Integrins mediate the lymphocyte migration into an infected tissue, and these cells are essential for controlling the multiplication of many intracellular parasites such as Trypanosoma cruzi, the causative agent of Chagas disease. Here, we explore LFA-1 and VLA-4 roles in the migration of specific CD8+ T cells generated by heterologous prime-boost immunization during experimental infection with T. cruzi. To this end, vaccinated mice were treated with monoclonal anti-LFA-1 and/or anti-VLA-4 to block these molecules. After anti-LFA-1, but not anti-VLA-4 treatment, all vaccinated mice displayed increased blood and tissue parasitemia, and quickly succumbed to infection. In addition, there was an accumulation of specific CD8+ T cells in the spleen and lymph nodes and a decrease in the number of those cells, especially in the heart, suggesting that LFA-1 is important for the output of specific CD8+ T cells from secondary lymphoid organs into infected organs such as the heart. The treatment did not alter CD8+ T cell effector functions such as the production of pro-inflammatory cytokines and granzyme B, and maintained the proliferative capacity after treatment. However, the specific CD8+ T cell direct cytotoxicity was impaired after LFA-1 blockade. Also, these cells expressed higher levels of Fas/CD95 on the surface, suggesting that they are susceptible to programmed cell death by the extrinsic pathway. We conclude that LFA-1 plays an important role in the migration of specific CD8+ T cells and in the direct cytotoxicity of these cells.

CD8 + T lymphocytes play an important role in controlling infections by intracellular pathogens. Chemokines and their receptors are crucial for the migration of CD8 + T-lymphocytes, which are the main IFNγ producers and cytotoxic effectors cells. Although the participation of chemokine ligands and receptors has been largely explored in viral infection, much less is known in infection by Trypanosoma cruzi , the causative agent of Chagas disease. After T . cruzi infection, CXCR3 chemokine receptor is highly expressed on the surface of CD8 + T-lymphocytes. Here, we hypothesized that CXCR3 is a key molecule for migration of parasite-specific CD8 + T-cells towards infected tissues, where they may play their effector activities. Using a model of induction of resistance to highly susceptible A/Sn mice using an ASP2-carrying DNA/adenovirus prime-boost strategy, we showed that CXCR3 expression was upregulated on CD8 + T-cells, which selectively migrated towards its ligands CXCL9 and CXCL10. Anti-CXCR3 administration reversed the vaccine-induced resistance to T . cruzi infection in a way associated with hampered cytotoxic activity and increased proapoptotic markers on the H2K K -restricted TEWETGQI-specific CD8 + T-cells. Furthermore, CXCR3 receptor critically guided TEWETGQI-specific effector CD8 + T-cells to the infected heart tissue that express CXCL9 and CXCL10. Overall, our study pointed CXCR3 and its ligands as key molecules to drive T . cruzi -specific effector CD8 + T-cells into the infected heart tissue. The unveiling of the process driving cell migration and colonization of infected tissues by pathogen-specific effector T-cells is a crucial requirement to the development of vaccine strategies.

Deficiency in memory formation and increased immunosenescence are pivotal features of Trypanosoma cruzi infection proposed to play a role in parasite persistence and disease development. The vaccination protocol that consists in a prime with plasmid DNA followed by the boost with a deficient recombinant human adenovirus type 5, both carrying the ASP2 gene of T. cruzi, is a powerful strategy to elicit effector memory CD8+ T-cells against this parasite. In virus infections, the inhibition of mTOR, a kinase involved in several biological processes, improves the response of memory CD8+ T-cells. Therefore, our aim was to assess the role of rapamycin, the pharmacological inhibitor of mTOR, in CD8+ T response against T. cruzi induced by heterologous prime-boost vaccine. For this purpose, C57BL/6 or A/Sn mice were immunized and daily treated with rapamycin for 34 days. CD8+ T-cells response was evaluated by immunophenotyping, intracellular staining, ELISpot assay and in vivo cytotoxicity. In comparison with vehicle-injection, rapamycin administration during immunization enhanced the frequency of ASP2-specific CD8+ T-cells and the percentage of the polyfunctional population, which degranulated (CD107a+) and secreted both interferon gamma (IFNγ) and tumor necrosis factor (TNF). The beneficial effects were long-lasting and could be detected 95 days after priming. Moreover, the effects were detected in mice immunized with ten-fold lower doses of plasmid/adenovirus. Additionally, the highly susceptible to T. cruzi infection A/Sn mice, when immunized with low vaccine doses, treated with rapamycin, and challenged with trypomastigote forms of the Y strain showed a survival rate of 100%, compared with 42% in vehicle-injected group. Trying to shed light on the biological mechanisms involved in these beneficial effects on CD8+ T-cells by mTOR inhibition after immunization, we showed that in vivo proliferation was higher after rapamycin treatment compared with vehicle-injected group. Taken together, our data provide a new approach to vaccine development against intracellular parasites, placing the mTOR inhibitor rapamycin as an adjuvant to improve effective CD8+ T-cell response.

Background: Although gene editing by CRISPR/Cas9 is a promising curative strategy for inherited and acquired diseases, potential off-targets remain a major concern. Most computational and biochemical methods for off-target search have focused on single genomes, not considering the impact of genetic diversity among human populations. Previous studies showed that genetic variants might significantly impact on- and off-target specificity. However, none of them explored the most extensive worldwide human genetic diversity dataset to examine the impact of population genetic variation on therapeutically relevant guide RNAs (gRNAs). Objective: To evaluate how genetic variation among human populations affects the predicted off-targets for gRNAs that have been used in clinical trials or had consistent results in preclinical studies. Methods: We selected 13 gRNAs tested for sickle cell disease (SCD), severe combined immunodeficiency, chronic granulomatous disease, X-linked hyper IgM syndrome, acquired immunodeficiency syndrome, and Leber congenital amaurosis. Off-target prediction was performed against the reference genome (hg38) and against population-specific genomes using CRISPRitz (https://github.com/pinellolab/CRISPRitz). Genomic variants with allele frequencies ≥1 % were obtained from both the entire gnomAD v3.0 dataset (76,156 whole-genome sequences) and each ethnic group separately. Off-target sequences were filtered considering up to four mismatches and bulges, and a cutting frequency determination (CFD) score ≥0.2 for those harbouring only mismatch events. Sequences were annotated with Annovar (https://annovar.openbioinformatics.org/). Results: A total of 1,727 off-target events were found against the reference genome for the 13 selected gRNAs. 134 additional events were detected with the gnomad_all dataset. When variants from different ethnic groups were considered 234 additional off-targets were observed. The distribution of CFD values was comparable between the population-specific/additional and the reference genome off-targets, as well as the distribution of their genomic localization. Most events mapped to intergenic regions (~50%), followed by intronic regions (~35%). The proportion of additional events varied for the different gRNAs (ranging from 7.8% to 27.7% increase), and the number of events was also variable among the populations. Considering the different ethnic groups, the highest number of events was found in African-ancestry populations (164) and the lowest in the non Finish European population (87), implying that some off-target events were found exclusively in one of the populations (91 in total). Although 219 (75%) of the variants within the population-specific/ additional off-targets have a frequency between 1-10%, 20 (6.8%) have a frequency higher than 80% in at least one of the populations analysed. Considering only the gRNAs used in gene therapy for SCD (n=6), 100 additional predicted off-targets were found in the African population of which 29 are exclusive, while only 77 were found in gnomad_all and 43 in the non-Finish European population, being 2 exclusive. Discussion: Rigorous analysis regarding off-target events is a necessary step to address the safety of gene therapy strategies. Although in silico analysis against the reference genome captures most predicted off-target events, we and others have demonstrated that an important proportion of additional events are lost if population-specific variants are not considered. Herein, we showed that the number of additional events is particularly higher when variants with allele frequencies >=1% in African populations are considered. This might occur because the African population is more genetically diverse and the reference genome was assembled based on Caucasian individuals. In addition, several off-target events are exclusively found in non-African populations, showing the importance of considering the genetic background of patients who undergo gene therapy. Additional caution should be taken for diseases that are more frequent in specific populations, such as SCD and β-thalassemia, for which there are several ongoing gene therapy trials. Disclosures No relevant conflicts of interest to declare.