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Lymphocyte Activation Gene 3 (LAG-3) modulates the ability of CD4 T-cells to be suppressed in vivo.

Durham NM, Nirschl CJ, Jackson CM, Elias J, Kochel CM, Anders RA, Drake CG - PLoS ONE (2014)

Bottom Line: Further studies also identified a role for LAG-3 in the induction/expansion of Treg.Finally, we found that LAG-3 blockade (or knockout) led to a relative skewing of naïve CD4 T-cells toward a TH1 phenotype both in vitro and in in vivo.Together, these data suggest that LAG-3 expression on Tconv cells makes them more susceptible to Treg based suppression, and also regulates the development of a TH1 T-cell response.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Lymphocyte Activation Gene - 3 (LAG-3) is an immune checkpoint molecule that regulates both T-cell activation and homeostasis. However, the molecular mechanisms underlying LAG-3's function are generally unknown. Using a model in which LAG-3 blockade or absence reliably augmented homeostatic proliferation in vivo, we found that IL-2 and STAT5 are critical for LAG-3 function. Similarly, LAG-3 blockade was ineffective in the absence of regulatory T-cells (Treg), suggesting an important role for LAG-3 in either the responsiveness of conventional T-cells (Tconv) to regulation, or a relative defect in the ability of LAG-3 KO regulatory T-cells (Treg) to suppress the proliferation of Tconv. In this model, LAG-3 KO Treg suppressed proliferation in a manner fairly similar to wild-type (WT) Treg, but LAG-3 KO Tconv were relatively resistant to suppression. Further studies also identified a role for LAG-3 in the induction/expansion of Treg. Finally, we found that LAG-3 blockade (or knockout) led to a relative skewing of naïve CD4 T-cells toward a TH1 phenotype both in vitro and in in vivo. Together, these data suggest that LAG-3 expression on Tconv cells makes them more susceptible to Treg based suppression, and also regulates the development of a TH1 T-cell response.

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WT Treg Cannot Completely Protect against LAG-3 KO Tresp in a Colitis Model.A) WT or LAG-3 KO Tresp were transferred into RAG KO mice at a ratio of 4∶1 with WT Treg. Mice were weighed 3 times weekly for 50 days. Percentage of initial body weight is reported. B) Percentage of initial body weight at Day 49. C) H & E staining of histological sections of colons from the 4 groups of mice. D) Blinded histological score of colitis in mouse groups. E) Total splenocytes as well as total CD4+ T-cells were counted and analyzed. F) Percentage of CD4+ T-cells that were FOXP3 or TBET positive. Data shown are representative of at least two independent experiments with n = 8–10 mice per group.
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pone-0109080-g007: WT Treg Cannot Completely Protect against LAG-3 KO Tresp in a Colitis Model.A) WT or LAG-3 KO Tresp were transferred into RAG KO mice at a ratio of 4∶1 with WT Treg. Mice were weighed 3 times weekly for 50 days. Percentage of initial body weight is reported. B) Percentage of initial body weight at Day 49. C) H & E staining of histological sections of colons from the 4 groups of mice. D) Blinded histological score of colitis in mouse groups. E) Total splenocytes as well as total CD4+ T-cells were counted and analyzed. F) Percentage of CD4+ T-cells that were FOXP3 or TBET positive. Data shown are representative of at least two independent experiments with n = 8–10 mice per group.

Mentions: To test whether LAG-3 modulates the ability of Tresp to be suppressed in a potentially more physiologically relevant context, we turned to the well-described in vivo model of induced colitis [34]. As a positive control we adoptively transferred WT responders into RAG1 KO mice. As shown in Figure 7A, these mice progressively lost weight, and upon sacrifice at day 50 had obvious colitis upon pathological examination (Figure 7C). As a control for Treg mediated prevention of colitis, WT responders were transferred along with WT Treg. These mice gained weight over the course of the experiment (Figure 7A) and showed only mild colitis at the experiments close. LAG-3 KO responder cells, by contrast, were relatively resistant to suppression this model; young mice gained only a minimal amount of weight over the course of the experiment (Figure 7B), and demonstrated an intermediate level of colitis upon histological examination (Figure 7C). In terms of overall colitis score, mice receiving LAG-3 KO responder cells plus Treg showed slightly more colitis then mice receiving WT responders and no Treg at all, although that increase was not statistically significant (Figure 7D). This intermediate phenotype was, however, reflected in overall cell numbers, since, mice receiving no Treg (WT responders only) had greatest total numbers of splenocytes, followed by mice receiving LAG-3 KO responders (+ suppressors), and then by mice receiving WT responders and suppressors (Figure 7E). We also tested whether these observed differences in colitis could be explained by an unexpected difference in the relative survival of the WT Treg population, but as shown in Figure 7F, the percentage of suppressors appeared to be relatively similar in mice the received either LAG-3 KO or WT responders. Interestingly, and consistent with the skewing data shown in Figures 5 and 6, the LAG-3 KO responders were more likely to display a TH1 (Tbet +) phenotype (Figure 7F). While these findings do not demonstrate the absolute susceptibility of LAG-3 KO responders, they do demonstrate that the LAG-3 KO responders are relatively more resistant to suppression than their WT counterparts in an in vivo colitis model, and confirm our previous in vitro results.


Lymphocyte Activation Gene 3 (LAG-3) modulates the ability of CD4 T-cells to be suppressed in vivo.

Durham NM, Nirschl CJ, Jackson CM, Elias J, Kochel CM, Anders RA, Drake CG - PLoS ONE (2014)

WT Treg Cannot Completely Protect against LAG-3 KO Tresp in a Colitis Model.A) WT or LAG-3 KO Tresp were transferred into RAG KO mice at a ratio of 4∶1 with WT Treg. Mice were weighed 3 times weekly for 50 days. Percentage of initial body weight is reported. B) Percentage of initial body weight at Day 49. C) H & E staining of histological sections of colons from the 4 groups of mice. D) Blinded histological score of colitis in mouse groups. E) Total splenocytes as well as total CD4+ T-cells were counted and analyzed. F) Percentage of CD4+ T-cells that were FOXP3 or TBET positive. Data shown are representative of at least two independent experiments with n = 8–10 mice per group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4220939&req=5

pone-0109080-g007: WT Treg Cannot Completely Protect against LAG-3 KO Tresp in a Colitis Model.A) WT or LAG-3 KO Tresp were transferred into RAG KO mice at a ratio of 4∶1 with WT Treg. Mice were weighed 3 times weekly for 50 days. Percentage of initial body weight is reported. B) Percentage of initial body weight at Day 49. C) H & E staining of histological sections of colons from the 4 groups of mice. D) Blinded histological score of colitis in mouse groups. E) Total splenocytes as well as total CD4+ T-cells were counted and analyzed. F) Percentage of CD4+ T-cells that were FOXP3 or TBET positive. Data shown are representative of at least two independent experiments with n = 8–10 mice per group.
Mentions: To test whether LAG-3 modulates the ability of Tresp to be suppressed in a potentially more physiologically relevant context, we turned to the well-described in vivo model of induced colitis [34]. As a positive control we adoptively transferred WT responders into RAG1 KO mice. As shown in Figure 7A, these mice progressively lost weight, and upon sacrifice at day 50 had obvious colitis upon pathological examination (Figure 7C). As a control for Treg mediated prevention of colitis, WT responders were transferred along with WT Treg. These mice gained weight over the course of the experiment (Figure 7A) and showed only mild colitis at the experiments close. LAG-3 KO responder cells, by contrast, were relatively resistant to suppression this model; young mice gained only a minimal amount of weight over the course of the experiment (Figure 7B), and demonstrated an intermediate level of colitis upon histological examination (Figure 7C). In terms of overall colitis score, mice receiving LAG-3 KO responder cells plus Treg showed slightly more colitis then mice receiving WT responders and no Treg at all, although that increase was not statistically significant (Figure 7D). This intermediate phenotype was, however, reflected in overall cell numbers, since, mice receiving no Treg (WT responders only) had greatest total numbers of splenocytes, followed by mice receiving LAG-3 KO responders (+ suppressors), and then by mice receiving WT responders and suppressors (Figure 7E). We also tested whether these observed differences in colitis could be explained by an unexpected difference in the relative survival of the WT Treg population, but as shown in Figure 7F, the percentage of suppressors appeared to be relatively similar in mice the received either LAG-3 KO or WT responders. Interestingly, and consistent with the skewing data shown in Figures 5 and 6, the LAG-3 KO responders were more likely to display a TH1 (Tbet +) phenotype (Figure 7F). While these findings do not demonstrate the absolute susceptibility of LAG-3 KO responders, they do demonstrate that the LAG-3 KO responders are relatively more resistant to suppression than their WT counterparts in an in vivo colitis model, and confirm our previous in vitro results.

Bottom Line: Further studies also identified a role for LAG-3 in the induction/expansion of Treg.Finally, we found that LAG-3 blockade (or knockout) led to a relative skewing of naïve CD4 T-cells toward a TH1 phenotype both in vitro and in in vivo.Together, these data suggest that LAG-3 expression on Tconv cells makes them more susceptible to Treg based suppression, and also regulates the development of a TH1 T-cell response.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Lymphocyte Activation Gene - 3 (LAG-3) is an immune checkpoint molecule that regulates both T-cell activation and homeostasis. However, the molecular mechanisms underlying LAG-3's function are generally unknown. Using a model in which LAG-3 blockade or absence reliably augmented homeostatic proliferation in vivo, we found that IL-2 and STAT5 are critical for LAG-3 function. Similarly, LAG-3 blockade was ineffective in the absence of regulatory T-cells (Treg), suggesting an important role for LAG-3 in either the responsiveness of conventional T-cells (Tconv) to regulation, or a relative defect in the ability of LAG-3 KO regulatory T-cells (Treg) to suppress the proliferation of Tconv. In this model, LAG-3 KO Treg suppressed proliferation in a manner fairly similar to wild-type (WT) Treg, but LAG-3 KO Tconv were relatively resistant to suppression. Further studies also identified a role for LAG-3 in the induction/expansion of Treg. Finally, we found that LAG-3 blockade (or knockout) led to a relative skewing of naïve CD4 T-cells toward a TH1 phenotype both in vitro and in in vivo. Together, these data suggest that LAG-3 expression on Tconv cells makes them more susceptible to Treg based suppression, and also regulates the development of a TH1 T-cell response.

Show MeSH
Related in: MedlinePlus