Natural Killer Cells Regulate Th17 Cells After Autologous Hematopoietic Stem Cell Transplantation for Relapsing Remitting Multiple Sclerosis

Abstract

In autoimmunity, the balance of different helper T (Th) cell subsets can influence the tissue damage caused by autoreactive T cells. Pro-inflammatory Th1 and Th17 T cells are implicated as mediators of several human autoimmune conditions such as multiple sclerosis (MS). Autologous hematopoietic stem cell transplantation (aHSCT) has been tested in phase 2 clinical trials for MS patients with aggressive disease. Abrogation of new clinical relapses and brain lesions can be seen after ablative aHSCT, accompanied by significant reductions in Th17, but not Th1, cell populations and activity. The cause of this selective decrease in Th17 cell responses following ablative aHSCT is not completely understood. We identified an increase in the kinetics of natural killer (NK) cell reconstitution, relative to CD4+ T cells, in MS patients post-aHSCT, resulting in an increased NK cell:CD4+ T cell ratio that correlated with the degree of decrease in Th17 responses. Ex vivo removal of NK cells from post-aHSCT peripheral blood mononuclear cells resulted in higher Th17 cell responses, indicating that NK cells can regulate Th17 activity. NK cells were also found to be cytotoxic to memory Th17 cells, and this toxicity is mediated through NKG2D-dependent necrosis. Surprisingly, NK cells induced memory T cells to secrete more IL-17A. This was preceded by an early rise in T cell expression of RORC and IL17A mRNA, and could be blocked with neutralizing antibodies against CD58, a costimulatory receptor expressed on NK cells. Thus, NK cells provide initial co-stimulation that supports the induction of a Th17 response, followed by NKG2D-dependent cytotoxicity that limits these cells. Together these data suggest that rapid reconstitution of NK cells following aHSCT contribute to the suppression of the re-emergence of Th17 cells. This highlights the importance of NK cells in shaping the reconstituting immune system following aHSCT in MS patients.

Trial registration: ClinicalTrials.gov NCT01099930.

Keywords: CD58; NKG2D; Th17 cells; aHSCT; multiple sclerosis; natural killer cells.

Figures

Figure 1

Rapid reconstitution kinetics of natural killer (NK) cell counts compared with CD4 T cells in aHSCT-treated multiple sclerosis (MS) patient blood. From freshly drawn blood samples, absolute number of NK cell counts (A) and CD4+ T cell counts (B) were determined at baseline (BL), and at 3 weeks (3 W), 3 months (3 M), 12 months (12 M), and 21 months (21 M) post-aHSCT. The ratio of NK cells to CD4+ T cells was calculated (C). N = 7 patients.

Figure 2

The proportions of CD56dim and CD56bright natural killer (NK) cells are higher post-aHSCT compared witho baseline (BL). Cryopreserved peripheral blood mononuclear cell (PBMC) from aHSCT-treated MS patients was analyzed by flow cytometry. Representative plots for BL (A,C), and month 12 [M12; (B,D)] stained for CD3 and CD56 (A,B), gated on CD3−CD56+, then shown as CD56 with CD16 (C,D). Example gates for CD56bright and CD56dim are shown. Additional time points include BL, and serial samples approximately every 3 months for up to 24 months post-aHSCT. The average proportions of total NK cells as well as CD56dim and CD56bright NK cell subsets expressed as a proportion of total PBMC (E). The ratio of NK cells with the CD56bright phenotype calculated as CD56bright/CD56dim NK cells (F). The proportions of NK cells with CD16+ or CD16− phenotype expressed as a proportion of total PBMC (G). The ratio of gated NK cells with the CD16− phenotype calculated as CD16−/CD16+(H). N = 7 patients. For statistical analysis, the time points were grouped in M3–M6, M9–M12, M15–M18, and M21–M24, followed by univariate one-way ANOVA with pairwise comparisons with the BL values.

Figure 3

Changes in blood natural killer (NK) cell populations correlate with changes in helper T (Th) cells populations following aHSCT in MS patients. MS patient peripheral blood mononuclear cell (PBMC) samples from baseline (BL) and 12 month post-aHSCT were activated in vitro with anti-CD3, anti-CD28, and Th17 polarizing factors for 4 days. Th17 and Th1 cells were assessed by analysis of cytokine production by intracellular flow cytometry (CD3+CD4+IL-17A+IFN-γ− or CD3+CD4+IL-17A−IFN-γ+, respectively). The change in frequency of Th17 cells (A) or Th1 cells (B) was plotted against the change in NK cell frequency, and linear regression was performed on the data points. N = 7 patients.

Figure 4

CD56+ cells suppress Th17 and Th1 cell responses in aHSCT. CD56+ cells were depleted from MS patient PBMC samples collected at 12 months post-aHSCT. Representative plots are shown for complete (A,B) and CD56-depleted samples (C,D). Cells were activated in vitro with anti-CD3, anti-CD28, and Th17 polarizing factors (Act) for 4 days. The proportions of Th17 and Th1 cells were assessed by analysis of cytokine production by intracellular flow cytometry. Representative plots are shown for complete samples (B) and CD56-depleted samples (D). The average proportion of Th17 (E) or Th1 cells (F) is shown. N = 3 patients.

Figure 5

Natural killer (NK) cells reduce the proportion of Th17 and Th1 cells in vitro. Memory CD4+ T cells and NK cells were isolated from healthy subject PBMC. Memory CD4+ T cells were CD3+CD4+CD45RO+CD45RA− as shown in representative dot plots (A,B). NK cells were CD3−CD56+ as shown in a representative dot plot (C). Memory CD4+ T cells were cultured without (D,E), or with NK cells at a 1:1 ratio (F,G) for 4 days with anti-CD3, anti-CD28, and Th17 polarizing factors for 4 days. Plots are shown for CD4 × CD56, which was used to gate CD4+ helper T (Th) cells. Expression of IL-17 and IFN-γ by CD4+ T cells was assessed by intracellular flow cytometry (Th17 = CD3+CD4+IL-17A+IFN-γ−, Th1 = CD3+CD4+IL-17A−IFN-γ+, and Th1/17 = CD3+CD4+IL-17A+IFN-γ+). Data pooled from 12 experiments showing the proportion (H) and absolute number of Th cells (I). A time-course analysis for Th17 cells (J) and Th1 cells (K) was performed for 5 days using intracellular cytokine staining. Open diamond = T cells and closed square = T cells + NK cells.

Figure 6

Activated memory CD4+ T cells express MICA and are sensitive to NKG2D-mediated natural killer (NK) cell cytotoxicity. Memory CD4+ T cells were obtained from healthy subject PBMC, as described in Figure 5, and activated with anti-CD3, anti-CD28, and Th17 polarizing factors for 4 days. Expression of CD4, MICA (Zenon labeled), IL-17A, and IFN-γ were assessed by intracellular cytokine staining and flow cytometry. Representative plots of FSC × SSC (A), CD4 × SSC (B), and IL-17A × IFN-γ (C) are shown. MICA expression on Th17 (D), Th1 (E), and the Th0 cells (F) is shown. Open histogram indicate MICA stained cells and closed histograms indicate an isotype control. The average mean fluorescent intensity of MICA minus the isotype control is shown [ΔMFI; (G)]. Expression of CD56 and NKG2D by NK cells was assessed by flow cytometry and a representative plot is shown (H). NK cells were cultured with memory CD4+ T cells and activated with anti-CD3, anti-CD28, and Th17 polarizing factors, at the same time treated without antibody, (NIL), anti-NKG2D neutralizing antibody, or isotype control antibody (I). N = 7 samples.

Figure 7

Natural killer (NK) cells are cytotoxic toward helper T (Th) cells through necrosis but not apoptosis. Memory CD4+ T cells from healthy subject PBMC were activated with anti-CD3, anti-CD28, and Th17 polarizing factors, either in the absence or presence of NK cells for 4 days. Samples were stained with CD3, CD56, 7AAD, and annexin V. Representative plots of T cells (CD4+CD56−) cultured without (A) or with NK cells (B) are shown. The average proportion of necrotic (7-AAD+annexinV+) T cells is shown (C). Open bars = memory CD4 T cells, closed bars = memory CD4+ T cells plus NK cells. N = 8 samples. A time-course analysis of apoptotic (7-AAD−annexin V+) T cells is shown (D). Open circles indicate memory CD4 T cells alone and filled squares indicate T cells plus NK cells.

Figure 8

Natural killer (NK) cells augment IL-17A and RORC levels in memory CD4 T cells. Purified memory CD4+ T cells from healthy subject PBMCs were activated with anti-CD3, anti-CD28, and Th17 polarizing factors without (open diamond) or with NK cells (closed square). Cytokines IL-17A (A) and IFN-γ (B) were measured in the supernatant by enzyme-linked immunosorbent assay. Expression of RORC(C) and IL17A(D) mRNA was measured by qPCR at the indicated time points. Data are representative of three samples. Groups included non-activated memory CD4+ T cells (T nil; closed circles), activated memory CD4+ T cells (T; open circle), activated memory CD4+ T cells with NK cells (T NK; closed squares), and NK cells cultured alone with IL-2 (NK IL-2; open squares). Representative plots of IL-17A and IFN-γ expression in NK cells (CD3−CD56+) are shown (E,F).

Figure 9

Natural killer (NK) cells support IL-17A expression by helper T (Th) cells by CD58 co-stimulation. A representative plot of CD58 expression by CD3−CD56+ NK cells is shown from healthy subject peripheral blood mononuclear cell (PBMC) (A). Memory CD4+ T cells from healthy subjects PBMC were activated with anti-CD3, anti-CD28, and Th17 polarizing factors with NK cells in the presence or absence of CD58 neutralizing or isotype control antibody for 4 days. The cytokine IL-17A was assessed by enzyme-linked immunosorbent assay from cell culture supernatants (B). Graph indicates mean IL-17A concentration for N = 3 samples.

Figure 10

CD58 expression levels on natural killer (NK) cells before and after aHSCT treatment of multiple sclerosis (MS) patients. Cryopreserved peripheral blood mononuclear cell (PBMC) from the aHSCT cohort of MS patients was stained for CD3, CD56, and CD58. Representative plots for CD56 and CD58 are shown for baseline (BL) (A) and month 21 [M21; (B)]. A time series of samples from BL until month 24 (M24) is presented (C). For statistical analysis, the time points were grouped in M3–M6, M9–M12, M15–M18, and M21–M24, followed by univariate one-way ANOVA with pairwise comparisons with the BL values. N = 7 patients.