scholarly journals Exogenous IFN-γ ex vivo shapes the alloreactive T-cell repertoire by inhibition of Th17 responses and generation of functional Foxp3+ regulatory T cells

2008 ◽  
Vol 38 (9) ◽  
pp. 2512-2527 ◽  
Author(s):  
Gang Feng ◽  
Wenda Gao ◽  
Terry B. Strom ◽  
Mohamed Oukka ◽  
Ross S. Francis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Ifn Γ ◽  
Alloreactive T Cell

Patients (Pts) Surviving Haploidentical Stem Cell Transplantation (SCT) after Ex Vivo Costimulatory Blockade To Induce Anergy Experience Few Long-Term Complications.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 599-599 ◽  
Author(s):  
Eva C. Guinan ◽  
John G. Gribben ◽  
Lisa L. Brennan ◽  
Lee M. Nadler
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Ex Vivo ◽  
Chronic Gvhd ◽  
T Cell Response ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Costimulatory Blockade ◽  
Cell Counts

Abstract Poor and delayed immune reconstitution remains a major stumbling block to successful SCT especially when alternative donors are used. Strategies to selectively remove or inactivate alloreactive cells while leaving the other donor T cell repertoire intact might address this problem. A functional T cell response requires an antigen (Ag)-specific MHC-restricted signal (signal 1) to the T cell receptor (TCR) by an Ag presenting cell (APC) as well as a second, Ag independent costimulatory signal (signal 2) provided in large part by B7 family members on APC to CD28 on T cells. Without signal 2, T cells develop tolerance to the specific Ag. Costimulation can be blocked by either CTLA4-Ig, a fusion of Ig with human CTLA4 (the T cell high affinity B7 ligand) or a combination of humanized IgG2 isotype mutated monoclonal antibodies to the APC molecules B7-1 and B7-2. In 2 pilot studies of patients (pts) undergoing haploidentical SCT, donor T cell replete BM was incubated ex vivo with recipient irradiated peripheral blood mononuclear cells with CTLA4-Ig (pilot 1) or anti-B7-1+anti-B7-2 (pilot 2) to induce alloAg specific tolerance. 19 pts age 7 mos-50 yrs (median 15 yrs) were enrolled on pilot 1 and 5 aged 4–12 (median 6) on pilot 2. 3 pts had congenital BM failure. 21 pts with malignancy, ALL (11), AML(7), NHL(2), MDS(1), were >CR1and 14/21 had progressive disease (PD). Pts received TBI based ablative conditioning. Pts received a median of 3.3x106/kg CD34+ cells (0.5–12.3) containing a median of 2.8x 107/kg CD3+ (0.7–6.8), 1.6x 107/kg CD4+ (0.4–4.1), and 1x107/kg CD8+ (0.2–3.7) T cells. One pt got additional anergized cells for slow recovery and engrafted fully. One AML pt had autologous persistence and graft failure (GF). Evaluable pts engrafted at median 21 d (range, 13–29) with full donor chimerism. Of the 21 evaluable pts, 9 (43%) had findings consistent with acute GVHD graded B (n=4), C (n=4) and D (n=1) despite inconsistent pathology. GVHD symptoms were largely isolated to the GI tract and resolved with observation or moderate steroids. No death was attributable to GVHD. 11 pts died early of a combination of bacterial or fungal infection and/or regimen-related toxicity at a median of 35 d (8–159). Of the remaining 13 pts, the GF pt died after 2nd SCT elsewhere, 1 pt had sudden death d 176 at home and 2 pts with extramedullary AML died d 60 and 149 with PD. One T-ALL pt died of late PD d 1758. All BM failure and 3/14 transplanted with PD survive. All 8 survivors (8/19 < 23 yrs) have 100% performance status at a median of 2423 d (1580–2875). None take medications or have chronic GVHD. 3 pts became CMV Ag + by d 100, (1 was transplanted with CMV), and responded to anti-viral therapy. Unlike many reported approaches to haploidentical SCT, aside from several CVL associated bacteremias, there have been no admissions for opportunistic infection and no late viral infections. All pts have good T cell counts, respond to vaccines and specific Ags and have good immunoglobulin levels. Costimulatory blockade, a method of limiting alloreactivity which leaves the remaining T cell repertoire intact, holds out promise as a method of overcoming alloreactivity while better preserving donor immune function and preserving anti-tumor activity. A new study combining costimulatory blockade and megadose stem cell SCT has been initiated.

Repertoire Analysis of Ex Vivo Polyclonally Expanded Regulatory T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3222-3222
Author(s):  
Jenny Zilberberg ◽  
Kira Goldgirsh ◽  
Robert Korngold ◽  
Thea M. Friedman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Clinical Situation ◽  
Post Transplantation ◽  
Cell Repertoire ◽  
Hematopoietic Stem ◽  
Pcr Products

Abstract CD4+CD25+ regulatory T cells (Treg) are essential for the maintenance of self-tolerance and have also been implicated in the control of alloreactive immune responses. Several studies using murine models of graft-vs.-host disease (GVHD) have shown that addition of equivalent numbers of Treg to the donor T cell inoculum at time of hematopoietic stem cell transplantation can significantly reduce the incidence of GVHD. In addition, in an MHC-matched, minor histocompatibility disparate model, the infusion of Treg ten days post-transplantation was shown to ameliorate the progression of GVHD while permitting a graft-versus-leukemia effect. However, because Treg constitute <5% of peripheral CD4+ T cells in humans, the use of freshly isolated Treg to treat and/or prevent GVHD, as well as other diseases in the clinical situation, is limited. Therefore, much effort is now under way to expand Treg in order to have sufficient numbers for therapeutic use. There is little available information regarding the repertoire complexity of ex vivo, polyclonally expanded regulatory T cells. We hypothesize that like their CD4+CD25− T cell counterparts, the diversity of the Treg T cell receptor (TCR) repertoire will also be complex. To this end, CD4+CD25− and CD4+CD25+ T cells from B10.BR mice were purified using fluorescence activated cell sorting; both populations were polyclonally expanded using CD3/CD28 paramagnetic microbeads in combination with high levels (100 IU/ml) of hrIL-2. After achieving a greater than 50 fold expansion, RNA from 1–1.5×107 cells was isolated for RT-PCR. The complexity of the T cell repertoire of expanded CD4+CD25− and CD4+CD25+ was determined using TCR Vb CDR3-size spectratype analysis. The PCR products were run on a sequencing gel and analyzed by the GeneMapper Software from Applied Biosystems. This comparison revealed that the number of resolvable Vb families is more heterogeneous in the CD25− populations. Whether this reflected a lack of complexity in the regulatory repertoire warrants further investigation. However, for the resolvable Vb families there were no significant differences in the complexity indexes between these two groups.

Spectratype Analysis of In Vitro Donor Anti-Host T Cell Repertoire Responses Predict Those of In Vivo Post-BMT.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2163-2163
Author(s):  
Thea M. Friedman ◽  
Kira Goldgirsh ◽  
Jenny Zilberberg ◽  
Stephanie A. Berger ◽  
Joanne Filicko-O’Hara ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Responses ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Post Transplant ◽  
Cell Responses ◽  
Repertoire Analysis ◽  
Alloreactive T Cell

Abstract Immunotherapeutic strategies have gained recognition as viable alternatives to more conventional modalities for the treatment of cancer. In this regard, adoptive T cell therapy through allogeneic blood and marrow transplantation (BMT) has provided the strongest evidence that anti-tumor effects could be achieved against hematological malignancies. However, the major complications of BMT still include graft failure, opportunistic infections, disease relapse and graft-versus-host disease (GVHD). The presence of mature donor T cells in the transplant inoculum reduces the incidence of the first three complications, while unfortunately increasing the risk of GVHD, which can be directed against either HLA or minor histocompatibilty antigen (miHA) disparities. Thus, a major objective in the field has been to develop tactics that could facilitate the separation of graft-versus-tumor (GVT) effects from the deleterious effects of GVHD. One such approach would be to selectively deplete donor alloreactive T cells in the donor inoculum while allowing residual T cells to provide some protection against infection and to support a tumor-specific GVT response. For a more targeted approach, delayed donor lymphocyte infusion (DLI) of positively-selected donor GVT-reactive T cells could be used weeks to months post-transplant, if these elements were identifiable. In this regard, TCR Vβ repertoire analysis by CDR3-size spectratyping can be a powerful tool for the characterization of alloreactive T cell responses. Theoretically, molecular analysis of T cell responses in vitro, given the high sensitivity of the PCR-based spectratyping technique, should identify the most potentially critical Vβ families involved in the later development of GVHD and GVT effects in patients. To this end, we tested the hypothesis that T cell repertoire analysis of HLA-matched sibling (SIB) or matched unrelated donors (URD) from in vitro, host-stimulated, mixed lymphocyte cultures (MLC) would be predictive of the TCR-Vβ spectratype analysis of the T cell repertoire in the patient following BMT. In this study, we examined 17 patient pairs and report that for the resolvable Vβ families, we observed overall 71.2 ± 11.9% (mean ± SD.; range 40%–85%) of the in vitro anti-host T cell responses were predictive of those in the patient post-transplant. Of the 28.8% non-predictive Vβ families, 6.9 ± 6.3% (range 0%–27%) exhibited skewing in the MLC but no skewing in the patient post-transplant repertoire, 9.3 ± 6.3% (range 0%–18.8%) exhibited skewing in different peaks within the same Vβ family, and 12.5 ± 10.8% (range 0%–40%) showed skewing in the patient post-transplant and none in the MLC. Taken together, these results suggest that the in vitro MLC T cell responses show good consistency with post-transplant patient responses. Thus, in vitro spectratyping may be useful for predicting the alloreactive T cell responses involved in GVHD and could be used to guide custom-designed select Vβ family T cell-depleted transplants to improve patient outcomes. The additional advantage of this approach is that minimization of GVHD risk can be obtained without any direct knowledge of the specific miHA involved in the individual donor-patient pair.

scholarly journals Human Tumor Antigen-Specific Helper and Regulatory T Cells Share Common Epitope Specificity but Exhibit Distinct T Cell Repertoire

2010 ◽  
Vol 184 (12) ◽  
pp. 6709-6718 ◽  
Author(s):  
Julien Fourcade ◽  
Zhaojun Sun ◽  
Pavol Kudela ◽  
Bratislav Janjic ◽  
John M. Kirkwood ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Tumor Antigen ◽  
Human Tumor ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Epitope Specificity

CD4+CD25+ Regulatory T Cells Enhance Immune Reconstitution Following Allogeneic Hematopoietic Cell Transplantation by Protecting Thymic and Lymphoid Compartments from Graft-Versus-Host Disease Damage without Impacting T Cell Repertoire Development.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 70-70 ◽  
Author(s):  
Vu H. Nguyen ◽  
Daisy Chang ◽  
Sumana Shashidhar ◽  
Michael Bachmann ◽  
Christopher H. Contag ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Graft Versus Host Disease ◽  
Immune Reconstitution ◽  
Hematopoietic Cell Transplantation ◽  
Effector T Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Graft Versus Host

Abstract Regulatory T cells (Treg) protect from acute graft-versus-host disease (GvHD) in murine models of major-MHC mismatched hematopoietic cell transplantation (HCT) presumably by dampening the proliferation of mature effector T cells. It is unclear whether the effect of Treg on effector T cells is a selective or nonselective process or if Treg regulate the process of intrathymic and peripheral T cell maturation and selection following HCT, particularly given the intrinsic link of GvHD and immune reconstitution. We previously showed that Treg improved the quantitative and functional lymphoid reconstitution in a murine model of HCT. In the current study, we hypothesize that Treg prevent thymic and lymphoid damage from GvHD, leading to enhanced lymphoid reconstitution. Lethally-irradiated adult thymectomized Balb/c (H2d) recipients were transplanted with wild-type FVB (H2q) T cell depleted bone marrow (TCD-BM) cells and CD4+/CD8+ cells (Tcon), the latter to induce GvHD, with or without donor Treg given at a 1:1 dose ratio with Tcon. At day 30, when all groups had reached full donor chimerism, transplant recipients were challenged with murine CMV (5×105 pfu/mouse) intraperitoneally. At day 90, survival for thymectomized groups with TCD-BM alone, with Tcon, or with Tcon+Treg was 78%, 0%, and 45%, respectively, compared to 100%, 0%, and 86% survival in their respective euthymic infected counterparts (p&lt;0.05 for thymectomized vs euthymic Treg groups). Elispot for Interferon-γ showed CMV-specific donor responses in all infected groups. CMV viral titers in the liver and kidney 2 weeks after infection was lower in recipients that received Treg compared to animals that received Tcon alone. Compared to euthymic transplant controls, thymectomized animals had higher viral titers in the liver, lungs, and kidneys in all groups. Uninfected thymectomized mice in the respective groups served as controls to separate the effect of CMV infection and GvHD on survival. All animals, infected or uninfected, that received Treg had no evidence of clinical GvHD while animals that received Tcon alone had significant GvHD. In euthymic recipients, gross and histologic examination confirmed the general preservation of thymic integrity and architecture in animals that received Treg compared with smaller involuted thymuses partially replaced by adipose tissue in animals that received Tcon alone. T cell repertoire assessed by V-beta TCR screening with FACS analysis showed a polyclonal distribution in animals with or without Treg. Spectratyping at day 30 post-transplantation showed that Treg had no significant impact on the TCR repertoire diversity in animals which received Tcon. Based on survival of a subset of infected thymectomized animals that received Treg, we evaluated the impact of Treg on secondary lymphoid organs following HCT. Animals without transferred Treg had significant splenic and lymph node fibrosis and hypoplasia with a reduction in T cell numbers due to GvHD. Our findings indicate that Treg indirectly enhance immune reconstitution by protecting the thymic and secondary lymphoid compartments from GvHD damage, allowing the generation and peripheral expansion of lymphoid cells without impacting the diversity of T cell repertoire.

scholarly journals CD1b tetramers bind αβ T cell receptors to identify a mycobacterial glycolipid-reactive T cell repertoire in humans

2011 ◽  
Vol 208 (9) ◽  
pp. 1741-1747 ◽  
Author(s):  
Anne G. Kasmar ◽  
Ildiko van Rhijn ◽  
Tan-Yun Cheng ◽  
Marie Turner ◽  
Chetan Seshadri ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptors ◽  
Direct Detection ◽  
Ex Vivo ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Cell Receptors ◽  
Cell Staining ◽  
Antigen Presenting

Microbial lipids activate T cells by binding directly to CD1 and T cell receptors (TCRs) or by indirect effects on antigen-presenting cells involving induction of lipid autoantigens, CD1 transcription, or cytokine release. To distinguish among direct and indirect mechanisms, we developed fluorescent human CD1b tetramers and measured T cell staining. CD1b tetramer staining of T cells requires glucose monomycolate (GMM) antigens, is specific for TCR structure, and is blocked by a recombinant clonotypic TCR comprised of TRAV17 and TRBV4-1, proving that CD1b–glycolipid complexes bind the TCR. GMM-loaded tetramers brightly stain a small subpopulation of blood-derived cells from humans infected with Mycobacterium tuberculosis, providing direct detection of a CD1b-reactive T cell repertoire. Polyclonal T cells from patients sorted with tetramers are activated by GMM antigens presented by CD1b. Whereas prior studies emphasized CD8+ and CD4−CD8− CD1b-restricted clones, CD1b tetramer-based studies show that nearly all cells express the CD4 co-receptor. These findings prove a cognate mechanism whereby CD1b–glycolipid complexes bind to TCRs. CD1b tetramers detect a natural CD1b-restricted T cell repertoire ex vivo with unexpected features, opening a new investigative path to study the human CD1 system.

scholarly journals Limits of the Human-PBL-SCID Mice Model: Severe Restriction of the Vβ T-Cell Repertoire of Engrafted Human T Cells

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 329-336 ◽  
Author(s):  
Sylvie Garcia ◽  
Gilles Dadaglio ◽  
Marie-Lise Gougeon
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Scid Mice ◽  
Phenotypic Characterization ◽  
T Cell Repertoire ◽  
Severe Restriction ◽  
Cell Repertoire ◽  
Activation Markers ◽  
Human T Cells

Abstract A recent study in the human-peripheral blood lymphocytes-severe combined immunodeficiency (hu-PBL-SCID) model, analyzing the specificity of the engrafted human T cells, showed that human T-cell lines and clones derived from engrafted cells presented a xenoreactivity toward murine host molecules. This observation raised the question of the influence of the SCID environment on the ex vivo repertoire and function on the human T cells reconstituting the murine host. We have characterized the human Vβ repertoire in the spleen of hu-PBL-SCID mice 1 to 3 months after their engraftment. Fluorescence-activated cell sorting (FACS) analysis of human Vβ T-cell representation showed that, for all chimeras, all tested Vβ subsets were submitted to underrepresentation and/or expansion upon engraftment. Importantly, these quantitative modifications of the T-cell repertoire were associated with a severe restriction in both the CDR3 size distribution pattern of the Vβ transcripts and the number of Jβ segments used by these transcripts. In addition, ex vivo phenotypic characterization of engrafted cells showed that 70% to 100% expressed the activation markers HLA-DR, CD45RO, and CD38. Taken together, these results suggest that, following their engraftment, human T cells were submitted to a massive antigenic selection. Moreover, we found that these activated T cells were unresponsive to in vitro mitogenic and superantigenic activation. The consequences of the skewed repertoire and altered function of engrafted human T cells on the validity of this humanized murine model are discussed.

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