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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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LAG-3 KO Treg suppress homeostatic proliferation, but LAG-3 KO responders are resistant to suppression.A) WT or LAG-3 KO Treg were transferred into RAG KO mice at a ratio of 1∶4 with WT responders. Responders alone received 4E6 WT Tresp with no Treg. B) WT or LAG-3 KO Treg were transferred into RAG KO mice at a ratio of 1∶4 with KO responders. Responders alone received 4E6 LAG3 KO Tresp with no Treg. C) Representative plots of FOXP3 expression in adoptively transferred cells. D) Summary of FOXP3 expression in adoptively transferred cells (n = 4). E) CD25 MFI expression on Treg with either WT or LAG-3 KO Responders. F) WT or LAG-3 KO CD4 Tresp were mixed at a 4∶1 ratio with FOXP3 GFP Treg, stimulated with CD3/CD28, and pulsed with H3-Thymadine after 72 hours. Total CPM counts are shown. Baseline activation of WT or LAG-3 KO Tresp without Treg was not different and is reported by a dashed line. Data shown are representative of at least two independent experiments with n = 4–5 mice per group.
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pone-0109080-g004: LAG-3 KO Treg suppress homeostatic proliferation, but LAG-3 KO responders are resistant to suppression.A) WT or LAG-3 KO Treg were transferred into RAG KO mice at a ratio of 1∶4 with WT responders. Responders alone received 4E6 WT Tresp with no Treg. B) WT or LAG-3 KO Treg were transferred into RAG KO mice at a ratio of 1∶4 with KO responders. Responders alone received 4E6 LAG3 KO Tresp with no Treg. C) Representative plots of FOXP3 expression in adoptively transferred cells. D) Summary of FOXP3 expression in adoptively transferred cells (n = 4). E) CD25 MFI expression on Treg with either WT or LAG-3 KO Responders. F) WT or LAG-3 KO CD4 Tresp were mixed at a 4∶1 ratio with FOXP3 GFP Treg, stimulated with CD3/CD28, and pulsed with H3-Thymadine after 72 hours. Total CPM counts are shown. Baseline activation of WT or LAG-3 KO Tresp without Treg was not different and is reported by a dashed line. Data shown are representative of at least two independent experiments with n = 4–5 mice per group.

Mentions: The results described above might be explained by either an inability of LAG-3 KO Treg to suppress HP, or by a relative resistance to suppression in LAG-3 KO responder cells. To distinguish between these two possibilities, we sorted Treg from WT or LAG-3 KO animals (Figure S2) and tested their ability to suppress HP by adoptively transferring them into RAG1 KO mice along with WT responders. In this model, WT Treg and Tconv both express similar levels of LAG-3 in vivo (Figures S3A and S3B). As shown in Figure 4A, we found that both WT and LAG-3 KO Treg were capable of suppressing the proliferation of whole splenocytes and total CD4+ T-cells during homeostatic proliferation. Therefore, in this HP model, both WT and LAG-3 KO Treg appeared to be fully functional suppressors. We next investigated the second possibility, i.e. that the absence of LAG-3 on responding cells decreases their ability to be suppressed by Treg during HP in vivo. To test this, we adoptively transferred LAG-3 KO responders into RAG1 KO mice, along with either WT or LAG-3 KO Treg. Surprisingly, neither WT nor LAG-3 KO Treg were able to significantly suppress proliferation of LAG-3 KO responders during HP (Figure 4B). In fact, the addition of WT Treg actually increased the number of total CD4 T-cells found in the spleen, though the reason for this increase is unclear. These data are further supported by the finding that LAG-3 blockade only increased Tconv cell proliferation, and had no affect on Treg division (Figures S3C and S3D). As shown in Figure 4C (top row), relative suppression during HP did not correlate well with total FOXP3 levels, as expression of FOXP3 was relatively increased in LAG-3 KO versus WT mediated suppression, despite the similar levels of suppression seen in Figure 4A. While there was a trend towards increased suppression with increased FoxP3 expression, this trend was not statistically significant in this model. Similarly, FOXP3 levels were also increased when LAG-3 KO cells were the targets of suppression (Figure 4C bottom row), despite their relative inability to be suppressed (Figure 4B). Since LAG-3 blockade did not seem to affect Treg cell division (Figure S3C), these data suggest that these increases in the expression of FOXP3 are due to increased cell survival. These findings are summarized in Figure 4D. We also found that CD25 MFI levels were increased in studies with LAG-3 KO responders (Figure 4E), here likely reflecting the relative activation of these cells and consistent with their inability to be suppressed during HP in vivo (Figure 4B). To further investigate whether LAG-3 KO cells were resistant to suppression, we performed in vitro suppression assays, using sorted FoxP3-GFP Treg as suppressors. As shown in Figure 4F, LAG-3 KO T-cells were also significantly more resistant to in vitro suppression than their wild type counterparts, confirming the in vivo results above.


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)

LAG-3 KO Treg suppress homeostatic proliferation, but LAG-3 KO responders are resistant to suppression.A) WT or LAG-3 KO Treg were transferred into RAG KO mice at a ratio of 1∶4 with WT responders. Responders alone received 4E6 WT Tresp with no Treg. B) WT or LAG-3 KO Treg were transferred into RAG KO mice at a ratio of 1∶4 with KO responders. Responders alone received 4E6 LAG3 KO Tresp with no Treg. C) Representative plots of FOXP3 expression in adoptively transferred cells. D) Summary of FOXP3 expression in adoptively transferred cells (n = 4). E) CD25 MFI expression on Treg with either WT or LAG-3 KO Responders. F) WT or LAG-3 KO CD4 Tresp were mixed at a 4∶1 ratio with FOXP3 GFP Treg, stimulated with CD3/CD28, and pulsed with H3-Thymadine after 72 hours. Total CPM counts are shown. Baseline activation of WT or LAG-3 KO Tresp without Treg was not different and is reported by a dashed line. Data shown are representative of at least two independent experiments with n = 4–5 mice per group.
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Related In: Results  -  Collection

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

pone-0109080-g004: LAG-3 KO Treg suppress homeostatic proliferation, but LAG-3 KO responders are resistant to suppression.A) WT or LAG-3 KO Treg were transferred into RAG KO mice at a ratio of 1∶4 with WT responders. Responders alone received 4E6 WT Tresp with no Treg. B) WT or LAG-3 KO Treg were transferred into RAG KO mice at a ratio of 1∶4 with KO responders. Responders alone received 4E6 LAG3 KO Tresp with no Treg. C) Representative plots of FOXP3 expression in adoptively transferred cells. D) Summary of FOXP3 expression in adoptively transferred cells (n = 4). E) CD25 MFI expression on Treg with either WT or LAG-3 KO Responders. F) WT or LAG-3 KO CD4 Tresp were mixed at a 4∶1 ratio with FOXP3 GFP Treg, stimulated with CD3/CD28, and pulsed with H3-Thymadine after 72 hours. Total CPM counts are shown. Baseline activation of WT or LAG-3 KO Tresp without Treg was not different and is reported by a dashed line. Data shown are representative of at least two independent experiments with n = 4–5 mice per group.
Mentions: The results described above might be explained by either an inability of LAG-3 KO Treg to suppress HP, or by a relative resistance to suppression in LAG-3 KO responder cells. To distinguish between these two possibilities, we sorted Treg from WT or LAG-3 KO animals (Figure S2) and tested their ability to suppress HP by adoptively transferring them into RAG1 KO mice along with WT responders. In this model, WT Treg and Tconv both express similar levels of LAG-3 in vivo (Figures S3A and S3B). As shown in Figure 4A, we found that both WT and LAG-3 KO Treg were capable of suppressing the proliferation of whole splenocytes and total CD4+ T-cells during homeostatic proliferation. Therefore, in this HP model, both WT and LAG-3 KO Treg appeared to be fully functional suppressors. We next investigated the second possibility, i.e. that the absence of LAG-3 on responding cells decreases their ability to be suppressed by Treg during HP in vivo. To test this, we adoptively transferred LAG-3 KO responders into RAG1 KO mice, along with either WT or LAG-3 KO Treg. Surprisingly, neither WT nor LAG-3 KO Treg were able to significantly suppress proliferation of LAG-3 KO responders during HP (Figure 4B). In fact, the addition of WT Treg actually increased the number of total CD4 T-cells found in the spleen, though the reason for this increase is unclear. These data are further supported by the finding that LAG-3 blockade only increased Tconv cell proliferation, and had no affect on Treg division (Figures S3C and S3D). As shown in Figure 4C (top row), relative suppression during HP did not correlate well with total FOXP3 levels, as expression of FOXP3 was relatively increased in LAG-3 KO versus WT mediated suppression, despite the similar levels of suppression seen in Figure 4A. While there was a trend towards increased suppression with increased FoxP3 expression, this trend was not statistically significant in this model. Similarly, FOXP3 levels were also increased when LAG-3 KO cells were the targets of suppression (Figure 4C bottom row), despite their relative inability to be suppressed (Figure 4B). Since LAG-3 blockade did not seem to affect Treg cell division (Figure S3C), these data suggest that these increases in the expression of FOXP3 are due to increased cell survival. These findings are summarized in Figure 4D. We also found that CD25 MFI levels were increased in studies with LAG-3 KO responders (Figure 4E), here likely reflecting the relative activation of these cells and consistent with their inability to be suppressed during HP in vivo (Figure 4B). To further investigate whether LAG-3 KO cells were resistant to suppression, we performed in vitro suppression assays, using sorted FoxP3-GFP Treg as suppressors. As shown in Figure 4F, LAG-3 KO T-cells were also significantly more resistant to in vitro suppression than their wild type counterparts, confirming the in vivo results above.

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