scholarly journals The insulin A-chain epitope recognized by human T cells is posttranslationally modified

2005 ◽  
Vol 202 (9) ◽  
pp. 1191-1197 ◽  
Author(s):  
Stuart I. Mannering ◽  
Leonard C. Harrison ◽  
Nicholas A. Williamson ◽  
Jessica S. Morris ◽  
Daniel J. Thearle ◽  
...  

The autoimmune process that destroys the insulin-producing pancreatic β cells in type 1 diabetes (T1D) is targeted at insulin and its precursor, proinsulin. T cells that recognize the proximal A-chain of human insulin were identified recently in the pancreatic lymph nodes of subjects who had T1D. To investigate the specificity of proinsulin-specific T cells in T1D, we isolated human CD4+ T cell clones to proinsulin from the blood of a donor who had T1D. The clones recognized a naturally processed, HLA DR4–restricted epitope within the first 13 amino acids of the A-chain (A1–13) of human insulin. T cell recognition was dependent on the formation of a vicinal disulfide bond between adjacent cysteine residues at A6 and A7, which did not alter binding of the peptide to HLA DR4. CD4+ T cell clones that recognized this epitope were isolated from an HLA DR4+ child with autoantibodies to insulin, and therefore, at risk for T1D, but not from two healthy HLA DR4+ donors. We define for the first time a novel posttranslational modification that is required for T cell recognition of the insulin A-chain in T1D.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4084-4084
Author(s):  
Marieke Griffioen ◽  
M. Willy Honders ◽  
Anita N. Stumpf ◽  
Edith D. van der Meijden ◽  
Cornelis A.M. van Bergen ◽  
...  

Abstract Abstract 4084 Poster Board III-1019 Donor lymphocyte infusion (DLI) can be an effective cellular immunotherapy for patients with hematological malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). The effect of DLI is mediated by donor derived T-cells recognizing minor histocompatibility antigens (mHags) encoded by single nucleotide polymorphisms (SNPs) on malignant cells of the recipient. Donor T-cells may also induce Graft-versus-Host Disease (GvHD) when directed against mHags with broad expression on non-malignant tissues. The aim of this study was to investigate the specificity and diversity of mHags recognized by T-cells in Graft-versus-Leukemia (GvL) reactivity. Activated (HLA-DR+) CD8+ and CD4+ T-cell clones were isolated from a patient successfully treated with DLI for relapsed chronic myeloid leukemia (CML) more than one year after HLA-matched alloSCT. GvL reactivity in this patient was accompanied with mild GvHD of the skin. Isolated T-cell clones were shown to recognize 13 different mHags. CD8+ T-cell clones were specific for HA-1 and HA-2 in HLA-A*0201, one unknown mHag in B*0801 and 4 unknown mHags in B*4001. CD4+ T-cell clones were specific for one unknown mHag in HLA-DQ and 5 unknown mHags in DR. By screening plasmid (class I) and bacteria (class II) cDNA libraries, we identified a mHag in HLA-DQ encoded by the PI4K2B gene (Griffioen et al., PNAS 2008), 4 mHags in HLA-DR encoded by the PTK2B, MR-1, LY75 and MTHFD1 genes (Stumpf et al., Blood 2009) and a mHag in B*4001 encoded by the TRIP10 gene. For the 3 T cell clones recognizing unknown mHags in B*4001, we performed Whole Genome Assocation scanning (WGAs). A panel of 60 EBV-LCL was retrovirally-transduced with B*4001 and tested for T-cell recognition. In parallel, genomic DNA was isolated and more than one million single nucleotide polymorphisms (SNPs) were determined by the Illumina beadchip array. Statistical analysis revealed significant association between T-cell recognition of EBV-LCL and the presence of coding SNPs in the SON DNA-binding protein and SWAP-70 genes. To get more insight into the role and potential use of the mHags in GvL reactivity and GvHD, all T-cell clones were analyzed in detail for reactivity against hematopoietic and non-hematopoietic cells. Hematopoietic cells included peripheral blood cells (monocytes, B-cells and T-cells), professional antigen presenting cells (APC) and leukemic cells (CML, ALL and AML). All CD8+ T-cell clones recognized (subsets of) peripheral blood cells as well as CML cells, except for the T-cell clone for TRIP10. Recognition of (subsets of) peripheral blood cells was also observed for all CD4+ T-cell clones, but CML cells were differentially recognized. CML cells were strongly recognized by the T-cell clones for MTHFD1 and the unknown mHag in HLA-DR, whereas no or low reactivity was observed for all other CD4+ T-cell clones. All CD8+ and CD4+ T-cell clones strongly recognized professional APC, including monocyte-derived dendritic cells and in vitro differentiated CML cells with APC phenotype. All T-cell clones were also capable of recognizing AML and ALL, except for the T-cell clone for TRIP10, which showed restricted recognition of AML-M4 and -M5 of monocytic origin. As non-hematopoietic cells, patient-derived fibroblasts were cultured with and without IFN-γ and tested for T-cell recognition. In the absence of IFN-γ, all T-cell clones failed to recognize fibroblasts, except for the T-cell clone for the unknown mHag in B*0801. After treatment with IFN-γ, additional reactivity was observed for the T-cell clones for SON DNA-binding protein and the unknown mHag in B*4001. Our data showed the specificity and diversity of mHags recognized by T-cells induced in a patient successfully treated with DLI for relapsed CML. The T-cell response was directed against 13 different mHags, of which 10 mHags in HLA class I and class II have now been identified by different techniques. Detailed analysis of T-cell recognition of hematopoietic and non-hematopoietic cells provides evidence that the mHags played different roles in the onset and execution of GvL and GvHD. Moreover, only one of the 10 identified mHags was expressed on fibroblasts after treatment with IFN-γ, indicating the characterization of mHags with potential relevance for T-cell based immunotherapy. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1330-1330
Author(s):  
Sanja Stevanovic ◽  
Bart Nijmeijer ◽  
Marianke LJ Van Schie ◽  
Roelof Willemze ◽  
Marieke Griffioen ◽  
...  

Abstract Abstract 1330 Poster Board I-352 Immunodeficient mice inoculated with human leukemia can be used as a model to investigate Graft-versus-Leukemia (GvL) effects of donor lymphocyte infusions (DLIs). In addition to GvL reactivity, treatment with DLI induces xenogeneic Graft-versus-Host Disease (GvHD) in mice, characterized by pancytopenia and weight loss. In patients treated with DLI for relapsed or residual leukemia after allogeneic stem cell transplantation, immune responses against non-leukemic cells may also cause GvHD. It has been suggested that GvL reactivity and GvHD, which co-develop in vivo, can be separated and that distinct T cells exist with the specific capacity to mediate GvL reactivity or GvHD. Since adoptive T cell transfer models that allow analysis of separation of GvL and GvHD are rare, we aimed to establish whether GvL reactivity and xenogeneic GvHD could be separated using our model of human leukemia-engrafted NOD/scid mouse after treatment with human donor T cells. In this study, non-conditioned NOD/scid mice engrafted with primary human acute lymphoblastic leukemic cells were treated with CD3+ DLI. Established tumors were effectively eliminated by emerging human T cells, but also induced xenogeneic GvHD. Flowcytometric analysis demonstrated that the majority of emerging CD8+ and CD4+ T cells were activated (HLA-DR+) and expressed an effector memory phenotype (CD45RA-CD45RO+CCR7-). To investigate whether GvL reactivity and xenogeneic GvHD were mediated by the same T cells showing reactivity against both human leukemic and murine cells, or displaying distinct reactivity against human leukemic and murine cells, we clonally isolated and characterized the T cells during the GvL response and xenogeneic GvHD. T cell clones were analyzed for reactivity against primary human leukemic cells and primary NOD/scid hematopoietic (BM and spleen cells) and non-hematopoietic (skin fibroblasts) cells in IFN-g ELISA. Isolated CD8+ and CD4+ T cell clones were shown to recognize either human leukemic or murine cells, indicating that GvL response and xenogeneic GvHD were mediated by different human T cells. Flowcytometric analysis demonstrated that all BM and spleen cells expressed MHC class I, whereas only 1-3 % of the cells were MHC class II +. Primary skin fibroblasts displayed low MHC class I and completely lacked MHC class II expression. Xeno-reactive CD8+ T cell clones were shown to recognize all MHC class I + target cells and xeno-reactive CD4+ T cells clones displayed reactivity only against MHC class II + target cells. To determine the MHC restriction of xeno-reactive T cell clones, NOD/scid bone marrow (BM) derived dendritic cells (DC) expressing high levels of murine MHC class I and class II were tested for T cell recognition in the presence or absence of murine MHC class I and class II monoclonal antibodies in IFN-g ELISA. Xeno-reactive CD8+ T cell clones were shown to be MHC class I (H-2Kd or H-2Db) restricted, whereas xeno-reactive CD4+ T cell clones were MHC class II (I-Ag7) restricted, indicating that xeno-reactivity reflects genuine human T cell response directed against allo-antigens present on murine cells. Despite production of high levels of IFN-gamma, xeno-reactive CD8+ and CD4+ T cell clones failed to exert cytolytic activity against murine DC, as determined in a 51Cr-release cytotoxicity assay. Absence of cytolysis by CD8+ T cell clones, which are generally considered as potent effector cells, may be explained by low avidity interaction between human T cells and murine DC, since flowcytometric analysis revealed sub-optimal activation of T cells as measured by CD137 expression and T cell receptor downregulation upon co-culture with murine DC, and therefore these results indicate that xenogeneic GvHD in this model is likely to be mediated by cytokines. In conclusion, in leukemia-engrafted NOD/scid mice treated with CD3+ DLI, we show that GvL reactivity and xenogeneic GvHD are mediated by separate human T cells with distinct specificities. All xeno-reactive T cell clones showed genuine recognition of MHC class I or class II associated allo-antigens on murine cells similar as GvHD-inducing human T cells. These data suggest that our NOD/scid mouse model of human acute leukemia may be valuable for studying the effectiveness and specificity of selectively enriched or depleted T cells for adoptive immunotherapy. Disclosures: No relevant conflicts of interest to declare.


1986 ◽  
Vol 164 (3) ◽  
pp. 897-910 ◽  
Author(s):  
A Finnegan ◽  
M A Smith ◽  
J A Smith ◽  
J Berzofsky ◽  
D H Sachs ◽  
...  

Previous studies (1) have indicated that the repertoire of murine T cells specific for a potentially complex protein antigen is in fact specific for a limited number of antigenic epitopes on that antigen in association with a given Ia molecule. Since those studies generally analyzed responses to antigens that differ in only a few amino acids from homologous murine molecules, it was possible that tolerance to self proteins was responsible for the limited T cell repertoire seen in responses to closely related proteins. It was therefore of interest to determine whether T cell recognition of a structurally and phylogenetically more distant protein molecule would also show specificity for a limited number of immunodominant peptides on that molecule. A series of experiments was designed to study the antigen fine specificity and MHC restriction of T cell clones specific for the bacterially derived antigen staphylococcal nuclease (Nase). T cell clones generated in (H-2b X H-2a)F1 (B6AF1) T cells were shown to be specific for Nase and to be restricted by either Ab alpha Ab beta or Ek alpha Ek beta. The fine specificity of these clones was then analyzed using cyanogen bromide and tryptic fragments and a series of overlapping 20-amino-acid synthetic peptides corresponding to and spanning the entire sequence of the Nase molecule. Two Ab alpha Ab beta-restricted clones were highly responsive to peptide 91-110, and not to other synthetic Nase peptides. In contrast, seven Ek alpha Ek beta-restricted clones were consistently responsive to peptide 81-100 and not to 91-110 or to other Nase peptides. Certain of these Ek alpha Ek beta-restricted T cells expressed an interesting crossreactivity, in that they responded to peptide 51-70 as well as to 81-100, although the response to 51-70 was characterized by a markedly shifted dose-response curve, indicating a reduced efficiency of activation by this peptide. Analysis of the amino acid sequences of these regions indicates that this unexpected crossreaction may have a structural basis. A single Nase-specific T cell line generated from BALB/c T cells was, in contrast to any of the B6AF1 clones studied, responsive only to peptide 61-80 and not to other peptides, including 81-100 or 91-110. Collectively, these findings show that Nase-specific T cells are responsive to discrete Nase peptides. Moreover, the present findings suggest that in T cell recognition of a complex and highly foreign protein antigen, a limited number of peptide epitopes are preferentially recognized by T cells in association with a given Ia molecule.


Diabetes ◽  
1996 ◽  
Vol 45 (3) ◽  
pp. 328-336 ◽  
Author(s):  
J. D. Peterson ◽  
K. Haskins

2020 ◽  
Vol 4 (21) ◽  
pp. 5343-5356
Author(s):  
Laura R. E. Becher ◽  
Wendy K. Nevala ◽  
Shari Lee Sutor ◽  
Megan Abergel ◽  
Michele M. Hoffmann ◽  
...  

Abstract Human cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(−) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.


2015 ◽  
Vol 89 (8) ◽  
pp. 4449-4456 ◽  
Author(s):  
Sumiti Jain ◽  
Matthew T. Trivett ◽  
Victor I. Ayala ◽  
Claes Ohlen ◽  
David E. Ott

ABSTRACTThe expression of xenogeneic TRIM5α proteins can restrict infection in various retrovirus/host cell pairings. Previously, we have shown that African green monkey TRIM5α (AgmTRIM5α) potently restricts both human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus mac239 (SIVmac239) replication in a transformed human T-cell line (L. V. Coren, et al., Retrovirology 12:11, 2015,http://dx.doi.org/10.1186/s12977-015-0137-9). To assess AgmTRIM5α restriction in primary cells, we transduced AgmTRIM5α into primary rhesus macaque CD4 T cells and infected them with SIVmac239. Experiments with T-cell clones revealed that AgmTRIM5α could reproducibly restrict SIVmac239replication, and that this restriction synergizes with an intrinsic resistance to infection present in some CD4 T-cell clones. AgmTRIM5α transduction of virus-specific CD4 T-cell clones increased and prolonged their ability to suppress SIV spread in CD4 target cells. This increased antiviral function was strongly linked to decreased viral replication in the AgmTRIM5α-expressing effectors, consistent with restriction preventing the virus-induced cytopathogenicity that disables effector function. Taken together, our data show that AgmTRIM5α restriction, although not absolute, reduces SIV replication in primary rhesus CD4 T cells which, in turn, increases their antiviral function. These results support priorin vivodata indicating that the contribution of virus-specific CD4 T-cell effectors to viral control is limited due to infection.IMPORTANCEThe potential of effector CD4 T cells to immunologically modulate SIV/HIV infection likely is limited by their susceptibility to infection and subsequent inactivation or elimination. Here, we show that AgmTRIM5α expression inhibits SIV spread in primary effector CD4 T cellsin vitro. Importantly, protection of effector CD4 T cells by AgmTRIM5α markedly enhanced their antiviral function by delaying SIV infection, thereby extending their viability despite the presence of virus. Ourin vitrodata support priorin vivoHIV-1 studies suggesting that the antiviral CD4 effector response is impaired due to infection and subsequent cytopathogenicity. The ability of AgmTRIM5α expression to restrict SIV infection in primary rhesus effector CD4 T cells now opens an opportunity to use the SIV/rhesus macaque model to further elucidate the potential and scope of anti-AIDS virus effector CD4 T-cell function.


1993 ◽  
Vol 90 (23) ◽  
pp. 10984-10988 ◽  
Author(s):  
P B Ehrhard ◽  
P Erb ◽  
U Graumann ◽  
U Otten

Recent evidence suggests that nerve growth factor (NGF), in addition to its neurotrophic functions, acts as an immunomodulator mediating "cross-talk" between neuronal and immune cells, including T lymphocytes. We have analyzed murine CD4+ T-cell clones for their ability to express transcripts encoding NGF, low-affinity NGF receptor, and trk protooncogene, the signal-transducing receptor subunit for NGF. We show that two CD4+ T-helper (Th) clones, Th0-type clone 8/37 and Th2-type clone D10.G4.1, express NGF and Trk mRNA after appropriate activation with mitogen or with antigen and antigen-presenting cells. NGF and trk induction occurred to a similar extent and over a similar time course in activated 8/37 T cells, raising the possibility that NGF and trk genes are under coordinate control. NGF and NGF receptor expression does not seem to be a universal property of all activated CD4+ T cells, since Th1-type clone 9/9 did not express any of the transcripts after either stimulation. The absence of low-affinity NGF receptor mRNA in resting and activated T cells implies that the low-affinity NGF receptor is not involved in NGF signal transduction in CD4+ T cells. Our finding that activated CD4+ T-cell clones not only express Trk but also synthesize and release biologically active NGF implicates NGF as an autocrine and/or paracrine factor in the development and regulation of immune responses.


1988 ◽  
Vol 168 (5) ◽  
pp. 1659-1673 ◽  
Author(s):  
F T Rotteveel ◽  
I Kokkelink ◽  
R A van Lier ◽  
B Kuenen ◽  
A Meager ◽  
...  

A large number of CD4+ T cell clones, obtained from peripheral blood T lymphocytes by direct limiting dilution, allowed us to address the question whether functional heterogeneity exists within the human CD4+ T cell subset. Cytotoxic capacity of cloned T cells was analyzed with the use of anti-CD3 antibodies and target cells bearing FcR for murine IgG. 6 of 12 CD4+ clones obtained were able to lyse Daudi or P815 cells in the presence of anti-CD3 antibodies. The remaining six CD4+ T cell clones tested did not display anti-CD3-mediated cytotoxic activity and did not acquire this cytotoxic capacity during a culture period of 20 wk. In the absence of anti-CD3 mAb, no lytic activity against Daudi, P815, and K562 target cells was observed under normal culture conditions. Phenotypic analysis of these two distinct types of CD4+ T cells did not reveal differences with regard to reactivity with CDw29 (4B4) and CD45R (2H4) mAbs that have been described to recognize antigens associated with helper suppressor/inducer (respectively) CD4+ cells. The CD4+ clones without anti-CD3-mediated cytotoxic activities (Th2) consistently showed a high expression level of CD28 antigens, whereas the cytotoxic clones (Th1) expressed low amounts of CD28. Th1 CD4+ clones did produce IL-2, IFN-gamma, and TNF-alpha/beta, whereas the Th2 T cell clones produced minimal amounts of IL-2 and only low levels of INF-gamma and TNF-alpha/beta in response to anti-CD3 mAbs and PMA. Although not all CD4+ clones did release IL-4, there was no correlation with cytotoxic activity. Moreover, as compared with the Th1 CD4+ clones, Th2 CD4+ T cell clones proliferated moderately in response to immobilized anti-CD3 mAbs. However, proliferation reached the level of the cytotoxic clones when anti-CD28 mABs were present during culture. Both CD4+ subsets provided help for B cell differentiation upon stimulation with anti-CD3 mAbs. Our data suggest that the human CD4+ subset, in analogy to the murine system, comprises two functionally distinct T cell subpopulations, both of which are able to exert helper activity for polyclonal B cell differentiation, but which differ in cytotoxic capacity, lymphokine production, and requirements for proliferation. A function for these two types of T cells in the immune response is discussed.


Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3665-3665
Author(s):  
Caroline E. Rutten ◽  
Simone A.P. van Luxemburg-Heijs ◽  
Inge Jedema ◽  
Mirjam Heemskerk ◽  
Roelof Willemze ◽  
...  

Abstract Mismatching for HLA-DPB1 in unrelated donor hematopoietic stem cell transplantation (URD-SCT) has been associated with a significant decreased risk of disease relapse, indicating that HLA-DP might be a target for a graft versus leukemia (GVL) effect in HLA-class II expressing hematological malignancies. To determine whether a specific GVL effect could be caused by allo-HLA-DP specific T cells, we analyzed the immune response in a patient with a refractory immunocytoma responding to donor lymphocyte infusion (DLI) after HLA-DP mismatched URD-SCT. Patient and donor were fully matched for HLA-A, -B, -C, -DR and -DQ, but differed for both HLA-DP alleles (donor HLA-DPB1*0402/0501; patient HLA-DPB*020102/0301). The patient received a T cell depleted URD-SCT after a non-myeloablative conditioning regimen, resulting in mixed chimerism (75% donor) without GVHD. Because of a hematological relapse, a single DLI was given 6 months after SCT, resulting in a profound anti-leukemic effect with only grade I GVHD, treated with topical corticosteroids. 6 weeks after DLI, malignant cells in peripheral blood (PB) had dropped from 72% to 47%. 7 weeks later, only 3% malignant cells were present, and after 4 months, complete remission and conversion to full donor chimerism in the absence of GVHD was observed. To determine whether allo-HLA-DP specific T cells were involved in the immune response, leukemia-reactive donor T cell clones were isolated from PB or bone marrow at different time points during the response to DLI. Patient derived T cells were overnight stimulated with irradiated leukemic cells harvested before transplantation, and clonal IFNγ producing T cells were sorted and expanded. 21 CD4+ T cell clones, 19 CD8+ T cell clones and 6 NK cell clones were tested for recognition of patient or donor derived cells as measured by IFNγ production and cytotoxic activity. The CD8+ or NK clones did not recognize patient leukemic cells. However, all 21 CD4+ clones produced INFγ in response to patient leukemic cells but not to donor cells. To determine whether these CD4+ T cell clones were capable of killing the leukemic cells, a CFSE based cytotoxicity assay was performed. 8 clones showed 30–90% lysis of the leukemic cell population. To further analyze the specificity of these CD4+ clones, blocking and panel studies were performed. Blocking with the HLA-DP specific mAb B7.21 abrogated IFNγ production by all clones, confirming HLA-DP restricted recognition. A panel study using 12 unrelated EBV-LCL expressing different HLA-DP alleles identified 18 clones specific for HLA-DPB1*0301, and 3 clones specific for HLA-DPB1*0201. To analyze the polyclonality of the immune response, the distribution of TCR Vβ chains was characterized by RT-PCR and sequence reactions. 7 different Vβs were found within the HLA-DPB1*0301 specific clones and 3 different Vβs within the HLA-DPB1*0201 specific clones. T cells using the same Vβ could be isolated at different time points during the clinical response, demonstrating the significance of this anti-HLA-DP response. In conclusion, we observed in a patient with an HLA-class II positive B cell malignancy a profound GVL effect without GVHD, caused by a polyclonal immune response comprising both T helper and cytotoxic CD4+ HLA-DP specific T cell clones directed against both HLA-DP alleles. These data indicate that in HLA-class II expressing hematological malignancies HLA-DP mismatched SCT may be preferable over a fully matched SCT making use of HLA-DP as a specific target for immunotherapy.


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