RESUMO
Immunodeficient mice reconstituted with immune systems from patients, or personalized immune (PI) mice, are powerful tools for understanding human disease. Compared with immunodeficient mice transplanted with human fetal thymus tissue and fetal liver-derived CD34+ cells administered i.v. (Hu/Hu mice), PI mice, which are transplanted with human fetal thymus and adult bone marrow (aBM) CD34+ cells, demonstrate reduced levels of human reconstitution. We characterized APC and APC progenitor repopulation in human immune system mice and detected significant reductions in blood, bone marrow (BM), and splenic APC populations in PI compared with Hu/Hu mice. APC progenitors and hematopoietic stem cells (HSCs) were less abundant in aBM CD34+ cells compared with fetal liver-derived CD34+ cell preparations, and this reduction in APC progenitors was reflected in the BM of PI compared with Hu/Hu mice 14-20 wk posttransplant. The number of HSCs increased in PI mice compared with the originally infused BM cells and maintained functional repopulation potential, because BM from some PI mice 28 wk posttransplant generated human myeloid and lymphoid cells in secondary recipients. Moreover, long-term PI mouse BM contained functional T cell progenitors, evidenced by thymopoiesis in thymic organ cultures. Injection of aBM cells directly into the BM cavity, transgenic expression of hematopoietic cytokines, and coinfusion of human BM-derived mesenchymal stem cells synergized to enhance long-term B cell and monocyte levels in PI mice. These improvements allow a sustained time frame of 18-22 wk where APCs and T cells are present and greater flexibility for modeling immune disease pathogenesis and immunotherapies in PI mice.
Assuntos
Medula Óssea , Transplante de Células-Tronco Hematopoéticas , Animais , Células da Medula Óssea , Células-Tronco Hematopoéticas , Humanos , Fígado , CamundongosRESUMO
Alginate is one of the most abundant polysaccharides in algae. Alginate lyase degrades alginate through a ß-elimination mechanism to produce alginate oligosaccharides with special bioactivities. Improving enzyme activity and thermal stability can promote the application of alginate lyase in the industrial preparation of alginate oligosaccharides. In this study, the recombinant alginate lyase cAlyM and its thermostable mutant 102C300C were expressed and characterized in Pichia pastoris. The specific activities of cAlyM and 102C300C were 277.1 U/mg and 249.6 U/mg, respectively. Both enzymes showed maximal activity at 50 °C and pH 8.0 and polyG preference. The half-life values of 102C300C at 45 °C and 50 °C were 2.6 times and 11.7 times the values of cAlyM, respectively. The degradation products of 102C300C with a lower degree of polymerization contained more guluronate. The oligosaccharides with a polymerization degree of 2-4 were the final hydrolytic products. Therefore, 102C300C is potentially valuable in the production of alginate oligosaccharides with specific M/G ratio and molecular weights.
Assuntos
Alginatos/metabolismo , Pichia/metabolismo , Polissacarídeo-Liases/metabolismo , Animais , Clonagem Molecular , TemperaturaRESUMO
Understanding the effect of conserved domains reconstruction of alginate lyases on action mode is essential for their application and in-depth study. We report the expression and action mode of recombinant alginate lyase (AlyM) and its conserved domain reconstruction forms (AlyMΔCBM, cAlyM, and AlyMΔ58C). The enzymatic activities of AlyM, AlyMΔCBM, cAlyM, and AlyMΔ58C were 61.77, 150.57, 388.97, and 308.21 U/mg, respectively. The transcription level of cAlyM was 49.89-fold of AlyM. cAlyM and AlyMΔ58C showed higher thermal stability than AlyM, indicating that the removal of F5_F8_type_C domain was beneficial for the increase of thermal stability. The enzymes were bifunctional alginate lyases and preferred polyG to polyM. The enzymes degraded alginate to produce unsaturated disaccharide, trisaccharide, and tetrasaccharide as the main end-products. Pentamannuronic acid and pentaguluronic acid were the smallest substrates that could be degraded by AlyM, with unsaturated trisaccharide/tetrasaccharide (40.61%/44.42%) and disaccharide/trisaccharide (10.57%/83.85%) as the main products, respectively. The action modes of enzymes remain unaffected after conserved domain reconstruction, but the affinity of AlyMΔ58C toward polyM increased. This study provides a new strategy for rational modification of alginate lyase based on conserved domain reconstruction.
Assuntos
Alginatos/metabolismo , Gammaproteobacteria/enzimologia , Expressão Gênica , Polissacarídeo-Liases/biossíntese , Polissacarídeo-Liases/metabolismo , Estabilidade Enzimática , Gammaproteobacteria/genética , Hidrólise , Polissacarídeo-Liases/química , Polissacarídeo-Liases/genética , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Transcrição GênicaRESUMO
Chondroitinase (ChSase), a type of glycosaminoglycan (GAG) lyase, can degrade chondroitin sulfate (CS) to unsaturate oligosaccharides, with various functional activities. In this study, ChSase AC II from a newly isolated marine bacterium Arthrobacter sp. CS01 was cloned, expressed in Pichia pastoris X33, purified, and characterized. ChSase AC II, with a molecular weight of approximately 100 kDa and a specific activity of 18.7 U/mg, showed the highest activity at 37 °C and pH 6.5 and maintained stability at a broad range of pH (5â»7.5) and temperature (below 35 °C). The enzyme activity was increased in the presence of Mn2+ and was strongly inhibited by Hg2+. Moreover, the kinetic parameters of ChSase AC II against CS-A, CS-C, and HA were determined. TLC and ESI-MS analysis of the degradation products indicated that ChSase AC II displayed an exolytic action mode and completely hydrolyzed three substrates into oligosaccharides with low degrees of polymerization (DPs). All these features make ChSase AC II a promising candidate for the full use of GAG to produce oligosaccharides.
Assuntos
Organismos Aquáticos/química , Arthrobacter/química , Proteínas de Bactérias/metabolismo , Condroitina Liases/metabolismo , Sulfatos de Condroitina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Condroitina Liases/química , Condroitina Liases/isolamento & purificação , Ensaios Enzimáticos , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Oligossacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Alginate lyase degrades alginate by the ß-elimination mechanism to produce oligosaccharides with special bioactivities. The low thermal stability of alginate lyase limits its industrial application. In this study, introducing the disulfide bonds while using the rational design methodology enhanced the thermal stability of alginate lyase cAlyM from Microbulbifer sp. Q7. Enzyme catalytic sites, secondary structure, spatial configuration, and molecular dynamic simulation were comprehensively analyzed. When compared with cAlyM, the mutants D102C-A300C and G103C-T113C showed an increase by 2.25 and 1.16 h, respectively, in half-life time at 45 °C, in addition to increases by 1.7 °C and 0.4 °C in the melting temperature, respectively. The enzyme-specific activity and kcat/Km values of D102C-A300C were 1.8- and 1.5-times higher than those of cAlyM, respectively. The rational design strategy that was used in this study provides a valuable method for improving the thermal stability of the alginate lyase.
Assuntos
Alginatos/química , Alteromonadaceae/química , Proteínas de Bactérias/química , Polissacarídeo-Liases/química , Domínio Catalítico , Estabilidade Enzimática/efeitos dos fármacos , Oligossacarídeos/química , Especificidade por Substrato , TemperaturaRESUMO
In humans receiving intestinal transplantation (ITx), long-term multilineage blood chimerism often develops. Donor T cell macrochimerism (≥4%) frequently occurs without graft-versus-host disease (GVHD) and is associated with reduced rejection. Here we demonstrate that patients with macrochimerism had high graft-versus-host (GvH) to host-versus-graft (HvG) T cell clonal ratios in their allografts. These GvH clones entered the circulation, where their peak levels were associated with declines in HvG clones early after transplant, suggesting that GvH reactions may contribute to chimerism and control HvG responses without causing GVHD. Consistently, donor-derived T cells, including GvH clones, and CD34+ hematopoietic stem and progenitor cells (HSPCs) were simultaneously detected in the recipients' BM more than 100 days after transplant. Individual GvH clones appeared in ileal mucosa or PBMCs before detection in recipient BM, consistent with an intestinal mucosal origin, where donor GvH-reactive T cells expanded early upon entry of recipient APCs into the graft. These results, combined with cytotoxic single-cell transcriptional profiles of donor T cells in recipient BM, suggest that tissue-resident GvH-reactive donor T cells migrated into the recipient circulation and BM, where they destroyed recipient hematopoietic cells through cytolytic effector functions and promoted engraftment of graft-derived HSPCs that maintain chimerism. These mechanisms suggest an approach to achieving intestinal allograft tolerance.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Intestinos/transplante , Linfopoese/imunologia , Transplante de Órgãos , Linfócitos T/imunologia , Quimeras de Transplante/imunologia , Aloenxertos , Feminino , Doença Enxerto-Hospedeiro/patologia , Humanos , Intestinos/imunologia , Intestinos/patologia , Masculino , Linfócitos T/patologiaRESUMO
Glucose oxidase (GOx) with high enzyme activity at low temperature (4°C) is potentially useful for food preservation, especially for aquatic products preservation. A cold-active GOx with approximately 83% similarity to known protein sequences, was isolated from Penicillium sp. MX3343 and expressed in Pichia pastoris X33. Through high cell density fermentation, the yield of recombinant enzyme (named GOxP5) reached 458.6 U/mL. GOxP5 showed optimal activity at 30°C and pH 5.5, and was stable at a broad pH range from pH 2-6. Moreover, GOxP5 could maintain 72% maximum activity at 4°C, suggesting its application for the preservation of aquatic products at low-temperatures. Importantly, GOxP5 showed a good antimicrobial effect against common fish pathogenic bacteria (Listeria monocytogenes and Vibrio parahaemolyticus). Moreover, sensory, microbiological (total bacterial count), and physicochemical (total volatile basic nitrogen and pH) systematic analyses proved GOxP5 to be an excellent freshness preserving agent in the context of the grass carp. These favorable enzymatic properties of GOxP5 make it potentially useful in food biopreservation, and the effect was better compared to the commonly used chemical preservatives.
RESUMO
ß-mannanase with high specific activity is a prerequisite for the industrial preparation of prebiotic mannooligosaccharides. Three mutants, namely MEI, MER, and MEIR, were constructed by cooperative substitution based on three predominant single-point site mutations (K291E, L211I, and Q112R, respectively). Heterologous expression was facilitated in Pichia pastoris and the recombinase was characterized completely. The specific activities of MER (7481.9 U mg-1) and MEIR (9003.1 U mg-1) increased by 1.07- and 1.29-fold from the initial activity of ME (6970.2U mg-1), respectively. MEIR was used for high-cell-density fermentation to further improve enzyme activity, and the expression levels achieved in the 10-L fermenter were significantly high (105,836 U mL-1). The prebiotic mannooligosaccharides (<2000 Da) were prepared by hydrolyzing konjac gum and locust bean gum with MEIR, with 100% and 76.40% hydrolysis rates, respectively. These characteristics make MEIR highly attractive for prebiotic development in food and related industries.
Assuntos
Amorphophallus , beta-Manosidase , Hidrólise , Pichia , PrebióticosRESUMO
An engineered thermophilic and acidophilic ß-mannanase (ManAK) from Aspergillus kawachii IFO 4308 was highly expressed in Pichia pastoris. Through high cell density fermentation, the maximum yield reached 11,600â¯U/mL and 15.5â¯g/L, which is higher than most extreme ß-mannanases. The recombinant ManAK was thermostable with a temperature optimum of 80⯰C, and acid tolerant with a pH optimum of 2.0. ManAK could efficiently degrade locust bean gum, konjac gum, and guar gum into small molecular mannooligosaccharide (<2000â¯Da), even at high initial substrate concentration (10%), and displayed different Mw distributions in their end products. Docking analysis demonstrated that the catalytic pocket of ManAK could only accommodate a galactopyranosyl residue in subsite -1, which might be responsible for the distinct hydrolysis product compositions from locust bean gum and guar gum. These superior properties of ManAK strongly facilitate mannooligosaccharide preparation and application in food and feed area.
Assuntos
Mananas , beta-Manosidase , Aspergillus , Concentração de Íons de Hidrogênio , Hidrólise , Oligossacarídeos , Pichia , Especificidade por SubstratoRESUMO
NOD2 (the nucleotide-binding oligomerization domain containing protein 2) is known to be involved in host recognition of bacteria, although its role in the host response to Staphylococcus aureus infection is unknown. NOD2-deficient (Nod2(-/-)) mice and wild-type (WT) littermate controls were injected intraperitoneally with S. aureus suspension (10(7) bacteria/g of body weight), and their survival was monitored. Cultured bone marrow-derived neutrophils were harvested from Nod2(-/-) and WT mice and tested for cytokine production and phagocytosis. Compared to WT mice, Nod2(-/-) mice were significantly more susceptible to S. aureus infection (median survival of 1.5 days versus >5 days; P = 0.003) and had a significantly higher bacterial tissue burden. Cultured bone marrow-derived neutrophils from Nod2(-/-) and WT mice had similar levels of peritoneal neutrophil recruitment and intracellular killing, but bone marrow-derived neutrophils from Nod2(-/-) mice had significantly reduced ability to internalize fluorescein-labeled S. aureus. Nod2(-/-) mice had significantly higher levels of Th1-derived cytokines in serum (tumor necrosis factor alpha, gamma interferon, and interleukin-2 [IL-2]) compared to WT mice, whereas the levels of Th2-derived cytokines (IL-1beta, IL-4, IL-6, and IL-10) were similar in Nod2(-/-) and WT mice. Thus, mice deficient in NOD2 are more susceptible to S. aureus. Increased susceptibility is due in part to defective neutrophil phagocytosis, elevated serum levels of Th1 cytokines, and a higher bacterial tissue burden.
Assuntos
Proteína Adaptadora de Sinalização NOD2/metabolismo , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/patogenicidade , Animais , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Proteína Adaptadora de Sinalização NOD2/genética , Fagocitose/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/isolamento & purificação , Células Th1/imunologiaRESUMO
Human intestinal transplantation often results in long-term mixed chimerism of donor and recipient blood in transplant patients. We followed the phenotypes of chimeric peripheral blood cells in 21 patients receiving intestinal allografts over 5 years. Donor lymphocyte phenotypes suggested a contribution of hematopoietic stem and progenitor cells (HSPCs) from the graft. Surprisingly, we detected donor-derived HSPCs in intestinal mucosa, Peyer's patches, mesenteric lymph nodes, and liver. Human gut HSPCs are phenotypically similar to bone marrow HSPCs and have multilineage differentiation potential in vitro and in vivo. Analysis of circulating post-transplant donor T cells suggests that they undergo selection in recipient lymphoid organs to acquire immune tolerance. Our longitudinal study of human HSPCs carried in intestinal allografts demonstrates their turnover kinetics and gradual replacement of donor-derived HSPCs from a circulating pool. Thus, we have demonstrated the existence of functioning HSPCs in human intestines with implications for promoting tolerance in transplant recipients.
Assuntos
Movimento Celular , Células-Tronco Hematopoéticas/citologia , Intestinos/citologia , Intestinos/transplante , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Quimerismo , Doença Enxerto-Hospedeiro/imunologia , Humanos , Tolerância Imunológica , Mucosa Intestinal/citologia , Fígado/citologia , Linfonodos/citologia , Camundongos , Nódulos Linfáticos Agregados/citologia , Fenótipo , Linfócitos T/citologia , Doadores de Tecidos , Transplante HomólogoRESUMO
Alginate lyases play an important role in preparation of alginate oligosaccharides. Although a large number of alginate lyases have been characterized, reports on directional preparation of alginate oligosaccharides by alginate lyases are still rather less. Here, a gene alyM encoding a new alginate lyase AlyM was cloned from Microbulbifer sp. Q7 and expressed in Escherichia coli. AlyM exhibited the maximumactivity at pH 7.0 and 55°C and showed special preference to poly-guluronic acid (polyG). Glycine promoted the extracellular secretion of AlyM by 3.6 times. PBS and glycerol significantly improved the thermal stability of AlyM, the enzyme activity remained 75 and 78% after heat-treatment at 45°C for 2 h, respectively. ESI-MS analysis suggested that AlyM mainly produced oligosaccharides with degrees of polymerization (DP) of 2-5. The results of 1H-NMR showed that guluronic acid (G) occupied the reducing end of the end products, indicating that AlyM preferred to degrade the glycosidic bond at the G-X linkage. HPLC analysis showed that the hydrolysis products with a lower degree of polymerization contained more G. Therefore, AlyM shows good potential to produce alginate oligosaccharides with specific M/G ratio and molecular weights.
RESUMO
Alloreactive T lymphocytes are the primary mediators of immune responses in transplantation, both in the graft-versus-host and host-versus-graft directions. While essentially all clones comprising the human T cell repertoire have been selected on self-peptide presented by self-human leukocyte antigens (self-HLAs), much remains to be understood about the nature of clones capable of responding to allo-HLA molecules. Quantitative tools to study these cells are critical to understand fundamental features of this important response; however, the large size and diversity of the alloreactive T cell repertoire in humans presents a great technical challenge. We have developed a high-throughput T cell receptor (TCR) sequencing approach to characterize the human alloresponse. We present a statistical method to model T cell clonal frequency distribution and quantify repertoire diversity. Using these approaches, we measured the diversity and frequency of distinct alloreactive CD4+ and CD8+ T cell populations in HLA-mismatched responder-stimulator pairs. Our findings indicate that the alloimmune repertoire is highly specific for a given pair of individuals, that most alloreactive clones circulate at low frequencies, and that a high proportion of TCRs is likely able to recognize alloantigens.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Antígenos HLA/imunologia , Isoantígenos/imunologia , Linfócitos T/imunologia , Adulto , Transplante de Medula Óssea/efeitos adversos , Simulação por Computador , Voluntários Saudáveis , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Transplante de Rim/efeitos adversos , Modelos Biológicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Transplante Homólogo/efeitos adversosRESUMO
Allograft tolerance, in which a graft is accepted without long-term immunosuppression, could overcome numerous obstacles in transplantation. Human allograft tolerance has been intentionally induced across HLA barriers via combined kidney and bone marrow transplantation (CKBMT) with a regimen that induces only transient chimerism. Tregs are enriched early after CKBMT. While deletional tolerance contributes to long-term tolerance, the role of Tregs remains unclear. We have optimized a method for identifying the donor-specific Treg repertoire and used it to interrogate the fate of donor-specific Tregs after CKBMT. We expanded Tregs with several different protocols. Using functional analyses and T cell receptor sequencing, we found that expanding sorted Tregs with activated donor B cells identified the broadest Treg repertoire with the greatest potency and donor specificity of suppression. This method outperformed both alloantigen stimulation with CTLA4Ig and sequencing of CFSElo cells from the primary mixed lymphocyte reaction. In 3 tolerant and 1 nontolerant CKBMT recipients, we sequenced donor-specific Tregs before transplant and tracked them after transplant. Preexisting donor-specific Tregs were expanded at 6 months after CKBMT in tolerant patients and were reduced in the nontolerant patient. These results suggest that early expansion of donor-specific Tregs is involved in tolerance induction following CKBMT.
Assuntos
Transplante de Rim , Linfócitos T Reguladores/transplante , Tolerância ao Transplante , Linfócitos B/imunologia , Linfócitos B/transplante , Transplante de Medula Óssea , Contagem de Linfócito CD4 , Antígeno CTLA-4/imunologia , Humanos , Teste de Cultura Mista de Linfócitos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Doadores de TecidosRESUMO
BACKGROUND: Little is known about innate lymphoid cell (ILC) populations in the human gut, and the turnover of these cells and their subsets after transplantation has not been described. METHODS: Intestinal samples were taken from 4 isolated intestine and 3 multivisceral transplant recipients at the time of any operative resection, such as stoma closure or revision. ILCs were isolated and analyzed by flow cytometry. The target population was defined as being negative for lineage markers and double-positive for CD45/CD127. Cells were further stained to define ILC subsets and a donor-specific or recipient-specific HLA marker to analyze chimerism. RESULTS: Donor-derived ILCs were found to persist greater than 8 years after transplantation. Additionally, the percentage of cells thought to be lymphoid tissue inducer cells among donor ILCs was far higher than that among recipient ILCs. CONCLUSIONS: Our findings demonstrate that donor-derived ILCs persist long-term after transplantation and support the notion that human lymphoid tissue inducer cells may form in the fetus and persist throughout life, as hypothesized in rodents. Correlation between chimerism and rejection, graft failure, and patient survival requires further study.
Assuntos
Imunidade Inata , Mucosa Intestinal/patologia , Intestinos/transplante , Linfócitos/patologia , Tecido Linfoide/patologia , Transplante de Órgãos , Adolescente , Adulto , Biópsia , Células Cultivadas , Pré-Escolar , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Mucosa Intestinal/imunologia , Intestinos/imunologia , Intestinos/patologia , Linfócitos/imunologia , Tecido Linfoide/imunologia , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Doadores de Tecidos , Quimeras de TransplanteRESUMO
B cells play a major role in antigen presentation and antibody production in the development of autoimmune diseases, and some of these diseases disproportionally occur in females. Moreover, immune responses tend to be stronger in female vs male humans and mice. Because it is challenging to distinguish intrinsic from extrinsic influences on human immune responses, we used a personalized immune (PI) humanized mouse model, in which immune systems were generated de novo from adult human hematopoietic stem cells (HSCs) in immunodeficient mice. We assessed the effect of recipient sex and of donor autoimmune diseases (type 1 diabetes [T1D] and rheumatoid arthritis [RA]) on human B-cell development in PI mice. We observed that human B-cell levels were increased in female recipients regardless of the source of human HSCs or the strain of immunodeficient recipient mice. Moreover, mice injected with T1D- or RA-derived HSCs displayed B-cell abnormalities compared with healthy control HSC-derived mice, including altered B-cell levels, increased proportions of mature B cells and reduced CD19 expression. Our study revealed an HSC-extrinsic effect of recipient sex on human B-cell reconstitution. Moreover, the PI humanized mouse model revealed HSC-intrinsic defects in central B-cell tolerance that recapitulated those in patients with autoimmune diseases. These results demonstrate the utility of humanized mouse models as a tool to better understand human immune cell development and regulation.
RESUMO
A paradigm in transplantation states that graft-infiltrating T cells are largely non-alloreactive "bystander" cells. However, the origin and specificity of allograft T cells over time has not been investigated in detail in animals or humans. Here, we use polychromatic flow cytometry and high throughput TCR sequencing of serial biopsies to show that gut-resident T cell turnover kinetics in human intestinal allografts are correlated with the balance between intra-graft host-vs-graft (HvG) and graft-vs-host (GvH) reactivities and with clinical outcomes. In the absence of rejection, donor T cells were enriched for GvH-reactive clones that persisted long-term in the graft. Early expansion of GvH clones in the graft correlated with rapid replacement of donor APCs by the recipient. Rejection was associated with transient infiltration by blood-like recipient CD28+ NKG2DHi CD8+ alpha beta T cells, marked predominance of HvG clones, and accelerated T cell turnover in the graft. Ultimately, these recipient T cells acquired a steady state tissue-resident phenotype, but regained CD28 expression during rejections. Increased ratios of GvH to HvG clones were seen in non-rejectors, potentially mitigating the constant threat of rejection posed by HvG clones persisting within the tissue-resident graft T cell population.
RESUMO
T cell responses to allogeneic major histocompatibility complex antigens present a formidable barrier to organ transplantation, necessitating long-term immunosuppression to minimize rejection. Chronic rejection and drug-induced morbidities are major limitations that could be overcome by allograft tolerance induction. Tolerance was first intentionally induced in humans via combined kidney and bone marrow transplantation (CKBMT), but the mechanisms of tolerance in these patients are incompletely understood. We now establish an assay to identify donor-reactive T cells and test the role of deletion in tolerance after CKBMT. Using high-throughput sequencing of the T cell receptor B chain CDR3 region, we define a fingerprint of the donor-reactive T cell repertoire before transplantation and track those clones after transplant. We observed posttransplant reductions in donor-reactive T cell clones in three tolerant CKBMT patients; such reductions were not observed in a fourth, nontolerant, CKBMT patient or in two conventional kidney transplant recipients on standard immunosuppressive regimens. T cell repertoire turnover due to lymphocyte-depleting conditioning only partially accounted for the observed reductions in tolerant patients; in fact, conventional transplant recipients showed expansion of circulating donor-reactive clones, despite extensive repertoire turnover. Moreover, loss of donor-reactive T cell clones more closely associated with tolerance induction than in vitro functional assays. Our analysis supports clonal deletion as a mechanism of allograft tolerance in CKBMT patients. The results validate the contribution of donor-reactive T cell clones identified before transplant by our method, supporting further exploration as a potential biomarker of transplant outcomes.