T Lymphocytes in Patients With Nijmegen Breakage Syndrome Demonstrate Features of Exhaustion and Senescence in Flow Cytometric Evaluation of Maturation Pathway

Abstract

Patients with Nijmegen Breakage Syndrome (NBS) suffer from recurrent infections due to humoral and cellular immune deficiency. Despite low number of T lymphocytes and their maturation defect, the clinical manifestations of cell-mediated deficiency are not as severe as in case of patients with other types of combined immune deficiencies and similar T cell lymphopenia. In this study, multicolor flow cytometry was used for evaluation of peripheral T lymphocyte maturation according to the currently known differentiation pathway, in 46 patients with genetically confirmed NBS and 46 sex and age-matched controls. Evaluation of differential expression of CD27, CD31, CD45RA, CD95, and CD197 revealed existence of cell subsets so far not described in NBS patients. Although recent thymic emigrants and naïve T lymphocyte cell populations were significantly lower, the generation of antigen-primed T cells was similar or even greater in NBS patients than in healthy controls. Moreover, the senescent and exhausted T cell populations defined by expression of CD57, KLRG1, and PD1 were more numerous than in healthy people. Although this hypothesis needs further investigations, such properties might be related to an increased susceptibility to malignancy and milder clinical course than expected in view of T cell lymphopenia in patients with NBS.

Keywords: Nijmegen Breakage Syndrome; T lymphocyte maturation; flow cytometry; immune exhaustion; immune senescence; primary immune deficiency.

Figure 1

Evaluation of basic lymphocyte subsets. Patients with NBS demonstrated significantly smaller general population of lymphocytes, with disturbed distribution of T (CD3⁺), T CD4⁺ (CD3⁺CD4⁺), T CD8⁺ (CD3⁺CD8⁺), NK (CD3⁻CD16⁺CD56⁺), and B (CD19⁺) lymphocyte subsets in comparison to healthy controls. Individual results in respective study cohorts are presented as circles. Median values are presented as bars with numerical values. Statistical significance: ** <0.01. (A) Relative counts and (B) absolute counts.

Figure 2

Gating strategy for evaluation of T lymphocyte maturation process. Lymphocyte subsets were identified by differential expression of CD27, CD31, CD45RA, CD95, and CD197. T lymphocytes were gated based on CD3 vs. side scatter characteristics. Identification of individual populations is presented for T helper cells. Identical strategy was used for T CD8⁺ cell subsets. (A) T helper and T suppressor cells were identified within T cell gate as CD3⁺CD4⁺ and CD3⁺CD8⁺, respectively. (B) RTE CD31⁺CD45RA⁺ were identified within the CD3⁺CD4⁺ gate. (C) Two additional gates: CD27⁺ and CD27⁻ were set. (D) Within CD27⁺ gate TN have been defined as CD27⁺CD45RA⁺CD197⁺, TCM as CD27⁺CD45RA⁻CD197⁺, TEM as CD27⁺CD45RA⁻CD197⁻, and low-differentiated effector RA⁺ L-TEMRA as CD27⁺CD45RA⁺CD197⁻). (E) High differentiated effector RA+ (H-TEMRA CD27⁻CD45RA⁺CD197⁻) and terminally differentiated (TD, CD27⁻CD45RA⁻CD197⁻) have been identified within the CD27⁻ gate. (F) CD95 gate was drawn within the T CD4⁺ gate. (G) Two populations were identified within CD95⁺ gate: TSCM (CD27⁺CD45RA⁺CD95⁺), and effector RA⁺ (TEMRA, CD27⁻CD45RA⁺CD95⁺). (H) Identification of cells with positive expression of CD57, KLRG1, and PD1 on the whole T CD4⁺ population.

Figure 3

Peripheral T helper cell maturation was significantly disturbed. Individual results in respective study cohorts are presented as circles. Median values are presented as bars with numerical values. Statistical significance: NS, not significant, ** <0.01. (A) Median relative counts of T helper cell subsets in NBS patients in relation to normal control. Patients with NBS demonstrated significantly lower proportions of RTE and naïve helper cells, and significantly higher proportions of TCM, TEM, H-TEMRA, TEMRA, and TD lymphocytes. There was no statistical difference between TSCM and L-TEMRA. (B) Median absolute counts of T helper cell subsets in relation to normal control. Patients with NBS demonstrated significantly different absolute counts of all analyzed T helper subsets, except for H-TEMRA and TEMRA.

Figure 4

Peripheral T CD8⁺ lymphocyte maturation was significantly disturbed. Individual results in respective study cohorts are presented as circles. Median values are presented as bars with numerical values. Statistical significance: NS, not significant, * <0.05, ** <0.01. (A) Relative counts of T CD8⁺ cell subsets in NBS patients in relation to normal control. Patients with NBS demonstrated significantly lower proportions of T CD8 cells with phenotype corresponding to RTE (CD31⁺CD45RA⁺) and naïve cells, and significantly higher proportions of TCM, H-TEMRA, TEMRA, and TD lymphocytes. There was no statistical difference in relative distribution of TSCM, TEM, and L-TEMRA. (B) Absolute counts of individual studied CD8⁺ T lymphocyte populations. Significantly smaller populations of CD31⁺CD45RA⁺, TN, TSCM, TEM, and L-TEMRA cells were observed in NBS patients in comparison to controls. TEM, H-TEMRA, TEMRA, and TD composed similar populations in NBS and healthy cohorts.

Figure 5

Expression of senescence (CD57, KLRG1) and senescence (PD1=CD279) cell markers. Statistical significance: NS, not significant, * <0.05, ** <0.01. (A) Patients with NBS demonstrated significantly elevated proportions of T lymphocytes (from both CD4⁺ and CD8⁺ subsets) with features of senescence (expression of CD57 and KLRG1), as well as exhaustion (CD279) than healthy controls. (B) T helper cells composed significantly more numerous populations of cells expressing (CD57, KLRG1, and CD279) in NBS patients than healthy controls. No statistical difference in absolute counts within the studied populations was found among T CD8⁺ lymphocytes.