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. 2015 Nov 1;195(9):4524-36.
doi: 10.4049/jimmunol.1401990. Epub 2015 Sep 28.

Regulation of Adaptive NK Cells and CD8 T Cells by HLA-C Correlates with Allogeneic Hematopoietic Cell Transplantation and with Cytomegalovirus Reactivation

Amir Horowitz  1 , Lisbeth A Guethlein  2 , Neda Nemat-Gorgani  2 , Paul J Norman  2 , Sarah Cooley  3 , Jeffrey S Miller  3 , Peter Parham  4
Affiliations

Regulation of Adaptive NK Cells and CD8 T Cells by HLA-C Correlates with Allogeneic Hematopoietic Cell Transplantation and with Cytomegalovirus Reactivation

Amir Horowitz et al. J Immunol. .

Abstract

Mass cytometry was used to investigate the effect of CMV reactivation on lymphocyte reconstitution in hematopoietic cell transplant patients. For eight transplant recipients (four CMV negative and four CMV positive), we studied PBMCs obtained 6 mo after unrelated donor hematopoietic cell transplantation (HCT). Forty cell-surface markers, distinguishing all major leukocyte populations in PBMC, were analyzed with mass cytometry. This group included 34 NK cell markers. Compared with healthy controls, transplant recipients had higher HLA-C expression on CD56(-)CD16(+) NK cells, B cells, CD33(bright) myeloid cells, and CD4CD8 T cells. The increase in HLA-C expression was greater for CMV-positive HCT recipients than for CMV negative recipients. Present in CMV-positive HCT recipients, but not in CMV-negative HCT recipients or controls, is a population of killer cell Ig-like receptor (KIR)-expressing CD8 T cells not previously described. These CD8 T cells coexpress CD56, CD57, and NKG2C. The HCT recipients also have a population of CD57(+)NKG2A(+) NK cells that preferentially express KIR2DL1. An inverse correlation was observed between the frequencies of CD57(+)NKG2C(+) NK cells and CD57(+)NKG2A(+) NK cells. Although CD57(+)NKG2A(+) NK cells are less abundant in CMV-positive recipients, their phenotype is of a more activated cell than the CD57(+)NKG2A(+) NK cells of controls and CMV-negative HCT recipients. These data demonstrate that HCT and CMV reactivation are associated with an increased expression of HLA-C. This could influence NK cell education during lymphocyte reconstitution. The increased inhibitory KIR expression by proliferating CMV-specific CD8 T cells suggests regulatory interactions between HLA-C and KIR might promote Graft-versus-Leukemia effects following transplantation.

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Figures

Figure 1
Figure 1. HCT recipient characteristics
A list of HCT recipients including information on conditioning regime, type of leukemia and CMV status. HCT recipients were aged 28–61, median age, 44.
Figure 2
Figure 2. HLA genotyping for CMV-negative controls and HCT recipients
Genotypes for HLA-A, -B and –C are provided for sixty CMV-negative controls (green font), four CMV seropositive controls (light blue font), four CMV seronegative (dark blue font) and four CMV-positive (red font) HCT recipients. HLA-A and –B alleles listed in red font indicate expression of the Bw4 epitope. HLA-C allotypes in purple font indicate expression of the C2 epitope.
Figure 3
Figure 3. Comparing NK cell receptor expression on NK cells between controls and HCT recipients
NK cells were defined by a gating strategy described in Fig. S1. Six patterns of perturbations in receptor expression between healthy controls and HCT recipients were observed and are labeled Group 1–6. CMV-neg Controls, Healthy CMV seronegative individuals; CMV-pos Controls, Healthy CMV seropositive individuals; CMV-negative, HCT recipients that are CMV seronegative; CMV-positive, HCT recipients seropositive with reactivated CMV; ns, not significant. Data represent means (± SE).
Figure 4
Figure 4. Comparison of phenotypes and function of adaptive CD57+NKG2C+ and CD57+NKG2A+ NK cells
(A) The frequency of total NK cells that are CD57−NKG2A+, CD57+NKG2A+, CD57−NKG2C+ and CD57+NKG2C+ and (B) mean signal intensity (MSI) of CD94 expression within each population from CMV-negative controls (black bars; N=40), CMV-positive controls (white bars; N=4), CMV-negative (green bars; N=4) and CMV-positive (blue bars; N=4) HCT recipients. (C) Heat maps comparing fold change in frequency of each receptor between CD57+NKG2A+ and CD57+NKG2C+ NK cells in CMV-negative (left column) and CMV-positive (right column) HCT recipients. Shaded boxes represent increased fold change differences biased towards CD57+NKG2C+ (red) and CD57+NKG2A+ (blue). (D) Frequencies of CD57+NKG2A+ NK cells expressing receptors shown to decrease (left column) or increase (right column) in CMV-positive HCT recipients compared to CMV-negative HCT recipients. (E–J) PBMC from CMV-negative (black circles) and CMV-positive (white circles) controls and from CMV-negative (green circles) and CMV- positive (blue circles) HCT recipients were challenged with (E–G) K562 target cells or with (H–J) Raji cells pre-treated with anti-human CD20 antibody. Graphs show the frequencies of NK cell subsets positive for intracellular IFN-γ (E and H), MIP-1β (F and I) and CD107a (G and J). The bars in the dot plots represent mean (± SE). The p values are derived from unpaired Mann Whitney tests. *p<0.05, **p<0.005, ***p<0.0001.
Figure 5
Figure 5. Expression of inhibitory KIR and NKG2A across T cell subsets
(A) The frequency of CD3+ T cells expressing KIR2DL1, KIR2DL2/3, KIR3DL1, KIR3DL2, NKG2A or LILRB1 in CMV-negative (black bars; N=40) and CMV-positive (white bars; N=4) controls and in CMV-negative (green bars; N=4) and CMV-positive (blue bars; N=4) HCT recipients. Data represent means (± SE). The p values are derived from unpaired Mann Whitney tests. *p<0.05, **p<0.005, ***p<0.0001. (B) Heat map summarizing the proportion of KIR+, NKG2A+ and LILRB1+ T cells that are skewed towards CD4 (blue boxes), CD8 (red bars) or balanced between CD4 and CD8 (black boxes) T cells in CMV-negative and seropositive controls and in CMV-negative and CMV-positive HCT recipients. (C) Spanning-tree Progression Analysis of Density-normalized Events (SPADE) for 3 representative samples (CMV-negative control, CMV-negative and CMV-positive HCT recipients) evaluating distribution of KIR2DL1 (left column), KIR2DL2/3 (second column from left), KIR3DL2 (third column from left) and LILRB1 (right column) expression on CD4, CD8 and CD4CD8 T cells. Node color represents signal intensity and size represents frequency. A SPADE analysis evaluating distribution of all other measured receptors is shown in Figs. S2 and S3.
Figure 6
Figure 6. Comparing the functions between Null CD56+ T cells and CD56+ T cells expressing inhibitory NK receptors
PBMC from CMV-negative controls (green), CMV-seropositive controls (light blue), CMV-negative (dark blue) and CMV-positive (red) HCT recipients were incubated with K562 cells. (A) Comparing the frequency of CD3 T cells expressing CD56 in controls and in HCT recipients. (B) Comparing the proportion of CD56+ T cells that stain positive or negative for inhibitory KIR, NKG2A or LILRB1 (Null). (C) Comparing the frequency of Null CD3 T cells producing IFN-γ (circles) or expressing CD107a (squares) in controls and in HCT recipients. (D) Heatmap summary for Boolean gating analysis of inhibitory KIR, NKG2A and LILRB1 expression on CD56+ T cells resulting in sixty-four possible subsets. Each subset is grouped and color-coded according to the number of inhibitory receptors expressed. The color of each box ranges from black to yellow (range: 0–30%) according to the scale bar beneath the heatmap. (E and F) The frequency of each CD56+ T cell subset producing IFN-γ (E) and expressing CD107a (F) in CMV-negative (light blue circles) and CMV-positive (red circles) HCT recipients. The bars underneath the dot plots represent mean (± SE). The p values are derived from unpaired Mann Whitney tests. *p<0.05, **p<0.005.
Figure 7
Figure 7. Expression of HLA-C across major cell lineages in peripheral blood associates with HLA-C allotype but also with CMV reactivation following transplantation
PBMC from controls (N=60), CMV-negative (N=4) and CMV-positive (N=4) HCT recipients were stained ex vivo and analyzed for expression of HLA-C. (A) Mean Signal intensity (MSI) of HLA-C from PBMC is plotted twice for each sample (once for each HLA-C allele). There is strong correlation between expression level of HLA-C and HLA-C allotypes in analysis of variance (p <0.0001). MSI HLA-C from CMV-negative (red circles) and CMV-positive HCT recipients (blue circles) is compared to healthy cmv-negative controls (black circles) matched for HLA-C gene content. (B) Histogram measuring MSI HLA-C on all major cell lineages from CMV-negative controls (grey bars), CMV-negative (green bars) and CMV-positive (blue bars) HCT recipients. Data represent means (± SE). The p values are derived from unpaired Mann Whitney tests. **p<0.005, ***p<0.0001. (C) SPADE clustering analysis for the same 3 representative samples used in Fig. 5 (Controls, CMV-negative and CMV-positive HCT recipients) for evaluating distribution of HLA-C expression across major cell lineages. Node color represents HLA-C signal intensity and size represents frequency.
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