RESUMEN
HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Linfocitos T CD8-positivos/inmunología , Glioma/inmunología , Glicoesfingolípidos/metabolismo , Glicosiltransferasas/metabolismo , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoterapia/métodos , Presentación de Antígeno , Ácido Aspártico Endopeptidasas/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glioma/mortalidad , Glicoesfingolípidos/inmunología , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Activación de Linfocitos , Transducción de Señal , Análisis de Supervivencia , Escape del TumorRESUMEN
ABSTRACT: Allogeneic stem cell transplantation (alloSCT) is a curative treatment for hematological malignancies. After HLA-matched alloSCT, antitumor immunity is caused by donor T cells recognizing polymorphic peptides, designated minor histocompatibility antigens (MiHAs), that are presented by HLA on malignant patient cells. However, T cells often target MiHAs on healthy nonhematopoietic tissues of patients, thereby inducing side effects known as graft-versus-host disease. Here, we aimed to identify the dominant repertoire of HLA-I-restricted MiHAs to enable strategies to predict, monitor or modulate immune responses after alloSCT. To systematically identify novel MiHAs by genome-wide association screening, T-cell clones were isolated from 39 transplanted patients and tested for reactivity against 191 Epstein-Barr virus transformed B cell lines of the 1000 Genomes Project. By discovering 81 new MiHAs, we more than doubled the antigen repertoire to 159 MiHAs and demonstrated that, despite many genetic differences between patients and donors, often the same MiHAs are targeted in multiple patients. Furthermore, we showed that one quarter of the antigens are cryptic, that is translated from unconventional open reading frames, for example long noncoding RNAs, showing that these antigen types are relevant targets in natural immune responses. Finally, using single cell RNA-seq data, we analyzed tissue expression of MiHA-encoding genes to explore their potential role in clinical outcome, and characterized 11 new hematopoietic-restricted MiHAs as potential targets for immunotherapy. In conclusion, we expanded the repertoire of HLA-I-restricted MiHAs and identified recurrent, cryptic and hematopoietic-restricted antigens, which are fundamental to predict, follow or manipulate immune responses to improve clinical outcome after alloSCT.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Antígenos de Histocompatibilidad Clase I , Antígenos de Histocompatibilidad Menor , Humanos , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/genética , Linfocitos T/inmunología , Estudio de Asociación del Genoma Completo , Trasplante Homólogo , Femenino , MasculinoRESUMEN
In the current era of T cell-based immunotherapies, it is crucial to understand which types of MHC-presented T cell antigens are produced by tumor cells. In addition to linear peptide antigens, chimeric peptides are generated through proteasome-catalyzed peptide splicing (PCPS). Whether such spliced peptides are abundantly presented by MHC is highly disputed because of disagreement in computational analyses of mass spectrometry data of MHC-eluted peptides. Moreover, such mass spectrometric analyses cannot elucidate how much spliced peptides contribute to the pool of immunogenic antigens. In this Perspective, we explain the significance of knowing the contribution of spliced peptides for accurate analyses of peptidomes on one hand, and to serve as a potential source of targetable tumor antigens on the other hand. Toward a strategy for mass spectrometry independent estimation of the contribution of PCPS to the immunopeptidome, we first reviewed methodologies to identify MHC-presented spliced peptide antigens expressed by tumors. Data from these identifications allowed us to compile three independent datasets containing 103, 74, and 83 confirmed T cell antigens from cancer patients. Only 3.9%, 1.4%, and between 0% and 7.2% of these truly immunogenic antigens are produced by PCPS, therefore providing a marginal contribution to the pool of immunogenic tumor antigens. We conclude that spliced peptides will not serve as a comprehensive source to expand the number of targetable antigens for immunotherapies.
Asunto(s)
Antígenos de Neoplasias , Antígenos de Histocompatibilidad Clase I , Neoplasias , Complejo de la Endopetidasa Proteasomal , Empalme de Proteína , Linfocitos T , Presentación de Antígeno , Antígenos de Neoplasias/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Neoplasias/inmunología , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Linfocitos T/inmunologíaRESUMEN
Allogeneic stem cell transplantation (alloSCT) can be curative for hemato-oncology patients due to effective graft-versus-tumor immunity. However, relapse remains the major cause of treatment failure, emphasizing the need for adjuvant immunotherapies. In this regard, post-transplantation dendritic cell (DC) vaccination is a highly interesting strategy to boost graft-versus-tumor responses. Previously, we developed a clinically applicable protocol for simultaneous large-scale generation of end-stage blood DC subsets from donor-derived CD34+ stem cells, including conventional type 1 and 2 DCs (cDC1s and cDC2s), and plasmacytoid DCs (pDCs). In addition, the total cultured end-product (DC-complete vaccine), also contains non-end-stage-DCs (i.e. non-DCs). In this study, we aimed to dissect the phenotypic identity of these non-DCs and their potential immune modulatory functions on the potency of cDCs and pDCs in stimulating tumor-reactive CD8+ T and NK cell responses, in order to obtain rationale for clinical translation of our DC-complete vaccine. The non-DC compartment was heterogeneous and comprised of myeloid progenitors and (immature) granulocyte- and monocyte-like cells. Importantly, non-DCs potentiated toll-like receptor-induced DC maturation, as reflected by increased expression of co-stimulatory molecules and enhanced cDC-derived IL-12 and pDC-derived IFN-α production. Additionally, antigen-specific CD8+ T cells effectively expanded upon DC-complete vaccination in vitro and in vivo. This effect was strongly augmented by non-DCs in an antigen-independent manner. Moreover, non-DCs did not impair in vitro DC-mediated NK cell activation, degranulation nor cytotoxicity. Notably, in vivo i.p. DC-complete vaccination activated i.v. injected NK cells. Together, these data demonstrate that the non-DC compartment potentiates DC-mediated activation and expansion of antigen-specific CD8+ T cells and do not impair NK cell responses in vitro and in vivo. This underscores the rationale for further clinical translation of our CD34+-derived DC-complete vaccine in hemato-oncology patients post alloSCT.
Asunto(s)
Linfocitos T CD8-positivos , Interleucina-12 , Humanos , Células Dendríticas , Activación de Linfocitos , Antígenos CD34 , Moléculas de Adhesión CelularRESUMEN
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.
Asunto(s)
Epítopos/inmunología , Antígenos HLA-D/inmunología , Cadenas beta de HLA-DP/inmunología , Histocompatibilidad/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Aloinjertos , Antígenos de Diferenciación de Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Endosomas/metabolismo , Epítopos/metabolismo , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Células HeLa , Trasplante de Células Madre Hematopoyéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Histocompatibilidad/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Espectrometría de Masas , Chaperonas Moleculares , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Donante no EmparentadoRESUMEN
CAR T cell therapy has shown great promise for the treatment of B cell malignancies. However, antigen-negative escape variants often cause disease relapse, necessitating the development of multi-antigen-targeting approaches. We propose that a T cell receptor (TCR)-based strategy would increase the number of potential antigenic targets, as peptides from both intracellular and extracellular proteins can be recognized. Here, we aimed to isolate a broad range of promising TCRs targeting multiple antigens for treatment of B cell malignancies. As a first step, 28 target genes for B cell malignancies were selected based on gene expression profiles. Twenty target peptides presented in human leukocyte antigen (HLA)-A∗01:01, -A∗24:02, -B∗08:01, or -B∗35:01 were identified from the immunopeptidome of B cell malignancies and used to form peptide-HLA (pHLA)-tetramers for T cell isolation. Target-peptide-specific CD8 T cells were isolated from HLA-mismatched healthy donors and subjected to a stringent stepwise selection procedure to ensure potency and eliminate cross-reactivity. In total, five T cell clones specific for FCRL5 in HLA-A∗01:01, VPREB3 in HLA-A∗24:02, and BOB1 in HLA-B∗35:01 recognized B cell malignancies. For all three specificities, TCR gene transfer into CD8 T cells resulted in cytokine production and efficient killing of multiple B cell malignancies. In conclusion, using this systematic approach we successfully identified three promising TCRs for T cell therapy against B cell malignancies.
Asunto(s)
Neoplasias , Receptores de Antígenos de Linfocitos T , Linfocitos T CD8-positivos , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Aberrant expression of certain glycosphingolipids (GSLs) is associated with the differentiation of acute myeloid leukemia (AML) cells. However, the expression patterns of GSLs in AML are still poorly explored because of their complexity, the presence of multiple isomeric structures, and tedious analytical procedures. In this study, we performed an in-depth GSL glycan analysis of 19 AML cell lines using porous graphitized carbon liquid chromatography-mass spectrometry revealing strikingly different GSL glycan profiles between the various AML cell lines. The cell lines of the M6 subtype showed a high expression of gangliosides with α2,3-sialylation and Neu5Gc, while the M2 and M5 subtypes were characterized by high expression of (neo)lacto-series glycans and Lewis A/X antigens. Integrated analysis of glycomics and available transcriptomics data revealed the association of GSL glycan abundances with the transcriptomics expression of certain glycosyltransferases (GTs) and transcription factors (TFs). In addition, correlations were found between specific GTs and TFs. Our data reveal TFs GATA2, GATA1, and RUNX1 as candidate inducers of the expression of gangliosides and sialylation via regulation of the GTs ST3GAL2 and ST8SIA1. In conclusion, we show that GSL glycan expression levels are associated with hematopoietic AML classifications and TF and GT gene expression. Further research is needed to dissect the regulation of GSL expression and its role in hematopoiesis and associated malignancies.
Asunto(s)
Glicoesfingolípidos , Leucemia Mieloide Aguda , Diferenciación Celular , Línea Celular , Glicómica/métodos , Glicoesfingolípidos/química , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Polisacáridos/metabolismoRESUMEN
CD4 T cells play a central role as helper cells in adaptive immunity. Presentation of exogenous antigens in MHC class II by professional antigen-presenting cells is a crucial step in induction of specific CD4 T cells in adaptive immune responses. For efficient induction of immunity against intracellular threats such as viruses or malignant transformations, antigens from HLA class II-negative infected or transformed cells need to be transferred to surrounding antigen-presenting cells to allow efficient priming of naive CD4 T cells. Here we show indirect antigen presentation for a subset of natural HLA class II ligands that are created by genetic variants and demonstrated that (neo)antigens can be transferred between cells by extracellular vesicles. Intercellular transfer by extracellular vesicles was not dependent on the T-cell epitope, but rather on characteristics of the full-length protein. This mechanism of (neo)antigen transfer from HLA class II-negative cells to surrounding antigen-presenting cells may play a crucial role in induction of anti-tumor immunity.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Vesículas Extracelulares/metabolismo , Variación Genética , Antígenos de Histocompatibilidad Clase II/genética , Neoplasias/inmunología , Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Vesículas Extracelulares/inmunología , Quinasa 2 de Adhesión Focal/genética , Quinasa 2 de Adhesión Focal/inmunología , Células HeLa , Humanos , Ligandos , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/inmunología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Immunotherapy for hematological malignancies or solid tumors by administration of monoclonal antibodies or T cells engineered to express chimeric antigen receptors or T-cell receptors (TCRs) has demonstrated clinical efficacy. However, antigen-loss tumor escape variants and the absence of currently targeted antigens on several malignancies hamper the widespread application of immunotherapy. We have isolated a TCR targeting a peptide of the intracellular B cell-specific transcription factor BOB1 presented in the context of HLA-B*07:02. TCR gene transfer installed BOB1 specificity and reactivity onto recipient T cells. TCR-transduced T cells efficiently lysed primary B-cell leukemia, mantle cell lymphoma, and multiple myeloma in vitro. We also observed recognition and lysis of healthy BOB1-expressing B cells. In addition, strong BOB1-specific proliferation could be demonstrated for TCR-modified T cells upon antigen encounter. Furthermore, clear in vivo antitumor reactivity was observed of BOB1-specific TCR-engineered T cells in a xenograft mouse model of established multiple myeloma. Absence of reactivity toward a broad panel of BOB1- but HLA-B*07:02+ nonhematopoietic and hematopoietic cells indicated no off-target toxicity. Therefore, administration of BOB1-specific TCR-engineered T cells may provide novel cellular treatment options to patients with B-cell malignancies, including multiple myeloma.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Linfoma no Hodgkin/inmunología , Mieloma Múltiple/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transactivadores/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Citometría de Flujo , Ingeniería Genética/métodos , Humanos , Ratones , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Adult B-lymphoblastic leukemia (B-ALL) is a hematological malignancy characterized by genetic heterogeneity. Despite successful remission induction with classical chemotherapeutics and novel targeted agents, enduring remission is often hampered by disease relapse due to outgrowth of a pre-existing subclone resistant against the treatment. In this study, we show that small glycophosphatidylinositol (GPI)-anchor deficient CD52-negative B-cell populations are frequently present already at diagnosis in B-ALL patients, but not in patients suffering from other B-cell malignancies. We demonstrate that the GPI-anchor negative phenotype results from loss of mRNA expression of the PIGH gene, which is involved in the first step of GPI-anchor synthesis. Loss of PIGH mRNA expression within these B-ALL cells follows epigenetic silencing rather than gene mutation or deletion. The coinciding loss of CD52 membrane expression may contribute to the development of resistance to alemtuzumab (ALM) treatment in B-ALL patients resulting in the outgrowth of CD52-negative escape variants. Additional treatment with 5-aza-2'-deoxycytidine may restore expression of CD52 and revert ALM resistance.
Asunto(s)
Linfocitos B/metabolismo , Antígeno CD52/deficiencia , Metilación de ADN/efectos de los fármacos , Regulación Leucémica de la Expresión Génica , Silenciador del Gen , Glicosilfosfatidilinositoles/deficiencia , Proteínas de la Membrana/genética , Proteínas de Neoplasias/deficiencia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Alemtuzumab/uso terapéutico , Antimetabolitos Antineoplásicos/farmacología , Antimetabolitos Antineoplásicos/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Linfocitos B/patología , Antígeno CD52/biosíntesis , Antígeno CD52/genética , Línea Celular Tumoral , Decitabina/farmacología , Decitabina/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Glicosilfosfatidilinositoles/biosíntesis , Glicosilfosfatidilinositoles/genética , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fenotipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genéticaRESUMEN
Cell-mediated immunity plays a key role in host control of viral infection. This is exemplified by life-threatening reactivations of e.g. herpesviruses in individuals with impaired T-cell and/or iNKT cell responses. To allow lifelong persistence and virus production in the face of primed immunity, herpesviruses exploit immune evasion strategies. These include a reduction in viral antigen expression during latency and a number of escape mechanisms that target antigen presentation pathways. Given the plethora of foreign antigens expressed in virus-producing cells, herpesviruses are conceivably most vulnerable to elimination by cell-mediated immunity during the replicative phase of infection. Here, we show that a prototypic herpesvirus, Epstein-Barr virus (EBV), encodes a novel, broadly acting immunoevasin, gp150, that is expressed during the late phase of viral replication. In particular, EBV gp150 inhibits antigen presentation by HLA class I, HLA class II, and the non-classical, lipid-presenting CD1d molecules. The mechanism of gp150-mediated T-cell escape does not depend on degradation of the antigen-presenting molecules nor does it require gp150's cytoplasmic tail. Through its abundant glycosylation, gp150 creates a shield that impedes surface presentation of antigen. This is an unprecedented immune evasion mechanism for herpesviruses. In view of its likely broader target range, gp150 could additionally have an impact beyond escape of T cell activation. Importantly, B cells infected with a gp150-null mutant EBV displayed rescued levels of surface antigen presentation by HLA class I, HLA class II, and CD1d, supporting an important role for iNKT cells next to classical T cells in fighting EBV infection. At the same time, our results indicate that EBV gp150 prolongs the timespan for producing viral offspring at the most vulnerable stage of the viral life cycle.
Asunto(s)
Presentación de Antígeno/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Evasión Inmune/inmunología , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas Virales/inmunología , Western Blotting , Citometría de Flujo , Herpesvirus Humano 4/inmunología , Humanos , Microscopía Confocal , Linfocitos T/inmunología , Transducción GenéticaRESUMEN
CD8+ T cell-depleted (TCD) donor lymphocyte infusion (DLI) after TCD allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with a reduced risk of graft-versus-host disease (GVHD) while preserving conversion to donor hematopoiesis and antitumor immunity, providing a rationale for exploring CD4+ T cell-based immunotherapy for hematologic malignancies. Here, we analyzed the clinical course and specificity of T cell immune responses in 2 patients with acute myeloid leukemia (AML) who converted to full-donor chimerism but developed severe acute GVHD after prophylactic CD4+ DLI after 10/10-HLA-matched, but HLA-DPB1-mismatched TCD-alloSCT. Clonal analysis of activated T cells isolated during GVHD demonstrated allo-reactivity exerted by CD4+ T cells directed against patient-mismatched HLA-DPB1 molecules on hematopoietic cells and skin-derived fibroblasts only when cultured under inflammatory conditions. At the time of CD4+ DLI, both patients contained residual patient-derived T cells, including cytomegalovirus (CMV)-specific T cells as a result of CMV reactivations. Once activated by CMV antigens, these CMV-specific T cells could stimulate HLA-DPB1-specific CD4+ T cells, which in turn could target nonhematopoietic tissues in GVHD. In conclusion, our data demonstrate that GVHD after HLA-DPB1-mismatched CD4+ DLI can be mediated by allo-reactive HLA-DPB1-directed CD4+ T cells and that ongoing viral infections inducing HLA class II expression on nonhematopoietic cells may increase the likelihood of GVHD development. This trial is registered at http://www.controlled-trials.com/ISRCTN51398568/LUMC as #51398568.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Enfermedad Injerto contra Huésped/inmunología , Antígenos HLA/inmunología , Antígenos HLA-DP/inmunología , Virosis/inmunología , Anciano , Donantes de Sangre , Linfocitos T CD4-Positivos/trasplante , Células Cultivadas , Citomegalovirus/inmunología , Femenino , Antígenos HLA/metabolismo , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Inmunoterapia Adoptiva/métodos , Masculino , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud , Trasplante Homólogo , Regulación hacia Arriba/inmunología , Virosis/virologíaRESUMEN
Hematological malignancies often express surface HLA class II, making them attractive targets for CD4+ T cell therapy. We previously demonstrated that HLA class II ligands can be divided into DM-resistant and DM-sensitive antigens. In contrast to presentation of DM-resistant antigens, presentation of DM-sensitive antigens is suppressed by HLA-DM but can be rescued by HLA-DO. We also showed that HLA-DO expression remains low in nonhematopoietic cells under inflammatory conditions, suggesting that DM-sensitive antigens may be ideal T cell targets with a low risk for graft-versus-host disease. Here, we demonstrated that B cell malignancies often express HLA-DO and that levels are in particular high in chronic lymphocytic leukemia. Moreover, we showed that surface presentation of DM-sensitive antigens is regulated by HLA-DO, and that DM-sensitive antigens are relevant T cell targets for B cell malignancies and, especially, chronic lymphocytic leukemia. These data open the perspective to target HLA class II ligands with specific processing and presentation behavior for CD4+ T cell therapy of hematological malignancies.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Regulación Leucémica de la Expresión Génica/inmunología , Antígenos HLA-D/inmunología , Leucemia Linfocítica Crónica de Células B/genética , Presentación de Antígeno/genética , Linfocitos B/patología , Linfocitos T CD4-Positivos/patología , Femenino , Antígenos HLA-D/genética , Prueba de Histocompatibilidad , Humanos , Inmunofenotipificación , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Cultivo Primario de CélulasRESUMEN
CD4(+) T cells play a central role in adaptive immunity. The acknowledgment of their cytolytic effector function and the finding that endogenous antigens can enter the HLA class II processing pathway make CD4(+) T cells promising tools for immunotherapy. Expression of HLA class II and endogenous antigen, however, does not always correlate with T-cell recognition. We therefore investigated processing and presentation of endogenous HLA class II epitopes that induced CD4(+) T cells during in vivo immune responses. We demonstrate that the peptide editor HLA-DM allowed antigen presentation of some (DM-resistant antigens) but abolished surface expression of other natural HLA class II epitopes (DM-sensitive antigens). DM sensitivity was shown to be epitope specific, mediated via interaction between HLA-DM and the HLA-DR restriction molecule, and reversible by HLA-DO. Because of the restricted expression of HLA-DO, presentation of DM-sensitive antigens was limited to professional antigen-presenting cells, whereas DM-resistant epitopes were expressed on all HLA class II-expressing cells. In conclusion, our data provide novel insights into the presentation of endogenous HLA class II epitopes and identify intracellular antigen processing and presentation as a critical factor for CD4(+) T-cell recognition. This opens perspectives to exploit selective processing capacities as a new approach for targeted immunotherapy.
Asunto(s)
Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/inmunología , Epítopos/inmunología , Antígenos HLA-D/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Células Presentadoras de Antígenos/inmunología , Biomarcadores/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Antígenos HLA-D/química , Antígenos HLA-D/metabolismo , Antígenos HLA-D/farmacología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fragmentos de Péptidos/inmunología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
It is well known that allo-reactive T cells play a crucial role in graft-versus-leukemia and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation (alloSCT). Allo-reactive CD4(+) T cells can mediate direct cytolysis, but may also stimulate production of IgG antibodies as helper cells. Immune complexes may subsequently be processed and presented by professional antigen presenting cells and stimulate induction of specific CD8(+) T cells. As such, proteins targeted in coordinated T- and B-cell responses may represent a class of immunodominant antigens in clinical responses after alloSCT. We previously identified LB-PTK2B-1T as HLA class II restricted polymorphic antigen in a patient treated with donor lymphocyte infusion for relapsed chronic myeloid leukemia after HLA-matched alloSCT. Since PTK2B has also been described as antibody target, we here investigated whether a coordinated T- and B-cell response against PTK2B was induced. Patient serum before and after alloSCT and donor lymphocyte infusion (DLI) was screened for antibodies, and we indeed observed development of a humoral immune response against PTK2B. Antibodies against PTK2B were only found after DLI and, in contrast to the CD4(+) T cells, recognized a monomorphic region of the protein. To our knowledge, this is the first description of a coordinated allo-reactive CD4(+) T-cell and auto-reactive antibody response against an autosomal antigen.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Quinasa 2 de Adhesión Focal/inmunología , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas , Inmunoglobulina G/inmunología , Isoanticuerpos/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva , Aloinjertos , Linfocitos B/metabolismo , Linfocitos B/patología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Femenino , Quinasa 2 de Adhesión Focal/genética , Quinasa 2 de Adhesión Focal/metabolismo , Enfermedad Injerto contra Huésped/enzimología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/patología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunoglobulina G/sangre , Isoanticuerpos/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , MasculinoRESUMEN
Allogeneic hematopoietic cell transplantation (alloHCT) provides a potential curative treatment for haematological malignancies. The therapeutic Graft-versus-Leukaemia (GvL) effect is induced by donor T cells attacking patient hematopoietic (malignant) cells. However, if healthy non-hematopoietic tissues are targeted, Graft-versus-Disease (GvHD) may develop. After HLA-matched alloHCT, GvL and GvHD are induced by donor T cells recognizing polymorphic peptides presented by HLA on patient cells, so-called minor histocompatibility antigens (MiHAs). The balance between GvL and GvHD depends on the tissue distribution of MiHAs and T-cell frequencies targeting these MiHAs. T cells against broadly expressed MiHAs induce GvL and GvHD, whereas those targeting MiHAs with hematopoietic-restricted expression induce GvL without GvHD. Recently, the MiHA repertoire identified in natural immune responses after alloHCT was expanded to 159 total HLA-I-restricted MiHAs, including 14 hematopoietic-restricted MiHAs. This review explores their potential relevance to predict, monitor, and manipulate GvL and GvHD for improving clinical outcome after HLA-matched alloHCT.
Asunto(s)
Enfermedad Injerto contra Huésped , Efecto Injerto vs Leucemia , Trasplante de Células Madre Hematopoyéticas , Antígenos de Histocompatibilidad Menor , Humanos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Enfermedad Injerto contra Huésped/inmunología , Antígenos de Histocompatibilidad Menor/inmunología , Antígenos de Histocompatibilidad Menor/genética , Efecto Injerto vs Leucemia/inmunología , Trasplante Homólogo , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/inmunología , Linfocitos T/inmunología , AloinjertosRESUMEN
Subtyping of acute myeloid leukaemia (AML) is predominantly based on recurrent genetic abnormalities, but recent literature indicates that transcriptomic phenotyping holds immense potential to further refine AML classification. Here we integrated five AML transcriptomic datasets with corresponding genetic information to provide an overview (n = 1224) of the transcriptomic AML landscape. Consensus clustering identified 17 robust patient clusters which improved identification of CEBPA-mutated patients with favourable outcomes, and uncovered transcriptomic subtypes for KMT2A rearrangements (2), NPM1 mutations (5), and AML with myelodysplasia-related changes (AML-MRC) (5). Transcriptomic subtypes of KMT2A, NPM1 and AML-MRC showed distinct mutational profiles, cell type differentiation arrests and immune properties, suggesting differences in underlying disease biology. Moreover, our transcriptomic clusters show differences in ex-vivo drug responses, even when corrected for differentiation arrest and superiorly capture differences in drug response compared to genetic classification. In conclusion, our findings underscore the importance of transcriptomics in AML subtyping and offer a basis for future research and personalised treatment strategies. Our transcriptomic compendium is publicly available and we supply an R package to project clusters to new transcriptomic studies.
Asunto(s)
Leucemia Mieloide Aguda , Proteínas Nucleares , Humanos , Proteínas Nucleares/genética , Transcriptoma/genética , Nucleofosmina , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Mutación , Perfilación de la Expresión Génica , PronósticoRESUMEN
DNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 1 and 2 (IDH1/2) are genes involved in epigenetic regulation, each mutated in 7-23% of patients with acute myeloid leukemia. Here, we investigated whether hotspot mutations in these genes encode neoantigens that can be targeted by immunotherapy. Five human B-lymphoblastoid cell lines expressing common HLA class I alleles were transduced with a minigene construct containing mutations that often occur in DNMT3A or IDH1/2. From these minigene-transduced cell lines, peptides were eluted from HLA class I alleles and analyzed using tandem mass spectrometry. The resulting data are available via ProteomeXchange under the identifier PXD050560. Mass spectrometry revealed an HLA-A*01:01-binding DNMT3AR882H peptide and an HLA-B*07:02-binding IDH2R140Q peptide as potential neoantigens. For these neopeptides, peptide-HLA tetramers were produced to search for specific T-cells in healthy individuals. Various T-cell clones were isolated showing specific reactivity against cell lines transduced with full-length DNMT3AR882H or IDH2R140Q genes, while cell lines transduced with wildtype genes were not recognized. One T-cell clone for DNMT3AR882H also reacted against patient-derived acute myeloid leukemia cells with the mutation, while patient samples without the mutation were not recognized, thereby validating the surface presentation of a DNMT3AR882H neoantigen that can potentially be targeted in acute myeloid leukemia via immunotherapy.
RESUMEN
The diagnostic spectrum for AML patients is increasingly based on genetic abnormalities due to their prognostic and predictive value. However, information on the AML blast phenotype regarding their maturational arrest has started to regain importance due to its predictive power for drug responses. Here, we deconvolute 1350 bulk RNA-seq samples from five independent AML cohorts on a single-cell healthy BM reference and demonstrate that the morphological differentiation stages (FAB) could be faithfully reconstituted using estimated cell compositions (ECCs). Moreover, we show that the ECCs reliably predict ex-vivo drug resistances as demonstrated for Venetoclax, a BCL-2 inhibitor, resistance specifically in AML with CD14+ monocyte phenotype. We validate these predictions using LUMC proteomics data by showing that BCL-2 protein abundance is split into two distinct clusters for NPM1-mutated AML at the extremes of CD14+ monocyte percentages, which could be crucial for the Venetoclax dosing patients. Our results suggest that Venetoclax resistance predictions can also be extended to AML without recurrent genetic abnormalities and possibly to MDS-related and secondary AML. Lastly, we show that CD14+ monocytic dominated Ven/Aza treated patients have significantly lower overall survival. Collectively, we propose a framework for allowing a joint mutation and maturation stage modeling that could be used as a blueprint for testing sensitivity for new agents across the various subtypes of AML.