This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory mice of the Brazilian National Council of Animal Experimentation (http://www.mctic.gov.br/mctic/opencms/textogeral/concea.html). The protocol was approved by the Ethical Committee for Animal Experimentation at the Federal University of Sao Paulo (Id # CEP 7559051115).

Female 5- to 8-week-old A/Sn or C57BL/6 mice were purchased from the Federal University of São Paulo. ICAM-1-deficient mice were kindly supplied by Dr. João Santana, Ribeirão Preto School of Medicine-FMPR. Parasites of the Y strain of T. cruzi were used in this study (2, 3). Blood trypomastigotes of the Y strain of T. cruzi were maintained by weekly passages in A/Sn mice at the Xenodiagnosis Laboratory of Dante Pazzenese Cardiology Institute. Bloodstream trypomastigotes were obtained from mice infected 7–28 days earlier with parasites of the Y strain. For in vivo experiments, each mouse was inoculated with 150 trypomastigotes (A/Sn) or 10⁴ trypomastigotes (C57BL/6) diluted in 0.2 mL phosphate-buffered saline (PBS) and administrated subcutaneously (s.c.) in the base of the tail. Parasitemia was determined by collecting 5 μL of blood, and parasites were counted on the light microscope (25).

In this study, we used the heterologous prime-boost immunization protocol with plasmid pIgSPCl.9 and the human replication-defective adenovirus type 5 containing the ASP-2 gene, as described previously (3, 26). Briefly, this immunization consists of a dose of plasmid DNA as a prime (pcDNA3 control or pIgSPClone9). The mice were intramuscularly inoculated (i.m.) with 50 µg of plasmid DNA into each tibialis anterioris muscle. Three weeks after the first immunization, mice were boosted with 2 × 10⁸ plaque-forming units of the adenoviral vectors Adβ-gal or AdASP-2. Both injections were performed via intramuscular route (tibialis anterior muscle).

TEWETGQI peptide was synthesized by GenScript and obtained at purity higher than 95%. The TEWETGQI epitope expressed on ASP-2 surface is target of CD8⁺ T cells and was identified previously (27). It was used for specific CD8⁺ T cell stimulation in vitro and ex vivo. The H2K^K-TEWETGQI multimer, labeled with fluorophore APC, was purchased from Immudex (Copenhagen, Denmark) and used for specific CD8⁺ T cell detection in tissues.

Anti-LFA-1 (anti-CD11a, clone M17-4) and anti-VLA-4 (anti-CD49d, clone PS/2) monoclonal antibodies were purchased from BioXcell; in addition, we used Rat IgG2a (clone 2A3) isotype control. The in vivo treatment was performed with 10 i.p. injections of 250 µg of mAb/mouse (every 48 h after infection, until day 20 after infection). The concentration of LFA-1 used for in vivo treatment was the same used by Reisman et al. (28). To evaluate the efficiency of LFA-1 integrin blockade, C57BL/6 mice were infected with 10⁴ trypomastigote forms of Y strain, and 12 days post infection, the splenocytes were harvested and incubated in vitro for 24 h at 30°C with monoclonal 250 µg/mL of 2A3 isotype control or anti-LFA-1 in complete medium [1% NEAA, 1% l-glutamine, 1% vitamins and 1% pyruvate, 0.1% 2-ME, 10% fetal bovine serum (FBS) (HyClone)]. After incubation, splenocytes were washed and labeled with anti-CD8 PerCP (clone 53-6.7, BD) and anti-CD11a FITC (clone 2D7, BD), fixed with 1% paraformaldehyde and analyzed by flow cytometry. Concomitantly, we also evaluated the blockade of the LFA-1 molecule stimulating splenocytes in vitro with 1 µg/mL anti-CD3 (clone 145-2C11, eBioscience) in complete medium for 72 h at 37°C and 5% CO₂. On the second day of incubation, 250 µg of 2A3 isotype control or anti-LFA-1 monoclonal antibodies were added to the culture. On the third day of culture, cells were harvested and labeled with anti-CD8 PerCP and anti-CD11a FITC for flow cytometric analysis. LFA-1 expression was performed on gated CD8⁺ T cells, according to Figures S1A,B in Supplementary Material, treatment with monoclonal anti-LFA-1 blocked most LFA-1 molecule expressed on activated CD8⁺ T cells and after anti-LFA-1 FITC staining there was a lower CD11a MFI on the surface of these cells, indicating that there is competition between anti-LFA-1 monoclonal antibodies used for in vivo blocking (clone M17-4) and anti-CD11a FITC (clone 2D7, BD) used for flow cytometry labeling.

Hearts, livers, and spleens from the T. cruzi-infected, immunized, and/or treated mice with anti-LFA-1 A/Sn were used for extracting DNA. The extraction protocol, the specific primers for a satellite DNA region of the parasite, and the RT-PCR reaction using the TaqMan Universal Master Mix II with UNG were adapted from Piron and colleagues (29). For the race plates, we used StepOnePlus (Applied Biosystems^®), and distilled water for negative control reaction.

Sterile PBS containing 10 µg/mL of anti-mouse IFN-γ monoclonal antibody (clone R4-6A2, Pharmingen) was added onto nitrocellulose 96-well flat-bottom plates; after 24 h, the plates were washed with RPMI and blocked with RPMI containing 10% FBS for 2 h. Following, 1 × 10⁶ responder cells from spleen, liver, or lymph node were incubated with 3 × 10⁵ antigen-presenting cells in complete medium [1% NEAA, 1% l-glutamine, 1% vitamins and 1% pyruvate, 0.1% 2-ME, 10% FBS (HyClone), and 20 U/mL mouse recombinant IL-2 (SIGMA)]. The plate was incubated in the presence or absence of 10 µM of peptide TEWETGQI. After 24 h, the plates were washed three times with PBS, and five times with PBS–Tween 20 (0.05% Tween). Each well received biotinylated anti-mouse monoclonal antibody (clone XMG1.2, Pharmingen) diluted in PBS-0.05% Tween 20 at a final concentration of 2 µg/mL. The plates were incubated with streptavidin-peroxidase (BD) and developed by adding peroxidase substrate (50 mM Tris–HCl, pH 7.5, containing 1 mg/mL DAB and 1 µL/mL 30% hydrogen peroxide, both from SIGMA). The number of IFN-γ-producing cells was determined using a stereoscope.

Two million cells from the spleen, lymph node, or liver were treated with ACK buffer (NH₄Cl, 0.15 M; KHCO₃, 10 mM; Na₂-EDTA 0.1 mM; pH = 7.4). ICS was performed after in vitro culture of splenocytes in presence or absence of 10 µM of peptide TEWETGQI as described previously (25). Cells were washed three times in plain RPMI and resuspended in cell culture medium consisting of RPMI 1640 medium supplemented with 10 mM HEPES, 0.2% sodium bicarbonate, 59 mg/L of penicillin, 133 mg/L of streptomycin, 10% HyClone FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, 55 µM 2-mercaptoethanol. The viability of the cells was evaluated using 0.2% trypan blue exclusion dye to discriminate between live and dead cells. Cell concentration was adjusted to 2 × 10⁶ cells/mL in cell culture medium containing CD107a FITC antibody (clone 1D4B, BD), anti-CD28 (clone 37.51, BD), BD Golgi-Plug (1 µL/mL), and monensin (5 µg/mL) and incubated no longer than 12 h in V-bottom 96-well plates (Corning) in a final volume of 200 µL in duplicate, at 37°C in a humid environment containing 5% CO₂. After 12 h incubation, cells were stained for surface markers with anti-CD8 PERCP antibody (clone 53-6.7, BD) on ice for 30 min. To detect IFN-γ, TNF or granzyme B by intracellular staining, cells were then washed twice in buffer containing PBS, 0.5% bovine serum albumin (BSA), and 2 mM EDTA, fixed and permeabilized with BD perm/wash buffer. After being washed twice with BD perm/wash buffer, cells were stained for intracellular markers using APC-labeled anti-IFN-γ (clone XMG1.2, BD), PE-labeled anti-TNF-α (clone MP6-XT22, BD), and anti-granzyme B PE (clone GB11, INVITROGEN) for 20 min on ice. Finally, cells were washed twice with BD perm/wash buffer and fixed in 1% PBS–paraformaldehyde. At least 700,000 cells were acquired on a BD FACS Canto II flow cytometer and then analyzed with FlowJo. Figures S3A,B in Supplementary Material shows the representative ICS gate strategies.

The perfused liver was lysed with collagenase buffer composed of 0.2 mg/mL collagenase IV (SIGMA), 0.02 mg/mL DNase (SIGMA), and 5% FBS. The leukocytes were separated on a 35% Percoll gradient (GE Healthcare), followed by centrifugation at 600 × g for 20 min and at 4°C. The pellet was suspended in RPMI 1640 (SIGMA) with 10% FBS (30). For the purification of the lymphocytes of the heart, we followed the protocol of Gutierrez et al. (31). Briefly, hearts collected from five mice at day 20 d.p.i. were minced, pooled, and incubated for 1 h at 37°C with RPMI 1640, supplemented with NaHCO₃, penicillin–streptomycin gentamicin, and 0.05 g/mL of liberase blendzyme CI (Roche, Basel, Switzerland). The organs were processed in a Medimachine (BD Biosciences) in PBS containing 0.01% BSA. After tissue digestion and washes, cell viability was assessed by trypan blue exclusion, counted in a hemocytometer.

Splenocytes were treated with ACK buffer for red cell lysis and washed with RPMI with 10% FBS. The spleen, heart, lymph node, and liver cells were stained with H2K^k-TEWETGQI multimer for 10 min at RT. The cell surface was stained for 30 min at 4°C. The following antibodies were used for surface staining: anti-CD3 APCcy7 (clone 145-2C11, BD), anti-CD8 PERCP or anti-CD8 PACIFIC BLUE (clone 53-6.7, BD), anti-CD11a FITC (clone 2D7, BD), anti-CD11c APCcy7 (clone HL3, BD), anti-CD44 FITC (clone IM7, BD), anti-CD62L PE (clone MEL-14, BD), anti-CXCR3 PERCP/Cy5.5 (clone 173, BioLegend), anti-CD27 FITC (clone LG3A10, BD), anti-CD4 PEcy7 (clone RM4-5, BD), anti-KLRG1 FITC (clone 2F1, eBioscience), anti-CD49d PEcy7 (clone R1-2, BD), anti-CD69 PERCP (clone H1.2F3, BD), anti-CD43 PEcy7 (1B11, BioLegend), anti-CD95 PEcy7 (clone JO2, BD), anti-CD25 FITC (clone LG3A10, BD), anti-CD127 PE (clone SB/199, BD), anti-CD122 FITC (clone TM-β1, BD), anti-CD38 PE (clone 90, BD), anti-β7 PERCP (clone FIB27, BioLegend), anti-CD31 FITC (clone MEC 13.3, BD), anti-CD272 PE (clone 8F4, eBioscience), anti-PD-1 FITC (clone J43, eBioscience), anti-CTLA-4 PE (clone UC10-4B9, eBioscience), and anti-CCR7 PE (clone 4B12, BD). At least 500,000 cells were acquired on a BD FACS Canto II flow cytometer and analyzed with FlowJo 8.7.

A/Sn were immunized with ASP-2 using the heterologous “prime-boost” vaccination regimen and infected with 150 trypomastigotes forms of T. cruzi. At the moment of infection, mice were treated with monoclonal antibodies (LFA-1 or 2A3 isotype control) and 2 mg of BrdU (5-bromo-2′-deoxyuridine, SIGMA) by route i.p., at every 48 h, until the 20th day after challenge. Then, 2 × 10⁶ splenocytes were treated with ACK buffer for red cell lysis, washed with RPMI plus 10% FBS, and stained with H2K^k-TEWETGQI multimer and anti-CD8 antibody. The specific CD8⁺ T cells were stained according BrdU-FITC Kit protocol (BD Pharmingen) for analysis of BrdU incorporation. A minimum of 700,000 cells were acquired on a BD FACS Canto II flow cytometer and analyzed with FlowJo 8.7.

For the in vivo cytotoxicity assays, splenocytes collected from naive A/Sn mice were treated with ACK buffer to lyse the red blood cells, as described by Silverio et al. (23). The cells were divided into two populations and were labeled with the fluorogenic dye carboxyfluorescein diacetate succinimidyl diester (CFSE; Molecular Probes, Eugene, OR, USA) at a final concentration of 10 µM (CFSE^high) or 1 µM (CFSE^low). CFSE^high cells were coated with 2.5 µM of the TEWETGQI ASP-2 peptide for 40 min at 37°C. CFSE^low cells remained uncoated. Subsequently, CFSE^high cells were washed and mixed with equal numbers of CFSE^low cells before intravenous injection (2 × 10⁷ cells per mouse) into T. cruzi-infected, immunized and/or treated mice with anti-LFA-1 A/Sn recipients that were sedated with diazepam (20 mg/kg). Spleen cells from the recipient mice were collected at 20 h after adoptive cell transfer and fixed with 1.0% paraformaldehyde. At least 100,000 cells were acquired on a BD FACS Canto II flow cytometer and analyzed with FlowJo 8.7. The percentage of specific lysis was determined using the following formula: % lysis=1−(%CFSEhigh infected/%CFSElow infected)(%CFSEhigh naive/%CFSElow naive)×100.

The mice’s heart, spleen, and liver were fixed in 10% formalin, and then dehydrated, embedded in paraffin blocks, and sectioned on a microtome. Staining was obtained with hematoxylin and eosin, and the number of amastigotes nests was quantified using a light microscope with 40× objective lens. Overall, 50 fields/group were counted. For immunohistochemistry the hearts of the animals were removed and frozen in Tissue-Tek O.C.T. (Sakura Finetek), and the 7 µm thickness cuts were made in the cryostat (Leica) and then fixed in ice-cold acetone for 15 min. The samples were stained with 20 µg of the biotinylated anti-CD8 antibody (clone 53-6.7, RD systems) for 12 h in the wet chamber, and after incubation was labeled with streptavidin Alexa Fluor^® 488 (Thermo Fischer) at the concentration of 0.5 mg/mL, diluted 1:100 for 1 h and room temperature. The DAPI (4′,6-diamidino-2-phenylindole, SIGMA) dye was used for labeling the 5 mg/mL cell nucleus, diluted 1:1,000 for 15 min at room temperature. The images were acquired in the Confocal Leica TCS SP8 CARS microscope of the Institute of Pharmacology and Molecular Biology (INFAR) of the Paulista School of Medicine of the Federal University of São Paulo. The images were obtained using the 63× objective and processed by the ImageJ program.

The number of parasites/mL corresponding to the peak of parasitemia, the number of IFN-γ-producing cells (ELISPOT), and the absolute number of CD8⁺ T cells were compared by analysis of unidirectional variance (ANOVA); subsequently, the Tukey’s HSD test was used. To compare the survival of mice after challenge with T. cruzi, we used the Log-rank test. The receptor expression was compared using MFI (mean fluorescence intensity), and the naive group MFI was taken as the baseline. MFI was determined by the FlowJo software. Differences were considered significant when P value was <0.05.

Previously, we demonstrated that treatment with FTY720, which retains CD8⁺ T cells in the lymph nodes via blockade of receptor S1Pr1, culminates in death of immunized mice. As LFA-1 and VLA-4 integrins were expressed on those specific CD8⁺ T cells we investigated the role of these molecules following immunization and T. cruzi infection. To this end, immunized and infected mice were treated with 250 µg of monoclonal antibodies anti-LFA-1 and/or anti-VLA-4 every 48 h to block those molecules. Initially, we analyzed blood parasitemia and, as shown in Figure 1A, mice treated with anti-LFA-1 (green) antibody had increased blood parasitemia when compared with the group only immunized and treated with the control isotype (red), whereas mice treated with anti-VLA-4 (yellow) had a parasite burden similar to the immunized (red). To examine whether these two integrins exhibit synergism, one group was treated with both antibodies simultaneously (Figure 1A, blue group). Simultaneous treatment resulted in increased blood parasitemia, but this increase was not significant when compared with the group treated with anti-LFA-1 only, indicating that LFA-1, but not VLA-4, is important to control blood parasites. With respect to survival (Figure 1B), all mice treated with anti-LFA-1 died after 26 days, whereas all anti-VLA-4-treated mice and isotype control treated mice survived. Therefore, no statistical differences were observed in the survival rate between the mice treated with anti-LFA-1 (green) and the mice treated with both antibodies (blue), but there were differences in survival rate between the mice treated with anti-LFA-1 and mice treated with isotype control. Therefore, during LFA-1 blockade, mice displayed increased blood parasitemia and succumbed after challenge with T. cruzi, while VLA-4 blockade does not interfere with parasitemia and survival of treated mice.

To confirm the role of LFA-1 during T. cruzi infection, C57BL/6 mice naturally resistant to T. cruzi infection were infected and treated. C57BL/6 mice treatment with anti-LFA-1 was able to control blood parasitemia until 12th day after infection, but after that, the blood parasitemia increased and all mice treated with anti-LFA-1 rapidly succumbed to infection (Figures 1C,D) when compare the mice treated with the isotype control.

Since LFA-1 blockade increased mouse susceptibility to infection by T. cruzi, we investigated the importance of the ICAM-1 integrin, a major ligand of LFA-1. To this end, genetically ICAM-1-deficient mouse was used. These mice were immunized and infected for parasitemia and survival analysis. Both C57BL/6 and ICAM-1 knockout mice displayed similar parasitemia, and the two groups immunized with the ASP-2 gene showed a decreased parasitemia when compared with the vector control immunized groups (Figure 1E). In addition, all mice survived the experimental challenge with T. cruzi (Figure 1F). Figure 1G shows the expression of CD54 (ICAM-1) on spleen of CD8⁺ T cells of WT and deficient mice, and, as expected, the latter ones have lower expression of CD54 compared with WT mice. Altogether, these results indicate that the absence of ICAM-1 does not increase susceptibility to T. cruzi infection and suggest that, even though ICAM-1 is a major ligand of LFA-1, there is another ligand (i.e., ICAM-2) that binds to LFA-1 allowing it to exert its functions.

Since anti-VLA-4 treatment did not interfere in the mice parasitemia and/or survival, all following experiments were performed by blocking LFA-1 integrin only. As the LFA-1 blockade leads to increased blood parasitemia and rapid death of the mice, we investigated whether the parasitic increase also occurs in the tissues of infected, immunized, and/or anti-LFA-1-treated A/Sn mice. The heart, liver, and spleen of these mice were extracted after the 20th day of infection for quantification of the parasite’s DNA by real-time PCR; in addition, the number of amastigote nests in the heart was quantified using hematoxylin–eosin staining. There was a statistical increase in the number of amastigote nests in the hearts of the LFA-1-treated mice compared with the immunized and infected group, and the largest amount of nests was found in the hearts of mice solely infected (Figures 2A,B). In addition, LFA-1 blockade resulted in the increase of parasites in the tissues analyzed compared with immunized, infected mice. The spleen showed higher parasite increase, followed by hearts and livers respectively (Figure 2C). These results demonstrate that treatment with anti-LFA-1 increases blood parasitemia, which will reflect on increased tissue parasite burden.

We analyzed whether LFA-1 blockade affects effector phenotype and activation of specific CD8⁺ T cells. To this end, splenocytes were stained with anti-CD8 and H2K^K TEWETQGI-multimer, and surface markers. In previous results obtained by our group, we demonstrated that immunization followed by infection induces specific CD8⁺ T cells with the phenotype of effector cells (TE), which is characterized by the expression of CD11a^high, CD44^high, CD62L^low, and CD127^low (25, 26). Anti-LFA-1 treatment increased the frequency and the absolute number of specific CD8⁺ T cells in the spleen (Figures 3A,B). In addition, as there is competition between the antibodies used for in vivo blockade and flow cytometric labeling, we observed a decrease in CD11a MFI in the anti-LFA-1-treated groups (Figure 3C). In general, the markers whose expression levels increased in the anti-LFA-1-treated group (Gr.3) in comparison with the infected group (Gr.1) or the immunized and infected group (Gr.2) were as follows: CD27, CD43, CD69, and CD95 (Figure 3C). Our group has also demonstrated that the cells generated by immunization followed by infection expressed lower levels of CD95 on the surface compared with infected only group, and these cells were also resistant to death induced by anti-CD95 (12). Moreover, markers CD183, CD38, and PD-1 also displayed increased expression levels on the surface of specific CD8⁺ T cells after treatment with anti-LFA-1 (Gr.3) compared with Gr.2 (Figure 3C). KLRG1, however, had similar MFI among the three groups, and these groups showed low expression of markers CD122, BTLA, CTLA-4, and CD25. These results suggest that anti-LFA-1 treatment does not impair specific CD8⁺ T cells in the spleen. Instead, there is a greater frequency and absolute number of these cells. In addition, the treatment did not alter the phenotype of effector CD8⁺ T cells (TE); however, we found in the spleen of treated mice that those CD8⁺ T cells expressed high levels of CD95.

After demonstrating that anti-LFA-1 treatment increases blood and tissue parasitemia and leads to mice death, our hypothesis was that specific CD8⁺ T cells cannot migrate into the infected tissues since LFA-1 is associated with leukocyte migration. To test our hypothesis, we measured in the spleen, lymph, blood, liver, and heart the frequency of specific CD8⁺ T cells. For that propose, the cells were labeled with anti-CD8 and H2K^K-TEWETGQI multimer. We found higher frequency and increment in the absolute numbers of specific CD8⁺ T cells in spleens and lymph nodes, but not in the blood and liver of the anti-LFA-1-treated group when compared with the infected and immunized group (Figures 4A–F). A dramatic influx reduction of specific CD8⁺ T cells after anti-LFA-1 treatment (Figure 4A) was observed in group 3 hearts. In addition, we found that specific CD8⁺ T cells in the spleen, blood, and heart of the infected group (Gr.1) and the immunized and infected group (Gr.2) expressed high levels of CD11a, whereas the cells in the immunized, infected, and anti-LFA-1-treated group (Gr.3) showed decreased CD11a MFI due to the competition described earlier (Figure 4G). We also evaluated the frequency of total CD8⁺ T cells in the spleen, blood, and heart and, as shown in Figure S2 in Supplementary Material, there was a reduction in the frequency of CD8⁺ T cells in the heart of the anti-LFA-1-treated group (Figures S2A–D in Supplementary Material). Furthermore, by immunohistochemistry, there is a decrease in the number of CD8⁺ T cells in the heart of animals treated with anti-LFA-1 (Figures S2E,F in Supplementary Material). The number of CD8⁺ T cells in the heart of the treated group was similar to the infected group, and in relation to the immunized group, treatment with anti-LFA-1 decreased the migration of CD8⁺ T cells to the heart, observed by the low number of these cells in the cardiac tissue. These results corroborate the decrease in specific CD8⁺ T cells in those organs. Therefore, during LFA-1 blockade, specific CD8⁺ T cells accumulate in the secondary lymphoid organs, such as spleen and lymph node, and cannot migrate into the heart, as observed by the lower frequency of these cells in that organ.

Having in mind the important role of IFN-γ during infection by T. cruzi (2), we analyzed whether the anti-LFA-1 treatment may alter production of IFN-γ by specific CD8⁺ T cells. We also analyzed the effector function of specific CD8⁺ T cells in the spleen, liver, and lymph nodes regarding TNF-α production and indirect cytotoxicity involving cell surface mobilization of CD107a. The gate strategy used to evaluate the production of cytokines and the polyfunctionality of specific CD8⁺ T cells is illustrated in Figures S3A,B in Supplementary Material. In the spleen of the anti-LFA-1-treated group, compared with the immunized and infected group, there was an increase in the percentage of polyfunctional specific CD8⁺ T cells, that is, cells that are capable of degranulating and, at the same time, producing IFN-γ and TNF-α. Such increase in polyfunctionality of specific CD8⁺ T cells after anti-LFA-1 treatment was also observed in lymph nodes and liver (Figures 5A,B). In addition, anti-LFA-1 treatment also culminated in an increase in the amplitude of the immune response, i.e., the percentage of specific CD8⁺ T cells producing IFN-γ or TNF-α or degranulating, in the spleen, lymph node, and liver (Figure 5C). The number of specific CD8⁺ T cells producing IFN-γ was higher in the anti-LFA-1-treated group (Gr.3), when compared with Gr.2, and this increase occurred in the spleen, the lymph node, and liver (Figure 5D). Altogether, these results show that LFA-1 blockade does not affect the effector function of specific CD8⁺ T cells regarding IFN-γ and TNF-α secretion and degranulation. The increase in the effector function is probably due to the accumulation of specific cells in spleen and lymph node after treatment with anti-LFA-1.

First, we tested whether treatment had impaired the frequency and absolute numbers of specific CD8⁺ T cells. As we can see, there was an increase in the frequency and absolute numbers of specific CD8⁺ T cells in the anti-LFA-1-treated group (Figures 6A,B). We concluded that the treatment did not interfere with specific CD8⁺ T cell expansion. Since specific CD8⁺ T cells are capable of secreting IFN-γ and TNF-α after LFA-1 blockade in the spleen, we assessed whether the treatment had impaired specific CD8⁺ T cells proliferative capacity. The proliferation of specific CD8⁺ T cells in the spleen was analyzed in vivo by thymidine BrdU analog incorporation. We found that a similar proportion of the H2K^K-TEWETGQI CD8⁺ cells incorporated BrdU in vivo in non-treated or treated mice indicating that the proliferative capacity of these cells was not significantly different (Gr. 2 and Gr. 3, Figure 6C). However, the infected mice have a greater proliferation in comparison with groups 2 and 3 (Figures 6C,D).

Another effector function triggered by specific CD8⁺ T cells is the direct cytotoxicity against the target cells. Here, we analyzed whether this function had been affected by the treatment. For that purpose, we used in vivo cytotoxicity assay. Figure 7A shows representative histograms containing two populations P1 (CFSE^low) and P2 (CFSE^high) showing the specific lysis of H2K^K-TEWETGQI peptide-labeled CFSE^high cells from Gr.1, Gr.2, and Gr.3 groups (Figure 7A). Surprisingly, we observed that the immunized mice treated with anti-LFA-1 had significantly decreased cytotoxicity when compared with the immunized group (Figure 7B). The specific CD8⁺ T cells from the immunized mice have 80% cytotoxicity whereas the anti-LFA-1-treated cells showed a 29% percentage drop. Figure 7C shows that the numbers of CFSE^low cells were similar across groups 1, 2, and 3, while the number of cells CFSE^high decreased in groups 2 (non-treated) and 3 (treated) compared with group 1 (Figure 7D).

The reduction of cytotoxicity did not affect the amount of granzyme B produced by specific CD8⁺ T cells in the anti-LFA-1-treated group. We observed that MFI and the percentage of specific CD8⁺ T cells that produce granzyme B were similar across the groups (Figures 7E,F). These results demonstrate that treatment with LFA-1 directly affects the cytotoxicity of specific CD8⁺ T cells and the impairment of this function may be one of the factors responsible for the reversal protection generated by immunization observed after LFA-1 blockade.