Limited Foxp3+ Regulatory T Cells Response During Acute Trypanosoma cruzi Infection Is Required to Allow the Emergence of Robust Parasite-Specific CD8+ T Cell Immunity

While it is now acknowledged that CD4+ T cells expressing CD25 and Foxp3 (Treg cells) regulate immune responses and, consequently, influence the pathogenesis of infectious diseases, the regulatory response mediated by Treg cells upon infection by Trypanosoma cruzi was still poorly characterized. In order to understand the role of Treg cells during infection by this protozoan parasite, we determined in time and space the magnitude of the regulatory response and the phenotypic, functional and transcriptional features of the Treg cell population in infected mice. Contrary to the accumulation of Treg cells reported in most chronic infections in mice and humans, experimental T. cruzi infection was characterized by sustained numbers but decreased relative frequency of Treg cells. The reduction in Treg cell frequency resulted from a massive accumulation of effector immune cells, and inversely correlated with the magnitude of the effector immune response as well as with emergence of acute immunopathology. In order to understand the causes underlying the marked reduction in Treg cell frequency, we evaluated the dynamics of the Treg cell population and found a low proliferation rate and limited accrual of peripheral Treg cells during infection. We also observed that Treg cells became activated and acquired a phenotypic and transcriptional profile consistent with suppression of type 1 inflammatory responses. To assess the biological relevance of the relative reduction in Treg cells frequency observed during T. cruzi infection, we transferred in vitro differentiated Treg cells at early moments, when the deregulation of the ratio between regulatory and conventional T cells becomes significant. Intravenous injection of Treg cells dampened parasite-specific CD8+ T cell immunity and affected parasite control in blood and tissues. Altogether, our results show that limited Treg cell response during the acute phase of T. cruzi infection enables the emergence of protective anti-parasite CD8+ T cell immunity and critically influences host resistance

IL-17RA-Signaling Modulates CD8+ T Cell Survival and Exhaustion During Trypanosoma cruzi Infection

The IL-17 family contributes to host defense against many intracellular pathogens by mechanisms that are not fully understood. CD8+ T lymphocytes are key elements against intracellular microbes, and their survival and ability to mount cytotoxic responses are orchestrated by several cytokines. Here, we demonstrated that IL-17RA-signaling cytokines sustain pathogen-specific CD8+ T cell immunity. The absence of IL-17RA and IL-17A/F during Trypanosoma cruzi infection resulted in increased tissue parasitism and reduced frequency of parasite-specific CD8+ T cells. Impaired IL-17RA-signaling in vivo increased apoptosis of parasite-specific CD8+ T cells, while in vitro recombinant IL-17 down-regulated the pro-apoptotic protein BAD and promoted the survival of activated CD8+ T cells. Phenotypic, functional, and transcriptomic profiling showed that T. cruzi-specific CD8+ T cells derived from IL-17RA-deficient mice presented features of cell dysfunction. PD-L1 blockade partially restored the magnitude of CD8+ T cell responses and parasite control in these mice. Adoptive transfer experiments established that IL-17RA-signaling is intrinsically required for the proper maintenance of functional effector CD8+ T cells. Altogether, our results identify IL-17RA and IL-17A as critical factors for sustaining CD8+ T cell immunity to T. cruzi

CD8+ T Cell Immunity Is Compromised by Anti-CD20 Treatment and Rescued by Interleukin-17A

Monoclonal antibody targeting the CD20 antigen on B cells is used to treat the majority of non-Hodgkin lymphoma patients and some autoimmune disorders. This therapy generates adverse effects, notably opportunistic infections and activation of viruses from latency. Here, using the infection murine model with the intracellular parasite Trypanosoma cruzi, we report that anti-CD20 treatment affects not only B cell responses but also CD8+ T cell responses, representing the most important immune effectors involved in control of intracellular pathogens. Anti-CD20 treatment, directly or indirectly, affects cytotoxic T cell number and function, and this deficient response was rescued by the cytokine IL-17A. The identification of IL-17A as the cytokine capable of reversing the poor response of CD8+ T cells provides information about a potential therapeutic treatment aimed at enhancing defective immunity induced by B cell depletion.Treatment with anti-CD20, used in many diseases in which B cells play a pathogenic role, has been associated with susceptibility to intracellular infections. Here, we studied the effect of anti-CD20 injection on CD8+ T cell immunity using an experimental model of Trypanosoma cruzi infection, in which CD8+ T cells play a pivotal role. C57BL/6 mice were treated with anti-CD20 for B cell depletion prior to T. cruzi infection. Infected anti-CD20-treated mice exhibited a CD8+ T cell response with a conserved expansion phase followed by an early contraction, resulting in a strong reduction in total and parasite-specific CD8+ T cell numbers at 20 days postinfection. Anti-CD20 injection increased the frequency of apoptotic CD8+ T cells, decreased the number of effector and memory CD8+ T cells, and reduced the frequency of proliferating and cytokine-producing CD8+ T cells. Accordingly, infected anti-CD20-treated mice presented lower cytotoxicity of T. cruzi peptide-pulsed target cells in vivo. All of these alterations in CD8+ T cell immunity were associated with increased tissue parasitism. Anti-CD20 injection also dampened the CD8+ T cell response, when this had already been generated, indicating that B cells were involved in the maintenance rather than the induction of CD8+ T cell immunity. Anti-CD20 injection also resulted in a marked reduction in the frequency of interleukin-6 (IL-6)- and IL-17A-producing cells, and recombinant IL-17A (rIL-17A) injection partially restored the CD8+ T cell response in infected anti-CD20-treated mice. Thus, anti-CD20 reduced CD8+ T cell immunity, and IL-17A is a candidate for rescuing deficient responses either directly or indirectly

Control of DT treatment toxicity and evaluation of its impact on biochemical markers of damage associated with the progression of infection.

A-B) Parasite counts (A) and TSKB20-specific CD8+ T cell frequencies (B) in blood from PBS or DT-treated T. cruzi infected WT littermate mice at 20 dpi. Data were collected from 3 independent experiments and are presented as mean ± SEM. Statistical significance was determined by Mann Whitney test. C) Parasites numbers counted after 24 h of culture with PBS, increased doses of DT or benznidazole (BZ). Data are presented as mean ± SD of technical triplicates from 1 experiment. D) Treg cell depletion effect on tissue damage markers: activities of glutamate-oxalacetic transaminase (GOT), glutamate-pyruvate transaminase (GPT), lactate dehydrogenase (LDH), creatine phosphokinase (CPK), and creatine phosphokinase of muscle and brain (CPK MB), as well as Glucose concentration in plasma of PBS or DT-treated DEREG mice at days 20 and 33 pi. Data were collected from 6 independent experiments at 20 dpi (n = 20–26) and from 1 experiment at 33 dpi (n = 4–5). Data are presented as mean ± SEM. Statistical significance was determined by Unpaired t test for GOT, LDH, CPK, and CPK MB activities and Glucose concentration, and by Mann Whitney test for GPT activity, according to data distribution. P values for pairwise comparisons at day 20 pi are indicated in the graphs. ns = not significant. (PDF)</p

Effect of early Treg cell depletion on CD8+ T cell function.

A) Gating strategy for assessing effector cytokine production and CD107a surface mobilization in gated CD8+ cells. B) Percentage of CD8+ T cells from the spleen of PBS or DT-treated DEREG mice at day 21 pi that exhibit different combinations of effector functions, including CD107a mobilization and/or IFN-γ and/or TNF production upon 5h of the indicated stimulation. Medium condition was used as a negative control, while PMA/Ionomycin (PMA/Iono) was used as a positive control for polyclonal CD8+ T cell stimulation. Similar results were obtained in 2 independent experiments. All data are presented as mean ± SEM. Each symbol represents one individual mouse. Statistical significance was determined by Mann Whitney test. (PDF)</p

Early Treg cell depletion promotes parasite-specific CD8+ T cell differentiation into SLEC during <i>T</i>. <i>cruzi</i> infection.

A) Representative flow cytometry plots showing KLRG-1+ CD127- (SLEC) and KLRG-1- CD127+ (MPEC) subsets within CD44+ gated TSKB20-specific CD8+ T cells in the indicated organs obtained from PBS or DT-treated DEREG mice at day 20 pi. B-C) Frequencies (left) and absolute numbers (right) of SLEC (B) and MPEC (C) subsets within CD44+ gated TSKB20-specific CD8+ T cells of mice in (A). Data were collected from 2 independent experiments. D) Percentages of CD8+ T cells that produce IFN-γ and exhibit CD107a mobilization together (left) or not (right) with TNF production in the spleen of PBS or DT-treated DEREG mice at day 21 pi. Medium condition was used as a negative control, while PMA/Ionomycin (PMA/Iono) was used as a positive control for polyclonal CD8+ T cell stimulation. Similar results were obtained in 2 independent experiments. All data are presented as mean ± SEM. In (B), (C) and (D) each symbol represents one individual mouse. Statistical significance was determined by Unpaired t test or Mann Whitney test, according to data distribution. P values for pairwise comparisons are indicated in the graphs.</p

Treg cell depletion increases <i>T</i>. <i>cruzi</i>-specific CD8+ T cell expansion and improves parasite control during acute infection.

A) Kinetics of blood parasite counts in PBS or DT-treated DEREG mice. B) Parasite load in heart, liver, and spleen of PBS or DT-treated DEREG mice at day 20 pi. Values were calculated using the ΔΔCT algorithm, with GAPDH utilized as a housekeeping control for normalization, and the sample of PBS-treated mice serving as a reference. C-E) Representative flow cytometry dot plots showing TSKB20-specific CD8+ T cell detection at day 20 pi (C), their frequency quantification in blood at different dpi (D), and in spleen and liver at day 20 pi (E) of PBS or DT-treated DEREG mice. F) Absolute numbers of TSKB20-specific CD8+ T cells in spleen and liver according to (E). All data are presented as mean ± SEM. In (A), (D), (E) and (F) data were collected from 1–3 independent experiments at most dpi, and 5–8 independent experiments at day 20 pi according to the analyzed tissue. In (B) data were pooled from 2–4 independent experiments. In (E-F) each symbol represents one individual mouse. A total of 4–42 mice per group were included. In (A) n = 11–12 at 8 dpi, n = 12 at 11 dpi, n = 11 at 14 dpi, n = 35–42 at 20 dpi, n = 7–9 at 25 dpi, n = 4–5 at 33 dpi. In (D) n = 12–13 at 10 dpi, n = 7 at 12 dpi, n = 16–18 at 20 dpi, n = 6 at 25 dpi, n = 12 at 33 dpi, n = 5–7 at 55 dpi. Statistical significance was determined by Unpaired t test or Mann Whitney test, according to data distribution. P values for pairwise comparisons are indicated in the graphs. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 and ns = not significant.</p

Depletion of Treg cells on days 11 and 12 pi had no impact on parasitemia levels or parasite-specific CD8+ T cell numbers.

A) Experimental scheme for DT treatment (created with BioRender.com). B-C) Treg cell frequencies in blood at different dpi (B) and in spleen at day 21 pi (C) from T. cruzi-infected DEREG mice treated with PBS or DT on days 11 and 12 pi. D) Parasitemia levels from mice in (A). E-F) TSKB20-specific CD8+ T cell frequencies in blood at different dpi (E) and in spleen at day 21 pi (F) of mice in (A). All data are presented as mean ± SEM. Data were collected from 1–2 independent experiments. A total of 2–6 mice per group were included. Statistical significance was determined by Unpaired t test or Mann Whitney test, according to data distribution. P values for pairwise comparisons are indicated in the graphs. * P ≤ 0.05. (PDF)</p

Treg cell depletion promotes the expansion and activation of Tconv cells in <i>T</i>. <i>cruzi</i> target organs.

A-B) Frequencies (A) and absolute numbers (B) of Tconv cells in blood, spleen, and liver from PBS or DT-treated DEREG mice at day 11 pi. C-D) Representative flow cytometry plots (C) and frequency (D) of CD44- CD62L+ (naïve), CD44+ CD62L+ (memory) and CD44+ CD62L- (effector) Tconv cell subsets in the spleen and liver from mice in (A). E-F) Representative flow cytometry plots (E) and frequency (F) of KLRG-1+ and CD25+ Tconv cells in the spleen from mice in (A). All data are presented as mean ± SEM. Each symbol represents one individual mouse. Data in (A), (B), (D) and (F) were pooled from 1–2 independent experiments. Statistical significance was determined by Unpaired t test or Mann Whitney test, according to data distribution. P values for pairwise comparisons are indicated in the graphs. In (D), P values PBS vs DT: + P≤ 0.05 and +++ P ≤ 0.001, naïve Tconv cells; # P<0.05, memory Tconv cells; ** P ≤ 0.01, effector T conv cells.</p

Treg cell depletion induces modest effects on APC populations and innate cells.

A) UMAP visualization of flow cytometry data from the spleen of PBS or DT-treated DEREG mice at day 7 pi and PBS-treated non-infected controls. B) Histograms showing the expression of different APC and innate cell activation markers in selected cell clusters. Samples from the three experimental groups (NI PBS, INF PBS and INF DT) were pooled together. C) Representative flow cytometry plots showing CD86+ cells in the indicated cell clusters as defined in (A). D) Frequency of CD86+ cells in the indicated cell clusters as defined in (A). Data are presented as mean ± SEM. Each symbol represents one individual mouse. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. Similar results were obtained in 3 independent experiments. * P ≤ 0.05, *** P ≤ 0.001.</p