Anti-trbc1 antibody-based flow cytometric detection of t-cell clonality: Standardization of sample preparation and diagnostic implementation

Noemí Muñoz-García; Margarida Lima; Neus Villamor; F. Javier Morán-Plata; Susana Barrena; Sheila Mateos; Carolina Caldas; Ana Balanzategui; Miguel Alcoceba; Alejandro Domínguez; Fabio Gómez; Anton W. Langerak; Jacques J.M. van Dongen; Alberto Orfao; Julia Almeida

doi:10.3390/cancers13174379

Anti-trbc1 antibody-based flow cytometric detection of t-cell clonality: Standardization of sample preparation and diagnostic implementation

Noemí Muñoz-García
, Margarida Lima
, Neus Villamor
, F. Javier Morán-Plata
, Susana Barrena
, Sheila Mateos
, Carolina Caldas
, Ana Balanzategui
, Miguel Alcoceba
, Alejandro Domínguez
, Fabio Gómez
, Anton W. Langerak
, Jacques J.M. van Dongen
, Alberto Orfao
, Julia Almeida^*

^*Corresponding author for this work

Immunology

Universidad de Salamanca
Instituto de Salud Carlos III
University Hospital Center of Santo António
University of Porto
Hospital Clinic de Barcelona
Hospital Clínico Universitario de Salamanca
Gerencia Regional de Salud de Castilla y León
Leiden University Medical Centre

Research output: Contribution to journal › Article › Academic › peer-review

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Abstract

A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant domains was identified as a potentially useful flow-cy-tometry (FCM) marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-cells and validated the method in 211 normal, reactive and pathological sam-ples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1⁺ and TRBC1⁻ monoclonal and polyclonal Tαβ-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1⁺/TRBC1⁻ ratios within different Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ fami-lies, whereas highly-variable TRBC1⁺/TRBC1⁻ ratios were observed in more mature vs. naïve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-cells of ≤10⁻⁴ in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαβ⁺ leukemia/lymphoma patients.

Original language	English
Article number	4379
Journal	Cancers
Volume	13
Issue number	17
DOIs	https://doi.org/10.3390/cancers13174379
Publication status	Published - 30 Aug 2021

Bibliographical note

Funding Information:
Funding: This work was supported by the CB16/12/00400 (CIBERONC) and PI20-01346 grants, from the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (Madrid, Spain) and FONDOS FEDER, and by the EuroFlow Foundation (Leiden, The Netherlands). N. Muñoz-García is supported by a pre-doctoral grant (Ref. IBPredoc17/00012) from IBSAL (Salamanca, Spain). M. Lima, N. Vil-lamor, A.W. Langerak, J.J.M. van Dongen, A. Orfao, and J. Almeida are members of the EuroFlow Consortium [29].

Funding Information:
This work was supported by the CB16/12/00400 (CIBERONC) and PI20-01346 grants, from the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovaci?n (Madrid, Spain) and FONDOS FEDER, and by the EuroFlow Foundation (Leiden, The Netherlands). N. Mu?oz-Garc?a is supported by a pre-doctoral grant (Ref. IBPredoc17/00012) from IBSAL (Salamanca, Spain). M. Lima, N. Vil-lamor, A.W. Langerak, J.J.M. van Dongen, A. Orfao, and J. Almeida are members of the EuroFlow Consortium [29].

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© 2021 by the authors. Licensee MDPI, Basel, Switzerland.

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Anti-TRBC1 Antibody-Based Flow Cytometric Detection of T-Cell ClonalityFinal published version, 1.53 MBLicence: CC BY

Cite this

Muñoz-García, N., Lima, M., Villamor, N., Morán-Plata, F. J., Barrena, S., Mateos, S., Caldas, C., Balanzategui, A., Alcoceba, M., Domínguez, A., Gómez, F., Langerak, A. W., van Dongen, J. J. M., Orfao, A., & Almeida, J. (2021). Anti-trbc1 antibody-based flow cytometric detection of t-cell clonality: Standardization of sample preparation and diagnostic implementation. Cancers, 13(17), Article 4379. https://doi.org/10.3390/cancers13174379

@article{345787dfd2fd4feabb89911fbf8c8909,

title = "Anti-trbc1 antibody-based flow cytometric detection of t-cell clonality: Standardization of sample preparation and diagnostic implementation",

abstract = "A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant domains was identified as a potentially useful flow-cy-tometry (FCM) marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-cells and validated the method in 211 normal, reactive and pathological sam-ples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1+ and TRBC1− monoclonal and polyclonal Tαβ-cells rearranged TRBJ1 in 44/47 (94\%) and TRBJ1+TRBJ2 in 48 of 48 (100\%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1+/TRBC1− ratios within different Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ fami-lies, whereas highly-variable TRBC1+/TRBC1− ratios were observed in more mature vs. na{\"i}ve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96\%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-cells of ≤10−4 in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαβ+ leukemia/lymphoma patients.",

author = "Noem{\'i} Mu{\~n}oz-Garc{\'i}a and Margarida Lima and Neus Villamor and Mor{\'a}n-Plata, \{F. Javier\} and Susana Barrena and Sheila Mateos and Carolina Caldas and Ana Balanzategui and Miguel Alcoceba and Alejandro Dom{\'i}nguez and Fabio G{\'o}mez and Langerak, \{Anton W.\} and \{van Dongen\}, \{Jacques J.M.\} and Alberto Orfao and Julia Almeida",

note = "Funding Information: Funding: This work was supported by the CB16/12/00400 (CIBERONC) and PI20-01346 grants, from the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovaci{\'o}n (Madrid, Spain) and FONDOS FEDER, and by the EuroFlow Foundation (Leiden, The Netherlands). N. Mu{\~n}oz-Garc{\'i}a is supported by a pre-doctoral grant (Ref. IBPredoc17/00012) from IBSAL (Salamanca, Spain). M. Lima, N. Vil-lamor, A.W. Langerak, J.J.M. van Dongen, A. Orfao, and J. Almeida are members of the EuroFlow Consortium [29]. Funding Information: This work was supported by the CB16/12/00400 (CIBERONC) and PI20-01346 grants, from the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovaci?n (Madrid, Spain) and FONDOS FEDER, and by the EuroFlow Foundation (Leiden, The Netherlands). N. Mu?oz-Garc?a is supported by a pre-doctoral grant (Ref. IBPredoc17/00012) from IBSAL (Salamanca, Spain). M. Lima, N. Vil-lamor, A.W. Langerak, J.J.M. van Dongen, A. Orfao, and J. Almeida are members of the EuroFlow Consortium [29]. Publisher Copyright: {\textcopyright} 2021 by the authors. Licensee MDPI, Basel, Switzerland.",

year = "2021",

month = aug,

day = "30",

doi = "10.3390/cancers13174379",

language = "English",

volume = "13",

journal = "Cancers",

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Muñoz-García, N, Lima, M, Villamor, N, Morán-Plata, FJ, Barrena, S, Mateos, S, Caldas, C, Balanzategui, A, Alcoceba, M, Domínguez, A, Gómez, F, Langerak, AW, van Dongen, JJM, Orfao, A & Almeida, J 2021, 'Anti-trbc1 antibody-based flow cytometric detection of t-cell clonality: Standardization of sample preparation and diagnostic implementation', Cancers, vol. 13, no. 17, 4379. https://doi.org/10.3390/cancers13174379

TY - JOUR

T1 - Anti-trbc1 antibody-based flow cytometric detection of t-cell clonality

T2 - Standardization of sample preparation and diagnostic implementation

AU - Muñoz-García, Noemí

AU - Lima, Margarida

AU - Villamor, Neus

AU - Morán-Plata, F. Javier

AU - Barrena, Susana

AU - Mateos, Sheila

AU - Caldas, Carolina

AU - Balanzategui, Ana

AU - Alcoceba, Miguel

AU - Domínguez, Alejandro

AU - Gómez, Fabio

AU - Langerak, Anton W.

AU - van Dongen, Jacques J.M.

AU - Orfao, Alberto

AU - Almeida, Julia

N1 - Funding Information: Funding: This work was supported by the CB16/12/00400 (CIBERONC) and PI20-01346 grants, from the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (Madrid, Spain) and FONDOS FEDER, and by the EuroFlow Foundation (Leiden, The Netherlands). N. Muñoz-García is supported by a pre-doctoral grant (Ref. IBPredoc17/00012) from IBSAL (Salamanca, Spain). M. Lima, N. Vil-lamor, A.W. Langerak, J.J.M. van Dongen, A. Orfao, and J. Almeida are members of the EuroFlow Consortium [29]. Funding Information: This work was supported by the CB16/12/00400 (CIBERONC) and PI20-01346 grants, from the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovaci?n (Madrid, Spain) and FONDOS FEDER, and by the EuroFlow Foundation (Leiden, The Netherlands). N. Mu?oz-Garc?a is supported by a pre-doctoral grant (Ref. IBPredoc17/00012) from IBSAL (Salamanca, Spain). M. Lima, N. Vil-lamor, A.W. Langerak, J.J.M. van Dongen, A. Orfao, and J. Almeida are members of the EuroFlow Consortium [29]. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2021/8/30

Y1 - 2021/8/30

N2 - A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant domains was identified as a potentially useful flow-cy-tometry (FCM) marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-cells and validated the method in 211 normal, reactive and pathological sam-ples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1+ and TRBC1− monoclonal and polyclonal Tαβ-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1+/TRBC1− ratios within different Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ fami-lies, whereas highly-variable TRBC1+/TRBC1− ratios were observed in more mature vs. naïve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-cells of ≤10−4 in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαβ+ leukemia/lymphoma patients.

AB - A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant domains was identified as a potentially useful flow-cy-tometry (FCM) marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-cells and validated the method in 211 normal, reactive and pathological sam-ples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1+ and TRBC1− monoclonal and polyclonal Tαβ-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1+/TRBC1− ratios within different Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ fami-lies, whereas highly-variable TRBC1+/TRBC1− ratios were observed in more mature vs. naïve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-cells of ≤10−4 in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαβ+ leukemia/lymphoma patients.

UR - https://www.scopus.com/pages/publications/85113879288

U2 - 10.3390/cancers13174379

DO - 10.3390/cancers13174379

M3 - Article

C2 - 34503189

AN - SCOPUS:85113879288

SN - 2072-6694

VL - 13

JO - Cancers

JF - Cancers

IS - 17

M1 - 4379

ER -

Anti-trbc1 antibody-based flow cytometric detection of t-cell clonality: Standardization of sample preparation and diagnostic implementation

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