Time series clustering of T cell subsets dissects heterogeneity in immune reconstitution and clinical outcomes among MUD-HCT patients receiving ATG or PTCy.

Introduction: Anti-T-lymphocyte globulin (ATG) or post-transplant cyclophosphamide (PTCy) prevent graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT), yet individual patients benefit differentially. Methods: Given the sparse comparative data on the impact of cellular immune reconstitution in this setting, we studied flow cytometry and clinical outcomes in 339 recipients of 10/10 matched-unrelated donor (MUD) HCT using either ATG (n=304) or PTCy (n=35) for in vivo T cell manipulation along with a haploidentical PTCy control cohort (n=45). Longitudinal cellular immune reconstitution data were analyzed conventionally and with a data science approach using clustering with dynamic time warping to determine the similarity between time-series of T cell subsets. Results: Consistent with published studies, no significant differences in clinical outcomes were observed at the cohort level between MUD-ATG and MUD-PTCy. However, cellular reconstitution revealed preferences for distinct T cell subpopulations associating with GVHD protection in each setting. Starting early after HCT, MUD-PTCy patients had higher regulatory T cell levels after HCT (p <0.0001), while MUD-ATG patients presented with higher levels of γδ T- or NKT cells (both p <0.0001). Time-series clustering further dissected the patient population's heterogeneity revealing distinct immune reconstitution clusters. Importantly, it identified phenotypes that reproducibly associated with impaired clinical outcomes within the same in vivo T cell manipulation platform. Exemplarily, patients with lower activated- and αß T cell counts had significantly higher NRM (p=0.032) and relapse rates (p =0.01). Discussion: The improved understanding of the heterogeneity of cellular reconstitution in MUD patients with T cell manipulation both at the cohort and individual level may support clinicians in managing HCT complications.

An HLA map of the world: A comparison of HLA frequencies in 200 worldwide populations reveals diverse patterns for class I and class II.

HLA frequencies show widespread variation across human populations. Demographic factors as well as selection are thought to have shaped HLA variation across continents. In this study, a worldwide comparison of HLA class I and class II diversity was carried out. Multidimensional scaling techniques were applied to 50 HLA-A and HLA-B (class I) as well as 13 HLA-DRB1 (class II) first-field frequencies in 200 populations from all continents. Our results confirm a strong effect of geography on the distribution of HLA class I allele groups, with principal coordinates analysis closely resembling geographical location of populations, especially those of Africa-Eurasia. Conversely, class II frequencies stratify populations along a continuum of differentiation less clearly correlated to actual geographic location. Double clustering analysis revealed finer intra-continental sub-clusters (e.g., Northern and Western Europe vs. South East Europe, North Africa and Southwest Asia; South and East Africa vs. West Africa), and HLA allele group patterns characteristic of these clusters. Ancient (Austronesian expansion) and more recent (Romani people in Europe) migrations, as well as extreme differentiation (Taiwan indigenous peoples, Native Americans), and interregional gene flow (Sámi, Egyptians) are also reflected by the results. Barrier analysis comparing DST and geographic location identified genetic discontinuities caused by natural barriers or human behavior explaining inter and intra-continental HLA borders for class I and class II. Overall, a progressive reduction in HLA diversity from African to Oceanian and Native American populations is noted. This analysis of HLA frequencies in a unique set of worldwide populations confirms previous findings on the remarkable similarity of class I frequencies to geography, but also shows a more complex development for class II, with implications for both human evolutionary studies and biomedical research.

Impact of the HLA Immunopeptidome on Survival of Leukemia Patients After Unrelated Donor Transplantation.

PURPOSE: Immunopeptidome divergence between mismatched HLA-DP is a determinant of T-cell alloreactivity and clinical tolerability after fully HLA-A, -B, -C, -DRB1, -DQB1 matched unrelated donor hematopoietic cell transplantation (UD-HCT). Here, we tested this concept in HLA-A, -B, and -C disparities after single class I HLA-mismatched UD-HCT. PATIENTS AND METHODS: We studied 2,391 single class I HLA-mismatched and 14,426 fully HLA-matched UD-HCT performed between 2008 and 2018 for acute leukemia or myelodysplastic syndromes. Hierarchical clustering of experimentally determined peptide-binding motifs (PBM) was used as a proxy for immunopeptidome divergence of HLA-A, -B, or -C disparities, allowing us to classify 1,629/2,391 (68.1%) of the HLA-mismatched UD-HCT as PBM-matched or PBM-mismatched. Risks associated with PBM-matching status were assessed by Cox proportional hazards models, with overall survival (OS) as the primary end point. RESULTS: Relative to full matches, bidirectional or unidirectional PBM mismatches in graft-versus-host (GVH) direction (PBM-GVH mismatches, 60.7%) were associated with significantly lower OS (hazard ratio [HR], 1.48; P < .0001), while unidirectional PBM mismatches in host-versus-graft direction or PBM matches (PBM-GVH matches, 39.3%) were not (HR, 1.13; P = .1017). PBM-GVH mismatches also had significantly lower OS than PBM-GVH matches in direct comparison (HR, 1.32; P = .0036). The hazards for transplant-related mortality and acute and chronic graft-versus-host disease but not relapse increased stepwise from full HLA matches to single PBM-GVH matches, and single PBM-GVH mismatches. A webtool for PBM-matching of single class I HLA-mismatched donor-recipient pairs was developed. CONCLUSION: PBM-GVH mismatches inform mortality risks after single class I HLA-mismatched UD-HCT, suggesting that prospective consideration of directional PBM-matching status might improve outcome. These findings highlight immunopeptidome divergence between mismatched HLA as a driver of clinical tolerability in UD-HCT.

Combined Analysis of Early CD4+ T Cell Counts and CMV Serostatus May Improve CMV Risk Assessment after Allogeneic Hematopoietic Cell Transplantation.

The incidence and severity of viral complications after cellular therapy are highly variable. Recent publications describe relevant interactions between the human Cytomegalovirus (CMV) and host immunity in recipients of allogeneic hematopoietic cell transplantation (HCT). Although immune monitoring is routinely performed in HCT patients, validated cut-off levels correlating with transplant outcomes such as survival or CMV reactivation are mostly limited to day +100, which is later than the median time for CMV reactivation in the absence of medical prophylaxis. To address this gap in early risk assessment, we applied an unsupervised machine learning technique based on clustering of day +30 CD4+ helper T cell count data, and identified relevant cut-off levels within the diverse spectrum of early CD4+ reconstitution. These clusters were stratified for CMV recipient serostatus to identify early risk groups that predict clinical HCT outcome. Indeed, the new risk groups predicted subsequent clinical events such as NRM, OS, and high CMV peak titers better than the most established predictor, i.e., the positive CMV recipient serostatus (R+). More specifically, patients from the R+/low CD4+ subgroup strongly associated with high CMV peak titers and increased 3-year NRM (subdistribution hazard ratio (SHR) 10.1, 95% CI 1.38-73.8, p = 0.023), while patients from the R-/very high CD4+ subgroup showed comparable NRM risks (SHR 9.57, 95% CI 1.12-81.9, p = 0.039) without such an association. In short, our study established novel cut-off levels for early CD4+ T cells via unsupervised learning and supports the integration of host cellular immunity into clinical risk-assessment after HCT in the context of CMV reactivation.

Cytomegalovirus kinetics after hematopoietic cell transplantation reveal peak titers with differential impact on mortality, relapse and immune reconstitution.

Even in the era of PCR-based monitoring, prophylaxis, and preemptive therapy, Cytomegalovirus (CMV) viremia remains a relevant cause of non-relapse mortality (NRM) after allogeneic hematopoietic cell transplantation (HCT). However, studies using binary analysis (presence/absence of CMV) reported contradicting data for NRM, overall survival and leukemia relapse. Here, we analyzed CMV replication kinetics in 11 508 whole blood PCR samples of 705 patients with HCT between 2012 and 2017. Using two independent models based on CMV peak titers and on the time point of first CMV reactivation, we stratified patients into risk cohorts. Each cohort had distinct cellular immune reconstitution profiles and differentiated for relevant clinical outcomes. Patients with high CMV peak titers had significantly reduced overall survival (HR 2.13, 95% CI 1.53-2.96; p < .0001), due to high NRM. Early impaired T cell reconstitution was a risk factor for high CMV peak titers, however relevant CMV viremia also related to boosted T cell reconstitution. Importantly, intermediate CMV peak titers associated with a significantly reduced relapse probability (HR 0.53, 95% CI 0.31-0.91; p = .022). In short, CMV kinetics models distinguished relevant clinical outcome cohorts beyond the R+ serostatus with distinct immune reconstitution patterns and resolve in part contradicting results of previous studies exclusively focused on the presence or absence of CMV.

Permissive HLA-DPB1 mismatches in HCT depend on immunopeptidome divergence and editing by HLA-DM.

In hematopoietic cell transplantation (HCT), permissive HLA-DPB1 mismatches between patients and their unrelated donors are associated with improved outcomes compared with nonpermissive mismatches, but the underlying mechanism is incompletely understood. Here, we used mass spectrometry, T-cell receptor-ß (TCRß) deep sequencing, and cellular in vitro models of alloreactivity to interrogate the HLA-DP immunopeptidome and its role in alloreactive T-cell responses. We find that permissive HLA-DPB1 mismatches display significantly higher peptide repertoire overlaps compared with their nonpermissive counterparts, resulting in lower frequency and diversity of alloreactive TCRß clonotypes in healthy individuals and transplanted patients. Permissiveness can be reversed by the absence of the peptide editor HLA-DM or the presence of its antagonist, HLA-DO, through significant broadening of the peptide repertoire. Our data establish the degree of immunopeptidome divergence between donor and recipient as the mechanistic basis for the clinically relevant permissive HLA-DPB1 mismatches in HCT and show that permissiveness is dependent on HLA-DM-mediated peptide editing. Its key role for harnessing T-cell alloreactivity to HLA-DP highlights HLA-DM as a potential novel target for cellular and immunotherapy of leukemia.

The immunogenetic diversity of the HLA system in Mexico correlates with underlying population genetic structure.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) allele groups and alleles by PCR-SSP based typing in a total of 15,318 mixed ancestry Mexicans from all the states of the country divided into 78 sample sets, providing information regarding allelic and haplotypic frequencies and their linkage disequilibrium, as well as admixture estimates and genetic substructure. We identified the presence of 4268 unique HLA extended haplotypes across Mexico and find that the ten most frequent (HF > 1%) HLA haplotypes with significant linkage disequilibrium (Δ'≥0.1) in Mexico (accounting for 20% of the haplotypic diversity of the country) are of primarily Native American ancestry (A*02~B*39~DRB1*04~DQB1*03:02, A*02~B*35~DRB1*08~DQB1*04, A*68~B*39~DRB1*04~DQB1*03:02, A*02~B*35~DRB1*04~DQB1*03:02, A*24~B*39~DRB1*14~DQB1*03:01, A*24~B*35~DRB1*04~DQB1*03:02, A*24~B*39~DRB1*04~DQB1*03:02, A*02~B*40:02~DRB1*04~DQB1*03:02, A*68~B*35~DRB1*04~DQB1*03:02, A*02~B*15:01~DRB1*04~DQB1*03:02). Admixture estimates obtained by a maximum likelihood method using HLA-A/-B/-DRB1 as genetic estimators revealed that the main genetic components in Mexico as a whole are Native American (ranging from 37.8% in the northern part of the country to 81.5% in the southeastern region) and European (ranging from 11.5% in the southeast to 62.6% in northern Mexico). African admixture ranged from 0.0 to 12.7% not following any specific pattern. We were able to detect three major immunogenetic clusters correlating with genetic diversity and differential admixture within Mexico: North, Central and Southeast, which is in accordance with previous reports using genome-wide data. Our findings provide insights into the population immunogenetic substructure of the whole country and add to the knowledge of mixed ancestry Latin American population genetics, important for disease association studies, detection of demographic signatures on population variation and improved allocation of public health resources.

Immunopeptidome Analysis of HLA-DPB1 Allelic Variants Reveals New Functional Hierarchies.

HLA-DP alleles can be classified into functional T cell epitope (TCE) groups. TCE-1 and TCE-2 are clearly defined, but TCE-3 still represents an heterogeneous group. Because polymorphisms in HLA-DP influence the presented peptidome, we investigated whether the composition of peptides binding in HLA-DP may be used to refine the HLA-DP group classification. Peptidomes of human HLA-DP-typed B cell lines were analyzed with mass spectrometry after immunoaffinity chromatography and peptide elution. Gibbs clustering was performed to identify motifs of binding peptides. HLA-DP peptide-binding motifs showed a clear association with the HLA-DP allele-specific sequences of the binding groove. Hierarchical clustering of HLA-DP immunopeptidomes was performed to investigate the similarities and differences in peptidomes of different HLA-DP molecules, and this clustering resulted in the categorization of HLA-DP alleles into 3-DP peptidome clusters (DPC). The peptidomes of HLA-DPB1*09:01, -10:01, and -17:01 (TCE-1 alleles) and HLA-DPB1*04:01, -04:02, and -02:01 (TCE-3 alleles) were separated in two maximal distinct clusters, DPC-1 and DPC-3, respectively, reflecting their previous TCE classification. HLA-DP alleles categorized in DPC-2 shared certain similar peptide-binding motifs with DPC-1 or DPC-3 alleles, but significant differences were observed for other positions. Within DPC-2, divergence between the alleles was observed based on the preference for different peptide residues at position 9. In summary, immunopeptidome analysis was used to unravel functional hierarchies among HLA-DP alleles, providing new molecular insights into HLA-DP classification.

Genetic diversity of HLA system in two populations from Chiapas, Mexico: Tuxtla Gutiérrez and rural Chiapas.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in 173 Mexicans from the state of Chiapas living in the city of Tuxtla Gutiérrez (N = 52) and rural communities (N = 121), to obtain information regarding allelic and haplotypic frequencies. We found that the most frequent haplotypes in Chiapas include 12 Native American and one European haplotype. Admixture estimates revealed that the main genetic components in Chiapas are Native American (71.61 ±â€¯0.58% by ML; 53.16% of Native American haplotypes) and European (26.39 ±â€¯5.05% by ML; 25.86% of European haplotypes), and a less prominent African genetic component (2.00 ±â€¯5.20% by ML; 9.77% of African haplotypes).

Genetic diversity of HLA system in two populations from Tamaulipas, Mexico: Ciudad Victoria and rural Tamaulipas.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in 148 Mexicans from the state of Tamaulipas living in Ciudad Victoria (N = 23) and rural communities (N = 125), to obtain information regarding allelic and haplotypic frequencies. We found that the most frequent haplotypes in the state of Tamaulipas include ten Native American, three European and one African haplotypes. Admixture estimates revealed that the main genetic components in the state of Tamaulipas are Native American (54.69 ±â€¯0.93% by ML; 47.65% of Native American haplotypes) and European (34.66 ±â€¯5.62% by ML; 33.56% of European haplotypes), and a relatively high African genetic component (10.65 ±â€¯5.05% by ML; 12.42% of African haplotypes).

Genetic diversity of HLA system in two populations from Hidalgo, Mexico: Pachuca and rural Hidalgo.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in 122 Mexicans from the state of Hidalgo living in the city of Pachuca (N = 41) and rural communities (N = 81), to obtain information regarding allelic and haplotypic frequencies. We find that the most frequent haplotypes in Hidalgo include eight Native American and one European haplotypes. Admixture estimates revealed that the main genetic components in Hidalgo are Native American (58.93 ±â€¯2.16% by ML; 54.51% of Native American haplotypes) and European (32.49 ±â€¯2.88% by ML; 28.69% of European haplotypes), and a relatively high African genetic component (8.58 ±â€¯0.93% by ML; 6.97% of African haplotypes).

Genetic diversity of HLA system in seven populations from Veracruz, Mexico: Veracruz city, Coatzacoalcos, Córdoba, Orizaba, Poza Rica, Xalapa and rural Veracruz.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in 1113 Mexicans from the state of Veracruz living in the cities of Coatzacoalcos (N = 55), Orizaba (N = 60), Córdoba (N = 56), Poza Rica (N = 45), Veracruz (N = 171), Xalapa (N = 187) and rural communities (N = 539) to obtain information regarding allelic and haplotypic frequencies. We found that the most frequent haplotypes include 12 Native American haplotypes. Admixture estimates revealed that the main genetic components are Native American (64.93 ±â€¯1.27% by ML; 55.10% of Native American haplotypes) and European (26.56 ±â€¯0.89% by ML; 28.38% of European haplotypes), and a relatively high African genetic component (8.52 ±â€¯1.82% by ML; 8.78% of African haplotypes).

Genetic diversity of HLA system in two populations from Campeche, Mexico: Campeche city and rural Campeche.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in 81 Mexicans from the state of Campeche living in the city of Campeche (N = 34) and rural communities (N = 47), to obtain information regarding allelic and haplotypic frequencies. We find that the most frequent haplotypes in the state of Campeche include ten Native American, three European, one African and one Asian haplotype. Admixture estimates revealed that the main genetic components in the state of Campeche are Native American (65.56 ±â€¯0.96% by ML; 51.24% of Native American haplotypes), European (34.44 ±â€¯10.94% by ML; 30.25% of European haplotypes), and a virtually absent African genetic component (0.00 ±â€¯10.31% by ML; 9.26% of African haplotypes).

Genetic diversity of HLA system in two populations from Oaxaca, Mexico: Oaxaca city and rural Oaxaca.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in 636 Mexicans from the state of Oaxaca living in the city of Oaxaca (N = 151) and rural communities (N = 485), to obtain information regarding allelic and haplotypic frequencies. We found that the 13 most frequent haplotypes in Oaxaca are all of putative Native American origin. Admixture estimates revealed that the main genetic components in the state of Oaxaca are Native American (73.12 ±â€¯2.77% by ML; 61.52% of Native American haplotypes) and European (17.36 ±â€¯2.07% by ML; 20.69% of European haplotypes), and a relatively high African genetic component (9.52 ±â€¯0.88% by ML; 8.94% of African haplotypes).

Genetic diversity of HLA system in two populations from Puebla, Mexico: Puebla city and rural Puebla.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in 2827 Mexicans from the state of Puebla living in the city of Puebla (N = 1994) and rural communities (N = 833), to obtain information regarding allelic and haplotypic frequencies. We found that the 16 most frequent haplotypes in Puebla are all of them Native American. Admixture estimates revealed that the main genetic components in the state of Puebla are Native American (72.21 ±â€¯1.25% by ML; 63.30% of Native American haplotypes) and European (21.05 ±â€¯1.92% by ML; 23.86% of European haplotypes), and a less prominent African genetic component (6.74 ±â€¯2.20% by ML; 6.20% of African haplotypes).

Genetic diversity of HLA system in two populations from Yucatán, Mexico: Mérida and rural Yucatán.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in 324 Mexicans from the state of Yucatán living in the city of Mérida (N = 192) and rural communities (N = 132), to obtain information regarding allelic and haplotypic frequencies. We found that the most frequent haplotypes in the state of Yucatán include 16 Native American and one European haplotype. Admixture estimates revealed that the main genetic components in Yucatán are Native American (81.54 ±â€¯4.99% by ML; 62.92% of Native American haplotypes) and European (11.50 ±â€¯15.43% by ML; 23.26% of European haplotypes), and a less prominent African genetic component (6.96 ±â€¯10.47% by ML; 5.93% of African haplotypes).

Genetic diversity of HLA system in two populations from Morelos, Mexico: Cuernavaca and rural Morelos.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in 112 Mexicans from the state of Morelos living in the city of Cuernavaca (N = 82) and rural communities (N = 30), to obtain information regarding allelic and haplotypic frequencies. The most frequent haplotypes in Morelos include seven Native American, one European, one African and one Asian haplotype. Admixture estimates revealed that the main genetic components in Morelos are Native American (60.43 ±â€¯2.22% by ML; 53.57% of Native American haplotypes) and European (39.58 ±â€¯3.70% by ML; 27.68% of European haplotypes), and a virtually absent African genetic component (0.00 ±â€¯4.93% by ML; but 11.16% of African haplotypes).

Genetic diversity of HLA system in two populations from Quintana Roo, Mexico: Cancún and rural Quintana Roo.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in 98 Mexicans from the state of Quintana Roo living in the city of Cancún (N = 48) and rural communities (N = 50), to obtain information regarding allelic and haplotypic frequencies and their linkage disequilibrium. We found that the most frequent haplotypes in Quintana Roo include ten Native American and two European haplotypes. Admixture estimates revealed that the main genetic components in Quintana Roo are Native American (80.85 ±â€¯3.70% by ML; 60.20% of Native American haplotypes) and European (15.19 ±â€¯14.25% by ML; 26.02% of European haplotypes), and a less prominent African genetic component (3.96 ±â€¯10.75% by ML; 6.63% of African haplotypes).

Genetic diversity of HLA system in two populations from Tlaxcala, Mexico: Tlaxcala city and rural Tlaxcala.

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in 1011 Mexicans from the state of Tlaxcala residing in the city of Tlaxcala (N = 181) and rural communities (N = 830), to obtain information regarding allelic and haplotypic frequencies. We find that the ten most frequent haplotypes in Tlaxcala are all of Native American origin. Admixture estimates revealed that the main genetic components are Native American (75.13 ±â€¯1.56% by ML; 69.24% based on of Native American haplotypes) and European (16.10 ±â€¯4.98% by ML; 19.74% of European haplotypes), with a less prominent African genetic component (8.78 ±â€¯4.09% by ML; 4.35% of African haplotypes).