCT) of T-cells with S-59 psoralen and long-wavelength UV-A light can inhibit their proliferative capacity and significantly decrease their ability to induce acute GVHD after allogeneic BMT. PCT donor T-cells have been shown to facilitate donor engraftment in a myeloablative BMT model. In this study, we examined whether donor T-cells subjected to PCT ex vivo could retain the ability to facilitate engraftment or increase donor chimerism after nonmyeloablative BMT or after establishment of mixed hematopoietic chimerism. In a transplantation model in which mice were conditioned for BMT with sublethal (600 cGy) TBI, an infusion of PCT donor T-cells was unable to facilitate engraftment of donor BM. A BMT model was used in which a mixture of allogeneic and syngeneic marrow cells was infused into lethally irradiated recipients for establishment of mixed hematopoietic chimerism. The goal was to determine whether PCT donor splenocytes could increase levels of donor chimerism. Recipients of splenocytes treated with UV-A light only (no S-59 psoralen) and given at the time of BMT or in a donor lymphocyte infusion (DLI) had significantly higher levels of donor chimerism than did recipients of BM only. Although PCT donor splenocytes given at the time of BMT modestly increased donor chimerism, PCT donor splenocytes given in a DLI did not increase donor chimerism. A nonmyeloablative BMT model was employed for determining whether DLI given relatively late after BMT could increase donor chimerism. Recipient mice were conditioned for BMT with a combination of low-dose TBI (50 or 100 cGy) and anti-CD154 (anti-CD40L) monoclonal antibody for achievement of low levels of mixed chimerism. When control mixed chimeras were given a DLI 71 days after BMT, donor chimerism was significantly increased. In contrast, PCT of the donor cells eliminated the ability of the cells to increase donor chimerism after infusion. Together results from these 3 distinct BMT models indicate that PCT of donor T-cells significantly inhibited the ability of the cells to facilitate donor engraftment after nonmyeloablative BMT or to increase donor chimerism in mixed hematopoietic chimeras when the cells were administered in a DLI.

Multilineage engraftment with minimal graft-versus-host disease following in utero transplantation of S-59 psoralen/ultraviolet a light-treated, sensitized T cells and adult T cell-depleted bone marrow in fetal mice.

Although engraftment following in utero stem cell transplantation can readily be achieved, a major limitation is the low level of donor chimerism. We hypothesized that a lack of space for donor cells in the recipient marrow was one of the primary reasons for failure to achieve significant engraftment, and that donor T cells could make space in an allogeneic mismatched setting. We found that 3 x 10(5) C57BL/6 (B6) naive CD3(+) cells coinjected with B6 T cell-depleted bone marrow (TCDBM) into 14- to 15-day-old BALB/c fetuses resulted in multilineage engraftment (median, 68.3%) associated with severe graft-vs-host disease (GvHD; 62 vs 0% with TCDBM alone). When 1.5 x 10(5) CD4(+) or CD8(+) cells were used, low levels of engraftment were seen vs recipients of 1.5 x 10(5) CD3(+) cells (2.4 +/- 1.1 and 6.6 +/- 3.9 vs 20.4 +/- 10.4%, respectively). To test the hypothesis that proliferation of T cells in response to alloantigen resulted in GvHD and increased engraftment, we pretreated naive T cells with photochemical therapy (PCT) using S-59 psoralen and UVA light to prevent proliferation. GvHD was reduced (60-0%), but was also associated with a significant reduction in engrafted donor cells (53.4 +/- 4.2 to 1.7 +/- 0.5%). However, when B6 T cells were sensitized to BALB/c splenocytes, treated with PCT, and coinjected with TCDBM, there was a partial restoration of engraftment (13.3 +/- 2.4% H2Kb(+) cells) with only one of nine animals developing mild to moderate GvHD. In this study we have shown that PCT-treated T cells that are cytotoxic but nonproliferative can provide an engraftment advantage to donor cells, presumably by destroying host hemopoietic cells without causing GvHD.