CD8+ T lymphocytes are sensitive to NKG2A/HLA-E licensing interaction: role in the survival of cancer patients

Figures & data

Table 1. Biological, clinical and evolutionary features of patients

	n	Age (Years)	Sex (male)	Follow-up (months)	Progression (rate)	Dead (rate)
Solid Cancer^a	528
Melanoma	311	60.2 ± 15.5	53.8%	60.3 ± 45.1	35.1%	9.4%
Bladder	193	71.1 ± 10.1	85.3%	28.3 ± 27.1	34.6%	20.0%
Ovarian	88	58.7 ± 10.7	0.00%	18.8 ± 21.9	42.0%	22.7%
Plasma cell neoplasm^a	322
MGUS	160	67.3 ± 11.5	53.1%	61.6 ± 29.8	55.0%	13.5%
Multiple myeloma	162	67.1 ± 12.8	52.3%	54.9 ± 37.6	45.0%	37.8%
Childhood acute leukemia^a	97
B Lymphoblastic	70	8.3 ± 4.9	60.0%	41.9 ± 32.9	2.8%	10.0%
T lymphoblastic	16	9.2 ± 4.5	75.0%	46.6 ± 30.6	12.5%	0.00%
Myeloid	16	7.7 ± 5.6	56.3%	37.7 ± 25.9	31.3%	31.3%

^aDetailed information on the histological characteristics for each type of cancer can be found in previous publications in which these patients were included.^1–5

MGUS: monoclonal gammopathy of undetermined significance.

Figure 1. Immunophenotype analysis after in vitro stimulation with anti-CD3/CD28. (a) Gaiting strategy consisted in the following steps: selecting total lymphocytes in an FSC/SSC dot plot, selecting singlets in an FSC-A/FSC-H dot plot, selecting 7-AAD negative and positive alive and dead cells in a dot plot 7-AAD/NKG2D, and selecting CD8⁺ T cells in a CD8/CFSE dot plot and then logical gating to identify singlet alive and dead CD8⁺ T cells. Histogram gates were set in CFSE to identify cells in the zero, first, second, third, fourthor fifth proliferation cycle for alive cells, and in the zero or >1 cycle for dead cells. (b) Dot plots showing CFSE staining in the FITC channel versus the other 11 fluorescences with CD8-BV605, CD226-PE, CD25-PECy7, CD27-APC, CD38-AF700, NKG2D-APCCy7), TIGIT-BV421, CD45RA-BV510, TIM-3-BV711 and NKG2A-BV786

Figure 2. CD226 expression is up-modulated on NK and T cells in presence of specific inhibitory-receptor/ligand interactions. (a) Mean fluorescence intensity (MFI) of CD226 in CD56^dim and CD56^bright NK cells and CD8⁺ T cells (upper plots) and in KIR2DL1⁺, KIR2DL2/L3⁺ and KIR3DL1⁺ NK cell subsets (lower plots) in presence of HLA C1, C2 or Bw4 ligands and −21 M/T dimorphism. P values indicate global estimation in the ANOVA test; * p < .05, ** p < .01, and *** p < .001 in the post hoc tests for each group. (b) Contingency tables for C1, C2 and Bw4 HLA-I ligands versus −21 M/T dimorphism in controls and cancer patients. P values estimated in the χ2 test

Figure 3. Methionine −21 of the HLA-B leader peptide is associated with longer survival in patients with different types of cancer. (a) Kaplan–Meier and log-rank test for overall survival according to the genotype TT, MT or MM of the position −21 of the HLA-B leader peptide in all cancer patients and in patients with solid cancer (melanoma, bladderand ovarian cancer), plasma cell neoplasms and child acute leukemia. Ten-year overall survival rate (10yOS) is indicated for each group of patients. (b) Cox regression analysis of overall survival for sex, age, type of cancer, HLA-B −21 M/T dimorphism, and C1, C2, and Bw4 HLA-ligands

Figure 4. Methionine −21 is associated with higher proliferation and lower expression of TIGIT after anti-CD3/CD28 stimulation of CD8⁺ T cells. (a) Percentage of proliferative CD8⁺ T cells after anti-CD3/CD28 in vitro stimulation according to the −21 M/T genotype in total cells (left) or in cells within the proliferative cycles (right). Cells for each donor were assayed in triplicate and results are shown as mean±SEM. (b) Mean fluorescence intensity (MFI) of TIGIT on CD8⁺ T cells in similar conditions as in A. (c) Expression (MFI) of CD8, CD25, CD27, CD38, CD45RA, CD226, NKG2A, NGK2D TIM3, and CD45RA in total CD8⁺ T cells after in vitro stimulation with anti-CD3/CD28. (d) Expression (MFI) of CD45RA and NKG2D on CD8⁺ T cells within the proliferative cycles. * < 0.05, ** < 0.01and *** < 0.001 in the ANOVA and post hoc tests comparing each MM, MTor TT donors with the other two groups

Figure 5. TIGIT suppresses proliferation of CD8⁺ T cells after anti-CD3/CD28 stimulation. Percentage of proliferative cells within CD8⁺TIGIT⁻ (light green) and CD8⁺TIGIT⁺ (dark green) T cells after anti-CD3/CD28 in vitro stimulation, according to the −21 M/T genotype. Cells for each donor were assayed in triplicate and results are shown as mean±SEM. ** < 0.01 and *** < 0.001 in the Student’s t test comparing CD8⁺TIGIT⁻ and CD8⁺TIGIT⁺ T cells. Representative proliferation histograms are shown for each genotype

short-legendFigure 6.

Guillamón CF, Gimeno L, Server G, Martıinez-Sanchez MV, Escudero JF, Lopez-Cubillana P, et al. Immunological risk stratification of bladder cancer based on peripheral blood natural killer cell biomarkers. European Urology Oncology. 2019;4:246-255. doi: 10.1016/j.euo.2019.04.009.

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