Umbilical cord blood T cells express multiple natural cytotoxicity receptors after IL-15 stimulation, but only NKp30 is functional

Abstract

The natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46 are thought to be NK lineage restricted. Herein we show that IL-15 induces NCR expression on umbilical cord blood (UCB) T cells. NCRs were mainly on CD8(+) and CD56(+) UCB T cells. Only NKp30 was functional as demonstrated by degranulation, IFN-gamma release, redirected killing, and apoptosis. Since NCRs require adaptor proteins for function, the expressions of these adaptors were determined. The adaptors used by NKp30 and NKp46, FcepsilonR1gamma and CD3zeta, were detected in UCB T cells. There was a near absence of DAP12, the adaptor for NKp44, consistent with a hypofunctional state. NKp46 was on significantly fewer UCB T cells, possibly accounting for its lack of function. Adult peripheral blood (PB) T cells showed minimal NCR acquisition after culture with IL-15. Since UCB contains a high frequency of naive T cells, purified naive T cells from adult PB were tested. Although NKp30 was expressed on a small fraction of naive PB T cells, it was nonfunctional. In contrast to UCB, PB T cells lacked FcepsilonR1gamma expression. These results demonstrate differences between UCB and PB T cells regarding NCR expression and function. Such findings challenge the concept that NCRs are NK cell specific.

Figure 1. Comparison of NCR acquisition on UCB or PB T cells induced by IL-15

CD3⁺ T cells from UCB and PB were isolated from healthy donors on day 0 and cultured with IL-15 (50ng/ml). At day 0, 7, 14 and 21 of culture, NKp30, NKp44 and NKp46 expression were examined with flow cytometry. Representative dot plots of NCR expression on UCB and PB T cells at (A) day 0 and (B) day 14 are shown. Percentages of NCR expressing cells are indicated. Data are summarized from 10 UCB donors and 5 PB donors (C). p value < 0.05 for the differences between UCB and PB T cells, except at day 0.

Figure 2. UCB T cells show de novo NKp30 expression and NCR acquisition is linked with cell division

(A) Freshly isolated UCB T cells were sorted into CD3⁺NKp30^- or CD3⁺NKp30⁺ cells subsets. NKp30 expression was measured with flow cytometry on cells right after sorting or 14 days culture with IL-15. Results are representative of 2 donors. (B) Freshly isolated UCB T cells were labeled with CFSE and cultured with media containing IL-15 (50 ng/ml). At day 14 cells were tested for NKp30, NKp44 and NKp46 expression vs. CFSE. Results are representative of three independent experiments.

Figure 3. Influence of different IL-2 family cytokines on NCR acquisition in UCB T cells

Freshly isolated CD3⁺ T cells from UCB were cultured in the presence of different concentrations of (A) IL-2, (B) IL-4, (C) IL-7, and (D) IL-15. At day 14, NKp30, NKp44 and NKp46 expression was tested by FACS. Percentages of positive cells are indicated. Results are representative of three independent experiments.

Figure 4. Comparison of NCR expression on UCB T cells subsets

(A) Representative dot plot of IL-15 cultured UCB T cells at day 14 of culture stained for CD4⁺ and CD8⁺ expression and histograms for NKp30, NKp44 and NKp46 expression on the gated populations. (C) Representative dot plot of CD3 vs. CD56 of cultured UCB T cells at day 14 of culture and histograms for NKp30, NKp44 and NKp46 expression on the gated populations. (E) Representative dot plot of CD3 vs. TCR γδ of cultured UCB T cells at day 14 of culture and histograms for NKp30, NKp44 and NKp46 expression on the gated populations. Results are representative of 4, 4 and 3 donors, respectively. Shaded histograms represent isotype control and percentages of gated NCR positive cells are indicated. Summary data from all donors were summarized and Mann-Whitney rank sum test was performed (B, D, F) with p values indicated.

Figure 5. Function of NCRs on UCB T cells

IL-15 expanded UCB T cells at day 14 of culture were triggered with different plate-bound mAbs then assayed for (A) degranulation (summary of three independent experiments), (B) apoptosis (representative of 5 independent experiments) and (C) IFN-y release (summary of 2 independent experiments). Tukey's method was used to make multiple comparisons with p values indicated. D, E, F and G) show redirected killing assay using IL-15 expanded UCB T cells at day 14 of culture and after labeling with agonist antibodies as described in the methods. mAb antibody coated UCB T cells were combined with ⁵¹Cr labeled P815 cells and ⁵¹Cr was measured after 4 hrs. Each plot is representative of > 3 independent experiments with separate donors.

Figure 6. Expression of DAP12, CD3ζ and FcεRIγ in IL-15 Expanded UCB T cells

A) RT-PCR for DAP12 (650bp), CD3-ζ (273bp), FcεRIγ (209bp) was performed on IL-15 expanded UCB T cells at day 14. Results are representative of three independent experiments. B) CD3ζ expression was examined with intracellular staining. The number indicates the percentage of cells in the quadrant gate. Protein extracts of IL-15 expanded UCB T cells at D+14 and NK cells were separated on 15% acrylamide, then transferred to PVDF membrane and probed with C) anti-DAP12, D) FcεRIγ. Actin was used as a loading control.

Figure 7. Acquisition of NCRs and Function Of NKp30 on Sorted PB T Cell Subsets

Freshly isolated PB T cells were sorted into Naïve, CM, EMRA+, EMRA- and Bulk (unsorted) subsets and cultured in IL-15 for 14 days as described in the methods. Cells were then assayed for NCR expression with FACS at D+14. A) Representative histograms are shown. Shaded plots represented isotype controls and percentage of positive cells was indicated. B) Summary of the percentage of NCR positive cells after 14 days of culture with IL-15 are shown from 5 donors. C) Redirected killing assay (reverse ADCC) for sorted, naïve PB T cells. Results representative of 3 donors. D) Expression CD3ζ by FACS in PB populations that were sorted at D0 and cultured for 14 days with IL-15. E) Expression of FcεRIγ in UCB NK cells, T cells and PB T cells.