Utility of Next-Generation Sequencing-Based Chimerism Analysis for Early Relapse Prediction following Allogenic Hematopoietic Cell Transplantation.

Chimerism monitoring following allogeneic hematopoietic cell transplantation (HCT) plays a pivotal role in evaluating engraftment status and identifying early indicators of relapse. Recent advancements in next-generation sequencing (NGS) technology have introduced AlloSeq HCT as a more sensitive alternative to short tandem repeat (STR) analysis. This study aimed to compare AlloSeq HCT with STR, focusing on the prediction of early relapse post-allogeneic HCT. Chimerism levels in 29 HCT recipients were assessed using both STR and NGS, employing a total of 125 whole blood or bone marrow aspirate samples (68 post-HCT and 57 pre-HCT samples from recipients or donors). AlloSeq HCT exhibited high concordance with STR and demonstrated the potential for early detection of chimeric changes, particularly at extremely low levels. The combined advantages of high sensitivity and automated data analysis offered by AlloSeq HCT substantiate its clinical adoption for effective chimerism monitoring.

Maternal Administration of Busulfan before in Utero Hematopoietic Cell Transplantation Improves Congenic Bone Marrow Cell Engraftment in a Murine Model.

In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative procedure that leads to donor cell chimerism and donor-specific tolerance. However, most clinical applications of IUHCT have failed because of low levels or even no engraftment of donor cells in immunologically normal fetuses. It is likely that the competition from the host hematopoietic compartment is the primary barrier to successful IUHCT, suggesting that conditioning methods that provide a competitive advantage to donor cells may lead to higher-level engraftment following IUHCT. This study aimed to research whether maternal administration of low-dose total body irradiation (TBI) or busulfan (BU) before IUHCT may result in increased donor cell chimerism in postnatal bone marrow transplantation in a congenic murine model. We first determined the birth and mortality rates after maternal administration of low-dose TBI (0, 2 or 4 Gy) or BU (5, 10, 15, or 20 mg/kg) before IUHCT in B6 mice. The mice that received 2 Gy TBI plus IUHCT showed significantly lower birth rate (23.3%) and a 100% 3-day mortality rate. The mice that received 10 mg/kg BU plus IUHCT had similar birth and 3-day mortality rates (58.6% and 0%) compared to mice that received IUHCT alone (61.1% and 4.55%). We then performed maternal administration of BU at 1 of 3 dosages (5, 10, or 15 mg/kg) at 24 hours before intrauterine transplantation of 2.5 × 105 B6GFP Sca-1+ bone marrow cells (BMCs) or 2.5 × 106 B6GFP BMCs on gestational day 14 (E14). Green fluorescent protein (GFP) chimerism in peripheral blood mononuclear cells (PBMCs), RBCs, and platelets of mice at 4 weeks of age was enhanced significantly with an increase in BU dose. Moreover, GFP chimerism of PBMCs from the B6GFP BMC group was significantly higher than that of the B6GFP Sca-1+ BMC group (22.56% versus 7.20%; P = .018). Finally, the pregnant mice were treated with 10 mg/kg of BU at E13, E14, or E15, followed by intrauterine transplantation of 2.5 × 106 B6GFP BMCs 24 hours later. Except for the short-term level of chimerism in PBMCs, which showed no significant difference among the 3 study groups, the results indicate that both short-term (age 4 weeks) and long-term (age 14 weeks) engraftment in PBMCs, RBCs, and platelets was higher in group E16 compared with groups E14 and E15. We also discovered that the engraftment was stable, multilineage, and increased with time. In conclusion, maternal administration of BU, but not of TBI, along with IUHCT could significantly enhance engraftment in a congenic murine model.

A novel flow-cytometric based method to assess post-HSCT donor chimerism exploiting RNA hybridization.

Analysis of donor-recipient chimerism after hematopoietic stem cell transplantation (HSCT) is of pivotal importance for patient's clinical management, especially in the context of mixed chimerism. Patients are routinely monitored for chimerism in sorted subsets of peripheral blood cells. However, measurement of chimerism in sorted immune cell subsets is technically challenging and time consuming. We here propose a novel, flow cytometry-based approach to detect donor cell chimerism in sex-mismatched HSCT. We exploit RNA PrimeFlow™ system, based on RNA hybridization, to detect mRNA from a lysine demethylase encoded by Y chromosome, KDM5D. This approach allows to distinguish male and female derived cells with around 1% sensitivity. The procedure can be coupled with multiparametric immunophenotyping to assess chimerism in specific immune cell subsets without the need for prior FACS-sorting. We apply this method to a cohort of HSCT patients (n = 10) and we show that it is consistent with standard PCR-based method. We also show that different T lymphocyte subsets display variable degrees of donor chimerism, especially in CD8+ T cell compartment where we observe an enrichment for recipient chimerism in central memory T cells. This method can be exploited to advance current knowledge on immune reconstitution focusing on specific subsets avoiding prior FACS-sorting.

Short Tandem Repeat Analysis in a Living Related Donor Adult Renal Transplant Recipient with Rare Natural Chimerism.

After renal transplant, immunosuppression therapy is used to reduce the risk of rejection. Here, we describe the case of an adult living related donor renal transplant recipient with rare natural chimerism, as discovered by short tandem repeat sequence analysis. In our process of matching transplant patients, we perform human leukocyte antigen testing and short tandem repeat chimerism testing to decide postoperative immunosuppression strategy for transplant patients. We analyzed the short tandem repeat chimerism status before renal transplant and determined that this patient represented a rare case of natural chimerism. Assessment of organ recipient chimerism can inform physicians regarding a dosage reduction of immunosuppressive agents. Short tandem repeat sequence analysis provides substantial information regarding existing polymorphisms and can identify chimerism, if present, and thereby guide immunosuppression strategies after renal transplant, which may improve the long-term immunosuppression-free survival of renal transplant recipients.

Mixed donor chimerism following stem cell transplantation for sickle cell disease.

Sickle cell disease is a debilitating hemoglobinopathy with high morbidity and mortality. Hematopoietic stem cell transplantation (HCT) is curative, but the presence of mixed donor/recipient chimerism post-HCT raises concerns about disease control long-term. Mixed donor/recipient chimerism is reported in significant numbers even after aggressive HCT conditioning regimens. Post-HCT, adequacy of donor erythropoiesis is crucial for disease control. This review explores the relationship between mixed donor/recipient chimerism and outcomes post-HCT. Serial chimerism analysis in lineage specific manner in erythroid or myeloid cells post-HCT predicts for disease control and HCT success. Adequate and stable donor-derived erythropoiesis is essential for reversing SCD manifestations. Myeloid lineage chimerism mirrors erythropoiesis is commercially available, and a reliable indicator of adequacy. Using this tool, the minimum threshold of donor chimerism is required to prevent SCD-related complications and maintain sickle hemoglobin less than 50% is approximately 20-25% even when a donor has Hb S trait. Curative interventions should, at a minimum, meet this goal long-term. Achieving a balance between successful engraftment while minimizing toxicity is important in patients vulnerable because of age or preexisting morbidity and is the objective of recent clinical trials. As HCT and gene therapies evolve, efficient long-term follow-up that includes durability assessment of mixed donor/recipient chimerism will be crucial.

Helical TomoTherapy Total Lymphoid Irradiation and Hematopoietic Cell Transplantation for Kidney Transplant Tolerance in Rhesus Macaques.

Development of a post-transplant kidney transplant tolerance induction protocol involving a novel total lymphoid irradiation (TLI) conditioning method in a rhesus macaque model is described. We examined the feasibility of acheiving tolerance to MHC 1-haplotype matched kidney transplants by establishing a mixed chimeric state with infusion of donor hematopoietic cells (HC) using TomoTherapy TLI. The chimeric state was hypothesized to permit the elimination of all immunosuppressive (IS) medications while preserving allograft function long-term without development of graft-versus-host-disease (GVHD) or rejection. An experimental group of 11 renal transplant recipients received the tolerance induction protocol and outcomes were compared to a control group (n = 7) that received the same conditioning but without donor HC infusion. Development of mixed chimerism and operational tolerance was accomplished in two recipients in the experimental group. Both recipients were withdrawn from all IS and continued to maintain normal renal allograft function for 4 years without rejection or GVHD. None of the animals in the control group achieved tolerance when IS was eliminated. This novel experimental model demonstrated the feasibility for inducing of long-term operational tolerance when mixed chimerism is achieved using a TLI post-transplant conditioning protocol in 1-haplotype matched non-human primate recipients of combined kidney and HC transplantation.

Microchimerism as Post-Transplant Marker of a Chronic Rejection Process.

The risk of losing a transplanted organ is high, and non-invasive markers to warn of this phenomenon are still being sought. We investigated the impact of post-transplant microchimerism on the function of the transplanted kidney. The study included 100 kidney transplant recipients, mostly women. All transplanted organs were from opposite-sex deceased donors. Microchimerism was assessed using multiplex PCR. Male DNA was detected in all urine samples from female recipients and in 13/56 blood samples from female kidney recipients. Female DNA was found in 31/44 urine samples from male recipients, but in none of the blood samples. Microchimerism in the urine of female recipients correlated positively with blood urea (Rs = 0.45; p = 5.84 × 10-4) and K+ ions (Rs = 0.29; p = 0.03), while microchimerism in the blood of female recipients also correlated positively with blood urea (Rs = 0. 28; p = 0.04), cystatin C (Rs = 0.31; p = 0.02) and the number of incompatible HLA alleles (Rs = 0.42; p = 0.01). A history of DGF was associated with higher urinary donor DNA concentrations in female recipients.: Post-transplant microchimerism may serve as a potential marker of chronic kidney rejection.

Chimerism Testing by Next Generation Sequencing for Detection of Engraftment and Early Disease Relapse in Allogeneic Hematopoietic Cell Transplantation and an Overview of NGS Chimerism Studies.

Chimerism monitoring after allogenic Hematopoietic Cell Transplantation (allo-HCT) is critical to determine how well donor cells have engrafted and to detect relapse for early therapeutic intervention. The aim of this study was to establish and detect mixed chimerism and minimal residual disease using Next Generation Sequencing (NGS) testing for the evaluation of engraftment and the detection of early relapse after allo-HCT. Our secondary aim was to compare the data with the existing laboratory method based on Short Tandem Repeat (STR) analysis. One hundred and seventy-four DNA specimens from 46 individuals were assessed using a commercially available kit for NGS, AlloSeq HCT NGS (CareDx), and the STR-PCR assay. The sensitivity, precision, and quantitative accuracy of the assay were determined using artificially created chimeric constructs. The accuracy and linearity of the assays were evaluated in 46 post-transplant HCT samples consisting of 28 levels of mixed chimerism, which ranged from 0.3-99.7%. There was a 100% correlation between NGS and STR-PCR chimerism methods. In addition, 100% accuracy was attained for the two external proficiency testing surveys (ASHI EMO). The limit of detection or sensitivity of the NGS assay in artificially made chimerism mixtures was 0.3%. We conducted a review of all NGS chimerism studies published online, including ours, and concluded that NGS-based chimerism analysis using the AlloSeq HCT assay is a sensitive and accurate method for donor-recipient chimerism quantification and minimal residual disease relapse detection in patients after allo-HCT compared to STR-PCR assay.

Evaluation of Immunocompetence and Biomarkers of Tolerance in Chimeric and Immunosuppression-free Kidney Allograft Recipients.

BACKGROUND: Thirty-seven patients have received a living-donor kidney transplant in a phase 2 study designed to induce tolerance with facilitated allogeneic hematopoietic stem cell transplant. The study protocol is based on tolerogenic CD8 + /T-cell receptor - facilitating cells (FCR001; also including hematopoietic stem cells and αß-T-cell receptor + T cells) and low-dose, nonmyeloablative conditioning. Persistent chimerism allowing full immunosuppression (IS) withdrawal was achieved in 26 patients (time off IS 36-123 mo). METHODS: We evaluated biomarkers of tolerance through urinary cell mRNA profiling and immunocompetence to respond to vaccination in these patients. We also assessed kidney function and metabolic parameters compared with standard-of-care patients on IS. RESULTS: Persistently chimeric patients retained chimerism after removal of IS and remained rejection free without donor HLA-specific antibody development. The presence of donor chimerism at >50% correlated with a signature of tolerance in urinary cell mRNA profiles, with a uniquely elevated increase in the ratio of cytotoxic T lymphocyte-associated protein 4 to granzyme B mRNA. Tolerance was associated with protection from recurrence of immune-mediated causes of kidney disease. Tolerant participants were safely vaccinated, developed protective immune responses, and did not lose chimerism after vaccination. When compared with kidney transplant recipients treated with standard IS, tolerant participants showed stable kidney function and reduced medication use for hypertension and hyperlipidemia. CONCLUSIONS: These results suggest that elimination of IS has distinct advantages in living-donor kidney allograft recipients.

Bias reduction improves accuracy and informativity of high-throughput sequencing chimerism assays.

BACKGROUND AND AIMS: Chimerism monitoring by means of high-throughput sequencing of biallelic polymorphisms has shown promising advantages for patient follow-up after hematopoietic stem cell transplantation. Yet, the presence of method bias precludes achievement of an assay's theoretically attainable informativity rate, as method bias necessitates the exclusion of some markers. This method bias arises because of preferential observation of one allele over the other, and for some allelic constellations because of stochasticity. RESULTS: This paper suggests how preferential allelic observation may lead to method bias, and when and why such bias necessitates the exclusion of markers. It is shown that also markers that remain informative suffer a reduction in trueness and precision due to method bias. A bias reduction approach in the data analysis phase is introduced and shown to improve trueness and precision under all circumstances, meriting its universal adoption. This bias reduction furthermore allows to achieve an assay's theoretically achievable informativity rate, though at the cost of reduced sensitivity. Several strategies to consider in the assay design phase that may lower biases are proposed. CONCLUSION: Improved design and data analysis of chimerism assays increase the accuracy, applicability, and cost-effectiveness of high-throughput sequencing chimerism assays.

One fits all: a highly sensitive combined ddPCR/pyrosequencing system for the quantification of microchimerism after hematopoietic and solid organ transplantation.

OBJECTIVES: A combined digital droplet PCR (ddPCR)/pyrosequencing assay system was developed that demonstrated advantages applicable to multiple qualitative and quantitative molecular genetic diagnostic applications. Data for characterizing this combined approach for hematologic stem cell transplantation (HSCT) and allele quantification from graft-derived cell-free (cf) DNA in solid organ transplantation (SOT) is presented. METHODS: ddPCR and pyrosequencing assays targeting 32 SNPs/markers were established. ddPCR results from 72 gDNAs of 55 patients after allogeneic HSCT and 107 plasma-cfDNAs of 25 liver transplant recipients were compared with established methods/markers, i.e. short-tandem-repeat PCR and ALT, respectively. RESULTS: The ddPCR results were in good agreement with the established marker. The limit of detection was 0.02 % minor allele fraction. The relationship between ddPCR and STR-PCR was linear with R2=0.98 allowing to transfer previously established clinical STR-PCR cut-offs to ddPCR; 50-fold higher sensitivity and a variation coefficient of <2 % enable the use of low DNA concentrations (e.g. pre-sorted cells). ddPCR detected liver allograft injury at least as sensitive as ALT suggesting that ddPCR is a reliable method to monitor the transplant integrity, especially when other biomarkers are lacking (e.g. kidney). CONCLUSIONS: Combining pyrosequencing for genotyping and ddPCR for minor allele quantification enhances sensitivity and precision for the patient after HSCT and SOT. The assay is designed for maximum flexibility. It is expected to be suitable for other applications (sample tracking, prenatal diagnostics, etc.).

Induced pluripotent stem cell-derived hematopoietic stem and progenitor cells induce mixed chimerism and donor-specific allograft tolerance.

In transplantation using allogeneic induced pluripotent stem cells (iPSCs), strategies focused on major histocompatibility complexes were adopted to avoid immune rejection. We showed that minor antigen mismatches are a risk factor for graft rejection, indicating that immune regulation remains one of the most important issues. In organ transplantation, it has been known that mixed chimerism using donor-derived hematopoietic stem/progenitor cells (HSPCs) can induce donor-specific tolerance. However, it is unclear whether iPSC-derived HSPCs (iHSPCs) can induce allograft tolerance. We showed that 2 hematopoietic transcription factors, Hoxb4 and Lhx2, can efficiently expand iHSPCs with a c-Kit+Sca-1+Lineage- phenotype, which possesses long-term hematopoietic repopulating potential. We also demonstrated that these iHSPCs can form hematopoietic chimeras in allogeneic recipients and induce allograft tolerance in murine skin and iPSC transplantation. With mechanistic analyses, both central and peripheral mechanisms were suggested. We demonstrated the basic concept of tolerance induction using iHSPCs in allogeneic iPSC-based transplantation.

Donor programmed cell death 1 ligand 1 is required for organ transplant tolerance in major histocompatibility complex-mismatched mixed chimeras although programmed cell death 1 ligand 1 and major histocompatibility complex class II are not required for inducing chimerism.

Induction of major histocompatibility complex (MHC) human leukocyte antigen (HLA)-mismatched mixed chimerism is a promising approach for organ transplantation tolerance; however, human leukocyte antigen-mismatched stable mixed chimerism has not been achieved in the clinic. Tolerogenic dendritic cell (DC) expression of MHC class II (MHC II) and programmed cell death 1 ligand 1 (PD-L1) is important for immune tolerance, but whether donor-MHC II or PD-L1 is required for the induction of stable MHC-mismatched mixed chimerism and transplant tolerance is unclear. Here, we show that a clinically applicable radiation-free regimen can establish stable MHC-mismatched mixed chimerism and organ transplant tolerance in murine models. Induction of MHC-mismatched mixed chimerism does not require donor cell expression of MHC II or PD-L1, but donor-type organ transplant tolerance in the mixed chimeras (MC) requires the donor hematopoietic cells and the organ transplants to express PD-L1. The PD-L1 expressed by donor hematopoietic cells and the programmed cell death 1 expressed by host cells augment host-type donor-reactive CD4+ and CD8+ T cell anergy/exhaustion and differentiation into peripheral regulatory T (pTreg) cells in association with the organ transplant tolerance in the MC. Conversely, host-type Treg cells augment the expansion of donor-type tolerogenic CD8+ DCs that express PD-L1. These results indicate that PD-L1 expressed by donor-type tolerogenic DCs and expansion of host-type pTreg cells in MHC-mismatched MCs play critical roles in mediating organ transplant tolerance.

Chimerism-based Tolerance Induction in Clinical Transplantation: Its Foundations and Mechanisms.

Hematopoietic chimerism remains the most promising strategy to bring transplantation tolerance into clinical routine. The concept of chimerism-based tolerance aims to extend the recipient's mechanisms of self-tolerance (ie, clonal deletion, anergy, and regulation) to include the tolerization of donor antigens that are introduced through the cotransplantation of donor hematopoietic cells. For this to be successful, donor hematopoietic cells need to engraft in the recipient at least temporarily. Three pioneering clinical trials inducing chimerism-based tolerance in kidney transplantation have been published to date. Within this review, we discuss the mechanisms of tolerance that are associated with the specific therapeutic protocols of each trial. Recent data highlight the importance of regulation as a mechanism that maintains tolerance. Insufficient regulatory mechanisms are also a likely explanation for situations of tolerance failure despite persisting donor chimerism. After decades of preclinical development of chimerism protocols, mechanistic data from clinical trials have recently become increasingly important. Better understanding of the required mechanisms for tolerance to be induced in humans will be a key to design more reliable and less invasive chimerism protocols in the future.

New methods for the quantification of mixed chimerism in transplantation.

Background: Quantification of chimerism showing the proportion of the donor in a recipient is essential for the follow-up of hematopoietic stem cell transplantation but can also be useful to document an immune tolerance situation after solid organ transplantation. Historically, chimerism has been quantified from genomic DNA, but with technological advances, chimerism from donor-derived cell-free DNA seems particularly relevant in solid organ transplantation. Methods: The reference method was until recently the short tandem repeat technique, but new innovative techniques as digital PCR (dPCR) and NGS, have revolutionized the quantification of chimerism, such as the so-called microchimerism analysis. After a short review of chimerism methods, a comparison of chimerism quantification data for two new digital PCR systems (QIAcuity™ dPCR (Qiagen®) and QuantStudio Absolute Q (ThermoFisher®) and two NGS-based chimerism quantification methods (AlloSeq HCT™ (CareDx®) and NGStrack™ (GenDX®)) was performed. Results: These new methods were correlated and concordant to routinely methods (r²=0.9978 and r²=0.9974 for dPCR methods, r²=0.9978 and r²=0.9988 for NGS methods), and had similar high performance (sensitivity, reproductibility, linearity). Conclusion: Finally, the choice of the innovative method of chimerism within the laboratory does not depend on the analytical performances because they are similar but mainly on the amount of activity and the access to instruments and computer services.

Inadvertent stem cell transfer demonstrating a way for tolerance induction.

In this issue, Sobrino et al. present a case of hematopoietic chimerism and subsequent tolerance after isolated kidney transplantation in a pediatric patient with syndromic combined immune deficiency. This report not only highlights the chimerism approach for tolerance induction in transplantation, but it is also the first evidence of hematopoietic stem cells in human kidneys. This commentary discusses the potency and risks of the chimerism approach for tolerance induction after solid organ transplantation, especially in (pediatric) patients with immune deficiencies.

Adoptive Transfer and Bone Marrow Chimera Models to Analyze Treg Function and Differentiation.

Cellular adoptive transfer and mixed bone marrow chimera are cornerstone experimental tools for immuno-biology. Here we describe protocols for adoptive transfer and bone marrow chimera to address the effect of a specific mutation on T regulatory cell (Treg) function and differentiation, respectively. Treg function can be quantitatively measured by analyzing the expansion of conventional CD4 T cells and their differentiation into helper cells. The quantitative measure of Treg differentiation is addressed by analyzing the number and phenotype of Foxp3-expressing cells. The use of congenic markers is instrumental for these approaches.

Evaluation of a quantitative PCR-based method for chimerism analysis of Japanese donor/recipient pairs.

Chimerism analysis is a surrogate indicator of graft rejection or relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Although short tandem repeat PCR (STR-PCR) is the usual method, limited sensitivity and technical variability are matters of concern. Quantitative PCR-based methods to detect single nucleotide polymorphisms (SNP-qPCR) are more sensitive, but their informativity and quantitative accuracy are highly variable. For accurate and sensitive chimerism analysis, a set of KMR kits (GenDx, Utrecht, Netherlands), based on detection of insertions/deletions (indels) by qPCR, have been developed. Here, we investigated informativity and validated the accuracy of KMR kits in Japanese donor/recipient pairs and virtual samples of DNA mixtures representative of Japanese genetic diversity. We found that at least one recipient-specific marker among 39 KMR-kit markers was informative in all of 65 Japanese donor/recipient pairs. Moreover, the percentage of recipient chimerism estimated by KMRtrack correlated well with ratios of mixed DNA in virtual samples and with the percentage of chimerism in HSCT recipients estimated by STR-PCR/in-house SNP-qPCR. Moreover, KMRtrack showed better sensitivity with high specificity when compared to STR-PCR to detect recipient chimerism. Chimerism analysis with KMR kits can be a standardized, sensitive, and highly informative method to evaluate the graft status of HSCT recipients.