In vitro expansion of antigen-specific CD8+ T cells distorts the T-cell repertoire

2014 ◽  
Vol 405 ◽  
pp. 199-203 ◽  
Author(s):  
Dan Koning ◽  
Ana I. Costa ◽  
Raiza Hasrat ◽  
Bart P.X. Grady ◽  
Sanne Spijkers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
In Vitro Expansion

scholarly journals 762 A combination of functional biomarkers improves identification of the tumor-specific reactive T cell repertoire

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A810-A810
Author(s):  
Arianna Draghi ◽  
Katja Harbst ◽  
Inge Svane ◽  
Marco Donia
Keyword(s):  
T Cells ◽  
T Cell ◽  
De Novo ◽  
Autologous Tumor ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Simple Method ◽  
Functional Biomarkers

BackgroundDetecting the entire repertoire of tumor-specific reactive T cells is essential for investigating the broad range of T cell functions in the tumor-microenvironment. At present, assays identifying tumor-specific functional activation measure either upregulation of specific surface molecules, de novo production of the most common antitumor cytokines or mobilization of cytotoxic granules.MethodsIn this study, we combined transcriptomic analyses of tumor-specific reactive tumorinfiltrating lymphocytes (TILs), TIL-autologous tumor cell co-cultures and commonly used established detection protocols to develop an intracellular flow cytometry staining method encompassing simultaneous detection of intracellular CD137, de novo production of TNF and IFNy and extracellular mobilization of CD107a.ResultsThis approach enabled the identification of a larger fraction of tumor-specific reactive T cells in vitro compared to standard methods, revealing the existence of multiple distinct functional clusters of tumor-specific reactive TILs. Publicly available datasets of fresh tumor single-cell RNA-sequencing from four cancer types were investigated to confirm that these functional biomarkers identified distinct functional clusters forming the entire repertoire of tumor-specific reactive T cells in situ.ConclusionsIn conclusion, we describe a simple method using a combination of functional biomarkers that improves identification of the tumor-specific reactive T cell repertoire in vitro and in situ.

scholarly journals Characterization of human cytomegalovirus peptide–specific CD8+ T-cell repertoire diversity following in vitro restimulation by antigen-pulsed dendritic cells

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 213-223 ◽  
Author(s):  
Karl Peggs ◽  
Stephanie Verfuerth ◽  
Arnold Pizzey ◽  
Jenni Ainsworth ◽  
Paul Moss ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Viral Replication ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Repertoire Diversity ◽  
Clonal Composition

Under conditions of impaired T-cell immunity, human cytomegalovirus (HCMV) can reactivate from lifelong latency, resulting in potentially fatal disease. A crucial role for CD8+ T cells has been demonstrated in control of viral replication, and high levels of HCMV-specific cytotoxic T-lymphocytes are seen in immunocompetent HCMV-seropositive individuals despite very low viral loads. Elucidation of the minimum portion of the anti-HCMV T-cell repertoire that is required to suppress viral replication requires further study of clonal composition. The ability of dendritic cells to take up and process exogenous viral antigen by constitutive macropinocytosis was used to study HCMV-specific T-cell memory in the absence of viral replication. The specificity and clonal composition of the CD8+ T-cell responses were evaluated using HLA tetrameric complexes and T-cell receptor β chain (TCRBV) spectratypic analyses. There was a skewed reactivity toward the matrix protein pp65, with up to 40-fold expansion of CD8+ T cells directed toward a single peptide-MHC combination. Individual expansions detected on TCRBV spectratype analysis were HCMV-specific and composed of single or highly restricted numbers of clones. There was preferential TCRBV gene usage (BV6.1/6.2, BV8, and BV13 in HLA-A*0201+ individuals) but lack of conservation of CDR3 length and junctional motifs between donors. While there was a spectrum of TCR repertoire diversity directed toward individual MHC-peptide combinations between donors, a relatively small number of clones appeared to predominate the response in each case. These data provide further insight into the range of anti-HCMV responses and will aid the design and monitoring of adoptive immunotherapy protocols.

Reconstitution of the T Cell Repertoire Following Treatment with Alemtuzumab in Patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2986-2986
Author(s):  
Mohammad R. Rezvany ◽  
Mahmood J. Tehrani ◽  
Claes Karlsson ◽  
Jeanette Lundin ◽  
Hodjattallah Rabbani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Long Term Follow Up

Abstract Background and Methods: B-cell chronic lymphocytic leukemia (B-CLL) occurs as a result of clonal accumulation of functionally abnormal B cells. Alemtuzumab is a humanized monoclonal antibody specific for the CD52 antigen, which is highly expressed on both B-CLL cells and normal lymphocytes, but not on hematopoietic (CD34) stem cells. Alemtuzumab has been shown to effectively deplete the blood and bone marrow of lymphocytes, including CD4 and CD8 T cells, which may lead to profound immunosuppression and make patients more susceptible to infections. We and others have previously shown that the CD4 T cells in B-CLL patients may be clonally distinct from the normal population in that they present a more clonal pattern of the T-cell receptor (TCR) repertoire (Rezvany et al, Blood2003;101:1063–1070). It is therefore of interest to study the T cell repertoire following alemtuzumab administration as well as factors affecting T cell reconstitution following CD52 targeted therapy. In this study, we evaluated in depth the T-cell receptor-beta-variable sequence (TCR BV) in CD4 and CD8 T cells by real-time PCR, before and repeatedly after/during long term follow-up, in 5 B-CLL patients who had received alemtuzumab as first-line therapy (Lundin et al, Blood2002;100:768–773). Also, an analysis was conducted of CDR3 length polymorphism to describe changes in the clonality pattern. Results: A decline in most of BV genes either in CD4 or CD8 T cells was observed shortly after alemtuzumab treatment, which was followed by a gradual increase in most of the BV genes during long-term follow up. CDR3 length polymorphism analysis shortly after treatment revealed an even more highly restricted pattern in CD4 T cells compared to baseline with a shift towards a monoclonal/oligoclonal pattern regardless of increased or decreased BV usage. Furthermore, in the analysis of the clonal spectrum that was expressed shortly after alemtuzumab therapy, the number of peaks was significantly reduced in CD4 (P <0.01) but not in CD8 T cells, which was followed by a gradual increase in diversity towards a polyclonal repertoire during long-term follow up. Conclusions: These results indicate that perturbations in the T cell repertoire following alemtuzumab are complex, and are not reflected by changes in CD4/CD8 T cell numbers only. The restricted CDR3 pattern present prior to therapy became even more restricted after end of treatment, followed by a normalization of CDR3 patterns in CD4 T-cells during long-term follow-up. These results further suggest a regulatory role for T cells in relation to the malignant B cell clone in patients with B-CLL.

T Cell Receptor Repertoire of Patients with Chronic Graft-Versus Host Disease.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5174-5174
Author(s):  
Olga Y. Azhipa ◽  
Scott D. Rowley ◽  
Michele L. Donato ◽  
Robert Korngold ◽  
Thea M. Friedman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Flow Cytometric Analysis ◽  
T Cell Responses ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Cell Responses ◽  
Flow Cytometric ◽  
Complexity Index

Abstract Chronic GVHD (cGVHD) is a major risk factor in patients receiving allogeneic hematopoietic cell transplantation (HCT), and is a complicated syndrome with a combination of autoimmune-like features and a range of multiorgan manifestations. Currently, efforts are being made to standardize the criteria for diagnosis and staging of cGVHD, but there is little understanding of the pathogenesis of the disease, associated biomarkers, and the immune perturbations that may result. Reconstitution of the T cell repertoire after allo-HCT often takes several months to a year, and may be significantly impaired or skewed in patients who develop cGVHD. We thus sought to assess the immune T cell status of cGVHD patients by TCR Vβ CDR3-size spectratype analysis. A cohort of 9 patients who underwent allo-HCT (PBMC n=7; BM n=2) were enrolled in the study. The underlying diseases in these patients were CML (n=1), AML (n=4), ALL (n=1), CLL (n=1), and MM (n=2). Patients received either reduced intensity or myeloablative conditioning before transplantation, and 8 of the 9 had a previous history of acute GVHD. Furthermore, the patients did not have evidence of infectious disease. PBMC was collected from each patient at one time point ranging from 2 wk to 3 yr from the time they were diagnosed with cGVHD. The onset of cGVHD ranged from 100 d to 3 yr post-HCT (median of 5 mo). Flow cytometric analysis was performed on peripheral blood lymphocytes from 7 of the 9 patients to analyze recovery of different subpopulations. PCR amplification of the CDR3 region of 21 TCR Vβ genes was used to analyze the diversity of the T cell repertoire. The PCR products were run on a sizing gel to separate the CDR3-lengths, and further analyzed by ABI GeneMapper software. Flow cytometric analysis revealed diverse percentages of CD4+ and CD8+ T cells among the 7 patients tested, which were correlated with the post-HCT period. Two patients who received HCT, 4 and 9 months before blood sampling, had only 3% and 4% CD4+ and 3% and 9% CD8+ T cells in their PBMC sample, respectively. On the other hand, the remaining 5 patients, who were all at later time points post-HCT, had CD4+ and CD8+ T cell percentages within normal range. One patient had a ratio close to the normal 2:1 CD4/CD8 ratio, two patients had a 1:1 ratio, and four had inverse CD4/CD8 ratios. Based on CDR3-size spectratype analysis, we determined the recipient TCR-Vβ complexity index within each resoluble family, which represented the percentage of the number of peaks found for each Vβ relative to that found in the average corresponding Vβ family of 10 healthy donors. We considered Vβ to be fully complex if the complexity index exceeded 85%. The results indicated that 41 to 88% of resolved Vβ in all 9 patients were fully complex, with the lower range corresponding to those patients sampled early post-HCT. Vβ 1, 2, 4, 6, 8, 12, and 13 families revealed the best recovery in all patients, even in patients after 4-mo post-HCT. Importantly, extensive skewing of the repertoire within most of the TCR Vβ families were found in all 9 recipients, suggesting that there were active heterogenous T cell responses in those patients with cGVHD. As to what these T cell responses were directed to remains to be seen, and could theoretically involve autoantigens, alloantigens, tumor antigens, or sub-detectable infectious agents. In any case, the presence of a wide-ranging T cell response in these patients may serve as an important new diagnostic indicator for cGVHD.

Evolution of the Donor T Cell Repertoire In Allogeneic Stem Cell Transplant Recipients In the Second Decade After Transplantation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 831-831
Author(s):  
Robert Q. Le ◽  
J. Joseph Melenhorst ◽  
Brenna Hill ◽  
Sarfraz Memon ◽  
Minoo Battiwalla ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
T Cell Receptor ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Cell Receptor ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Significant Difference

Abstract Abstract 831 After allogeneic stem cell transplantation (SCT), donor T lymphocyte immune function is slowly re-established in the recipient through reconstruction of the donor's post-thymic T cell repertoire and from T cell neogenesis in the thymus. Although long-term survivors from SCT appear healthy, their immune repertoire and differences from that of their donors have not been characterized. We studied 38 healthy patients surviving more than 10 years from a myeloablative SCT for hematological malignancy (median follow-up 12 years, range 10–16 years). T cell and natural killer (NK) cell repertoires in these patients were compared with cells from their stem cell donors cryopreserved at time of transplant and from the same donors at 10 year after SCT. The median age of both recipients and their sibling donors at time of transplant was identical (36 years). Patients received cyclosporine GVHD prophylaxis and delayed add-back of donor lymphocytes 30–90 days post transplant. Only one patient was on continued immunosuppressive treatment at the time of study. Compared with the donor pre-transplant counts there was no significant difference in the absolute lymphocyte, neutrophil, monocyte, CD4+ and CD8+ T cell, NK cell, and B cell subset counts. However, compared to their donors, recipients had a) significantly fewer naïve CD4+ and CD8+ T cells; b) lower T cell receptor excision circles levels; c) fewer CD4+ central memory T cells; d) more effector CD8+ T cells; e) and more FOXP3+ regulatory T cells. These data suggest that the patient had a persistent deficiency on T cell neogenesis. Molecular examination of the T cell receptor Vbeta (TCRBV) repertoire by spectratype analysis showed that there was no significant difference in total complexity score, defined as the sum of the number of discrete peaks for each Vbeta subfamily, between the patients and their donors. TCRBV subfamily spectratyping profiles of patients and donors, however, had diverged, with both gains and losses of peaks identifiable in both patient and donor. In conclusion, patients surviving 10 or more years after allogeneic SCT still show a T cell repertoire that reflects expansion of the donor-derived post thymic T cell compartment, with a limited contribution by new T cell generation and persistently increased Tregs. It therefore appears that a diverse TCRBV repertoire predominantly derived from the memory T cell pool is compatible with good health. Disclosures: No relevant conflicts of interest to declare.

Prospective Molecular Identification of Alloreactive CTL Clones in Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4972-4972
Author(s):  
Christine L. O’Keefe ◽  
Ronald Sobecks ◽  
Alexander Rodriguez ◽  
Julie Curtis ◽  
Elizabeth Kuckowski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Molecular Analysis ◽  
Cd8 T Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Activation Markers ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Donor Cells

Abstract The process of immune recovery after allogeneic HSCT can be characterized by an often profound oligoclonality of the TCR spectrum which may reflect: 1) A decreased diversity within the T cell population or 2) Expansion of individual clones that may be caused by specific antigenic drive exerted by pathogens (e.g., CMV) or alloantigens during the process of GvHD. Novel technologies based on the molecular analysis of the TCR repertoire can be applied to study clonal responses, including multiplex amplification of rearranged TCR VB chains followed by sequencing and quantitation of their contribution to the entire T cell repertoire. We initially studied the T cell repertoire after allogeneic HSCT in sibling (N=20) and matched unrelated (N=9) transplants. VB spectratyping was performed on CD8+ T cells in 22 patients; of the expanded VB families tested, 61.2% (30 of 49) were mono- or oligoclonal by genotyping. The clonal size and structure was determined by sequencing. Immunodominant clones contributed up to 5.4% (avg. 1.4%; range 0.1–5.4%) of all CD8+ T cells, indicating that certain stimuli may drive expansion of immunodominant clones. We originally hypothesized that these expanded clones were allospecific and likely played a role in GvHD; however, we found no correlation between the presence of significant expansions and grade III/IV GvHD. Therefore, in order to identify alloreactive CTL clones and their clonotypic markers, an alternative approach was devised. The proposed technique utilizes an allostimulation step: recipient cells serve as targets to induce activation of allospecific donor cells. Donor alloreactive cells are identified by their expression of activation markers, such as CD25 or CD69. After sorting, allospecific T cells are used as a source of cDNA for identification and quantitation of allospecific clonotypes. In this fashion, we have analyzed patients undergoing allogeneic sibling and matched unrelated donor grafting (N=7). Prior to transplant, allostimulation was performed and alloreactive CD8-derived clonotypes were subjected to molecular analysis. VB families represented within alloresponsive CTL populations that were oligoclonal by genotyping were subcloned and a large number of CDR3 clones were sequenced to identify the immunodominant clonotypes. Sequences have been derived from activated CD8+ donor cells in 6 cases; an average of 4 (range 1–7) VB families per pair have been characterized.. Although the presence of multiple VB families with a diversified CDR3 spectrum suggests the polyclonal nature of alloresponsive clones, immunodominant clones were identified. A total of 13 immunodominant clonotypes have been identified in 5 patients. Five such clones were identified in one donor/recipient pair; in each pair at least one immunodominant clonotype was isolated. Up to 18 clones per VB family were sequenced, and the average expansion contributed 56% to the entire VB family (range 15–100%). Clonotype-specific primers have been designed from two expanded clones and used to detect the allospecific clones in post-transplant blood samples in one patient/donor pair. In sum, molecularly defined marker clonotypes indicative of alloresponsive CTLs in HSCT can be individually and prospectively isolated. Such clonotypes may find application in tissue and blood diagnosis of GvHD.

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 Influences of antigen processing on the expression of the T cell repertoire. Evidence for MHC-specific hindering structures on the products of processing.

1988 ◽  
Vol 168 (1) ◽  
pp. 357-373 ◽  
Author(s):  
S J Brett ◽  
K B Cease ◽  
J A Berzofsky
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Processing ◽  
Sperm Whale ◽  
T Cell Response ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Similar Dose ◽  
Sperm Whale Myoglobin

Two lines of evidence in the current study indicate that antigen processing is a major factor, in addition to MHC binding and T cell repertoire, that determines Ir gene responsiveness and epitope immunodominance. First, immunization with synthetic peptides of myoglobin sequences revealed new reactivities that had not appeared after priming with native myoglobin. For example, B10.S mice (H-2S) immune to equine myoglobin predominantly responded to peptide 102-118, whereas there was little, if any, response to this peptide in B10.BR (H-2k) mice immunized with native equine myoglobin. However, after immunization with the 102-118 peptide, both strains responded to the peptide. After in vitro restimulation, B10.BR T cells responded as well as B10.S T cells. Similarly, some individual 102-118-specific T cell clones from mice of both haplotypes showed similar dose responses and fine specificity patterns. Thus, low responsiveness to this site is due neither to a hole in the repertoire nor to a failure to bind to the appropriate MHC molecule. An alternative explanation was suggested by the observation that, whereas B10.S T cells from peptide 102-118-immune mice responded almost as well to whole myoglobin as to the peptide, the B10.BR T cells from peptide immune mice, while responding well to peptide, were poorly stimulated by whole myoglobin. Thus, the product of natural processing of equine myoglobin probably has hindering structures in the regions flanking the core epitope 102-118 that interfere with presentation by I-Ak but not I-AS. The second line of evidence that processing of native myoglobin may influence the apparent specificity of the T cell response was obtained using the I-Ad-restricted sperm whale myoglobin 102-118-specific clone 9.27. This clone discriminated readily between whole sperm whale myoglobin and equine myoglobin, but it did not distinguish between peptides corresponding to 102-118 of the sperm whale and equine sequences. This distinction between equine peptide and native equine myoglobin could be overcome by artificial "processing" of equine myoglobin with cyanogen bromide. In both sets of experiments, F1 APCs that present the same epitope well to T cells of another haplotype failed to overcome the defect, which was therefore not due to the availability of different processed cleavage fragments in APC of different haplotypes, as would be expected if there were MHC-linked processing. Thus, the differential responses to peptides versus native molecule for both I-Ad- and I-Ak-restricted clones appeared to depend on the restricting molecule used.(ABSTRACT TRUNCATED AT 400 WORDS)

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