RESUMO
Inflammatory intestinal diseases are characterized by abnormal immune responses and affect distinct locations of the gastrointestinal tract. Although the role of several immune subsets in driving intestinal pathology has been studied, a system-wide approach that simultaneously interrogates all major lineages on a single-cell basis is lacking. We used high-dimensional mass cytometry to generate a system-wide view of the human mucosal immune system in health and disease. We distinguished 142 immune subsets and through computational applications found distinct immune subsets in peripheral blood mononuclear cells and intestinal biopsies that distinguished patients from controls. In addition, mucosal lymphoid malignancies were readily detected as well as precursors from which these likely derived. These findings indicate that an integrated high-dimensional analysis of the entire immune system can identify immune subsets associated with the pathogenesis of complex intestinal disorders. This might have implications for diagnostic procedures, immune-monitoring, and treatment of intestinal diseases and mucosal malignancies.
Assuntos
Doença Celíaca/imunologia , Doença de Crohn/imunologia , Citometria por Imagem/métodos , Mucosa Intestinal/imunologia , Subpopulações de Linfócitos/imunologia , Linfócitos/imunologia , Linfócitos/fisiologia , Linfoma de Células T/imunologia , Adulto , Idoso , Doença Celíaca/diagnóstico , Estudos de Coortes , Biologia Computacional , Doença de Crohn/diagnóstico , Feminino , Células HEK293 , Humanos , Testes Imunológicos , Linfoma de Células T/diagnóstico , Masculino , Pessoa de Meia-Idade , Monitorização Imunológica , Especificidade de Órgãos , Análise de Célula ÚnicaRESUMO
Celiac disease is a human leukocyte antigen (HLA)-DQ2- and/or DQ8-associated T cell-mediated disorder that is induced by dietary gluten. Although it is established how gluten peptides bind HLA-DQ8 and HLA-DQ2, it is unclear how such peptide-HLA complexes are engaged by the T cell receptor (TCR), a recognition event that triggers disease pathology. We show that biased TCR usage (TRBV9(∗)01) underpins the recognition of HLA-DQ8-α-I-gliadin. The structure of a prototypical TRBV9(∗)01-TCR-HLA-DQ8-α-I-gliadin complex shows that the TCR docks centrally above HLA-DQ8-α-I-gliadin, in which all complementarity-determining region-ß (CDRß) loops interact with the gliadin peptide. Mutagenesis at the TRBV9(∗)01-TCR-HLA-DQ8-α-I-gliadin interface provides an energetic basis for the Vß bias. Moreover, CDR3 diversity accounts for TRBV9(∗)01(+) TCRs exhibiting differing reactivities toward the gliadin epitopes at various deamidation states. Accordingly, biased TCR usage is an important factor in the pathogenesis of DQ8-mediated celiac disease.
Assuntos
Doença Celíaca/imunologia , Gliadina/imunologia , Antígenos HLA-DQ/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Sequência de Aminoácidos , Epitopos de Linfócito T/imunologia , Antígenos HLA-DQ/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos T/químicaRESUMO
Refractory celiac disease type II (RCDII) is a severe complication of celiac disease (CD) characterized by the presence of an enlarged clonal population of innate intraepithelial lymphocytes (IELs) lacking classical B-, T-, and natural killer (NK)-cell lineage markers (Lin-IELs) in the duodenum. In â¼50% of patients with RCDII, these Lin-IELs develop into a lymphoma for which no effective treatment is available. Current evidence indicates that the survival and expansion of these malignant Lin-IELs is driven by epithelial cell-derived IL-15. Like CD, RCDII is strongly associated with HLA-DQ2, suggesting the involvement of HLA-DQ2-restricted gluten-specific CD4+ T cells. We now show that gluten-specific CD4+ T cells isolated from CD duodenal biopsy specimens produce cytokines able to trigger proliferation of malignant Lin-IEL lines as powerfully as IL-15. Furthermore, we identify TNF, IL-2, and IL-21 as CD4+ T-cell cytokines that synergistically mediate this effect. Like IL-15, these cytokines were found to increase the phosphorylation of STAT5 and Akt and transcription of antiapoptotic mediator bcl-xL Several small-molecule inhibitors targeting the JAK/STAT pathway blocked proliferation elicited by IL-2 and IL-15, but only an inhibitor targeting the PI3K/Akt/mTOR pathway blocked proliferation induced by IL-15 as well as the CD4+ T-cell cytokines. Confirming and extending these findings, TNF, IL-2, and IL-21 also synergistically triggered the proliferation of freshly isolated Lin-IELs and CD3-CD56+ IELs (NK-IELs) from RCDII as well as non-RCDII duodenal biopsy specimens. These data provide evidence implicating CD4+ T-cell cytokines in the pathogenesis of RCDII. More broadly, they suggest that adaptive immune responses can contribute to innate IEL activation during mucosal inflammation.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Citocinas/farmacologia , Linfócitos Intraepiteliais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Doença Celíaca/genética , Doença Celíaca/metabolismo , Proliferação de Células/genética , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Sinergismo Farmacológico , Duodeno/metabolismo , Humanos , Interleucina-15/genética , Interleucina-15/metabolismo , Interleucina-15/farmacologia , Interleucina-2/genética , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Interleucinas/genética , Interleucinas/metabolismo , Interleucinas/farmacologia , Linfócitos Intraepiteliais/metabolismo , Proteínas Recombinantes/farmacologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Knowledge of human NK cells is based primarily on conventional CD56(bright) and CD56(dim) NK cells from blood. However, most cellular immune interactions occur in lymphoid organs. Based on the coexpression of CD69 and CXCR6, we identified a third major NK cell subset in lymphoid tissues. This population represents 30-60% of NK cells in marrow, spleen, and lymph node but is absent from blood. CD69(+)CXCR6(+) lymphoid tissue NK cells have an intermediate expression of CD56 and high expression of NKp46 and ICAM-1. In contrast to circulating NK cells, they have a bimodal expression of the activating receptor DNAX accessory molecule 1. CD69(+)CXCR6(+) NK cells do not express the early markers c-kit and IL-7Rα, nor killer cell Ig-like receptors or other late-differentiation markers. After cytokine stimulation, CD69(+)CXCR6(+) NK cells produce IFN-γ at levels comparable to CD56(dim) NK cells. They constitutively express perforin but require preactivation to express granzyme B and exert cytotoxicity. After hematopoietic stem cell transplantation, CD69(+)CXCR6(+) lymphoid tissue NK cells do not exhibit the hyperexpansion observed for both conventional NK cell populations. CD69(+)CXCR6(+) NK cells constitute a separate NK cell population with a distinct phenotype and function. The identification of this NK cell population in lymphoid tissues provides tools to further evaluate the cellular interactions and role of NK cells in human immunity.
Assuntos
Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Tecido Linfoide/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígeno CD56/metabolismo , Separação Celular , Células Cultivadas , Citometria de Fluxo , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunofenotipagem , Interferon gama/metabolismo , Lectinas Tipo C/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Receptores CXCR6 , Receptores de Quimiocinas/metabolismo , Receptores Virais/metabolismoRESUMO
In HLA-DQ8-associated celiac disease (CD), the pathogenic T cell response is directed toward an immunodominant α-gliadin-derived peptide (DQ8-glia-α1). However, our knowledge of TCR gene usage within the primary intestinal tissue of HLA-DQ8 (+) CD patients is limited. We identified two populations of HLA-DQ8-glia-α1 tetramer(+) CD4(+) T cells that were essentially undetectable in biopsy samples from patients on a gluten-free diet but expanded rapidly and specifically after antigenic stimulation. Distinguished by expression of TRBV9, both T cell populations displayed biased clonotypic repertoires and reacted similarly against HLA-DQ8-glia-α1. In particular, TRBV9 paired most often with TRAV26-2, whereas the majority of TRBV9(-) TCRs used TRBV6-1 with no clear TRAV gene preference. Strikingly, both tetramer(+)/TRBV9(+) and tetramer(+)/TRBV9(-) T cells possessed a non-germline-encoded arginine residue in their CDR3α and CDR3ß loops, respectively. Comparison of the crystal structures of three TRBV9(+) TCRs and a TRBV9(-) TCR revealed that, as a result of distinct TCR docking modes, the HLA-DQ8-glia-α1 contacts mediated by the CDR3-encoded arginine were almost identical between TRBV9(+) and TRBV9(-) TCRs. In all cases, this interaction centered on two hydrogen bonds with a specific serine residue in the bound peptide. Replacement of serine with alanine at this position abrogated TRBV9(+) and TRBV9(-) clonal T cell proliferation in response to HLA-DQ8-glia-α1. Gluten-specific memory CD4(+) T cells with structurally and functionally conserved TCRs therefore predominate in the disease-affected tissue of patients with HLA-DQ8-mediated CD.
Assuntos
Doença Celíaca/imunologia , Seleção Clonal Mediada por Antígeno/imunologia , Gliadina/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Doença Celíaca/genética , Doença Celíaca/metabolismo , Linhagem Celular , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos HLA-DQ/química , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/imunologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunofenotipagem , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica/imunologia , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismoRESUMO
OBJECTIVE: Coeliac disease (CD), a gluten-induced enteropathy, alters the composition and function of duodenal intraepithelial T cells. The intestine also harbours four types of CD3-negative intraepithelial lymphocytes (IELs) with largely unknown function: CD56(-)CD127(-), CD56(-)CD127(+), CD56(+)CD127(-) and CD56(+)CD127(+). Here we aimed to gain insight into the potential function of these innate IELs in health and disease. DESIGN: We determined the phenotypes, relative abundance and differentiation potential of these innate IEL subsets in duodenal biopsies from controls and patients with CD or patients with refractory CD type II (RCDII). RESULTS: Hierarchical clustering analysis of the expression of 15 natural killer and T cell surface markers showed that innate IELs differed markedly from innate peripheral blood lymphocytes and divided innate IEL subsets into two main branches: a CD127(-) branch expressing high levels of interleukin (IL) 2/15Rß but no IL-21R, and a CD127(+) branch with the opposite phenotype. While CD was characterised by the contraction of all four innate IEL subsets, a selective expansion of CD56(-)CD127(-) and CD56(-)CD127(+) innate IEL was detected in RCDII. In vitro, in the presence of IL-15, CD56(-)CD127(-) IEL from controls and patients with CD, but not from patients with RCDII, differentiated into functional natural killer and T cells, the latter largely dependent on notch-signalling. Furthermore, compared with non-coeliac controls, CD56(-)CD127(-) IEL from patients with CD expressed more intracellular CD3ε and CD3γ and gave more pronounced T cell differentiation. CONCLUSIONS: Thus, we demonstrate previously unappreciated diversity and plasticity of the innate IEL compartment and its loss of differentiation potential in patients with RCDII.
Assuntos
Complexo CD3/análise , Doença Celíaca , Duodeno/patologia , Mucosa Intestinal , Peptídeos e Proteínas de Sinalização Intracelular/análise , Subpopulações de Linfócitos T , Doença Celíaca/imunologia , Doença Celíaca/patologia , Diferenciação Celular/imunologia , Linhagem Celular , Citocinas/imunologia , Humanos , Subunidade alfa de Receptor de Interleucina-7/análise , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , RNA Polimerase I , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologiaRESUMO
In patients with celiac disease, gluten consumption causes inflammation of the duodenum, and, to a lesser extent, the proximal jejunum. Immune-dominant gluten peptides are modified by the enzyme TG2, leading to their high-affinity binding to HLA-DQ2 or HLA-DQ8 molecules, present in people with a predisposition to celiac disease. Gluten peptide-loaded HLA-DQ2 or HLA-DQ8 molecules are recognized by highly conserved receptors on CD4(+) T cells in the lamina propria. B cells specific for TG2 and modified gluten peptides are also abundant in the lamina propria of patients with celiac disease. In the epithelium, interleukin-15 activates intraepithelial lymphocytes that promote destruction of epithelial cells. However, it is not clear how the immune responses in the lamina propria and the epithelium, separated by a basement membrane, are linked. We review the immune processes that occur in the lamina propria and their potential effects on epithelial pathology in celiac disease.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Doença Celíaca/imunologia , Comunicação Celular , Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Animais , Autoanticorpos/sangue , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Doença Celíaca/genética , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Proteínas de Ligação ao GTP , Predisposição Genética para Doença , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/imunologia , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Fenótipo , Proteína 2 Glutamina gama-Glutamiltransferase , Transdução de Sinais , Transglutaminases/imunologiaRESUMO
OBJECTIVE: Increased numbers of IL-17-producing CD4(+) T cells have been observed in AS. However, it is not known whether these CD4(+) T cells are already present in early disease or if this is a late disease phenomenon only. Therefore we aimed to investigate whether IL-17-producing CD4(+) T cells are involved in early active axial SpA, including patients without imaging abnormalities, by determining the frequency and phenotype of IL-17-producing CD4(+) T cells in these patients. METHODS: Flow cytometry was used to analyse cytokine production and surface marker expression of peripheral blood mononuclear cells from 31 patients suffering from early active HLA-B27-positive axial SpA fulfilling the Assessment of SpondyloArthritis International Society criteria with or without MRI abnormalities and 21 healthy controls. RESULTS: Patients with early active axial SpA showed an increased percentage of IL-17-producing CD4(+) T cells compared with healthy controls (mean 1.1% vs 0.4%, respectively; P = 0.013). The percentage of IL-17-producing CD4(+) T cells was equally increased in patients with and without MRI abnormalities (1.2% vs 1.1%, respectively; P = 0.81). These IL-17-producing CD4(+)T cells expressed the αß T cell receptor but not the γδ T cell receptor, exhibited a memory phenotype and expressed CD161, but only sporadically expressed killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2). CONCLUSION: IL-17-producing CD4(+) T cells are increased in patients with early active axial SpA both with and without MRI abnormalities. This finding shows that the frequency of IL-17-producing CD4(+) T cells is enhanced in the early stages of disease.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interleucina-17/imunologia , Sacroileíte/imunologia , Coluna Vertebral/patologia , Espondilartrite/imunologia , Adulto , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Humanos , Interleucina-17/metabolismo , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores KIR3DL2/imunologia , Sacroileíte/patologia , Espondilartrite/patologia , Adulto JovemRESUMO
Refractory coeliac disease type II (RCDII) is characterized by a continuous gluten-independent duodenal immune activation with an extremely high risk of malignant transformation. It is therefore considered as an indolent lymphoma. RCDII is characterized by the presence of villous atrophy (Marsh III A-C) in combination with an aberrant intra- epithelial lymphocyte (IEL) population consisting of >20% sCD3-CD7+iCD3+ IELs. The sCD3-CD7+iCD3+ IELs are a heterogeneous lineage-negative cell population, consisting of cells that do or do not express CD127/IL7Rα. Experiments using IEL from non-RCDII patients have indicated that while the CD127- cells are IL-15 responsive, the CD127+ cells are not. Together with the observation that some patients express an aberrant (monoclonal) TCRγδ phenotype, this confirms the heterogeneity of the aberrant IEL population in RCDII and suggests that the aberrant cells are heterogeneous with respect to their response to common γ-chain cytokines. Although cladribine with or without autologous stem cell transplantation is effective in the treatment of signs and symptoms of RCDII and improves survival as compared to symptomatic topical steroid therapy, cladribine failures still bear a high risk of malignant transformation, and the rate of enteropathy-associated T-cell lymphoma (EATL) development in this subgroup is extremely high. It might be hypothesized that the heterogenous nature of aberrant IEL and the high risk of malignant transformation require a treatment strategy which is effective despite this heterogeneity. RCDII should be seen more in the light of a low-grade/no mass lymphoma or pre-EATL. We would suggest an upfront combination therapy approach integrating inhibition of downstream Jak-STAT signalling pathways with conventional therapy (2-CDA) to hopefully effectively treat signs and symptoms of RCDII and accomplish a more effective EATL prevention.
Assuntos
Doença Celíaca/terapia , Terapia Combinada , Citocinas/metabolismo , Heterogeneidade Genética , HumanosRESUMO
NK cells use NK cell receptors to be able to recognize and eliminate infected, transformed, and allogeneic cells. Human NK cells are prevented from killing autologous healthy cells by virtue of inhibitory NKRs, primarily killer cell Ig-like receptors (KIR) that bind "self" HLA class I molecules. Individual NK cells stably express a selected set of KIR, but it is currently disputed whether the fraction of NK cells expressing a particular inhibitory KIR is influenced by the presence of the corresponding HLA ligand. The extreme polymorphism of the KIR and HLA loci, with wide-ranging affinities for individual KIR and HLA allele combinations, has made this issue particularly hard to tackle. In this study, we used a transgenic mouse model to investigate the effect of HLA on KIR repertoire and function in the absence of genetic variation inside and outside the KIR locus. These H-2K(b-/-) and H-2D(b-/-) mice lacked ligands for inhibitory Ly49 receptors and were transgenic for HLA-Cw3 and a KIR B haplotype. In this reductionist system, the presence of HLA-Cw3 reduced the frequency of KIR2DL2(+) cells, as well as the surface expression levels of KIR2DL2. In addition, in the presence of HLA-Cw3, the frequency of NKG2A(+) cells and the surface expression levels of NKG2A were reduced. In line with these findings, both transgene-encoded KIR and endogenous NKG2A contributed to the rejection of cells lacking HLA-Cw3. These findings support the idea that HLA influences the human KIR repertoire.
Assuntos
Antígenos HLA-C/fisiologia , Modelos Imunológicos , Receptores KIR2DL2/antagonistas & inibidores , Receptores KIR/antagonistas & inibidores , Animais , Antígenos H-2/genética , Antígenos HLA-C/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Subfamília C de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília C de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília C de Receptores Semelhantes a Lectina de Células NK/genética , Receptores KIR/biossíntese , Receptores KIR/genética , Receptores KIR2DL2/genética , Receptores KIR2DL2/metabolismoRESUMO
OBJECTIVE: Refractory coeliac disease type II (RCDII) is a severe complication of coeliac disease (CD) characterised by aberrant intraepithelial lymphocytes (IELs) of unknown origin that display an atypical CD3(-)CD7(+)icCD3(+) phenotype. In approximately 40% of patients with RCDII these lymphocytes develop into an invasive lymphoma. In the current study we aimed to identify the physiological counterpart of these cells. DESIGN: RCDII cell lines were compared with T-cell receptor positive (TCR(+)) IEL (T-IEL) lines by microarray analysis, real-time quantitative PCR and flow cytometry. This information was used to identify cells with an RCDII-associated phenotype in duodenal biopsies from non-refractory individuals by multicolour flow cytometry. RESULTS: RCDII lines were transcriptionally distinct from T-IEL lines and expressed higher levels of multiple natural killer (NK) cell receptors. In addition to the CD3(-)CD7(+)icCD3(+) phenotype, the RCDII lines were distinguishable from other lymphocyte subsets by the absence of CD56, CD127 and CD34. Cells matching this surface lineage-negative (Lin(-)) CD7(+)CD127(-)CD34(-) phenotype expressed a functional interleukin-15 (IL-15) receptor and constituted a significant proportion of IELs in duodenal specimens of patients without CD, particularly children, and were also found in the thymus. In patients without CD, the Lin(-)CD7(+)CD127(-)CD34(-) subset was one of four subsets within the CD3(-)CD7(+)icCD3(+) population that could be distinguished on the basis of differential expression of CD56 and/or CD127. CONCLUSION: Our studies indicate that the CD3(-)CD7(+)icCD3(+) population is heterogeneous and reveal the existence of a Lin(-) subset that is distinct from T, B, NK and lymphoid tissue inducer cells. We speculate that this IL-15 responsive population represents the physiological counterpart of aberrant cells expanded in RCDII and transformed in RCDII-associated lymphoma.
Assuntos
Doença Celíaca/imunologia , Doença Celíaca/patologia , Duodeno/imunologia , Duodeno/patologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Linfócitos/imunologia , Linfócitos/patologia , Antígenos CD/imunologia , Biomarcadores/análise , Biópsia , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Interleucina-15/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T/imunologia , Análise Serial de TecidosRESUMO
CD4+ T helper antigens are essential components of cancer vaccines, but the relevance of the source of these MHC class II-restricted antigens remains underexplored. To compare the effectiveness of tumor-specific versus tumor-unrelated helper antigens, we designed three DNA vaccines for the murine MC-38 colon carcinoma, encoding CD8+ T cell neoantigens alone (noHELP) or in combination with either "universal" helper antigens (uniHELP) or helper neoantigens (neoHELP). Both types of helped vaccines increased the frequency of vaccine-induced CD8+ T cells, and particularly uniHELP increased the fraction of KLRG1+ and PD-1low effector cells. However, when mice were subsequently injected with MC-38 cells, only neoHELP vaccination resulted in significantly better tumor control than noHELP. In contrast to uniHELP, neoHELP-induced tumor control was dependent on the presence of CD4+ T cells, while both vaccines relied on CD8+ T cells. In line with this, neoHELP variants containing wild-type counterparts of the CD4+ or CD8+ T cell neoantigens displayed reduced tumor control. These data indicate that optimal personalized cancer vaccines should include MHC class II-restricted neoantigens to elicit tumor-specific CD4+ T cell help.
RESUMO
In refractory celiac disease (RCD), intestinal epithelial damage persists despite a gluten-free diet. Characteristic for RCD type II (RCD II) is the presence of aberrant surface TCR-CD3(-) intraepithelial lymphocytes (IELs) that can progressively replace normal IELs and eventually give rise to overt lymphoma. Therefore, RCD II is considered a malignant condition that forms an intermediate stage between celiac disease (CD) and overt lymphoma. We demonstrate in this study that surface TCR-CD3(-) IEL lines isolated from three RCD II patients preferentially lyse epithelial cell lines. FACS analysis revealed that DNAM-1 was strongly expressed on the three RCD cell lines, whereas other activating NK cell receptors were not expressed on all three RCD cell lines. Consistent with this finding, cytotoxicity of the RCD cell lines was mediated mainly by DNAM-1 with only a minor role for other activating NK cell receptors. Furthermore, enterocytes isolated from duodenal biopsies expressed DNAM-1 ligands and were lysed by the RCD cell lines ex vivo. Although DNAM-1 on CD8(+) T cells and NK cells is known to mediate lysis of tumor cells, this study provides, to our knowledge, the first evidence that (pre)malignant cells themselves can acquire the ability to lyse epithelial cells via DNAM-1. This study confirms previous work on epithelial lysis by RCD cell lines and identifies a novel mechanism that potentially contributes to the gluten-independent tissue damage in RCD II and RCD-associated lymphoma.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Doença Celíaca/imunologia , Citotoxicidade Imunológica/imunologia , Células Epiteliais/imunologia , Linfócitos/imunologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Células CACO-2 , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células Epiteliais/metabolismo , Citometria de Fluxo , Células HT29 , Humanos , Cadeias alfa de Integrinas/imunologia , Cadeias alfa de Integrinas/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Células K562 , Linfócitos/metabolismoRESUMO
Because susceptibility to celiac disease is associated strongly with HLA-DQ2 (DQA1*05/DQB1*02) and weakly with HLA-DQ8 (DQA1*03/DQB1*03), a subset of patients carries both HLA-DQ2 and HLA-DQ8. As a result, these patients may express two types of mixed HLA-DQ2/8 transdimers (encoded by DQA1*05/DQB1*03 and DQA1*03/DQB1*02) in addition to HLA-DQ2 and HLA-DQ8. Using T cells from a celiac disease patient expressing HLA-DQ8trans (encoded by DQA*0501/DQB*0302), but neither HLA-DQ2 nor HLA-DQ8, we demonstrate that this transdimer is expressed on the cell surface and can present multiple gluten peptides to T cell clones isolated from the duodenum of this patient. Furthermore, T cell clones derived from this patient and HLA-DQ2/8 heterozygous celiac disease patients respond to gluten peptides presented by HLA-DQ8trans, as well as HLA-DQ8, in a similar fashion. Finally, one gluten peptide is recognized better when presented by HLA-DQ8trans, which correlates with preferential binding of this peptide to HLA-DQ8trans. These results implicate HLA-DQ8trans in celiac disease pathogenesis and demonstrate extensive T cell cross-reactivity between HLA-DQ8 and HLA-DQ8trans. Because type 1 diabetes is strongly associated with the presence of HLA-DQ8trans, our findings may bear relevance to this disease as well.
Assuntos
Epitopos de Linfócito T/imunologia , Gliadina/imunologia , Gliadina/metabolismo , Antígenos HLA-DQ/imunologia , Antígenos HLA-DQ/metabolismo , Cadeias beta de HLA-DQ/metabolismo , Subpopulações de Linfócitos T/imunologia , Apresentação de Antígeno/imunologia , Doença Celíaca/imunologia , Doença Celíaca/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Células Clonais , Reações Cruzadas/imunologia , Epitopos de Linfócito T/metabolismo , Células HEK293 , Antígenos HLA-DQ/química , Cadeias beta de HLA-DQ/química , Humanos , Multimerização Proteica , Subpopulações de Linfócitos T/metabolismoRESUMO
SARS-CoV-2 is the third zoonotic coronavirus to cause a major outbreak in humans in recent years, and many more SARS-like coronaviruses with pandemic potential are circulating in several animal species. Vaccines inducing T cell immunity against broadly conserved viral antigens may protect against hospitalization and death caused by outbreaks of such viruses. We report the design and preclinical testing of 2 T cell-based pan-sarbecovirus vaccines, based on conserved regions within viral proteins of sarbecovirus isolates of human and other carrier animals, like bats and pangolins. One vaccine (CoVAX_ORF1ab) encoded antigens derived from nonstructural proteins, and the other (CoVAX_MNS) encoded antigens from structural proteins. Both multiantigen DNA vaccines contained a large set of antigens shared across sarbecoviruses and were rich in predicted and experimentally validated human T cell epitopes. In mice, the multiantigen vaccines generated both CD8+ and CD4+ T cell responses to shared epitopes. Upon encounter of full-length spike antigen, CoVAX_MNS-induced CD4+ T cells were responsible for accelerated CD8+ T cell and IgG Ab responses specific to the incoming spike, irrespective of its sarbecovirus origin. Finally, both vaccines elicited partial protection against a lethal SARS-CoV-2 challenge in human angiotensin-converting enzyme 2-transgenic mice. These results support clinical testing of these universal sarbecovirus vaccines for pandemic preparedness.
Assuntos
Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Vacinas de DNA , Humanos , Camundongos , Animais , Linfócitos T CD8-Positivos , Imunidade Celular , SARS-CoV-2/genética , Epitopos de Linfócito T/genéticaRESUMO
Pyroptosis is a recently discovered form of inflammatory programmed necrosis characterized by caspase-1-mediated and gasdermin D-dependent cell death leading to the release of pro-inflammatory cytokines such as Interleukin-1 beta (IL-1ß). Here, we evaluated whether pyroptosis could be exploited in DNA vaccination by incorporating a constitutively active variant of caspase-1 to the antigen-expressing DNA. In vitro, transfection with constitutively active caspase-1 DNA induced pro-IL-1ß maturation and IL-1ß release as well as gasdermin D-dependent cell death. To test active caspase-1 as a genetic adjuvant for the induction of antigen-specific T cell responses, mice were vaccinated intradermally with a DNA vaccine consisting of the active caspase-1 plasmid together with a plasmid encoding an ovalbumin-derived CD8 T cell epitope. Active caspase-1 accelerated and amplified antigen-specific CD8 T cell responses when administered simultaneously with the DNA vaccine at an equimolar dose. Moreover, upon challenge with melanoma cells expressing ovalbumin, mice vaccinated with the antigen vaccine adjuvanted with active caspase-1 showed significantly better survival compared to the non-adjuvanted group. In conclusion, we have developed a novel genetic adjuvant that for the first time employs the pyroptosis pathway to improve DNA vaccination against cancer.
Assuntos
Piroptose , Vacinas de DNA , Animais , Caspase 1/metabolismo , Inflamação , Interleucina-1beta , Camundongos , Ovalbumina , VacinaçãoRESUMO
Human cytomegalovirus (HCMV) is an ubiquitous herpesvirus that can cause serious morbidity and mortality in immunocompromised or immune-immature individuals. A vaccine that induces immunity to CMV in these target populations is therefore highly needed. Previous attempts to generate efficacious CMV vaccines primarily focused on the induction of humoral immunity by eliciting neutralizing antibodies. Current insights encourage that a protective immune response to HCMV might benefit from the induction of virus-specific T cells. Whether addition of antiviral T cell responses enhances the protection by antibody-eliciting vaccines is however unclear. Here, we assessed this query in mouse CMV (MCMV) infection models by developing synthetic vaccines with humoral immunity potential, and deliberately adding antiviral CD8+ T cells. To induce antibodies against MCMV, we developed a DNA vaccine encoding either full-length, membrane bound glycoprotein B (gB) or a secreted variant lacking the transmembrane and intracellular domain (secreted (s)gB). Intradermal immunization with an increasing dose schedule of sgB and booster immunization provided robust viral-specific IgG responses and viral control. Combined vaccination of the sgB DNA vaccine with synthetic long peptides (SLP)-vaccines encoding MHC class I-restricted CMV epitopes, which elicit exclusively CD8+ T cell responses, significantly enhanced antiviral immunity. Thus, the combination of antibody and CD8+ T cell-eliciting vaccines provides a collaborative improvement of humoral and cellular immunity enabling enhanced protection against CMV.
Assuntos
Anticorpos Antivirais/sangue , Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/imunologia , Citomegalovirus/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Infecções por Citomegalovirus/imunologia , Epitopos/imunologia , Imunidade Celular , Imunidade Humoral , Imunização Secundária/métodos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Organismos Livres de Patógenos Específicos , Vacinação , Vacinas de DNA/administração & dosagem , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
Although it is well established that human NK cells are able to detect the absence of autologous HLA class I in vitro by virtue of inhibitory killer Ig-like receptors (KIR), direct evidence that KIR can mediate "missing self" recognition in vivo is lacking. To test this, we generated mice transgenic for a human KIR B-haplotype and HLA-Cw3 on a C57BL/6 background. NK cells in these mice expressed multiple KIR in a stochastic manner, including the HLA-Cw3-specific inhibitory receptor KIR2DL2. KIR and HLA transgenic mice rejected wild-type C57BL/6 spleen cells upon i.v. injection. This rejection was dependent on the presence of the KIR transgene in the host and on the absence of HLA-Cw3 from the injected target cells. Hence, the KIR transgene mediated "missing self" recognition in vivo. We anticipate that this KIR and HLA transgenic mouse will help shed light on KIR and HLA effects in disease and transplantation.
Assuntos
Apresentação de Antígeno/imunologia , Autoantígenos/metabolismo , Receptores KIR2DL2/fisiologia , Animais , Apresentação de Antígeno/genética , Autoantígenos/genética , Autoantígenos/imunologia , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Antígenos HLA-C/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília A de Receptores Semelhantes a Lectina de Células NK/genética , Receptores KIR2DL2/deficiência , Receptores KIR2DL2/genética , Baço/citologia , Baço/imunologia , Baço/metabolismo , Baço/transplante , Processos EstocásticosRESUMO
Although very few CD4(+) T cells express killer Ig receptors (KIR), a large proportion of CD4(+) T cells with a late memory phenotype, characterized by the absence of CD28, does express KIR. Here, we show that KIR expression on CD4(+) T cells is also associated with memory T cell function, by showing that the frequency of CMV-specific cells is higher in CD4(+)KIR(+) than CD4(+)KIR(-) T cells. In addition, engagement of an inhibitory KIR inhibited the CMV-specific proliferation of these CD4(+)KIR(+) memory T cells, but had no detectable effect on cytokine production. Our data reveal that, in marked contrast with CD8(+) T cells, the activity of a subset of CMV-specific CD4(+) T cells is modulated by HLA class I-specific KIR. Thus, the CMV-induced down-regulation of HLA class I may in fact enhance memory CMV-specific CD4(+) T cell responses restricted by HLA class II.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citomegalovirus/imunologia , Memória Imunológica , Receptores KIR/imunologia , Subpopulações de Linfócitos T/imunologia , Idoso , Artrite Reumatoide/imunologia , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Receptores KIR/metabolismo , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/virologiaRESUMO
Killer Ig-like receptors (KIR) are expressed by human NK cells and T cells. Although Ag-specific cytolytic activity and cytokine production of KIR(+) T cells can be inhibited by KIR ligation, the effect of KIR on proliferation is unclear. KIR(+) T cells have been reported to have a general proliferative defect. To investigate whether KIR(+) T cells represent end-stage dysfunctional T cells, we characterized KIR(+) CMV-specific T cells in allogeneic stem cell transplantation patients and healthy donors. In both patients and healthy donors, a significant percentage KIR(+) T cells was detected at various time points. All stem cell transplantation patients studied showed KIR expression on CMV-specific T cells, while not all donors had KIR-expressing CMV-specific T cells. From two of the patients and one donor KIR(+) CMV-specific T clones were isolated and analyzed functionally. T cells were detected that expressed KIR that could not encounter their corresponding KIR ligands in vivo, illustrating that KIR expression by these T cells was not based on functional selection but a random process. Our data demonstrate that KIR(+) T cells are fully functional T cells that are only restricted in effector functions and proliferation upon KIR ligation. The level of KIR-mediated inhibition of the effector functions and proliferation depended on the strength of TCR stimulation. We observed no diminished general proliferative capacity and therefore we conclude that these T cells do not represent end-stage dysfunctional T cells.