Functional impairment of Tax-specific but not cytomegalovirus-specific CD8+ T lymphocytes in a minor population of asymptomatic human T-cell leukemia virus type 1-carriers

Abstract

Background: Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T-cell response, an important host defense system. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) already showed impaired T-cell responses against the major target antigen, Tax. However, it is unclear whether the impaired HTLV-1 Tax-specific T-cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, in order to characterize the impaired HTLV-1-specific T-cell response, we investigated the function of Tax-specific CD8+ T-cells in various clinical status of HTLV-1 infection.

Results: By using tetramers consisting of HLA-A*0201, -A*2402, or -A*1101, and corresponding Tax epitope peptides, we detected Tax-specific CD8+ T-cells in the peripheral blood from 87.0% of ACs (n = 20/23) and 100% of HAM/TSP patients (n = 18/18) tested. We also detected Tax-specific CD8+ T-cells in 38.1% of chronic type ATL (cATL) patients (n = 8/21), although its frequencies in peripheral blood CD8+ T cells were significantly lower than those of ACs or HAM/TSP patients. Tax-specific CD8+ T-cells detected in HAM/TSP patients proliferated well in culture and produced IFN-γ when stimulated with Tax peptides. However, such functions were severely impaired in the Tax-specific CD8+ T-cells detected in cATL patients. In ACs, the responses of Tax-specific CD8+ T-cells were retained in most cases. However, we found one AC sample whose Tax-specific CD8+ T-cells hardly produced IFN-γ, and failed to proliferate and express activation (CD69) and degranulation (CD107a) markers in response to Tax peptide. Importantly, the same AC sample contained cytomegalovirus (CMV) pp65-specific CD8+ T-cells that possessed functions upon CMV pp65 peptide stimulation. We further examined additional samples of two smoldering type ATL patients and found that they also showed dysfunctions of Tax-specific but not CMV-specific CD8+ T-cells.

Conclusions: These findings indicated that Tax-specific CD8+ T-cells were scarce and dysfunctional not only in ATL patients but also in a limited AC population, and that the dysfunction was selective for HTLV-1-specifc CD8+ T-cells in early stages.

Figure 1

Incidence and frequency of Tax-specific CD8⁺ T-cells in ACs, and HAM/TSP and cATL patients. (A) Whole blood or PBMCs from AC (top), and HAM/TSP (middle) and cATL (bottom) patients were stained with Tax/HLA tetramer. Number indicates the percentage of tetramer⁺ cells in CD8⁺ T-cells. (B) The percentage of Tax-tetramer⁺ CD8⁺ T-cells in AC (n = 23), and HAM/TSP (n = 18) and cATL (n = 21) patients. P value was determined by the Mann-Whitney U test. Horizontal bars indicate the average percentage of Tax-tetramer⁺ CD8⁺ T-cells for the group.

Figure 2

IFN-γ production and proliferation of Tax-specific CD8⁺ T-cells in HAM/TSP and cATL patients. (A) PBMCs from HAM/TSP and cATL patients were stimulated with or without 10 μM Tax peptide for 6 hrs. The number indicates the percentage of IFN-γ-producing cells in tetramer⁺ cells. (B) For CFSE-based T-cell proliferation, CFSE-labeled PBMCs from HAM/TSP and cATL patients were cultured in the presence or absence of 100 nM Tax peptide for 6 days. The number indicates the percentage of dividing (CFSE^low) cells in tetramer⁺ cells. The percentage of tetramer⁺ cells among CD8⁺ T cells in fresh blood is indicated in parenthesis under the patient ID.

Figure 3

IFN-γ production and cell proliferation of Tax-specific CD8⁺ T-cells in ACs. (A, B) IFN-γ production (A) and cell proliferation (B) of Tax-specific CD8⁺ T-cells in PBMCs from 4 ACs were assessed as in Figure 2. The number given in parenthesis shows mean fluorescence intensity (MFI) of IFN-γ expression in the IFN-γ⁺ tetramer⁺ cells. (C, D) Relation between the percentage of IFN-γ⁺ (C) or dividing (D) Tax-specific CD8⁺ T-cells and proviral loads (PVL) in ACs. Dots represent individual ACs. The Spearman rank correlation test was used to determine correlations and P values.

Figure 4

Dysfunction of Tax-specific CD8⁺ T-cells and inefficient CD8⁺ cell-mediated HTLV-1 control in AC#287. (A) For antigen-specific T-cell proliferation, PBMCs from #313 and #287 were cultured for 13 days with or without Tax peptide in the presence or absence of 0.1 μg/ml LPS. The number indicates the percentage of tetramer⁺ cells in CD8⁺ T-cells. (B, C) PBMCs were stimulated with or without 10 μM Tax peptide for 6 hrs. The expression of CD69 (B) and CD107a (C) in Tax-specific CD8⁺ T-cells was analyzed by flow cytometry. (B) Bar indicates the percentage of CD69⁺ cells in Tax-specific CD8⁺ T-cells. (C) The number represents the percentage of CD107a⁺ cells in Tax-specific CD8⁺ T-cells. (D) Whole PBMCs and CD8-depleted fractions in ACs (#287 and #313) were cultured for 7 days and HTLV-1 p19 in the supernatants were measured by HTLV-1 p19 ELISA. P value was determined by the unpaired t test.

Figure 5

Phenotypic analysis of functional and dysfunctional Tax-specific CD8⁺ T-cells. (A) Differentiation memory phenotype, based on the expression of CD45RA, CCR7, and CD27 and (B) PD-1 expression of Tax-specific CD8⁺ T-cells from ACs were examined by flow cytometry. The number represents the percentage of indicated marker-positive or -negative cells in tetramer⁺ CD8⁺ T-cells. The number given in parenthesis shows MFI of PD-1 expression on the PD-1⁺ tetramer⁺ cells.

Figure 6

Conserved functions of CMV-specific CD8⁺ T-cells in AC#287. (A) For antigen-specific T-cell proliferation, PBMCs from #287 were cultured for 13 days with or without 100 nM CMV peptide. The number indicates the percentage of CMV tetramer⁺ cells in CD8⁺ T-cells. (B-D) PBMCs were stimulated with or without 10 μM CMV peptide for 6 hrs. IFN-γ production (B), CD69 (C) and CD107a (D) expression of CMVpp65-specific CD8⁺ T-cells in #287 was analyzed by flow cytometry. (B, D) The number represents the percentage of the indicated marker-positive cells in CMVpp65-specific CD8⁺ T-cells. (C) Bar indicates the percentage of CD69⁺ cells in CMV-specific CD8⁺ T-cells.

Figure 7

Impaired proliferation of Tax-specific but not CMVpp65-specific CD8⁺ T-cells in sATL patients. For antigen-specific T-cell proliferation, PBMCs from sATL (#110; square, #353; triangle) and cATL (#224; circle) patients were cultured for 13 days with 100 nM Tax (A) or CMV (B) peptide. Each dot indicates the percentage of tetramer⁺ cells in CD8⁺ T-cells at day 0 and day 13 after culture. Clinical information on ATL patients used here is as follows; sATL#110: age; 40 s, gender; F, WBC#; 11,000/μL {lymphocyte (lym); 39%, abnormal lymphocytes (ably); 4%}, cATL#224: age; 50 s, gender; F, WBC#; 7900/μL (lym; 30%, ably; 33%), sATL#353: age; 60 s, gender; M, WBC#; 4620/μL (lym; 39%, ably; 5%).