Sex-mismatched Stem Cell Transplantation Research Articles

ASXL1 Truncating Mutations Drive Leukemic Resistance to T Cell Attack

Restoration of graft-versus leukemia activity underlies the efficacy of donor lymphocyte infusion (DLI), a curative immunotherapy for leukemic relapse following allogeneic stem cell transplant (SCT). We found that while specific exhausted T cell subsets defined DLI response, no pattern of T cell dysfunction appeared in nonresponders (NRs) [PMID: 34758319]. We hypothesized that leukemic molecular pathways define DLI resistance (DLIr). We performed whole exome sequencing on bone marrow samples collected before and after DLI from 11 patients (pts) with relapsed chronic myeloid leukemia (CML) at Dana-Farber Cancer Institute. We used MuTect2 to discover somatic alterations using genomic DNA from recipient-derived mesenchymal stromal cells and donor peripheral blood cells as germline sources. The burden of nonsynonymous mutations and mutational spectra was similar between responders (Rs) and non-responders (NRs). Known non-Philadelphia (Ph) chromosome cancer drivers were found in 0/7 Rs and 4/4 NRs (RUNX1 (1/4), IKZF1 (2/4), and truncating exon 12 ASXL1 mutations (ASXL1 m) (3/4)). We profiled 6 more DLI-treated pts with relapsed CML from the MD Anderson Cancer Center with a targeted mutation panel that identified ASXL1 m in 4/5 NRs. Altogether, we found ASXL1 m in 78% (7/9) of NRs and 0% (0/10) of Rs (p=7e-4, two-sided Fisher's test), suggesting ASXL1 m as a driver of DLIr. To discover molecular pathways of DLIr, we interrogated unpublished non T-cell single cell (sc)RNA-seq data in our DLI cohort (8 R, 6 NR). Leukemic cells were identified for patients (n=8) with sex-mismatched SCT by male sex-defining genes, which correlated with Ph chromosomal positivity (Pearson correlation = 0.85). These cells were primarily confined to the myeloid population, so we performed a scRNA myeloid reclustering (138,152 cells) with Harmony batch correction and identified 5 populations enriched in R vs NR; 4 clusters in NR, and 1 monocyte cluster in R (p=0.039, 0.005, 0.038, 0.043, and 0.025, respectively). These NR clusters were enriched in progenitor cell identities, notably the LSC17 [PMID: 27926740] signature (p<2e-16). Interrogation of immune pathways in the LSC cluster showed downregulation of HLA-I and multiple antigen processing/presentation (APP) genes in NRs (p<2e-16), while HLA-II remained unchanged. Thus, ASXL1 m may mediate immune escape through suppression of HLA-I expression and antigen presentation. To directly test this hypothesis, we CRISPR corrected the endogenous ASXL1 m (Y591*) in a BCR-ABL-driven leukemic cell line (K562). This wildtype reversion (ASXL1 c) increased HLA-I expression at the RNA (RNA-seq; HLA-B/C, log2foldchange= -4.77, q=7e-42) and protein level (flow cytometry; 49.0% vs 1.62%, q=2e-4); here too, HLA-II expression remained unaffected by ASXL1 mutation status. Gene-set enrichment analysis also showed increased APP expression in ASXL1 c K562 (NES=-1.75, q=0.01). We then sought to identify whether correction of ASXL1m acted through known regulators of HLA-I. Treatment with IFNɣ or EPZ011989, an inhibitor of enhancer of zeste-homolog 2 (EZH2), resulted in greater HLA-I in K562 ASXL1 c (85.0% vs 50.2% and 89.5% vs 39.3%, respectively, q<0.001) suggesting that ASXL1 cacts orthogonally to IFNɣ or EZH2. ASXL1 c also mediated increased T-cell cytotoxicity towards K562 ASXL1 c vs ASXL1 m without (43% vs 16%, q=2e-4) and with IFNɣ pre-treatment (71% vs 34%, q=1e-6) at 10:1 target to effector ratios. Finally, to study other disease contexts, we examined published sc chromatin accessibility and scRNA-seq data from a chronic myelomonocytic leukemia cohort [PMID: 35301312] and an acute myeloid leukemia cohort [PMID:32649887], respectively. We found both chromatin and transcriptional states enriched in ASXL1m samples to have decreased accessibility and expression, respectively, of HLA-I and APP genes (p<2E-16). In summary, we integrated molecular analyses with immuno-functional studies to define a novel oncogenetic pathway of immune evasion. Specifically, we link DLIr to ASXL1 m that suppresses transcription of HLA-I and antigen presentation machinery in myeloid diseases; moreover, its correction restores anti-leukemic CD8+ T-cell cytotoxicity. Ongoing studies will examine additional clinical contexts and the epigenetic mechanisms by which ASXL1m regulates HLA-I expression.

Chromosome Y–encoded antigens associate with acute graft-versus-host disease in sex-mismatched stem cell transplant

AbstractAllogeneic hematopoietic stem cell transplantation (allo-HCT) is a curative option for blood cancers, but the coupled effects of graft-versus-tumor and graft-versus-host disease (GVHD) limit its broader application. Outcomes improve with matching at HLAs, but other factors are required to explain residual risk of GVHD. In an effort to identify genetic associations outside the major histocompatibility complex, we conducted a genome-wide clinical outcomes study on 205 acute myeloid leukemia patients and their fully HLA-A–, HLA-B–, HLA-C–, HLA-DRB1–, and HLA-DQB1–matched (10/10) unrelated donors. HLA-DPB1 T-cell epitope permissibility mismatches were observed in less than half (45%) of acute GVHD cases, motivating a broader search for genetic factors affecting clinical outcomes. A novel bioinformatics workflow adapted from neoantigen discovery found no associations between acute GVHD and known, HLA-restricted minor histocompatibility antigens (MiHAs). These results were confirmed with microarray data from an additional 988 samples. On the other hand, Y-chromosome–encoded single-nucleotide polymorphisms in 4 genes (PCDH11Y, USP9Y, UTY, and NLGN4Y) did associate with acute GVHD in male patients with female donors. Males in this category with acute GVHD had more Y-encoded variant peptides per patient with higher predicted HLA-binding affinity than males without GVHD who matched X-paralogous alleles in their female donors. Methods and results described here have an immediate impact for allo-HCT, warranting further development and larger genomic studies where MiHAs are clinically relevant, including cancer immunotherapy, solid organ transplant, and pregnancy.

In Situ Detection of HY-Specific T Cells in Acute Graft-versus-Host Disease–Affected Male Skin after Sex-Mismatched Stem Cell Transplantation

HY-specific T cells are presumed to play a role in acute graft-versus-host disease (aGVHD) after female-to-male stem cell transplantation (SCT). However, infiltrates of these T cells in aGVHD-affected tissues have not yet been reported. We evaluated the application of HLA-A2/HY dextramers for the in situ detection of HY-specific T cells in cryopreserved skin biopsy specimens. We applied the HLA-A2/HY dextramers on cryopreserved skin biopsy specimens from seven male HLA-A2(+) pediatric patients who underwent stem cell transplantation with confirmed aGVHD involving the skin. The dextramers demonstrated the presence of HY-specific T cells. In skin biopsy specimens of three male recipients of female grafts, 68% to 78% of all skin-infiltrating CD8(+) T cells were HY-specific, whereas these cells were absent in biopsy specimens collected from sex-matched patient-donor pairs. Although this study involved a small and heterogeneous patient group, our results strongly support the hypothesis that HY-specific T cells are actively involved in the pathophysiology of aGVHD after sex-mismatched stem cell transplantation.

Automated detection of residual cells after sex-mismatched stem-cell transplantation – evidence for presence of disease-marker negative residual cells

BackgroundA new chimerism analysis based on automated interphase fluorescence in situ hybridization (FISH) evaluation was established to detect residual cells after allogene sex-mismatched bone marrow or blood stem-cell transplantation.Cells of 58 patients were characterized as disease-associated due to presence of a bcr/abl-gene-fusion or a trisomy 8 and/or a simultaneous hybridization of gonosome-specific centromeric probes. The automatic slide scanning platform Metafer with its module MetaCyte was used to analyse 3,000 cells per sample.ResultsOverall 454 assays of 58 patients were analyzed. 13 of 58 patients showed residual recipient cells at one stage of more than 4% and 12 of 58 showed residual recipient cells less than 4%, respectively. As to be expected, patients of the latter group were associated with a higher survival rate (48 vs. 34 month). In only two of seven patients with disease-marker positive residual cells between 0.1–1.3% a relapse was observed. Besides, disease-marker negative residual cells were found in two patients without relapse at a rate of 2.8% and 3.3%, respectively.ConclusionThe definite origin and meaning of disease-marker negative residual cells is still unclear. Overall, with the presented automatic chimerism analysis of interphase FISH slides, a sensitive method for detection of disease-marker positive residual cells is on hand.

Evaluation of hematopoietic chimerism following allogeneic peripheral blood stem cell transplantation with amelogenin marker.

Monitoring the engraftment of donor cells after allogeneic stem cell transplantation is important for the early diagnosis of graft failure or relapse of disease. The objective of the present study was to evaluate the application of the amelogenin gene for the assessment of chimerism in samples of patients who had received a sex-mismatched stem cell transplantation. A polymerase chain reaction technique was developed using a set of amelogenin gene primers alone and/or in combination with short tandem repeats primers and was preformed on blood and/or bone marrow aspiration samples of 30 recipient patients after transplantation. The technique was then set up as a routine procedure, from September 2000 through April 2006, more than 1400 samples taken from 300 stem cell transplantation patients suffering from different types of leukemia and nonmalignant hematologic disorders were evaluated for detection of chimerism after transplantation. The sensitivity of the test was as low as 1 - 2%. The ratio of X/Y fragments was as the mixed chimerism. In 90% of the patients, amelogenin marker was as informative as short tandem repeats markers, as confirmed by the clinical outcome. In 5% of the patients, when there was no pre- bone marrow transplantation sample from either donor or recipient, the applicability of this assay became crucial to our treating physicians. The application of the amelogenin marker alone or in combination with the short tandem repeats system can be used for relative quantitative analysis of mixed chimerism and for observing kinetics of engraftment in patients who have sex-mismatched bone marrow transplantation. Amelogenin polymerase chain reaction analysis showed an excellent correlation with the short tandem repeats-polymerase chain reaction results.

Mesenchymal Stem Cells in Stem Cell Transplant Recipients Are Damaged and Remain of Host Origin

The aim of this study was to investigate the expansion capacity and origin of bone marrow-derived mesenchymal stem cells (MSCs) in 34 patients who received a sex-mismatched stem cell transplant (SCT). Polymerase chain reaction (PCR) analysis of the amelogenin gene (AMEL) was used to detect donor-derived MSCs. Cultured MSCs were hybridized with fluorescence in situ hybridization (FISH) probes for chromosomes X and Y to distinguish cells of donor origin from those of host origin. The MSCs of 31 of the 34 patients showed confluent stroma, and the MSCs from 24 of these 31 patients were successfully passaged more than 5 times and were able to be used for PCR and FISH analyses. The colony-forming unit-fibroblast, confluence time, and passage numbers of the MSCs and the colony-forming capacity of the hematopoietic progenitor cells of the patients were significantly different from those of 30 healthy control subjects. Flow cytometry results showed that the proportion of CD14(+)CD45(+) cells, which are regarded as monocytes/macrophages, in cultured MSCs (fifth passage) was less than 0.04%. PCR and FISH analyses revealed that the MSC-derived cells in all 24 patients were from the host. In conclusion, the expansion capacity of MSCs in patients who receive an SCT is damaged, and the MSCs originate from the host.

X chromosome methylation-based chimerism assay for sex-mismatched hematopoietic stem cell transplantation.

Analysis of hematopoietic chimerism is important for monitoring engraftment, graft failure, and disease recurrence. Although several techniques are now available, their sensitivity is unsatisfactory. In sex-mismatched stem cell transplantation (SCT) with a female donor, Y chromosome-specific sequences have proven the most sensitive marker. However, in the case of a male donor, no such reliable marker has been available to date. In this study, we report a novel method we developed to detect microchimerism in female recipients who receive SCT from male donors. The X-linked human androgen receptor gene (HUMARA) contains a highly polymorphic CAG trinucleotide repeat. Near this polymorphic site are methyl-sensitive HpaII restriction enzyme sites. After HpaII digestion, unmethylated male HUMARA sequences are completely digested, while methylated female ones remain intact among the male origin cells. This allows a highly efficient detection of a small number of female cells. Combined with the nested PCR technique, the X chromosome methylation-based chimerism assay could attain a 10(-4) level of sensitivity, which is 1000-fold higher than that of conventional assays. The applicability of the method was confirmed in two transplant cases. This highly sensitive method can also be applied to detect minimal residual disease or microchimerism in conditions other than hematopoietic SCT.

UTY gene codes for an HLA-B60–restricted human male-specific minor histocompatibility antigen involved in stem cell graft rejection: characterization of the critical polymorphic amino acid residues for T-cell recognition

Rejection of a graft after human leukocyte antigen (HLA)-identical stem cell transplantation (SCT) can be caused by recipient's immunocompetent T lymphocytes recognizing minor histocompatibility antigens on donor stem cells. During rejection of a male stem cell graft by a female recipient, 2 male (H-Y)–specific cytotoxic T lymphocyte (CTL) clones were isolated from peripheral blood. One CTL clone recognized an HLA-A2–restricted H-Y antigen, encoded by the SMCY gene. Another CTL clone recognized an HLA-B60–restricted H-Y antigen. In this study UTY was identified as the gene coding for the HLA-B60–restricted H-Y antigen. The UTY-derived H-Y antigen was characterized as a 10-amino acid residue peptide, RESEEESVSL. Although the epitope differed by 3 amino acids from its X-homologue, UTX, only 2 polymorphisms were essential for recognition by the CTL clone HLA-B60 HY. These results illustrate that CTLs against several H-Y antigens derived from different proteins can contribute simultaneously to graft rejection after HLA-identical, sex-mismatched SCT. Moreover, RESEEESVSL-specific T cells could be isolated from a female HLA-B60+ patient with myelodysplastic syndrome who has been treated with multiple blood transfusions, but not from control healthy HLA-B60+ female donors. This may indicate that RESEEESVSL-reactive T cells are more common in sensitized patients.

UTY gene codes for an HLA-B60–restricted human male-specific minor histocompatibility antigen involved in stem cell graft rejection: characterization of the critical polymorphic amino acid residues for T-cell recognition

AbstractRejection of a graft after human leukocyte antigen (HLA)-identical stem cell transplantation (SCT) can be caused by recipient's immunocompetent T lymphocytes recognizing minor histocompatibility antigens on donor stem cells. During rejection of a male stem cell graft by a female recipient, 2 male (H-Y)–specific cytotoxic T lymphocyte (CTL) clones were isolated from peripheral blood. One CTL clone recognized an HLA-A2–restricted H-Y antigen, encoded by the SMCY gene. Another CTL clone recognized an HLA-B60–restricted H-Y antigen. In this study UTY was identified as the gene coding for the HLA-B60–restricted H-Y antigen. The UTY-derived H-Y antigen was characterized as a 10-amino acid residue peptide, RESEEESVSL. Although the epitope differed by 3 amino acids from its X-homologue, UTX, only 2 polymorphisms were essential for recognition by the CTL clone HLA-B60 HY. These results illustrate that CTLs against several H-Y antigens derived from different proteins can contribute simultaneously to graft rejection after HLA-identical, sex-mismatched SCT. Moreover, RESEEESVSL-specific T cells could be isolated from a female HLA-B60+ patient with myelodysplastic syndrome who has been treated with multiple blood transfusions, but not from control healthy HLA-B60+ female donors. This may indicate that RESEEESVSL-reactive T cells are more common in sensitized patients.