Mass cytometry reveals single-cell kinetics of cytotoxic lymphocyte evolution in CMV-infected renal transplant patients

Abstract

Cytomegalovirus (CMV) infection is associated with graft rejection in renal transplantation. Memory-like natural killer (NK) cells expressing NKG2C and lacking FcεRIγ are established during CMV infection. Additionally, CD8⁺ T cells expressing NKG2C have been observed in some CMV-seropositive patients. However, in vivo kinetics detailing the development and differentiation of these lymphocyte subsets during CMV infection remain limited. Here, we interrogated the in vivo kinetics of lymphocytes in CMV-infected renal transplant patients using longitudinal samples compared with those of nonviremic (NV) patients. Recipient CMV-seropositive (R+) patients had preexisting memory-like NK cells (NKG2C⁺CD57⁺FcεRIγ^-) at baseline, which decreased in the periphery immediately after transplantation in both viremic and NV patients. We identified a subset of prememory-like NK cells (NKG2C⁺CD57⁺FcεRIγ^low-dim) that increased during viremia in R+ viremic patients. These cells showed a higher cytotoxic profile than preexisting memory-like NK cells with transient up-regulation of FcεRIγ and Ki67 expression at the acute phase, with the subsequent accumulation of new memory-like NK cells at later phases of viremia. Furthermore, cytotoxic NKG2C⁺CD8⁺ T cells and γδ T cells significantly increased in viremic patients but not in NV patients. These three different cytotoxic cells combinatorially responded to viremia, showing a relatively early response in R+ viremic patients compared with recipient CMV-seronegative viremic patients. All viremic patients, except one, overcame viremia and did not experience graft rejection. These data provide insights into the in vivo dynamics and interplay of cytotoxic lymphocytes responding to CMV viremia, which are potentially linked with control of CMV viremia to prevent graft rejection.

Keywords: NK cell; cytomegalovirus; cytotoxic lymphocyte; mass cytometry; renal transplantation.

Fig. 1.

Timetable of blood sampling. Time points of blood sampling (triangles) are shown. A pink triangle indicates detection of CMV viremia by PCR. Viremic patients had four or five sampling time points (day 0 of transplantation, previremia, ∼1 wk, ∼1 mo, and late [>1 mo] postviremia). NV patients had three sampling time points (day 0 and 50 and 180 d posttransplant). Viremic patients (n = 11) and NV patients (n = 9) used for longitudinal analysis are described. CMV serostatus in recipient (R) and donor (D) is shown. An R–/D– patient was not on CMV prophylaxis medication. V, viremic patient; MMF, mycophenolate mofetil.

Fig. 2.

Longitudinal changes of count and frequency of each lymphocyte subset in CMV infection. The x axis indicates time points. The y axis indicates count or frequency of each lymphocyte subset. Data at days 50 and 180 in NV patients were plotted at pre- and postviremia time points in viremic patients, respectively. Data represent the average count or percentage ± SEM from CMV viremic patients (pink, n = 11) and NV patients (green, n = 9). Statistical significance was analyzed with the Student’s t test, relative to the previremia time point. n.s., not significant. *P < 0.05; **P < 0.01.

Fig. 3.

Longitudinal kinetics of NK cells subsets in R+ patients. (A) Schema diagrams shown for NK cell subsets identified by t-SNE analysis in R+ patients (Left) and R– patients (Right). Bold line-shaped subsets are significantly expanded subsets in CMV viremic patients. The horizontal line indicates maturity as indicated by CD57 expression. The table defines the signature phenotypes representative of each NK cell subset. The colored plots in the table correspond to the subsets in the schema. New memory-like NK cells are observed only in R+ viremic patients and exhibit FcεRIγ^– to low intermediate level between preexisting memory-like and prememory-like NK cells. CD57⁺KIR⁺ NK cells in R– patients are a cluster composed of NKG2C⁺ (green) and NKG2C^– (gold) cells. (B) Line graphs showing longitudinal changes of the frequencies of six NK cell subsets. Data represent the average from R+ viremic patients (n = 5) and R+ NV patients (n = 6). Statistical significance was analyzed with the Student’s t test, relative to the day 0 time point. (C) Representative t-SNE plots showing longitudinal change of phenotypes in an R+ viremic patient. Colored shapes delineate old (preexisting) memory-like (red), new memory-like (orange), prememory-like (green), and NKG2C⁺CD57⁻ (pink) NK cells. (D) Line graphs showing longitudinal change of the frequencies of the three memory-like NK cells. Data represent the average ± SEM from R+ viremic patients (n = 3). (E) Representative single-cell scatterplots showing Ki67 and FcεRIγ expression of the three memory-like NK cells. The Pearson product–moment correlation coefficient was used to evaluate the association. (F) Box plots showing single-cell Ki67 and FcεRIγ expression of the three memory-like NK cells compiled from R+ viremic (n = 5) and R+ NV patients (n = 6). (G) Heat map showing each marker expression in an R+ viremic patient. Colored boxes indicate marker profiles of the three memory-like NK cells. A dotted line box highlights cytotoxic molecules and intracellular molecules. (H) Line graphs showing longitudinal changes of Ki67 and FcεRIγ expression of six NK cell subsets. Data represent the average from R+ viremic patients (n = 5) and R+ NV patients (n = 6). Statistical significance was analyzed with the Student’s t test, relative to the day 0 time point. n.s., not significant. *P < 0.05; **P < 0.01.

Fig. 4.

Longitudinal kinetics of NK cell subsets in R− patients. (A) Line graphs showing longitudinal changes of frequencies and Ki67 and FcεRIγ expression of seven NK cell subsets. Data represent the average from R– viremic (n = 6) and R– NV patients (n = 3). Statistical significance was analyzed with the Student’s t test, relative to the day 0 time point. (B) Representative t-SNE plots showing longitudinal change of phenotypes in an R– viremic patient. Colored shapes delineate memory-like (red), NKG2C⁺CD57⁻ (pink), and CD57⁺KIR⁺ NK cells, which are a cluster composed of NKG2C⁺ (green) and NKG2C^– (gold). *P < 0.05.

Fig. 5.

Single-cell inhibitory KIR repertoire analysis of NK cells. (A) Heat map showing the frequencies of NK cells with each inhibitory KIR profile in CD57⁺ NK cells without memory-like NK cells (Left) and with memory-like NK cells (Right). Data represent viremic patients (n = 11) and NV patients (n = 9). HLA-C genotypes are described on the top of the heat map. A pink box highlights single KIR2DL3⁺ NK cells. Comparison of the frequency between viremic patients and NV patients was analyzed with the Student’s t test. **P < 0.01. (B) Line graphs showing longitudinal changes of the frequencies of NK cells with each inhibitory KIR profile in a representative R+ viremic patient. Colored boxes and lines highlight single KIR2DL3⁺ (pink), KIR3DL1⁺ (green), and KIR3DL2⁺ (blue) NK cells. (C) Pie charts showing longitudinal changes of inhibitory KIR repertoires of memory-like NK cells in the same patient as in B. The value above the pie chart is the percentage of memory-like NK cells in total NK cells at each time point.

Fig. 6.

Interplay of NK cells, CD8⁺ T cells, and γδ T cells in viremic patients. (A) Representative t-SNE plots showing phenotypes of NKG2C⁺ T cells (red) and triple cytotoxic (granulysin, perforin, granzyme B) γδ T cells (orange). (B) Heat map showing marker profiles of NKG2C⁺ T cells (red) and triple cytotoxic γδ T cells (orange). (C) Frequencies of NKG2C⁺CD8⁺ T cells and triple cytotoxic γδ T cells in viremic (n = 11) and NV (n = 9) patients. Statistical significance was analyzed with the Student’s t test. (D) Three-dimensional line graphs showing longitudinal changes of three different lymphocytes. (Left) Frequencies of NK cells, CD8⁺ T cells, and γδ T cells in viremic (pink) and NV (green) patients. (Right) Frequencies of prememory-like NK cells (in R+ patients) or NKG2C⁺CD57⁺ NK cells (in R– patients), NKG2C⁺CD8⁺ T cells, and triple cytotoxic γδ T cells in R+ (red) and R– (white) viremic patients. Variations from previremia to postviremia time points are depicted. Viremic patient identifications are labeled above the plot. (E) Line graphs showing longitudinal changes of frequencies of the three different cytotoxic lymphocytes in R+ (Upper) and R– (Lower) viremic patients. Data represent the average percentage ± SEM from R+ (n = 5) and R– (n = 6) viremia patients. Statistical significance was analyzed with the Student’s t test, relative to the day 0 time point. (F) Line graphs showing longitudinal changes of cell counts and Ki67 expression of the three different cytotoxic lymphocytes in R+ (Upper) and R– (Lower) viremic patients. Scales of cell counts and Ki67 expression are described on the left and right sides, respectively. Cell count data represent individual (gray) and the average (green) ± SEM from R+ (n = 5) and R– (n = 6) viremic recipients. Ki67 expression represents the average (orange). The statistical significance of the mean slope of linear model was evaluated with the Student’s t test, relative to the previremia time point. *P < 0.05; **P < 0.01.

Fig. 7.

Longitudinal change of CD16-activating anti-gB antibodies in renal transplant patients. Line graphs showing longitudinal changes of CD16-activating anti-gB antibodies in R+ patients (Left) and R– patients (Right) as measured by using a CD16 LacZ BWZ cell reporter assay. Patient identifications of R+ viremic patients are labeled. Red lines indicate viremia, and gray lines indicate NV. Bold lines represent the average ± SEM. Data were from R+ viremic (n = 5), R+ NV (n = 6), R– viremic (n = 6), and R– NV (n = 3) patients. Statistical significance was analyzed with the Student’s t test.