T cells fail to develop in the human skin-cell explants system; an inconvenient truth

Abstract

Background: Haplo-identical hematopoietic stem cell (HSC) transplantation is very successful in eradicating haematological tumours, but the long post-transplant T-lymphopenic phase is responsible for high morbidity and mortality rates. Clark et al. have described a skin-explant system capable of producing host-tolerant donor-HSC derived T-cells. Because this T-cell production platform has the potential to replenish the T-cell levels following transplantation, we set out to validate the skin-explant system.

Results: Following the published procedures, while using the same commercial components, it was impossible to reproduce the skin-explant conditions required for HSC differentiation towards mature T-cells. The keratinocyte maturation procedure resulted in fragile cells with minimum expression of delta-like ligand (DLL). In most experiments the generated cells failed to adhere to carriers or were quickly outcompeted by fibroblasts. Consequently it was not possible to reproduce cell-culture conditions required for HSC differentiation into functional T-cells. Using cell-lines over-expressing DLL, we showed that the antibodies used by Clark et al. were unable to detect native DLL, but instead stained 7AAD+ cells. Therefore, it is unlikely that the observed T-lineage commitment from HSC is mediated by DLL expressed on keratinocytes. In addition, we did confirm expression of the Notch-ligand Jagged-1 by keratinocytes.

Conclusions: Currently, and unfortunately, it remains difficult to explain the development or growth of T-cells described by Clark et al., but for the fate of patients suffering from lymphopenia it is essential to both reproduce and understand how these co-cultures really "work". Fortunately, alternative procedures to speed-up T-cell reconstitution are being established and validated and may become available for patients in the near future.

Figure 1

Differentiation of keratinocytes results in a mixture of immature and semi mature cells. Keratinocytes of a representative donor were differentiated in chamber-slides for 6 days. Following fixation, cells were stained for the indicated cytokeratins (CK) using a panel of monoclonal (CK10, CK14, CK16) and polyclonal antibodies (pan). Duration of fluorescence detection was kept constant. Though variable, all cells were positive for CK14, while staining of CK10 and CK16 was limited to areas with blast-like keratinocytes.

Figure 2

Analyses of DLL expression by Immunoblot and FACS using cell-lines over-expressing DLL. (A) Protein blots were prepared from TSt-4 thymic stromal cell-lines over-expressing human DLL1 or 4, and incubated with polyclonal antibodies specific for DLL. Both H20 and H265 only stained lanes containing TSt-4-DLL1 extracts, with a main band at 78 kDa. H265 stained additional bands at 50 kDa and 230 kDa, which could represent processed and glycosylated DLL-1, respectively. In contrast to H20, H265 also heavily stained the marker-lane, indicating that the specificity of this polyclonal antibody preparation is not limited to DLL. (B) Indicated cells were harvested using trypsin-EDTA, and incubated with H265 and H20 + control polyclonal antibodies specific for IRF1 and ACE. Control antibodies were selected based on species-origin and specificity for non-surface antigens. Both H265 and H20 showed weak, yet specific staining of TSt4-DLL1.

Figure 3

7AAD⁺ Keratinocytes show intense staining with any polyclonal primary antibody. Keratinocytes of donor C were harvested following high-density culture for 6 days, either in standard keratinocyte (iKer) or maturation medium (dKer). (A) All cells being stained with indicated rabbit pan antibodies were 7AAD⁺. Left column shows FACS plots as used by Clark et al. [15], right column contains additional information regarding viability. Within experiments, the percentage of positively stained cells was always similar with any pan-antibody. Insert shows background staining by the secondary antibody used. Even though staining intensity was lower after incubation with the secondary antibody, when compared with unstained cells (LL quadrant of the right column), the percentage of cells with signal was similar. This staining-pattern was observed for all donors. (B) When gated on 7AAD^- cells, any pan antibody stained a low percentage of keratinocytes (donor C, donor D showed similar inconclusive staining profile). (C) Almost all cells that were stained with H265, co-stained with H20. A nearly similar plot could be obtained with any pan rabbit (eg ACE) and pan goat antibody combination (eg, NOS2), and invariably involved 7AAD⁺ cells (donor C, donor D showed similar co-staining).

Figure 4

Keratinocytes have very low expression of DLL. (A): Next to FACS, Clark et al. [15] used immuno-fluorescence to demonstrate DLL expression in immature keratinocyte-cultures. While the secondary antibody did not give any background staining (LL), there were occasional H265-FITC positive cells (UR), but these always proved to be DAPI negative. In contrast to DLL, immature keratinocytes were reliably stained for the Notch-ligand Jagged-1. (B) For PCR, 4 dilutions (5x-20x-80x-320x) were prepared from cDNA of mKer from 3 donors (A, B, and C), iKer from donor C. Positive controls were prepared from murine TSt-4 cells constitutively expressing DLL-1 or DLL-4. At 35 cycles, DLL4-specific PCR product was only detected in the 5x diluted samples, while mRNA encoding for Jagged-1 was easily amplified in 30 cycles.

Figure 5

Keratinocytes are out-competed by fibroblasts. (A) Matrix was stained for keratinocytes and fibroblasts using panCK and CD90, respectively, six days after matrices were seeded at a 2:1 ratio. At this stage, matrices were carefully seeded with HSC. (B) After 4 weeks, matrices were completely covered with CD90⁺ fibroblasts. HSC-derived cells could only be retrieved from the matrix following collagenase-dispase treatment. Fibroblasts from all donors grew similarly.

Figure 6

HSC expand, but do not differentiate in the skin-cell explants. HSC from donor V1 and V2 were seeded into skin-cell explants from donor A and B. As controls, HSC were expanded for 1 week using a HSC expansion-mix, followed by maintenance with IL-7. For reasons unknown, cell-numbers declined after 2-3 weeks in culture with skin-cell explants (not shown). The phenotype of the hematopoietic cells was analyzed at various time-points after seeding, and the results of week 2 and 4 are depicted. Of markers associated with T-lineage commitment, only CD7 was detected on 20-30%/60% of the cells in skin-cell explants/controls, and its expression-level increased from week 2 to 4.

Figure 7

Skin-cell explants cannot maintain T-lineage committed cells. HSC from indicated donors were pre-differentiated on monolayers of thymic stromal cells expressing huDLL-1 (see figure 2). After 4 weeks, 55-65% of the cells were CD5⁺CD7⁺ (see insert UL), and > 90% iCD3⁺CD45RA⁺ (NS). 1 × 10⁵ cells were seeded in skin-cell explants and cultured for 2 and 4 weeks. In the presence of IL15, the population expanded 10-15 ×. Already at 2 weeks, all cells had lost expression of CD5 and become CD56⁺ NK-lineage committed cells. The same phenotype was observed when IL-15 was omitted, although overall expansion was strongly reduced to 1-1,5 ×.