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Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities

View Article: PubMed Central - PubMed

ABSTRACT

Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord blood hematopoietic progenitor cells (HPCs) that were transduced to express an antigen receptor (AR): either a CAR or a TCR with or without built-in CD28 co-stimulatory domains. These AR-transgenic HPCs were culture-expanded on an OP9-DL1 feeder layer and subsequently differentiated to CD5+CD7+ T-lineage precursors, to CD4+ CD8+ double positive cells and finally to mature AR+ T cells. The AR+ T cells were largely naive CD45RA+CD62L+ T cells. These T cells had mostly germline TCRα and TCRβ loci and therefore lacked surface-expressed CD3/TCRαβ complexes. The CD3− AR-transgenic cells were mono-specific, functional T cells as they displayed specific cytotoxic activity. Cytokine production, including IL-2, was prominent in those cells bearing ARs with built-in CD28 domains. Data sustain the concept that cord blood HPC derived, in vitro generated allogeneic CD3− AR+ T cells can be used to more effectively eliminate malignant cells, while at the same time limiting the occurrence of GvHD.

No MeSH data available.


TCRα and TCRβ rearrangements in CD34+ HPC derived transgenic AR+ T cells. (A) Expression of the various components of the CD3/TCRαβ complex at the mRNA level. RT-PCR was performed on the JY B cell line as negative control, on a CAR:28ζ transgenic PBMC-derived T cell line (PBMC) as a positive control and on CAR transgenic HPC-derived cell lines of OP9 cultures transduced to express either the CAR:ζ (CAR:ζ) or the CAR:28ζ (CAR:28ζ). (B) TCR:ζ and TCR:28ζ transgenic CD3-negative HPC-derived T-cell lines were transduced to express the TCRα chain of a CMV-specific TCR and GFP as marker, the TCRβ chain with truncated NGFR as marker or with both TCR chains. Three days later, cells were gated for GFP+, NGFR+ or double positive cells and the CD3/TCRαβ expression was measured. Note that the TCRαβ antibody does not bind CD3-negative TCR:ζ nor TCR:28ζ complex although it binds to wtTCR/CD3 complexes. Percentage CD3/TCR positive cells is indicated in the upper right quadrant. (C) Histograms of read counts per CDR3 nucleotide length. CDR3α and CDR3β histograms are shown for wtTCR, TCR:ζ, TCR:28ζ, CAR:ζ and CAR:28ζ transgenic HPC-derived cell lines and as a control CAR:28ζ transgenic PBMC-derived T-cell line. All samples were spiked with Jurkat T cell line mRNA and CDR3α and CDR3β sequences of each transgenic cell line were determined by next-gen sequencing. Asterisk denotes the CDR3 length of the transgenic reads: CDR3α of 48 nucleotides encoding CAASTSGGTSYGKLTF and CDR3β of 39 nucleotides encoding CASSLGSSYEQYF. Arrow points at the CDR3 length of spiked Jurkat CDR reads: CDR3α of 51 nucleotides encoding CAVSDLEPNSSASKIIF and CDR3β of 48 nucleotides encoding CASSFSTCSANYGYTF).
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f0004: TCRα and TCRβ rearrangements in CD34+ HPC derived transgenic AR+ T cells. (A) Expression of the various components of the CD3/TCRαβ complex at the mRNA level. RT-PCR was performed on the JY B cell line as negative control, on a CAR:28ζ transgenic PBMC-derived T cell line (PBMC) as a positive control and on CAR transgenic HPC-derived cell lines of OP9 cultures transduced to express either the CAR:ζ (CAR:ζ) or the CAR:28ζ (CAR:28ζ). (B) TCR:ζ and TCR:28ζ transgenic CD3-negative HPC-derived T-cell lines were transduced to express the TCRα chain of a CMV-specific TCR and GFP as marker, the TCRβ chain with truncated NGFR as marker or with both TCR chains. Three days later, cells were gated for GFP+, NGFR+ or double positive cells and the CD3/TCRαβ expression was measured. Note that the TCRαβ antibody does not bind CD3-negative TCR:ζ nor TCR:28ζ complex although it binds to wtTCR/CD3 complexes. Percentage CD3/TCR positive cells is indicated in the upper right quadrant. (C) Histograms of read counts per CDR3 nucleotide length. CDR3α and CDR3β histograms are shown for wtTCR, TCR:ζ, TCR:28ζ, CAR:ζ and CAR:28ζ transgenic HPC-derived cell lines and as a control CAR:28ζ transgenic PBMC-derived T-cell line. All samples were spiked with Jurkat T cell line mRNA and CDR3α and CDR3β sequences of each transgenic cell line were determined by next-gen sequencing. Asterisk denotes the CDR3 length of the transgenic reads: CDR3α of 48 nucleotides encoding CAASTSGGTSYGKLTF and CDR3β of 39 nucleotides encoding CASSLGSSYEQYF. Arrow points at the CDR3 length of spiked Jurkat CDR reads: CDR3α of 51 nucleotides encoding CAVSDLEPNSSASKIIF and CDR3β of 48 nucleotides encoding CASSFSTCSANYGYTF).

Mentions: We previously reported that transgenic expression of a wtTCRαβ in HPCs completely suppresses rearrangements of the endogenous TCRβ locus and that agonist selection by TCR stimulation suppresses rearrangements of the endogenous TCRα locus, resulting in mature T cells that express only the transgenic TCR.27. In line with this, we showed evidence in the previous section that the CAR:ζ, CAR:28ζ, TCR:ζ and TCR:28ζ expressing cells were CD3 negative, suggesting the absence of endogenous rearrangements of the TCR loci. To further substantiate the absence of endogenous rearrangements, we measured mRNA levels coding for the different components of the CD3/TCR complex in the (TCR-negative) CAR transgenic HPC-derived cell lines. Transcripts encoding CD3γ, CD3δ, CD3ε and CD3ζ were detected, whereas TCRα and TCRβ transcripts were selectively lacking in the HPC-derived cell lines (Fig. 4A). Next, we analyzed whether a second transduction to express a transgenic (CMV-specific) wtTCRα or a wtTCRβ chain or both could induce membrane expression of the CD3 complex in TCR:ζ and TCR:28ζ transgenic HPC-derived cell lines. As expected, when both wtTCR chains were introduced, membrane CD3 expression was observed in both cell lines, indicating that all components required for TCR expression were present. However, introduction of only the TCRα chain failed to induce membrane CD3 expression, indicating that no endogenous TCRβ chains were expressed. About 0.9–1.2% of the cells expressed CD3 upon transgenic TCRβ introduction, indicating, as previously reported, that low levels of early TCRα rearrangements may occur during the DP differentiation stage.27Figure 4.


Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities
TCRα and TCRβ rearrangements in CD34+ HPC derived transgenic AR+ T cells. (A) Expression of the various components of the CD3/TCRαβ complex at the mRNA level. RT-PCR was performed on the JY B cell line as negative control, on a CAR:28ζ transgenic PBMC-derived T cell line (PBMC) as a positive control and on CAR transgenic HPC-derived cell lines of OP9 cultures transduced to express either the CAR:ζ (CAR:ζ) or the CAR:28ζ (CAR:28ζ). (B) TCR:ζ and TCR:28ζ transgenic CD3-negative HPC-derived T-cell lines were transduced to express the TCRα chain of a CMV-specific TCR and GFP as marker, the TCRβ chain with truncated NGFR as marker or with both TCR chains. Three days later, cells were gated for GFP+, NGFR+ or double positive cells and the CD3/TCRαβ expression was measured. Note that the TCRαβ antibody does not bind CD3-negative TCR:ζ nor TCR:28ζ complex although it binds to wtTCR/CD3 complexes. Percentage CD3/TCR positive cells is indicated in the upper right quadrant. (C) Histograms of read counts per CDR3 nucleotide length. CDR3α and CDR3β histograms are shown for wtTCR, TCR:ζ, TCR:28ζ, CAR:ζ and CAR:28ζ transgenic HPC-derived cell lines and as a control CAR:28ζ transgenic PBMC-derived T-cell line. All samples were spiked with Jurkat T cell line mRNA and CDR3α and CDR3β sequences of each transgenic cell line were determined by next-gen sequencing. Asterisk denotes the CDR3 length of the transgenic reads: CDR3α of 48 nucleotides encoding CAASTSGGTSYGKLTF and CDR3β of 39 nucleotides encoding CASSLGSSYEQYF. Arrow points at the CDR3 length of spiked Jurkat CDR reads: CDR3α of 51 nucleotides encoding CAVSDLEPNSSASKIIF and CDR3β of 48 nucleotides encoding CASSFSTCSANYGYTF).
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f0004: TCRα and TCRβ rearrangements in CD34+ HPC derived transgenic AR+ T cells. (A) Expression of the various components of the CD3/TCRαβ complex at the mRNA level. RT-PCR was performed on the JY B cell line as negative control, on a CAR:28ζ transgenic PBMC-derived T cell line (PBMC) as a positive control and on CAR transgenic HPC-derived cell lines of OP9 cultures transduced to express either the CAR:ζ (CAR:ζ) or the CAR:28ζ (CAR:28ζ). (B) TCR:ζ and TCR:28ζ transgenic CD3-negative HPC-derived T-cell lines were transduced to express the TCRα chain of a CMV-specific TCR and GFP as marker, the TCRβ chain with truncated NGFR as marker or with both TCR chains. Three days later, cells were gated for GFP+, NGFR+ or double positive cells and the CD3/TCRαβ expression was measured. Note that the TCRαβ antibody does not bind CD3-negative TCR:ζ nor TCR:28ζ complex although it binds to wtTCR/CD3 complexes. Percentage CD3/TCR positive cells is indicated in the upper right quadrant. (C) Histograms of read counts per CDR3 nucleotide length. CDR3α and CDR3β histograms are shown for wtTCR, TCR:ζ, TCR:28ζ, CAR:ζ and CAR:28ζ transgenic HPC-derived cell lines and as a control CAR:28ζ transgenic PBMC-derived T-cell line. All samples were spiked with Jurkat T cell line mRNA and CDR3α and CDR3β sequences of each transgenic cell line were determined by next-gen sequencing. Asterisk denotes the CDR3 length of the transgenic reads: CDR3α of 48 nucleotides encoding CAASTSGGTSYGKLTF and CDR3β of 39 nucleotides encoding CASSLGSSYEQYF. Arrow points at the CDR3 length of spiked Jurkat CDR reads: CDR3α of 51 nucleotides encoding CAVSDLEPNSSASKIIF and CDR3β of 48 nucleotides encoding CASSFSTCSANYGYTF).
Mentions: We previously reported that transgenic expression of a wtTCRαβ in HPCs completely suppresses rearrangements of the endogenous TCRβ locus and that agonist selection by TCR stimulation suppresses rearrangements of the endogenous TCRα locus, resulting in mature T cells that express only the transgenic TCR.27. In line with this, we showed evidence in the previous section that the CAR:ζ, CAR:28ζ, TCR:ζ and TCR:28ζ expressing cells were CD3 negative, suggesting the absence of endogenous rearrangements of the TCR loci. To further substantiate the absence of endogenous rearrangements, we measured mRNA levels coding for the different components of the CD3/TCR complex in the (TCR-negative) CAR transgenic HPC-derived cell lines. Transcripts encoding CD3γ, CD3δ, CD3ε and CD3ζ were detected, whereas TCRα and TCRβ transcripts were selectively lacking in the HPC-derived cell lines (Fig. 4A). Next, we analyzed whether a second transduction to express a transgenic (CMV-specific) wtTCRα or a wtTCRβ chain or both could induce membrane expression of the CD3 complex in TCR:ζ and TCR:28ζ transgenic HPC-derived cell lines. As expected, when both wtTCR chains were introduced, membrane CD3 expression was observed in both cell lines, indicating that all components required for TCR expression were present. However, introduction of only the TCRα chain failed to induce membrane CD3 expression, indicating that no endogenous TCRβ chains were expressed. About 0.9–1.2% of the cells expressed CD3 upon transgenic TCRβ introduction, indicating, as previously reported, that low levels of early TCRα rearrangements may occur during the DP differentiation stage.27Figure 4.

View Article: PubMed Central - PubMed

ABSTRACT

Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord blood hematopoietic progenitor cells (HPCs) that were transduced to express an antigen receptor (AR): either a CAR or a TCR with or without built-in CD28 co-stimulatory domains. These AR-transgenic HPCs were culture-expanded on an OP9-DL1 feeder layer and subsequently differentiated to CD5+CD7+ T-lineage precursors, to CD4+ CD8+ double positive cells and finally to mature AR+ T cells. The AR+ T cells were largely naive CD45RA+CD62L+ T cells. These T cells had mostly germline TCRα and TCRβ loci and therefore lacked surface-expressed CD3/TCRαβ complexes. The CD3− AR-transgenic cells were mono-specific, functional T cells as they displayed specific cytotoxic activity. Cytokine production, including IL-2, was prominent in those cells bearing ARs with built-in CD28 domains. Data sustain the concept that cord blood HPC derived, in vitro generated allogeneic CD3− AR+ T cells can be used to more effectively eliminate malignant cells, while at the same time limiting the occurrence of GvHD.

No MeSH data available.