Antitumor efficacy of combined CTLA4/PD-1 blockade without intestinal inflammation is achieved by elimination of FcγR interactions

Background Programmed cell death protein 1 (PD-1) and CTLA4 combination blockade enhances clinical efficacy in melanoma compared with targeting either checkpoint alone; however, clinical response improvement is coupled with increased risk of developing immune-related adverse events (irAE). Delineating the mechanisms of checkpoint blockade-mediated irAE has been hampered by the lack of animal models that replicate these clinical events.Methods We have developed a mouse model of checkpoint blockade-mediated enterocolitis via prolonged administration of an Fc-competent anti-CTLA4 antibody.Results Sustained treatment with Fc-effector, but not Fc-mutant or Fc-null, anti-CTLA4 antagonist for 7 weeks resulted in enterocolitis. Moreover, combining Fc-null or Fc-mutant CTLA4 antagonists with PD-1 blockade results in potent antitumor combination efficacy indicating that Fc-effector function is not required for combination benefit.Conclusion These data suggest that using CTLA4 antagonists with no Fc-effector function can mitigate gut inflammation associated with anti-CTLA4 antibody therapy yet retain potent antitumor activity in combination with PD-1 blockade

Lymphocyte Activation Gene 3 (LAG-3) Modulates the Ability of CD4 T-cells to Be Suppressed <i>In Vivo</i>

<div><p>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 <i>in vivo,</i> 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 T_H1 phenotype both <i>in vitro</i> and in <i>in vivo</i>. 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 T_H1 T-cell response.</p></div

IL-2 is required for LAG-3 blockade to augment homeostatic proliferation <i>in vivo</i>.

<p>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 <i>in vivo</i>. E) LAG-3 antibody staining of LAG-3 on STAT5 KO cells <i>in vivo</i>. Data shown are representative of at least two independent experiments with n = 3 mice per group.</p

WT Treg Cannot Completely Protect against LAG-3 KO Tresp in a Colitis Model.

<p>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.</p

LAG-3 blockade augments homeostatic proliferation <i>in vivo</i>.

<p>A) 1E6 WT or LAG-3 KO CD4+ T-cells were adoptively transferred into RAG KO mice and harvested on day 10. Splenocytes were then counted and analyzed. B) 1E6 WT CD4+ T-cells were transferred into RAG KO mice. Isotype control antibody or LAG-3 blocking antibody given every 2 days. Splenocytes were then counted and analyzed. C) LAG-3 antibody staining of LAG-3 <i>in vivo</i>. D) Serum IL-2 from RAG KO mice with 1E6 WT or LAG-3 KO CD4+ T-cells on Day 7. E) Percentage of CD4+ T-cells that express FOXP3. F) Total number of adoptively transferred CD4+ T-cells expressing FoxP3. Data shown are representative of at least two independent experiments with n = 3–6 mice per group.</p

LAG-3 KO Treg suppress homeostatic proliferation, but LAG-3 KO responders are resistant to suppression.

<p>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.</p

Decreased FOXP3 Treg induction in LAG-3 KO cells <i>in vitro</i>.

<p>WT or LAG-3 KO 6.5 TCR Transgenic CD4+ T-cells skewed in Th1 or Treg Conditions and analyzed for A) FOXP3 or B) TBET Expression. C) 6.5 TCR transgenic CD4+ T-cells were isolated and mixed with matched splenocytes 1∶3 and stimulated with 1 or 10 µM HA peptide for 3 days. WT or LAG-3 KO 6.5 CD4+ T-cells were treated with αLAG-3 or isotype control antibody at 50 µg/mL and cells were stained for P-STAT5. D) Summary graph of two experiments. Data shown are representative of at least two independent experiments.</p

Decreased FOXP3 Treg induction in LAG-3 KO T-cells in an <i>in vivo</i> self-tolerance.

<p>A) WT or LAG-3 KO 6.5 TCR Transgenic CD4+ T-cells adoptively transferred into C3-HA expressing mice. B) FOXP3 and C) Tbet expression was analyzed and representative graphs shown. C–D) Summary of FOXP3 expression in adoptively transferred cells. E–F) Summary of TBET expression in adoptively transferred cells. Data shown are representative of at least two independent experiments where n = 4 mice per group.</p

Building a network for multicenter, prospective research of central nervous system infections in South America: Process and lessons learned

Multicenter collaborative networks are essential for advancing research and improving clinical care for a variety of conditions. Research networks are particularly important for central nervous system infections, which remain difficult to study due to their sporadic occurrence and requirement for collection and testing of cerebrospinal fluid. Establishment of long-term research networks in resource-limited areas also facilitates diagnostic capacity building, surveillance for emerging pathogens, and provision of appropriate treatment where needed. We review our experience developing a research network for encephalitis among twelve hospitals in five Peruvian cities since 2009. We provide practical suggestions to aid other groups interested in advancing research on central nervous system infections in resource-limited areas. Keywords: Central nervous system infections, Encephalitis, Epidemiology, Herpes simplex virus, Virolog