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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.


Related in: MedlinePlus

Phenotype and endogenous TCR expression of CD34+ HPC-derived transgenic AR+ T cells. Flow cytometric analysis of the AR-transgenic T cells. (A) CAR-transgenic GFP+ cells of cultures transduced to express either the CAR:ζ or the CAR:28ζ were analyzed on day 26 of OP9-DL1 culture for CD3 and TCRαβ expression. As a control, GFP− cells are shown from the OP9-DL1 culture transduced to express the CAR:ζ (N = 5). (B) Dot plots show CD3 expression of cells from the OP9-DL1 cultures transgenic for the wtTCR, TCR:ζ and TCR:28ζ. Vβ14 staining is used to mark transgene expression, as no GFP is expressed by the transgenic cells (N = 5). (C) Surface and cytoplasmic staining for CD3 of in vitro generated mature T cells that were expanded for one cycle on feeder cells in the presence of cytokines. (D) Expression of various membrane markers by the CD27+CD1a− mature T cells at the end of OP9-DL1 culture (46 d) (N = 2). (E) Day 0: fresh cord blood after MACS CD34 enrichment sorted using the sorting window shown. Day 13: cord blood cells cultured on OP9-DL1 were sorted for CD5 CD7 double positive cells, using the indicated sorting window. The cells were then transduced to express CAR:28ζ and further differentiated on OP9-DL1 feeder layer. Day 21: analysis of the transgenic GFP+ cultured cells for DP cells and CD27+CD1a− mature cells. (F) Flow cytometric analysis of GFP+ CAR:28ζ-transgenic cultures, gated on GFP+ CD27+ CD1a− mature AR+ cells (N = 2).
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f0003: Phenotype and endogenous TCR expression of CD34+ HPC-derived transgenic AR+ T cells. Flow cytometric analysis of the AR-transgenic T cells. (A) CAR-transgenic GFP+ cells of cultures transduced to express either the CAR:ζ or the CAR:28ζ were analyzed on day 26 of OP9-DL1 culture for CD3 and TCRαβ expression. As a control, GFP− cells are shown from the OP9-DL1 culture transduced to express the CAR:ζ (N = 5). (B) Dot plots show CD3 expression of cells from the OP9-DL1 cultures transgenic for the wtTCR, TCR:ζ and TCR:28ζ. Vβ14 staining is used to mark transgene expression, as no GFP is expressed by the transgenic cells (N = 5). (C) Surface and cytoplasmic staining for CD3 of in vitro generated mature T cells that were expanded for one cycle on feeder cells in the presence of cytokines. (D) Expression of various membrane markers by the CD27+CD1a− mature T cells at the end of OP9-DL1 culture (46 d) (N = 2). (E) Day 0: fresh cord blood after MACS CD34 enrichment sorted using the sorting window shown. Day 13: cord blood cells cultured on OP9-DL1 were sorted for CD5 CD7 double positive cells, using the indicated sorting window. The cells were then transduced to express CAR:28ζ and further differentiated on OP9-DL1 feeder layer. Day 21: analysis of the transgenic GFP+ cultured cells for DP cells and CD27+CD1a− mature cells. (F) Flow cytometric analysis of GFP+ CAR:28ζ-transgenic cultures, gated on GFP+ CD27+ CD1a− mature AR+ cells (N = 2).

Mentions: Since CARs do not require CD3 for membrane expression, we checked whether the cells generated in these cultures had TCR/CD3 complexes expressed on the membrane. In Fig. 3A, it is shown that >7% of the untransduced GFP− cells express CD3 in combination with either a TCRαβ or a TCRγδ receptor. In contrast, the GFP+ CAR transgenic cells in the same cultures are largely CD3 negative, despite the presence of a higher percentage of mature CD27+ CD1a− cells (Fig. 1). This was the case for both CAR:ζ and CAR:28ζ transgenic cultures. A similar phenomenon was observed in TCR-transgenic cultures (Fig. 3B). wtTCR transgenic cells expressed CD3 as well as the transgenic TCR, as evidenced by the Vβ14 expression. In contrast, Vβ14-expressing TCR:ζ and TCR:28ζ transgenic cells expressed the AR in the absence of CD3 membrane expression16,20. These transgenic cells expressed no CD3. A small population of Vβ14+ cells expressed CD3, but these are most likely untransduced cells that have rearranged the endogenous TCR at the Vβ14 gene segment. To exclude that the CD3/TCR complex was not expressed on the membrane of CAR-transgenic T cells due to the absence of CD3 protein expression, we performed cytoplasmic CD3ε staining. Fig. 3C shows that cCD3 was present at high levels in the cytoplasm, but not on the cell surface (sCD3), which is likely due to the absence of rearranged endogenous TCRs rather than due to the non-T-cell nature of the cells. Cytoplasmic expression of CD3 suggests that the CAR+, TCR/CD3− cells are bona fide T cells. However, natural killer (NK) cells generated from cord blood on OP9-DL1 feeder cells may also express cytoplasmic CD3.29 We therefore analyzed the mature CAR+ cells for other T and NK-lineage markers. The CAR transgenic cells were positive for CD5, CD7 and CD2 (Fig. 3D). The combination of these three markers is exclusively expressed by T cells, whereas NK cells are consistently CD5−. While NK cells are defined as CD3− CD56+ cells, the CAR+ cells generated in vitro were to a large degree negative for the NK cell marker CD56. Only a minor population of the cells was CD56+, a marker that is also present on activated T cells. NKG2D, a marker for both NK cells and mature CD8+ single positive T cells, was expressed on these cells.Figure 3.


Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities
Phenotype and endogenous TCR expression of CD34+ HPC-derived transgenic AR+ T cells. Flow cytometric analysis of the AR-transgenic T cells. (A) CAR-transgenic GFP+ cells of cultures transduced to express either the CAR:ζ or the CAR:28ζ were analyzed on day 26 of OP9-DL1 culture for CD3 and TCRαβ expression. As a control, GFP− cells are shown from the OP9-DL1 culture transduced to express the CAR:ζ (N = 5). (B) Dot plots show CD3 expression of cells from the OP9-DL1 cultures transgenic for the wtTCR, TCR:ζ and TCR:28ζ. Vβ14 staining is used to mark transgene expression, as no GFP is expressed by the transgenic cells (N = 5). (C) Surface and cytoplasmic staining for CD3 of in vitro generated mature T cells that were expanded for one cycle on feeder cells in the presence of cytokines. (D) Expression of various membrane markers by the CD27+CD1a− mature T cells at the end of OP9-DL1 culture (46 d) (N = 2). (E) Day 0: fresh cord blood after MACS CD34 enrichment sorted using the sorting window shown. Day 13: cord blood cells cultured on OP9-DL1 were sorted for CD5 CD7 double positive cells, using the indicated sorting window. The cells were then transduced to express CAR:28ζ and further differentiated on OP9-DL1 feeder layer. Day 21: analysis of the transgenic GFP+ cultured cells for DP cells and CD27+CD1a− mature cells. (F) Flow cytometric analysis of GFP+ CAR:28ζ-transgenic cultures, gated on GFP+ CD27+ CD1a− mature AR+ cells (N = 2).
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f0003: Phenotype and endogenous TCR expression of CD34+ HPC-derived transgenic AR+ T cells. Flow cytometric analysis of the AR-transgenic T cells. (A) CAR-transgenic GFP+ cells of cultures transduced to express either the CAR:ζ or the CAR:28ζ were analyzed on day 26 of OP9-DL1 culture for CD3 and TCRαβ expression. As a control, GFP− cells are shown from the OP9-DL1 culture transduced to express the CAR:ζ (N = 5). (B) Dot plots show CD3 expression of cells from the OP9-DL1 cultures transgenic for the wtTCR, TCR:ζ and TCR:28ζ. Vβ14 staining is used to mark transgene expression, as no GFP is expressed by the transgenic cells (N = 5). (C) Surface and cytoplasmic staining for CD3 of in vitro generated mature T cells that were expanded for one cycle on feeder cells in the presence of cytokines. (D) Expression of various membrane markers by the CD27+CD1a− mature T cells at the end of OP9-DL1 culture (46 d) (N = 2). (E) Day 0: fresh cord blood after MACS CD34 enrichment sorted using the sorting window shown. Day 13: cord blood cells cultured on OP9-DL1 were sorted for CD5 CD7 double positive cells, using the indicated sorting window. The cells were then transduced to express CAR:28ζ and further differentiated on OP9-DL1 feeder layer. Day 21: analysis of the transgenic GFP+ cultured cells for DP cells and CD27+CD1a− mature cells. (F) Flow cytometric analysis of GFP+ CAR:28ζ-transgenic cultures, gated on GFP+ CD27+ CD1a− mature AR+ cells (N = 2).
Mentions: Since CARs do not require CD3 for membrane expression, we checked whether the cells generated in these cultures had TCR/CD3 complexes expressed on the membrane. In Fig. 3A, it is shown that >7% of the untransduced GFP− cells express CD3 in combination with either a TCRαβ or a TCRγδ receptor. In contrast, the GFP+ CAR transgenic cells in the same cultures are largely CD3 negative, despite the presence of a higher percentage of mature CD27+ CD1a− cells (Fig. 1). This was the case for both CAR:ζ and CAR:28ζ transgenic cultures. A similar phenomenon was observed in TCR-transgenic cultures (Fig. 3B). wtTCR transgenic cells expressed CD3 as well as the transgenic TCR, as evidenced by the Vβ14 expression. In contrast, Vβ14-expressing TCR:ζ and TCR:28ζ transgenic cells expressed the AR in the absence of CD3 membrane expression16,20. These transgenic cells expressed no CD3. A small population of Vβ14+ cells expressed CD3, but these are most likely untransduced cells that have rearranged the endogenous TCR at the Vβ14 gene segment. To exclude that the CD3/TCR complex was not expressed on the membrane of CAR-transgenic T cells due to the absence of CD3 protein expression, we performed cytoplasmic CD3ε staining. Fig. 3C shows that cCD3 was present at high levels in the cytoplasm, but not on the cell surface (sCD3), which is likely due to the absence of rearranged endogenous TCRs rather than due to the non-T-cell nature of the cells. Cytoplasmic expression of CD3 suggests that the CAR+, TCR/CD3− cells are bona fide T cells. However, natural killer (NK) cells generated from cord blood on OP9-DL1 feeder cells may also express cytoplasmic CD3.29 We therefore analyzed the mature CAR+ cells for other T and NK-lineage markers. The CAR transgenic cells were positive for CD5, CD7 and CD2 (Fig. 3D). The combination of these three markers is exclusively expressed by T cells, whereas NK cells are consistently CD5−. While NK cells are defined as CD3− CD56+ cells, the CAR+ cells generated in vitro were to a large degree negative for the NK cell marker CD56. Only a minor population of the cells was CD56+, a marker that is also present on activated T cells. NKG2D, a marker for both NK cells and mature CD8+ single positive T cells, was expressed on these cells.Figure 3.

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.


Related in: MedlinePlus