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Comparison of T-cell receptor repertoire restriction in blood and tumor tissue of colorectal cancer patients

Journal of Translational Medicine volume 8, Article number: 35 (2010) Cite this article

Abstract

Several immunotherapeutic approaches rely on antigen-specific T-cells. Restrictions in the T-cell receptor (TCR) repertoire were reported as indicator of anti-tumor cytotoxic T-lymphocyte (CTL) response in various tumor entities. It is unclear yet whether a TCR restriction in peripheral blood mirrors the tumor compartment. We compared the expression of TCR Vβ-families for the quantification of TCR repertoire alterations in blood and tissue samples from patients with colorectal carcinoma. Blood samples from patients with colorectal carcinoma and healthy volunteers and tissue samples of normal colonic mucosa and colorectal carcinoma were analyzed. Relative Vβ-family quantification was performed based on quantitative reverse transcribed PCR. Standard deviation and average mean of the single families were determined. Two variables describing the degree of Vβ-repertoire restriction were defined. Forty-eight blood samples and 37 tissue samples were analyzed. TCR repertoire restriction was higher in blood of tumor patients than in blood of healthy controls (p < 0.05). No difference in the degree of TCR repertoire restriction was found between carcinoma and unaffected colon tissue. We found no corresponding elevated TCR families among the different compartments blood, normal colon, and carcinoma tissue of the same patient. In conclusion, we observed a repertoire restriction in peripheral blood as well as in tumor tissue of cancer patients. However, in tumor tissue, repertoire alterations were comparable to normal mucosa, suggesting compartment-specific TCR distribution rather than alterations due to tumor-T-cell interaction questioning the presence of highly restricted clonal T-cell expansions in colorectal cancer as they have been described in other, assumingly more immunogenic tumor entities.

Background

Understanding the interaction between tumor and immune system might help improving immunotherapeutic approaches for malignant diseases. T-cells directed against tumor associated antigens (TAA) could play a key role in the surveillance of and in the defense against tumor cells [1]. In fact, spontaneous T-cell responses against TAAs have been described in peripheral blood, lymph nodes, and bone marrow of patients with various malignant diseases prior to immunotherapy [2].

In colorectal cancer (CRC), spontaneous T-cell responses against several TAAs have been detected in peripheral blood, particularly in patients with metastatic disease [3, 4]. No evidence was found that these spontaneous, peripheral TAA-specific T-cells have an impact on survival of CRC patients [5]. Therefore, the focus of interest has moved to tumor-infiltrating T cells. CD8+ T-cell infiltration of CRC is known to be associated with a better prognosis, but it is still unknown whether these infiltrating T cells, in fact, represent expanded tumor specific T-cell clones [6–13].

In case of unknown or multiple epitopes, the analysis of TCR repertoire both by FACS and PCR based methods offers the opportunity to detect oligoclonal expansion of specific T-cells [14–16]. The dimeric transmembrane T-cell receptor (TCR) is the central mediator of epitope specific cytotoxic T-cell activation. Consisting of an α- and a β-chain in most of the cases, diversity is generated during T-cell evolution by recombinations of the gene segments V (variable), in case of the β-chain D (diversity), and J (joining) to a constant chain gene C[17]. V-genes are grouped in families consisting of genes with sequence homology of at least 50% [18]. For analysis of the TCR repertoire, the β-chain is often preferred because of the lower number of families even if a higher overall variability of sequence compared to the α-chain has been described [19]. Alterations in TCR repertoire can be evaluated either by length or sequence analysis of the highly variable part of the α- or β-chain for each V-family [14, 20–22] or by quantification of the single families by southern blot, FACS, or quantitative reverse transcribed PCR (qRT PCR) [23–27].

In cancer research, a restricted TCR repertoire has been found at the tumor site of various malignant diseases [28–36], and in case of melanoma, a highly restricted repertoire may be linked to regression during cytokine therapy [37]. However, it is still a matter of debate whether a restricted TCR repertoire in peripheral blood of tumor patients exists and whether such a peripheral restriction mirrows oligoclonal expansions of specific T-cells in the tumor compartment [36, 38–42].

We used a qRT PCR-based relative Vβ-family quantification approach [27] for analysis of TCR Vβ-family expression. Especially in the gut, lymphocytes bearing γδ TCR are abundant, which are potentially involved in an antitumoral response in an MHC-independent manner [43]. Assessing Vβ-family restriction, clonal expansions of γδ T-cells are not addressed. Aim of the study was the application of mathematical markers to describe the global restriction of the αβ TCR repertoire in the different compartments rather than the detection of single expanded T-cell clones. From this general point of view we evaluated whether or not significant differences of TCR repertoire restriction can be detected in samples from carcinoma patients and healthy controls as well as in tumor tissue compared to unaffected colonic mucosa.

Materials and methods

Specimen collection

Peripheral blood samples were drawn from patients and healthy volunteers. Tissue samples both of carcinoma and unaffected mucosal tissue were collected from patients affected by CRC undergoing tumor resection. RNA was extracted from the macroscopic center of the tumor and from unaffected colonic mucosa at least 5 cm from the macroscopic border of the malignant lesion. Age, sex, and in CRC patients TNM and UICC stages were assessed. Both patients and controls had given informed consent for the use of their specimens before sampling.

RNA extraction, cDNA synthesis

Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) or fresh tissue using TRIzol® (Invitrogen, Carlsbad, CA, USA) or RNeasy® Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed with Omniscript Reverse Transcriptase® (Qiagen) as described previously [44]. Samples were stored at -20°C.

Quantification of TCR expression

For determination of general TCR expression, the Cα-chain was quantified on a LightCycler® instrument (Roche, Basel, Switzerland). Values were normalized using the low-abundance housekeeping gene porphobilinogen deaminidase (PBGD) as previously described [27]. For each cDNA synthesis reaction (+RT), a control reaction without reverse transcriptase was performed (-RT control). Samples with -RT/+RT ratio >0.1 for Cα-chain or PBGD indicating DNA contamination and/or RNA degradation were excluded from further analysis. The aim of our study was the detection of TCR repertoire restriction due to T-cell expansions associated with significant TCR expression of the expanded clones. Low overall TCR transcription in tissue could lead to putative oligoclonality due to the generally high sensitivity of PCR based approaches. Consequently, samples with HAC/PBGD < 0.1 were excluded from analysis to avoid this bias.

Relative quantification of Vβ-families

Relative quantification of the expression of a single TCR Vβ-chain was performed as described [27]. Shortly, qRT PCRs were performed with a universal reverse primer and TaqMan probe, both annealing at the constant part of the β-chain (Cβ), and 28 Vβ-family-specific forward primers (Vβ-family 1 to 24, Vβ-families 5, 6, 12, 13 subdivided into two subgroups each). Slope of each family specific reaction was estimated analyzing a dilution series spanning three orders of magnitude of a cDNA mixture of diagnostic samples. Calculation of the relative concentration P j [%] of a Vβ-family j was carried out using the formula

with Cp [amplification cycles] is the Crossing point and [ΔCp/log(concentration)] is the average slope of all families.

Normalization, quantification of Vβ-restriction

The approach calculating relative concentrations of different Vβ-families regarding slopes and crossing points, as a matter of fact, leads to per-sample normalized values. A per-family normalization step was added to circumvent different weighting of family alterations due to different physiological family expressions and PCR amplification efficacies. For this purpose, the mean percentage and standard deviation SD j of every Vβ-family j of all analyzed samples k were determined. It was decided not only to use PBMCs from blood of healthy donors but all analyzed samples for normalization leading to more reliable results comparing repertoire restriction of tissue with blood. Per-family normalized relative concentration P' jk of a family j of sample k was calculated as follows:

For estimation of the general degree of alteration in the Vβ-family repertoire, we defined a variable CD ('cumulative deviation'). For each family j of a sample k, the value of deviation from the mean percentage of all analyzed samples was expressed as multitude of SD j of this family. CD k of a sample was defined as sum of the moduli of these normalized deviations:

To accommodate the fact, that in tumor immunological settings, a mono- or oligoclonal T-cell response is postulated [16], which is supposed to be associated with the elevation of only a single or very few increased families, we defined a second marker n(F). For each sample k, the number n(F) of families was determined, which were expressed higher than the mean percentage plus two standard deviations SD j (P' j > 2). In contrast to CD depicting the sum of expression deviations from the average of all Vβ-families, only significantly elevated families were relevant for the value of the second marker n(F). The applied normalization procedure is depicted exemplary in Figure 1.

Figure 1
figure 1

Normalization of the relative concentrations P j . Two exemplary samples are shown (PBMCs from peripheral blood of a healthy control: left, carcinoma tissue: right). Shown are the single steps of normalization: relative concentration P j (A), P j minus the average value of Family j of all analyzed samples (B), and deviation from average given in fold of standard deviation of the respective family (C). The sum of the modulus' normalized concentrations of all families of the samples (CD) and the number of families elevated more than average plus two SD n(F) are indicated at the bottom (D)

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Statistical methods

All statistical tests were performed as two-tailed tests and a p-value < 0.05 was considered significant. Correlations were tested by calculation of the Pearson coefficient. Mann-Whitney U test was applied to unpaired samples, Wilcoxon test was used for paired samples.

Results

Patients and specimens

Fifty-one samples from peripheral blood (21 samples of healthy controls and 30 samples from carcinoma patients) and 58 tissue specimens both from normal colon tissue of CRC patients and colon carcinoma tissue were analyzed. Fourteen samples (three from peripheral blood, five samples of unaffected colon, and six samples originating from carcinoma tissue) had -RT/+RT ratios > 0.1. Six tissue samples had a HAC/PBGD ratio < 0.1 corresponding to minimal TCR expression (one sample of unaffected colon, five samples of tumor tissue). Altogether 19 Samples fulfilling one or both precondition were excluded from further analysis. Altogether, 48 blood and 42 tissue samples were evaluable. In total, samples of 19 healthy donors (peripheral blood) and 40 CRC patients (peripheral blood: n = 29, healthy colon: n = 24, carcinoma: n = 18) were analyzed. Characteristics of patients and healthy controls are depicted in Table 1.

Table 1 Characteristics of patients and healthy controls
Full size table

To test the reliability of our normalization approach, we calculated the theoretically expected percentage of samples with at least one family elevated more than two SD (n(F) >0) and compared that value with results from all samples analyzed. Assuming a symmetric Gaussian distribution of relative concentrations within a family, the expected percentage of samples with n(F) >0 would be 49%. Of all 90 samples analyzed, 46 (51%) showed n(F) >0 demonstrating high reliability of our method. Families with frequently low expression were tested negative more often than others (negative correlation between average relative concentrations and the number of negative PCRs of a family j in all evaluable samples, R p = -0.4286, p = 0.0229). However, after normalization, the absolute number a family j was considered as elevated (n(F) >0) in all evaluable samples was independent of the average relative expression of that family (data not shown).

TCR Cα expression in blood and tissue specimens

As expected, Cα (HAC/PBGD) was expressed significantly higher in peripheral blood than in tissue (p < 0.0001). HAC/PBGD was higher in blood samples of healthy volunteers compared to carcinoma patients; but no difference in Cα-expression was found between unaffected colon and tumor tissue in a paired analysis (Figure 2). These results have to be interpreted with caution because of the fact that HAC/PBGD was used as criteria to select samples for further repertoire analysis. Without excluding samples with HAC/PBGD < 0.1, Cα concentration was lower in the carcinoma samples than in samples of unaffected mucosa (paired, p = 0.047).

Figure 2
figure 2

Relative C α concentrations (HAC/PBGD) of the analyzed samples. (A) HAC/PBGD was significantly higher in blood samples (healthy controls and carcinoma patients) compared to tissue samples (unaffected colon and carcinoma tissue, ** p < 0.0001). (B) HAC/PBGD was higher in blood samples of healthy controls than in samples of carcinoma patients (* p = 0.0235, unpaired). No difference in HAC/PBGD was detected comparing tissue samples of unaffected colon with samples of carcinoma tissue (p = 0.1147, paired).

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According to the differences of expression between blood and tissue specimens, analyzing all evaluable samples irrespective of origin, we found a negative correlation between HAC/PBGD and CD (Figure 3) as well as HAC/PBGD and n(F) (not shown). However, separate analysis of the samples originating from blood and originating from tissue samples resulted in no significant correlations (Figure 3 and not shown).

Figure 3
figure 3

Correlation between relative C α expression (HAC/PBGD) and cumulative deviation CD. According to the higher HAC/PBGD expression in blood samples compared to tissue specimens, we found a strong negative correlation between HAC/PBGD and CD analyzing all specimens irrespective of origin (regression line, R p = -0.4004, p = 0.0003). Regarding samples from blood (empty diamonds) and tissue (filled diamonds) separately, no correlations between the mentioned parameters were determined (Rp, blood= -0.1108, p blood = 0.4585, Rp, tissue= 0.01344, p tissue = 0.9286).

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Vβ repertoire restriction in peripheral blood

TCR repertoire analysis of PBMCs from 19 healthy donors and 29 colon carcinoma patients showed in 16 out of 48 samples one or more families with P j ' >2 (2 out of 19 samples from healthy donors (11%), 14 out of 29 samples from cancer patients (48%)) reflecting an non-normalized expression of one or more families higher than the mean percentage plus two standard deviations SD j (n(F) >0). In the statistical analysis, both CD and n(F) were higher in PBMCs from cancer patients than in PBMCs from healthy volunteers (unpaired, p CD = 0.0316, pn(F)= 0.0195, Figure 2B).

Vβ repertoire in tissue samples of CRC patients

Twenty-four samples of normal colon tissue and 18 carcinoma samples were evaluable. The statistical analysis of both CD and n(F) comparing all tissue samples (carcinoma and unaffected colon) with all blood samples (healthy controls and carcinoma patients) showed a significantly higher repertoire restriction in tissue than in blood (unpaired, p CD < 0.001, pn(F)< 0.001, Figure 4A). However, no difference was observed between samples of unaffected colon and carcinoma tissue (paired, p CD = 0.471, pn(F)= 0.2783, Figure 4C).

Figure 4
figure 4

Statistical comparison of repertoire restriction degree measured in CD and n(F). Compared were samples from blood with tissue (both normal colon and carcinoma, A), blood from healthy controls with blood from carcinoma patients (B), and tumor free colon with carcinoma samples both originating from CRC patients (C). * p < 0.05, ** p < 0.01.

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Comparison of elevated families in different compartments

For 16 patients, samples of tumor and tumor free colon were available. In the carcinoma samples, altogether 30 families were elevated (0-5 families per patient). In the corresponding samples of unaffected colon tissue, 14 families were elevated (0-4 families per patient). In only two of these patients a single family was elevated in both tumor tissue and corresponding unaffected colon. For nine CRC patients, blood and tumor samples were available. Four of these patients had elevated families in peripheral blood, but none of these families was elevated in the corresponding tumor sample. Please refer to figure 4.

Discussion

In this study, TCR Vβ-family repertoire restrictions in blood and tissue of patients with colorectal carcinoma were compared using high throughput relative quantification of TCR Vβ-families based on qRT PCR technology. While a multitude of mathematical approaches have been established to describe the degree of repertoire restriction in single V-families from spectratype/immunoscope data (reviewed in [45]), to our knowledge, a model to describe global TCR repertoire restriction based on V-family quantification has not been published so far.

Colorectal carcinoma is thought to be of limited immunogenicity. Nevertheless, global Vβ-repertoire restriction degree in blood reflected by the CD and n(F) values was significantly higher in carcinoma patients confirming the results of former studies investigating Vβ-repertoire restrictions in blood of patients with other tumor entities, which have been interpreted as indication for the induction of specific clones reactive to autologous tumor [26, 42]. In fact, this TCR repertoire restriction in tumor patients might be attributed to cumulative Vβ-alterations caused by discrete epitope-specific T-cell expansions triggered by several different antigens. Because of the epitope-independency of the assay, unspecific antigen confrontation due to barrier disruption as postulated in the context of other tumor entities [28, 32] causing additional repertoire alterations can not be excluded. We observed a difference of Cα-chain expression between healthy controls and carcinoma patients, which could have impact on the TCR Vβ restriction grade. It remains elusive whether or not this difference is a result of the malignant disease or the higher median age of the patient cohort compared to the healthy control group.

Corresponding significant TCR Vβ-family elevations in blood and tumor tissue were not found in the present study. From previous analyses in colorectal cancer, we know, that TAA-specific T-cells circulated with frequencies lower than 1% of CD8+ cells without proven clonality [3]. As anticipated, the proportion of peripheral TAA-specific T-cells was too low to be detected by TCR Vβ-quantification due to physiological variation of the relative Vβ-family expressions. Assuming several TAAs, expansions of T-cell clones would affect various Vβ-families additionally reducing sensitivity of the relative Vβ-quantification regarding single family percentages. It is purely speculative to assume that such a specific T cell clone might be trapped at the tumor site and is therefore, not detectable in the peripheral blood.

In carcinoma tissues, we did not observe an elevated repertoire restriction compared to corresponding tissue of unaffected colon. Infiltration of CTLs in colon carcinoma tissue had been identified as prognostic factor suggesting TAA associated CTL activation [12]. Interestingly, we found no difference in the expression of Cα comparing healthy colon and carcinoma tissue. These inconsistent observations could be explained by recent results of Salama et al., who showed, that CD8+ CTL density is reduced but CD4+/CD25+/FoxP3+ T regulator cells are elevated in colon cancer compared to normal colon tissue [46, 47]. The fact that clonal CTL expansions are not principally associated with restrictions in Vβ-family usage of the T regulatory cell population of the same compartment [42, 47] might explain the absence of TCR restriction differences between healthy colon and CRC tissue. Independently of these considerations, we have to conclude that the present molecular quantitative TCR analysis is not suitable for the identification of local expansions in colorectal cancer tissue (if they exist) compared to normal colon tissue. This may differ in other compartments and/or for other tumor entities.

Regarding p-values comparing the different sample groups (blood of healthy controls, blood of carcinoma patients, unaffected colonic mucosa, carcinoma tissue) one has to keep in mind the differences in sample sizes and applied tests. The highly significant difference in CD between blood and tissue samples but the absence of a significant difference between unaffected colon and tumor tissue does not necessarily mean hat there would be no difference to detect if we would compare the same sample size of tissue samples as we used for comparing tissue with blood samples (48 vs 42 instead of 24 vs 18). Regarding the repertoire restriction in blood (healthy vs carcinoma patients) and tissue (unaffected colon mucosa vs carcinoma tissue), the results of the two statistical tests cannot directly be compared because of differences in distribution, range, and dependency of the data sets. However comparing tumor tissue to unaffected mucosa, we observed an even lower median value in carcinoma tissue. Consequently, the absence of a significantly higher Vβ restriction in tumor tissue must not be attributed to the slightly smaller sample size compared to the blood samples.

Taken together, we observed an increased TCR repertoire restriction in blood of colorectal carcinoma patients compared to blood of healthy controls using qRT-PCR based Vβ-family quantification. A similar degree of TCR restriction in colorectal carcinoma tissue in comparison to normal colon tissue contrasts with the phenomenon of high proliferative oligoclonal expansions of specific T-cell populations as described for highly immunogenic malignancies such as melanoma [16].

References

  1. Zitvogel L, Tesniere A, Kroemer G: Cancer despite immunosurveillance: immunoselection and immunosubversion. Nat Rev Immunol. 2006, 6: 715-27. 10.1038/nri1936.

    Article  PubMed  CAS  Google Scholar 

  2. Nagorsen D, Scheibenbogen C, Marincola FM, Letsch A, Keilholz U: Natural T cell immunity against cancer. Clin Cancer Res. 2003, 9: 4296-303.

    PubMed  CAS  Google Scholar 

  3. Nagorsen D, Keilholz U, Rivoltini L, Schmittel A, Letsch A, Asemissen AM, Berger G, Buhr HJ, Thiel E, Scheibenbogen C: Natural T-cell response against MHC class I epitopes of epithelial cell adhesion molecule, her-2/neu, and carcinoembryonic antigen in patients with colorectal cancer. Cancer Res. 2000, 60: 4850-4.

    PubMed  CAS  Google Scholar 

  4. Arlen P, Tsang KY, Marshall JL, Chen A, Steinberg SM, Poole D, Hand PH, Schlom J, Hamilton JM: The use of a rapid ELISPOT assay to analyze peptide-specific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines. Cancer Immunol Immunother. 2000, 49: 517-29. 10.1007/s002620000145.

    Article  PubMed  CAS  Google Scholar 

  5. Nagorsen D, Scheibenbogen C, Letsch A, Germer CT, Buhr HJ, Hegewisch-Becker S, Rivoltini L, Thiel E, Keilholz U: T cell responses against tumor associated antigens and prognosis in colorectal cancer patients. J Transl Med. 2005, 3: 3-10.1186/1479-5876-3-3.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Ropponen KM, Eskelinen MJ, Lipponen PK, Alhava E, Kosma VM: Prognostic value of tumour-infiltrating lymphocytes (TILs) in colorectal cancer. J Pathol. 1997, 182: 318-24. 10.1002/(SICI)1096-9896(199707)182:3<318::AID-PATH862>3.0.CO;2-6.

    Article  PubMed  CAS  Google Scholar 

  7. Naito Y, Saito K, Shiiba K, Ohuchi A, Saigenji K, Nagura H, Ohtani H: CD8+ T cells infiltrated within cancer cell nests as a prognostic factor in human colorectal cancer. Cancer Res. 1998, 58: 3491-3494.

    PubMed  CAS  Google Scholar 

  8. Oberg A, Samii S, Stenling R, Lindmark G: Different occurrence of CD8+, CD45R0+, and CD68+ immune cells in regional lymph node metastases from colorectal cancer as potential prognostic predictors. Int J Colorectal Dis. 2002, 17: 25-9. 10.1007/s003840100337.

    Article  PubMed  CAS  Google Scholar 

  9. Diederichsen AC, Hjelmborg JB, Christensen PB, Zeuthen J, Fenger C: Prognostic value of the CD4+/CD8+ ratio of tumour infiltrating lymphocytes in colorectal cancer and HLA-DR expression on tumour cells. Cancer Immunol Immunother. 2003, 52: 423-8. 10.1007/s00262-003-0388-5.

    Article  PubMed  CAS  Google Scholar 

  10. Funada Y, Noguchi T, Kikuchi R, Takeno S, Uchida Y, Gabbert HE: Prognostic significance of CD8+ T cell and macrophage peritumoral infiltration in colorectal cancer. Oncol Rep. 2003, 10: 309-13.

    PubMed  Google Scholar 

  11. Chiba T, Ohtani H, Mizoi T, Naito Y, Sato E, Nagura H, Ohuchi A, Ohuchi K, Shiiba K, Kurokawa Y, Satomi S: Intraepithelial CD8+ T-cell-count becomes a prognostic factor after a longer follow-up period in human colorectal carcinoma: possible association with suppression of micrometastasis. Br J Cancer. 2004, 91: 1711-7.

    PubMed  CAS  PubMed Central  Google Scholar 

  12. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pagès C, Tosolini M, Camus M, Berger A, Wind P, Zinzindohoué F, Bruneval P, Cugnenc PH, Trajanoski Z, Fridman WH, Pagès F: Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006, 313: 1960-4. 10.1126/science.1129139.

    Article  PubMed  CAS  Google Scholar 

  13. Loddenkemper C, Schernus M, Noutsias M, Stein H, Thiel E, Nagorsen D: In situ analysis of FOXP3+ regulatory T cells in human colorectal cancer. J Transl Med. 2006, 4: 52-10.1186/1479-5876-4-52.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Pilch H, Höhn H, Freitag K, Neukirch C, Necker A, Haddad P, Tanner B, Knapstein PG, Maeurer MJ: Improved assessment of T-cell receptor (TCR) VB repertoire in clinical specimens: combination of TCR-CDR3 spectratyping with flow cytometry-based TCR VB frequency analysis. Clin Diagn Lab Immunol. 2002, 9: 257-66.

    PubMed  CAS  PubMed Central  Google Scholar 

  15. Nagorsen D, Wang E, Marincola FM, Even J: Transcriptional analysis of tumor-specific T-cell responses in cancer patients. Crit Rev Immunol. 2002, 22: 449-62.

    Article  PubMed  CAS  Google Scholar 

  16. Thor Straten P, Schrama D, Andersen MH, Becker JC: T-cell clonotypes in cancer. J Transl Med. 2004, 2: 11-10.1186/1479-5876-2-11.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Davis MM, Bjorkman PJ: T cell antigen receptor genes and T-cell recognition. Nature. 1988, 334: 395-402. 10.1038/334395a0.

    Article  PubMed  CAS  Google Scholar 

  18. Arden B, Clark SP, Kabelitz D, Mak TW: Human T-cell receptor variable gene segment families. Immunogenetics. 1995, 42: 455-500.

    PubMed  CAS  Google Scholar 

  19. Maciejewski JP, O'Keefe C, Gondek L, Tiu R: Immune-mediated bone marrow failure syndromes of progenitor and stem cells: molecular analysis of cytotoxic T cell clones. Folia Histochem Cytobiol. 2007, 45: 5-14.

    PubMed  CAS  Google Scholar 

  20. Long SA, Khalili J, Ashe J, Berenson R, Ferrand C, Bonyhadi M: Standardized analysis for the quantification of Vbeta CDR3 T-cell receptor diversity. J Immunol Methods. 2006, 317: 100-113. 10.1016/j.jim.2006.09.015.

    Article  PubMed  CAS  Google Scholar 

  21. Thor Straten P, Barfoed A, Seremet T, Saeterdal I, Zeuthen J, Guldberg P: Detection and characterization of alpha-beta-T-cell clonality by denaturing gradient gel electrophoresis (DGGE). Biotechniques. 1998, 25: 244-250.

    PubMed  CAS  Google Scholar 

  22. Collette A, Cazenave P-A, Pied S, Six A: New methods and software tools for high throughput CDR3 spectratyping. Application to T lymphocyte repertoire modifications during experimental malaria. J Immunol Methods. 2003, 278: 105-116. 10.1016/S0022-1759(03)00225-4.

    Article  PubMed  CAS  Google Scholar 

  23. Willhauk M, Möhler T, Scheibenbogen C, Pawlita M, Brossart P, Schmier JW, Keilholz U: T-cell receptor beta variable region diversity in melanoma metastases after interleukin 2-based immunotherapy. Clin Cancer Res. 1996, 2: 767-772.

    Google Scholar 

  24. Brewer JL, Ericson SG: An improved methodology to detect human T cell receptor beta variable family gene expression patterns. J Immunol Methods. 2005, 302: 54-67. 10.1016/j.jim.2005.04.014.

    Article  PubMed  CAS  Google Scholar 

  25. Lim A, Baron V, Ferradini L, Bonneville M, Kourilsky P, Pannetier C: Combination of MHC-peptide multimer-based T cell sorting with the Immunoscope permits sensitive ex vivo quantitation and follow-up of human CD8+ T cell immune responses. J Immunol Methods. 2002, 261: 177-194. 10.1016/S0022-1759(02)00004-2.

    Article  PubMed  CAS  Google Scholar 

  26. Degauque N, Schadendorf D, Brouard S, Guillet M, Sébille F, Höhn H, Pallier A, Ruiz C, Dupont A, Chapin S, Hofmann U, Maeurer M, Soulillou JP: Blood T-cell Vbeta transcriptome in melanoma patients. Int J Cancer. 2004, 110: 721-729. 10.1002/ijc.20149.

    Article  PubMed  CAS  Google Scholar 

  27. Ochsenreither S, Fusi A, Busse A, Nagorsen D, Schrama D, Becker J, Thiel E, Keilholz U: Relative quantification of TCR Vbeta-chain families by real time PCR for identification of clonal T-cell populations. J Transl Med. 2008, 6: 34-10.1186/1479-5876-6-34.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Ikeda H, Sato N, Matsuura A, Kikuchi K: Analysis of T-cell receptor V region gene usage of cytotoxic T-lymphocytes and tumor-infiltrating lymphocytes derived from human autologous gastric signet ring cell carcinomas. Cancer Res. 1993, 53: 3078-84.

    PubMed  CAS  Google Scholar 

  29. Weidmann E, Elder EM, Trucco M, Lotze MT, Whiteside TL: Usage of T-cell receptor V beta chain genes in fresh and cultured tumor-infiltrating lymphocytes from human melanoma. Int J Cancer. 1993, 54: 383-90. 10.1002/ijc.2910540306.

    Article  PubMed  CAS  Google Scholar 

  30. Merlo A, Filgueira L, Zuber M, Juretic A, Harder F, Gratzl O, De Libero G, Heberer M, Spagnoli GC: T-cell receptor V-gene usage in neoplasms of the central nervous system. A comparative analysis in cultured tumor infiltrating and peripheral blood T cells. J Neurosurg. 1993, 78: 630-7. 10.3171/jns.1993.78.4.0630.

    Article  PubMed  CAS  Google Scholar 

  31. Ohmen JD, Moy RL, Zovich D, Lieberman A, Wyzykowski RJ, Sullivan L, Modlin RL, Uyemura K: Selective accumulation of T cells according to T-cell receptor V beta gene usage in skin cancer. J Invest Dermatol. 1994, 103: 751-7. 10.1111/1523-1747.ep12412288.

    Article  PubMed  CAS  Google Scholar 

  32. Chikamatsu K, Eura M, Nakano K, Kanzaki Y, Matsuoka H, Masuyama K, Ishikawa T: Analysis of T cell receptor variability in fresh tumor-infiltrating lymphocytes from human head and neck cancer. Jpn J Cancer Res. 1994, 85: 626-32.

    Article  PubMed  CAS  Google Scholar 

  33. Zeuthen J, Birck A, Straten PT: Analysis of T cell receptor alpha beta variability in tumor-infiltrating lymphocytes in primary and metastatic melanoma. Arch Immunol Ther Exp (Warsz). 1995, 43: 123-33.

    CAS  Google Scholar 

  34. Gaudin C, Dietrich PY, Robache S, Guillard M, Escudier B, Lacombe MJ, Kumar A, Triebel F, Caignard A: In vivo local expansion of clonal T cell subpopulations in renal cell carcinoma. Cancer Res. 1995, 55: 685-90.

    PubMed  CAS  Google Scholar 

  35. Mouri T, Nakamura S, Ohyama Y, Matsuzaki G, Shinohara M, Kishihara K, Hiroki A, Oka M, Shirasuna K, Nomoto K: T cell receptor V alpha and V beta gene usage by tumour-infiltrating lymphocytes in oral squamous cell carcinoma. Cancer Immunol Immunother. 1996, 43 (1): 10-8. 10.1007/s002620050297. Erratum in: Cancer Immunol Immunother 1997, 45:61

    Article  PubMed  CAS  Google Scholar 

  36. Echchakir H, Asselin-Paturel C, Dorothee G, Vergnon I, Grunenwald D, Chouaib S, Mami-Chouaib F: Analysis of T-cell-receptor beta-chain-gene usage in peripheral-blood and tumor-infiltrating lymphocytes from human non-small-cell lung carcinomas. Int J Cancer. 1999, 81: 205-13. 10.1002/(SICI)1097-0215(19990412)81:2<205::AID-IJC7>3.0.CO;2-M.

    Article  PubMed  CAS  Google Scholar 

  37. Möhler T, Willhauck M, Scheibenbogen C, Pawlita M, Bludau H, Brossart P, Hunstein W, Keilholz U: Restriction of T cell receptor V beta repertoire in melanoma metastasis responding to immunotherapy. Melanoma Res. 1995, 5 (2): 129-32. 10.1097/00008390-199504000-00010.

    Article  PubMed  Google Scholar 

  38. Velotti F, Chopin D, Gil-Diez S, Maille P, Abbou CC, Kourilsky P, Even J: Clonality of tumor-infiltrating lymphocytes in human urinary bladder carcinoma. J Immunother. 1997, 20: 470-8. 10.1097/00002371-199711000-00007.

    Article  PubMed  CAS  Google Scholar 

  39. Zhang XY, Chan WY, Whitney BM, Fan DM, Chow JH, Liu Y, Ng EK, Chung SC: T cell receptor Vbeta repertoire expression reflects gastric carcinoma progression. Clin Immunol. 2001, 101: 3-7. 10.1006/clim.2001.5100.

    Article  PubMed  CAS  Google Scholar 

  40. Weidmann E, Whiteside TL, Giorda R, Herberman RB, Trucco M: The T-cell receptor V beta gene usage in tumor-infiltrating lymphocytes and blood of patients with hepatocellular carcinoma. Cancer Res. 1992, 52: 5913-20.

    PubMed  CAS  Google Scholar 

  41. Banér J, Marits P, Nilsson M, Winqvist O, Landegren U: Analysis of T-cell receptor V beta gene repertoires after immune stimulation and in malignancy by use of padlock probes and microarrays. Clin Chem. 2005, 51: 768-75. 10.1373/clinchem.2004.047266.

    Article  PubMed  Google Scholar 

  42. Albers AE, Visus C, Tsukishiro T, Ferris RL, Gooding W, Whiteside TL, De Leo AB: Alterations in the T-cell receptor variable beta gene-restricted profile of CD8+ T lymphocytes in the peripheral circulation of patients with squamous cell carcinoma of the head and neck. Clin Cancer Res. 2006, 12: 2394-403. 10.1158/1078-0432.CCR-05-1818.

    Article  PubMed  CAS  Google Scholar 

  43. Corvaisier M, Moreau-Aubry A, Diez E, Bennouna J, Mosnier JF, Scotet E, Bonneville M, Jotereau F: V gamma 9V delta 2 T cell response to colon carcinoma cells. J Immunol. 2005, 15;175 (8): 5481-8.

    Article  Google Scholar 

  44. Keilholz U, Goldin-Lang P, Bechrakis NE, Max N, Letsch A, Schmittel A, Scheibenbogen C, Heufelder K, Eggermont A, Thiel E: Quantitative detection of circulating tumor cells in cutaneous and ocular melanoma and quality assessment by real-time reverse transcriptase-polymerase chain reaction. Clin Cancer Res. 2004, 10: 1605-1612. 10.1158/1078-0432.CCR-0610-3.

    Article  PubMed  CAS  Google Scholar 

  45. Miqueu P, Guillet M, Degauque N, Doré JC, Soulillou JP, Brouard S: Statistical analysis of CDR3 length distributions for the assessment of T and B cell repertoire biases. Mol Immunol. 2007, 44: 1057-64. 10.1016/j.molimm.2006.06.026.

    Article  PubMed  CAS  Google Scholar 

  46. Pacholczyk R, Kern J: The T-cell receptor repertoire of regulatory T cells. Immunology. 2008, 125: 450-8. 10.1111/j.1365-2567.2008.02992.x.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  47. Salama P, Phillips M, Grieu F, Morris M, Zeps N, Joseph D, Platell C, Iacopetta B: Tumor-Infiltrating FOXP3 T Regulatory Cells Show Strong Prognostic Significance in Colorectal Cancer. J Clin Oncol. 2009, 27 (2): 186-92. 10.1200/JCO.2008.18.7229.

    Article  PubMed  Google Scholar 

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Acknowledgements

The work was supported by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG NA 716/1-1 to DN).

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Authors and Affiliations

  1. Charité, Campus Benjamin Franklin, Department of Hematology and Oncology, Hindenburgdamm 30, 12200, Berlin, Germany

    Sebastian Ochsenreither, Alberto Fusi, Susanne Wojtke, Antonia Busse, Eckhard Thiel, Ulrich Keilholz & Dirk Nagorsen

  2. Department of General and Visceral Surgery, Klinikum Neuperlach, Städtisches Klinikum München, Oskar-Maria-Graf Ring 51, 81737, Munich, Germany

    Natascha C Nüssler

  3. Department of Hematology/Oncology, University of Heidelberg, Medizinische Fakultät Mannheim, Theodor-Kutzer-Ufer 1, 68167, Mannheim, Germany

    Dirk Nagorsen

  4. Micromet AG, Staffelseestr 2, 81477, Munich, Germany

    Dirk Nagorsen

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  1. Sebastian Ochsenreither
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  2. Alberto Fusi
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  3. Susanne Wojtke
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Correspondence to Dirk Nagorsen.

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Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SO conceived of the study, developed and supervised the molecular analyses carried out in this trial, performed the statistical analysis and drafted the manuscript.

AF carried out molecular measurements and analyses, and statistical analyses.

SW collected samples and carried out molecular measurements.

AB carried out molecular measurements and analyses.

NCN collected samples and participated in the conduct of the study.

ET participated in design and conduct of the study.

UK participated in design and conduct of the study.

DN conceived of the study, coordinated the study and drafted the manuscript.

All authors have read and approved the final manuscript.

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Ochsenreither, S., Fusi, A., Wojtke, S. et al. Comparison of T-cell receptor repertoire restriction in blood and tumor tissue of colorectal cancer patients. J Transl Med 8, 35 (2010). https://doi.org/10.1186/1479-5876-8-35

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Keywords

Journal of Translational Medicine

ISSN: 1479-5876

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