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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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IL-2 is required for LAG-3 blockade to augment homeostatic proliferation in vivo.A) 1E6 WT or IL-2 KO CD4 T-cells transferred into RAG KO mice. Isotype control antibody or LAG-3 blocking antibody given every 2 days. Splenocytes counted and analyzed. B) 1E6 WT or IL-2 KO CD4+ T-cells were transferred into RAG KO/IL-2 KO mice. C) 1E6 WT or STAT5 KO CD4+ T-cells transferred into RAG KO mice. Isotype control antibody or LAG-3 blocking antibody was given every 2 days. Splenocytes were then counted and analyzed. D) LAG-3 antibody staining of LAG-3 on IL-2 KO cells in vivo. E) LAG-3 antibody staining of LAG-3 on STAT5 KO cells in vivo. Data shown are representative of at least two independent experiments with n = 3 mice per group.
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pone-0109080-g002: IL-2 is required for LAG-3 blockade to augment homeostatic proliferation in vivo.A) 1E6 WT or IL-2 KO CD4 T-cells transferred into RAG KO mice. Isotype control antibody or LAG-3 blocking antibody given every 2 days. Splenocytes counted and analyzed. B) 1E6 WT or IL-2 KO CD4+ T-cells were transferred into RAG KO/IL-2 KO mice. C) 1E6 WT or STAT5 KO CD4+ T-cells transferred into RAG KO mice. Isotype control antibody or LAG-3 blocking antibody was given every 2 days. Splenocytes were then counted and analyzed. D) LAG-3 antibody staining of LAG-3 on IL-2 KO cells in vivo. E) LAG-3 antibody staining of LAG-3 on STAT5 KO cells in vivo. Data shown are representative of at least two independent experiments with n = 3 mice per group.

Mentions: Based on the finding that RAG1 KO recipients of LAG-3 KO CD4+ T-cells exhibit increased levels of serum IL-2 as compared to their counterparts receiving WT CD4 cells, we examined whether the increased HP observed in recipients of LAG-3 KO T-cells was dependent upon autocrine IL-2 signaling. To test for such a role, CD4+ T-cells were isolated from either IL-2 KO or WT donors, and adoptively transferred into RAG1 KO mice. Animals were treated every 2 days with αLAG-3 or isotype control antibody; and 10 days after transfer, HP was quantified using flow cytometry. Interestingly, LAG-3 blockade increased the total splenocyte number, as well as the total CD4+ T-cell number, regardless of the ability of transferred cells to secrete IL-2 (Figure 2A), arguing against a role for autocrine IL-2 in LAG-3 mediated control of homeostatic proliferation. However, since IL-2 is not totally absent in these animals, it remained possible that exogenous IL-2 (secreted by host cells) was required for LAG-3 to modulate HP. To test whether cell-extrinsic IL-2 is required for LAG-3 blockade to augment HP, we adoptively transferred CD4+ T-cells from IL-2 KO or WT mice into RAG1/IL-2 double knockout (DKO) recipients. In this experiment, DKO mice receiving WT CD4+ T-cells would be exposed only to autocrine IL-2, while DKO recipients receiving IL-2 KO T-cells would have no IL-2 whatsoever. As above, recipient mice were treated with either control antibody or αLAG-3 antibody, and splenocytes were harvested on Day 8. As shown in Figure 2B, LAG-3 blockade increased both the total splenocyte number as well as the total CD4 T-cell number in IL-2 KO mice that received WT cells, demonstrating that cell intrinsic IL-2 was sufficient for LAG-3 blockade to augment HP. However, when neither transferred nor host T-cells were capable of secreting IL-2, LAG-3 blockade was completely without effect in terms of HP. To confirm that the lack of effect of blocking antibody was not due to diminished LAG-3 expression, we stained IL-2 KO CD4+ T-cells in the IL-2 KO hosts (Figure 2D), and found that LAG-3 was still expressed, and that the blocking antibody successfully blocked staining. Taken together, these data show that LAG-3 blockade requires the presence of IL-2 to augment HP, but that either cell-extrinsic or cell intrinsic IL-2 is sufficient. Because IL-2 is known to signal through STAT5-dependent pathways [35], [36], we next sought to determine whether STAT5 signaling is required for LAG-3 to modulate homeostatic proliferation. To perform these studies, we adoptively transferred WT or STAT5 KO CD4 T-cells into RAG1 KO recipients. These mice were treated with αLAG-3 or isotype control antibody, sacrificed on day 10, and splenocytes were counted and analyzed by flow cytometry. Although, as expected, STAT5 KO CD4 T-cells did not expand to the same degree as WT-cells did, LAG-3 blockade did not augment homeostatic proliferation in the STAT5 KO CD4 T-cells (Figure 2C). This was not due to a lack of LAG-3 expression, as STAT5 KO CD4 T-cells still expressed significant levels of LAG-3 (Figure 2E). These data suggest that STAT-5 signaling is required for LAG-3 blockade to affect homeostatic proliferation in vivo, and that one of LAG-3’s functions may be to modulate the STAT5/IL-2 signaling axis during homeostatic proliferation.


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)

IL-2 is required for LAG-3 blockade to augment homeostatic proliferation in vivo.A) 1E6 WT or IL-2 KO CD4 T-cells transferred into RAG KO mice. Isotype control antibody or LAG-3 blocking antibody given every 2 days. Splenocytes counted and analyzed. B) 1E6 WT or IL-2 KO CD4+ T-cells were transferred into RAG KO/IL-2 KO mice. C) 1E6 WT or STAT5 KO CD4+ T-cells transferred into RAG KO mice. Isotype control antibody or LAG-3 blocking antibody was given every 2 days. Splenocytes were then counted and analyzed. D) LAG-3 antibody staining of LAG-3 on IL-2 KO cells in vivo. E) LAG-3 antibody staining of LAG-3 on STAT5 KO cells in vivo. Data shown are representative of at least two independent experiments with n = 3 mice per group.
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pone-0109080-g002: IL-2 is required for LAG-3 blockade to augment homeostatic proliferation in vivo.A) 1E6 WT or IL-2 KO CD4 T-cells transferred into RAG KO mice. Isotype control antibody or LAG-3 blocking antibody given every 2 days. Splenocytes counted and analyzed. B) 1E6 WT or IL-2 KO CD4+ T-cells were transferred into RAG KO/IL-2 KO mice. C) 1E6 WT or STAT5 KO CD4+ T-cells transferred into RAG KO mice. Isotype control antibody or LAG-3 blocking antibody was given every 2 days. Splenocytes were then counted and analyzed. D) LAG-3 antibody staining of LAG-3 on IL-2 KO cells in vivo. E) LAG-3 antibody staining of LAG-3 on STAT5 KO cells in vivo. Data shown are representative of at least two independent experiments with n = 3 mice per group.
Mentions: Based on the finding that RAG1 KO recipients of LAG-3 KO CD4+ T-cells exhibit increased levels of serum IL-2 as compared to their counterparts receiving WT CD4 cells, we examined whether the increased HP observed in recipients of LAG-3 KO T-cells was dependent upon autocrine IL-2 signaling. To test for such a role, CD4+ T-cells were isolated from either IL-2 KO or WT donors, and adoptively transferred into RAG1 KO mice. Animals were treated every 2 days with αLAG-3 or isotype control antibody; and 10 days after transfer, HP was quantified using flow cytometry. Interestingly, LAG-3 blockade increased the total splenocyte number, as well as the total CD4+ T-cell number, regardless of the ability of transferred cells to secrete IL-2 (Figure 2A), arguing against a role for autocrine IL-2 in LAG-3 mediated control of homeostatic proliferation. However, since IL-2 is not totally absent in these animals, it remained possible that exogenous IL-2 (secreted by host cells) was required for LAG-3 to modulate HP. To test whether cell-extrinsic IL-2 is required for LAG-3 blockade to augment HP, we adoptively transferred CD4+ T-cells from IL-2 KO or WT mice into RAG1/IL-2 double knockout (DKO) recipients. In this experiment, DKO mice receiving WT CD4+ T-cells would be exposed only to autocrine IL-2, while DKO recipients receiving IL-2 KO T-cells would have no IL-2 whatsoever. As above, recipient mice were treated with either control antibody or αLAG-3 antibody, and splenocytes were harvested on Day 8. As shown in Figure 2B, LAG-3 blockade increased both the total splenocyte number as well as the total CD4 T-cell number in IL-2 KO mice that received WT cells, demonstrating that cell intrinsic IL-2 was sufficient for LAG-3 blockade to augment HP. However, when neither transferred nor host T-cells were capable of secreting IL-2, LAG-3 blockade was completely without effect in terms of HP. To confirm that the lack of effect of blocking antibody was not due to diminished LAG-3 expression, we stained IL-2 KO CD4+ T-cells in the IL-2 KO hosts (Figure 2D), and found that LAG-3 was still expressed, and that the blocking antibody successfully blocked staining. Taken together, these data show that LAG-3 blockade requires the presence of IL-2 to augment HP, but that either cell-extrinsic or cell intrinsic IL-2 is sufficient. Because IL-2 is known to signal through STAT5-dependent pathways [35], [36], we next sought to determine whether STAT5 signaling is required for LAG-3 to modulate homeostatic proliferation. To perform these studies, we adoptively transferred WT or STAT5 KO CD4 T-cells into RAG1 KO recipients. These mice were treated with αLAG-3 or isotype control antibody, sacrificed on day 10, and splenocytes were counted and analyzed by flow cytometry. Although, as expected, STAT5 KO CD4 T-cells did not expand to the same degree as WT-cells did, LAG-3 blockade did not augment homeostatic proliferation in the STAT5 KO CD4 T-cells (Figure 2C). This was not due to a lack of LAG-3 expression, as STAT5 KO CD4 T-cells still expressed significant levels of LAG-3 (Figure 2E). These data suggest that STAT-5 signaling is required for LAG-3 blockade to affect homeostatic proliferation in vivo, and that one of LAG-3’s functions may be to modulate the STAT5/IL-2 signaling axis during homeostatic proliferation.

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