Cytotoxic cell populations developed during treatment with tyrosine kinase inhibitors protect autologous CD4+ T cells from HIV-1 infection

Abstract

Tyrosine kinase inhibitors (TKIs) are successfully used in clinic to treat chronic myeloid leukemia (CML). Our group previously described that CD4+ T cells from patients with CML on treatment with TKIs such as dasatinib were resistant to HIV-1 infection ex vivo. The main mechanism for this antiviral activity was primarily based on the inhibition of SAMHD1 phosphorylation, which preserves the activity against HIV-1 of this innate immune factor. Approximately 50% CML patients who achieved a deep molecular response (DMR) may safely withdraw TKI treatment without molecular recurrence. Therefore, it has been speculated that TKIs may induce a potent antileukemic response that is maintained in most patients even one year after treatment interruption (TI). Subsequent to in vitro T-cell activation, we observed that SAMHD1 was phosphorylated in CD4+ T cells from CML patients who withdrew TKI treatment more than one year earlier, which indicated that these cells were now susceptible to HIV-1 infection. Importantly, these patients were seronegative for HIV-1 and seropositive for cytomegalovirus (CMV), but without CMV viremia. Although activated CD4+ T cells from CML patients on TI were apparently permissive to HIV-1 infection ex vivo, the frequency of proviral integration was reduced more than 12-fold on average when these cells were infected ex vivo in comparison with cells isolated from untreated, healthy donors. This reduced susceptibility to infection could be related to an enhanced NK-dependent cytotoxic activity, which was increased 8-fold on average when CD4+ T cells were infected ex vivo with HIV-1 in the presence of autologous NK cells. Enhanced cytotoxic activity was also observed in CD8 + T cells from these patients, which showed 8-fold increased expression of TCRγδ and more than 18-fold increased production of IFNγ upon activation with CMV peptides. In conclusion, treatment with TKIs induced a potent antileukemic response that may also have antiviral effects against HIV-1 and CMV, suggesting that transient use of TKIs in HIV-infected patients could develop a sustained antiviral response that would potentially interfere with HIV-1 reservoir dynamics.

Keywords: Chronic myeloid leukemia; HIV-1; SAMHD1; Src tyrosine kinases; Viral reservoir.

Figure 1.

Expression of activation markers in CD4+ T cells from patients with CML on TFR. (A) Analysis by flow cytometry of the expression of activation markers CD25 and CD69 in resting and activated CD4+ T cells from CML patients on TFR in comparison with cells from healthy donors. T cell activation was performed with PHA and IL-2 for 3 days. (B) SAMHD1 phosphorylation was analyzed in the same cells by flow cytometry after intracellular staining. Each dot corresponds to one sample and lines represent mean ± standard error of the mean (SEM). Statistical significance was calculated using unpaired, non-parametric Mann-Whitney t test. *p < 0.05; *p < 0.01.

Figure 2.

PBMCs from CML patients on TFR showed resistance to HIV-1 proviral integration and transcription. (A) Analysis by qPCR of proviral integration in PBMCs infected in vitro with NL4–3_Renilla for 7 days and (B) quantification of the synthesis of renilla by chemiluminescence in the same cells. Each dot corresponds to one sample and lines represent mean ± SEM. Statistical significance was calculated using unpaired, non-parametric Mann-Whitney t test. *** p < 0.001; **** p < 0.0001.

Figure 3.

Phenotype and cytotoxic activity of NK cell populations in patients with CML on TFR. (A) Analysis by flow cytometry of the expression levels of CD56 and CD16 in NK cells from PBMCs of patients compared with healthy donors. (B) Percentage of intracellular levels of IFNγ produced by NK cells with CD3-CD56+CD16± phenotype from PBMCs of CML patients on TFR compared with healthy donors. (C) Expression levels of NK cells with memory phenotype CD3-CD56+CD57+ in PBMCs from CML patients on TFR compared with healthy donors. Each dot corresponds to one sample and lines represent mean ± SEM. Statistical significance was calculated using unpaired, non-parametric Mann-Whitney t test. * p < 0.05; ** p < 0.01.

Figure 4.

Phenotype and cytotoxic activity of CD8+ T cell populations in patients with CML on TFR. Analysis of cell populations expressing TCRγδ that were CD3+CD8+ (A) or CD8- (B) in patients with CML on TFR compared with healthy donors. (C) Fold levels of IgG against CMV in plasma from CML patients on TFR, compared with healthy donors. (D) Production of IFNγ in CD3+CD8+ T cells from CML patients on TFR was analyzed by flow cytometry after stimulation with CMV peptides, compared with PBMCs from patients who went under renal transplant and suffered afterwards CMV reactivation and from healthy donors. Each dot corresponds to one sample and lines represent mean ± SEM. Statistical significance was calculated using unpaired, non-parametric Mann-Whitney t test. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 5.

Antiviral effect of NK cells from CML patients on TFR against HIV-1 infection ex vivo of autologous CD4+ T cells. (A) Fold of HIV-1 proviral integration in CD4+ T cells isolated from patients with CML on TFR compared with cells from healthy donors, in the presence or absence of autologous NK cells, and (B) fold of cytotoxic activity of these NK cells. Each dot corresponds to one sample and lines represent mean ± SEM. Statistical significance was calculated using unpaired, non-parametric Mann-Whitney t test. *** p < 0.001; **** p < 0.0001.