Antigen Specificity and Immunoregulation in Syngeneic Graft-Versus-Host Disease.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4973-4973
Author(s):  
Allan D. Hess ◽  
Christopher J. Thoburn ◽  
Emilie C. Bright ◽  
Yuji Miura
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Mhc Class Ii ◽  
Ex Vivo ◽  
Class Ii ◽  
Mrna Levels ◽  
Mrna Transcript ◽  
Antigen Specificity ◽  
Transcript Levels ◽  
Host Disease

Abstract Syngeneic graft-vs-host disease (SGVHD) is a T cell dependent autoaggression syndrome induced by administering Cyclosporine following syngeneic bone marrow transplantation. The SGVHD autoreactive T cells recognize the MHC class II-invariant chain peptide complex (MHC class II-CLIP) and can be separated into functional subsets based on their differential dependence on the N- and C-terminal peptide flanking domains of CLIP. The present studies were undertaken to determine whether the N- or C-terminal flanking domain dependent subsets of CLIP reactive T cells reside within the CD4+CD25+ regulatory compartment. Multi-color flow cytometry was used to identify and isolate CD4+ (FITC) CD25+ (PE) T cells. Antigen-specific T cells within this compartment were identified with a soluble MHC class II-Ig chimeric construct (bioinylated, Cychrome avidin counterstaining) loaded with variants of CLIP containing the MHC class II binding domain and having either the N- or C-terminal flanking regions (N-CLIP, CLIP-C). Approximately 8.5% of the cells within the normal CD4+ lymphocyte population were CD25+. Both N-CLIP (1.1%) and CLIP-C (4.8%) reactive T cells coould be detected in the CD4+CD25+ population. Assessment of CD28, CTLA4, B7.1 and B7.2 mRNA expression levels by quantitative PCR directly ex vivo, revealed remarkable differences between the N-CLIP and CLIP-C specific CD4+CD25+ T cells. Although CD28 mRNA levels were comparable for both subsets, B7.2 and CTLA4 mRNA transcript levels were significantly increased (>50 fold) in the CLIP-C+CD4+CD25+ T cells compared to the N-CLIP specific subset. On the other hand, levels of B7.1 mRNA were increased >10 fold in N-CLIP+CD4+CD25+ T cells. Additional studies assessing mRNA transcript levels for the regulatory transcription factor Foxp3, also revealed a disparity between the N-CLIP and C-CLIP specific subsets. mRNA transcript levels for Foxp3 were markedly increased (>35 fold) in the CLIP-C dependent subset compared to the levels detected in N-CLIP+CD4+CD25+ T cells. Low levels of cytokine (IL-2, IL-4, interferon-γ) mRNA transcripts were detected in both subsets. Interestingly, intradermal immunization of normal animals with the peptides presented on dendritic cells increased mRNA transcript levels for type 1 cytokines in the N-CLIP reactive subset and type 2 cytokines in the CLIP-C dependent subset. Taken together, the results indicate that the CLIP-C antigen specific CD4+CD25+ cells have a profile consistent with regulatory T cells whereas the profile of the N-CLIP+CD4+CD25+ lymphocytes is more characteristic of activated T helper cells. The ability to identify and isolate regulatory T cells ex vivo and to modify their activity by immunization provides opportunities to both enhance and monitor the re-establishment of self-tolerance.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

scholarly journals Antidiabetogenic MHC class II promotes the differentiation of MHC-promiscuous autoreactive T cells into FOXP3+regulatory T cells

2013 ◽  
Vol 110 (9) ◽  
pp. 3471-3476 ◽  
Author(s):  
Sue Tsai ◽  
Pau Serra ◽  
Xavier Clemente-Casares ◽  
Jun Yamanouchi ◽  
Shari Thiessen ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Autoreactive T Cells

scholarly journals CD4+ and CD8+ Regulatory T Cells Generated Ex Vivo with IL-2 and TGF-β Suppress a Stimulatory Graft-versus-Host Disease with a Lupus-Like Syndrome

2004 ◽  
Vol 172 (3) ◽  
pp. 1531-1539 ◽  
Author(s):  
Song Guo Zheng ◽  
Ju Hua Wang ◽  
Michael N. Koss ◽  
Francisco Quismorio ◽  
J. Dixon Gray ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Graft Versus Host Disease ◽  
Ex Vivo ◽  
Host Disease ◽  
Graft Versus Host

Host Plasmacytoid or Conventional Dendritic Cells Alone Are Sufficient To Initiate Graft-Versus-Host Disease.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2164-2164
Author(s):  
Motoko Koyama ◽  
Daigo Hashimoto ◽  
Kazutoshi Aoyama ◽  
Ken-ichi Matsuoka ◽  
Kennosuke Karube ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
B Cells ◽  
Mhc Class Ii ◽  
Graft Versus Host Disease ◽  
Class Ii ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Host Disease ◽  
Graft Versus Host

Abstract Graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic stem cell transplantation. Alloantigen expression on host dendritic cells (DCs) is critical to initiate GVHD. DCs can be divided into two main subpopulations; conventional DCs (cDCs) and plasmacytoid DCs (pDCs), however, the contribution of each DC subset to elicit GVHD remains unclear. We examined the ability of cDCs and pDCs to initiate GVHD. pDCs, cDCs and B cells were isolated from C57BL/6 (B6: H–2b) mice treated with Flt3 ligand in order to expand DCs. pDCs were enriched from bone marrow by depleting CD3+, CD19+, CD11b+, and CD49b+ cells, followed by a FACS sorting of CD11cint B220+ cells. cDCs and B cells were sorted from splenocytes as CD11chi B220− cells and CD11c− B220+ cells, respectively. Isolated pDCs showed plasmacytoid morphology, produced IFN-α in response to CpG oligonucleotide. Although pDCs stimulated allogeneic T cells far less potently than cDCs, stimulation with CpG enhanced their allostimulatory capacity as potent as cDCs. We compared the ability of each DC subset to initiate GVHD by an add-back study of MHC class II-expressing DCs into MHC class II-deficient (II−/−) mice that were resistant to CD4-dependent GVHD. Lethally irradiated II−/− B6 mice were injected with 2 × 106 pDCs, cDCs or B cells from wild-type (II+/+) B6 mice on day -1 and injected with 2 × 106 CD4+ T cell from BALB/c (H–2d) mice on day 0. A flow cytometric analysis of the mesenteric lymph nodes on day +5 demonstrated significantly greater expansion of donor CD4+ T cells in recipients of pDCs or cDCs than those of B cells (Table). While injection of B cells did not cause any sign of GVHD, injection of pDCs or cDCs alone was sufficient to produce clinical and pathological GVHD (Table), thus breaking GVHD resistance of II−/− mice. We next examined the ability of pDCs to induce CD8-dependent GVHD in MHC-matched transplant using mice deficient in functional MHC class I expression (β2m−/−). Again, injection of pDCs or cDCs alone was sufficient to cause expansion of donor CD8+ T cells (p<0.05). We next asked whether signaling through Toll-like receptors (TLRs) could be required for pDCs to initiate GVHD. However, injection of pDCs isolated from MyD88/TRIF-double deficient mice was able to initiate GVHD as potent as wild-type pDCs, thus demonstrating that pDCs initiate GVHD in a TLR signaling independent manner. These results provide important information for developing strategies aimed at inactivating host DCs to prevent GVHD. Impact of each APC subpopulation on GVHD APC Donor CD4 expansion (×103±SE) Clinical GVHD score (mean±SE) Pathological GVHD score (mean±SE) *p<0.05 compared with B cells B cell 0.1 ± 0.0 2.1 ± 0.2 2.1 ± 0.2 pDC 5.3 ± 2.4* 4.3 ± 0.3* 7.4 ± 0.5* cDC 9.7 ± 3.8 * 3.8 ± 0.5 * 7.2 ± 0.7*

Adoptive Transfer Of Ex Vivo “Educated” CD4+CD25+FoxP3+ Regulatory T Cells Effectively Treats Acute Graft Versus Host Disease Preserving Graft Versus Tumor Effect

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4485-4485
Author(s):  
Antonio Pierini ◽  
Dominik Schneidawind ◽  
Mareike Florek ◽  
Maite Alvarez ◽  
Yuqiong Pan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Tumor Cells ◽  
Graft Versus Host Disease ◽  
Adoptive Transfer ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Host Disease ◽  
Graft Versus Host

Donor derived regulatory T cells (Tregs) effectively prevent graft versus host disease (GVHD) in mouse models and in early phase clinical trials. Interleukin 2 (IL-2) therapy in patients with chronic GVHD (cGVHD) can increase Treg number and the Treg/CD4+ T cell ratio resulting in organ damage reduction and symptom relief. Less is known regarding Treg-based treatment for acute GVHD (aGVHD). In this study we evaluated the role of donor Treg cellular therapy for aGVHD treatment in well established murine models. T cell depleted bone marrow (TCD BM) from C57BL/6 mice was transplanted into lethally irradiated (8 Gy) BALB/C recipients together with 7.5x105 to 1x106/animal donor derived luc+ Tcons. Naturally occurring CD4+CD25+FoxP3+ donor type Tregs (nTregs) were purified from C57BL/6 donor mice. 2.5x105/mouse nTregs were injected at day 6 or 7 after transplant in mice that showed clear clinical signs of aGVHD and Tcon proliferation assessed by bioluminescence imaging (BLI). Survival analysis showed a favorable trend for nTreg treated mice, but the impact of this treatment was modest and not statistically significant (p 0.08). aGVHD is a disease characterized by the activation and rapid proliferation of alloreactive donor conventional T cells (Tcons) directed against host antigens, so one of the major obstacles of this approach is to overcome the large number and effector function of activated Tcons. Several studies have utilized ex vivo expansion of Tregs to increase their number with the goal of maintaining suppressive function. We developed a different strategy with the intent to “educate” Tregs to specifically suppress the reactive Tcon population. We incubated 2.5x105 donor derived Tregs with irradiated (3000 cGy) blood of aGVHD affected mice for 20 hours without further stimulation and injected the entire pool of these cells, termed educated Treg (eTregs), at day 7 or 8 after transplant and Tcon injection. Interestingly eTregs significantly improved aGVHD affected mouse survival (p = 0.0025 vs Tcons alone). BLI showed no difference between the groups (p = 0.85) because the treatment intervened after Tcon proliferation and activation was initiated. To evaluate eTreg impact on graft versus tumor (GVT) effects, we transplanted BALB/C mice with C57BL/6 TCD BM and 1x104/mouse luc+ A20 tumor cells along with 1x106/mouse donor Tcons and 2.5x105 eTregs. Mice that received TCD BM and A20 tumor cells alone died from progressive tumor growth, while mice that received Tcons died from GVHD without tumor engraftment. Further animals that received both Tcon and eTreg treatment did not have tumor engraftment demonstrating that eTregs do not impact Tcon mediated GVT effects. Further studies are ongoing to characterize the eTreg population as compared to nTreg, with respect to expression of activation markers and in functional assays. Our observations indicate that Tregs can be ex vivo educated to suppress in vivo reactive and proliferating Tcons. Moreover our data demonstrate that eTreg adoptive transfer is clinically feasible and promising. These findings may be relevant for the development of clinical grade Treg based cellular therapy for the treatment of conditions caused by immune dysregulation such as aGVHD and autoimmune diseases and for transplant tolerance induction. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inhibition of the Alloimmune Response through the Generation of Regulatory T Cells by a MHC Class II-Derived Peptide

2008 ◽  
Vol 181 (11) ◽  
pp. 7499-7506 ◽  
Author(s):  
Weiping Zang ◽  
Marvin Lin ◽  
Safa Kalache ◽  
Nan Zhang ◽  
Bernd Krüger ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Mhc Class Ii ◽  
Class Ii

scholarly journals Effector and regulatory T?cells derived from the same T?cell clone differ in MHC class II-peptide multimer binding

2004 ◽  
Vol 34 (12) ◽  
pp. 3359-3369 ◽  
Author(s):  
Esther?N.?M. Nolte-?t Hoen ◽  
Maria?Grazia Amoroso ◽  
Jetty Veenstra ◽  
Mayken?C. Grosfeld-Stulemeyer ◽  
Willem van Eden ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Mhc Class Ii ◽  
Cell Clone ◽  
Class Ii ◽  
T Cell Clone

scholarly journals Deciphering the Plasmodium falciparum malaria-specific CD4+ T cell response: ex vivo detection of high frequencies of PD-1+TIGIT+ EXP1-specific CD4+ T cells using a novel HLA-DR11-restricted MHC class II tetramer

2021 ◽  
Author(s):  
Sophia Schulte ◽  
Janna Heide ◽  
Christin Ackermann ◽  
Sven Peine ◽  
Michael Ramharter ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cell Response ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Class Ii ◽  
T Cell Response ◽  
Cd4 T Cell ◽  
Inhibitory Receptors

Abstract Relatively little is known about the ex vivo frequency and phenotype of the P. falciparum-specific CD4+ T cell response in humans. The exported protein 1 (EXP1) is expressed by plasmodia at both, the liver stage and blood stage, of infection making it a potential target for CD4+ and CD8+ effector T cells. Here, a fluorochrome-labelled HLA-DRB1*11:01-restriced MHC class II tetramer derived from the P. falciparum EXP1 (aa62-74) was established for ex vivo tetramer analysis and magnetic bead enrichment in ten patients with acute malaria. EXP1-specific CD4+ T cells were detectable in nine out of ten (90%) malaria patients expressing the HLA-DRB1*11 molecule with an average ex vivo frequency of 0.11% (0-0.22%) of total CD4+ T cells. The phenotype of EXP1-specific CD4+ T cells was further assessed using co-staining with activation (CD38, HLA-DR, CD26), differentiation (CD45RO, CCR7, KLRG1, CD127), senescence (CD57) and co-inhibitory (PD-1, TIGIT, LAG-3, TIM-3) markers as well as the ectonucleotidases CD39 and CD73. EXP1-specific tetramer+ CD4+ T cells had a distinct phenotype compared to bulk CD4+ T cells and displayed a highly activated effector memory phenotype with elevated levels of co-inhibitory receptors and activation markers: EXP1-specific CD4+ T cells universally expressed the co-inhibitory receptors PD-1 and TIGIT as well as the activation marker CD38 and showed elevated frequencies of CD39. These results demonstrate that MHC class II tetramer enrichment is a sensitive approach to investigate ex vivo antigen-specific CD4+ T cells in malaria patients that will aid further analysis of the role of CD4+ T cells during malaria.

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