Impaired Thymic Output Can Be Related to the Low Immune Reconstitution and T Cell Repertoire Disturbances in Relapsing Visceral Leishmaniasis Associated HIV/AIDS Patients

Abstract

Background: Visceral leishmaniasis/HIV-co-infected patients (VL/HIV) accounts for around 8% of VL reported cases in Brazil. Relapses of Leishmania infection after anti-leishmanial treatment constitute a great challenge in the clinical practice because of the disease severity and drug resistance. We have shown that non-relapsing-VL/HIV (NR-) evolved with increase of CD4⁺ T-cell counts and reduction of activated CD4⁺ and CD8⁺ T cells after anti-leishmanial treatment. This immune profile was not observed in relapsing-VL/HIV patients (R-), indicating a more severe immunological compromising degree. Elevated activation status may be related to a deficient immune reconstitution and could help to explain the frequent relapses in VL/HIV co-infection. Our aim was to evaluate if this gain of T cells was related to changes in the peripheral TCRVβ repertoire and inflammatory status, as well as the possible thymus involvement in the replenishment of these newly formed T lymphocytes. Methods: VL/HIV patients, grouped into non-relapsing (NR- = 6) and relapsing (R- = 12) were evaluated from the active phase up to 12 months post-treatment (mpt). HIV-infected patients (non-VL) and healthy subjects (HS) were included. The TCRVβ repertoire was evaluated ex vivo by flow cytometry, whereas the plasmatic cytokine levels were assessed by Luminex assay. To evaluate the thymic output, DNA was extracted from PBMCs for TCR rearrangement excision circles (TREC) quantification by qPCR. Results: VL/HIV cases presented an altered mobilization profile (expansions or retractions) of the TCRVβ families when compared to HS independent of the follow-up phase (p < 0.05). TCRVβ repertoire on CD4⁺ T-cells was more homogeneous in the NR-VL/HIV cases, but heterogeneous on CD8⁺ T-cells, since different Vβ-families were mobilized. NR-VL/HIV had the inflammatory pattern reduced after 6 mpt. Importantly, VL/HIV patients showed number of TREC copies lower than controls during all follow-up. An increase of recent thymic emigrants was observed in NR-VL/HIV individuals at 10 mpt compared to R- patients (p < 0.01), who maintained lower TREC contents than the HIV controls. Conclusions: VL/HIV patients that maintain the thymic function, thus generating new T-cells, seem able to replenish the T lymphocyte compartment with effector cells, then enabling parasite control.

Keywords: TCRVβ repertoire; immune response; relapses; thymic output; visceral leishmaniasis/HIV-1 co-infection.

Figure 1

Percentages of the Vβ families on CD4⁺ T lymphocytes among non-relapsing (NR) and relapsing (R) visceral leishmaniasis/HIV (VL/HIV) co-infected patients. The mobilization percentages of Vβ3 (A), Vβ9 (B), Vβ23 (C), Vβ18 (D), Vβ7.2 (E), and Vβ13.6 (F) in NR-VL/HIV co-infected patients in comparison to R-VL/HIV group. The blue asterisk represents the statistical differences in relation to healthy subjects (HS). The column bar represents the median values with interquartile range. *p < 0.05 **p < 0.005.

Figure 2

Percentages of the Vβ families on CD8⁺ T lymphocytes among non-relapsing (NR) and relapsing (R) visceral leishmaniasis/HIV (VL/HIV) co-infected patients. The mobilization percentages of Vβ3 (A), Vβ18 (B), Vβ2 (C), Vβ23 (D), and Vβ7.2 (E) in NR-VL/HIV co-infected patients in comparison to R-VL/HIV group. The blue asterisk represents the statistical differences in relation to healthy subjects (HS). The column bar represents the median values with interquartile range. *p < 0.05 **p < 0.005.

Figure 3

Individual pattern of Vβ repertoire mobilization on CD4⁺ and CD8⁺ T cells of the visceral leishmaniasis/HIV (VL/HIV) co infected patients during all the follow-up by heatmap analysis. To this analysis, the mobilization index was calculated so that the percentage of a given Vβ family presented by each VL/HIV patient was divided by mobilization mean of this Vβ family presented by healthy subjects. Each Vβ family is demonstrated in the line (n = 24) and each VL/HIV patient is represented in the column (NR- 1 to 5; R- 1 to 8) during all phases of follow-up (A, P, 6 and 12). The Vβ families were clustered in accordance with the similarity, using appropriate distance and clustering methods. The red and green scales represent a lower and higher mobilization index, respectively, of a given Vβ family on CD4⁺ T (A) and CD8⁺ T (B) cells for each VL/HIV patient seen individually. Active phase (A); Early post-treatment (P); 6 months post-treatment (6; 6 mpt); 12 months post-treatment (12; 12 mpt).

Figure 4

Anti- and pro-inflammatory cytokines levels and parasite load of the non-relapsing and relapsing-visceral leishmaniasis/HIV (VL/HIV) co-infected patients during clinical follow-up. Plasma cytokine levels of IL-8 (A), TNF (B), IFN-γ (C), IL-6 (D), IL-10 (E) and the parasite load (F) in NR-VL/HIV and R-VL/HIV co-infected patients in the active phase, early post-treatment, 6 and 12 mpt. The cytokines results were represented in Median Fluorescence Intensity (MFI). The cytokine levels were assessed by Luminex assay and the parasite load quantification by qPCR. Each point represents a VL/HIV co-infected patient and each color represents the same patient in the different phases of the follow-up. The horizontal bar represents the median values. Early Post-treat (early post-treatment); 6 mpt (6 months post-treatment); 12 mpt (12 months post-treatment). *p < 0.05 **p < 0.005.

Figure 5

Number of T cell receptor excision circles (TREC) copies/10⁶ PBMC in visceral leishmaniasis/HIV (VL/HIV) co-infected patients during the prospective follow-up and its correlation with number of VL relapses. TREC copy numbers in the VL/HIV-co-infected patients (A) and TRECS copy numbers in non-relapsing-VL/HIV (NR-) and relapsing-VL/HIV (R-) group (B) during the clinical follow-up. The number of TREC copies was evaluated from 1 to 5 × 10⁶ cells/mL obtained of the peripheral blood of all VL/HIV co-infected patients in the active, early post-treatment and 10 mpt phases, as well as HIV mono-infected patients (CHIV) and healthy subjects (HS). Negative correlation (C) between the number of TREC copies at 10 mpt and the total number of relapses presented by VL/HIV patients (Spearman correlation, r = −0.545; p < 0.05). The green and red colors represent the NR-VL/HIV and R- patients, respectively (C). The dashed lines represent the median of TREC copies/10⁶ PBMC of the HIV mono-infected patients and HS (A,B). Each point represents a patient and each color represents the same patient in the different phases of follow-up. The horizontal bar represents the median values. 10 mpt (10 months post-treatment). **p < 0.005.