--- a
+++ b/Fig5G_CD70_CRISPR_T cell_stimulation.R
@@ -0,0 +1,79 @@
+
+# Analyze JE6-TPR stimulation experiments with SUDHL5 CD70 knockout co-culture (Figure 5G)
+
+library(data.table)
+library(readxl)
+library(dplyr)
+library(ggplot2)
+library(cowplot)
+library(gridExtra)
+library(RColorBrewer)
+library(ggpubr)
+
+
+# load data
+data <- fread("cd70_crispr_flow_cytometry.txt", data.table = F)
+
+# calculate percentages of NFAT/NFkB-activated T cells
+# LL (lower left) = double negative
+# LR (lower right) = NFAT only
+# UL (upper left) = NFkB only
+# UR (upper right) = double positive
+
+df <- data %>%
+  mutate(condition = paste(TPR, TCS, SUDHL5, sep = "_")) %>%
+  mutate(NFAT_NFkB_LL_percentage = 100*Count_of_NFAT_NFkB_LL/Count_of_TPR,
+         NFAT_NFkB_LR_percentage = 100*Count_of_NFAT_NFkB_LR/Count_of_TPR,
+         NFAT_NFkB_UL_percentage = 100*Count_of_NFAT_NFkB_UL/Count_of_TPR,
+         NFAT_NFkB_UR_percentage = 100*Count_of_NFAT_NFkB_UR/Count_of_TPR)
+
+
+# Barplot of % NFkB only-activated Jurkat-CD27 cells stimulated with TCS-Ctrl (stimulator cells with anti-CD3) + SUDHL5 with sgCD70/Ctrl sgRNAs
+
+PARAMETER = "NFAT_NFkB_UL_percentage"
+
+df_values <- df[df$TPR == "TPR-CD27" & (df$TCS=="TCS-Ctrl"|(df$TCS=="no_TCS"&df$SUDHL5=="no_SUDHL5")), c("TPR", "TCS", "SUDHL5", PARAMETER)]
+colnames(df_values) <- c("TPR", "TCS", "SUDHL5", "parameter")
+  
+  df_values <- df_values %>%
+    mutate(condition = ifelse(TCS=="no_TCS"&SUDHL5=="no_SUDHL5", "no stimulation", gsub(" \\+ no SUDHL5", "", gsub("_|\\.", " ", gsub("Ctrl", "Control", paste("anti-CD3 +", SUDHL5))))))
+  
+  df_stats <- df_values %>%
+    group_by(condition) %>% 
+    summarize(mean = mean(parameter),
+              sd = sd(parameter))
+  
+  df_plot <- merge(df_values, df_stats) %>%
+    mutate(condition = factor(condition, levels = c("no stimulation", "anti-CD3", "anti-CD3 + SUDHL5 sgControl", "anti-CD3 + SUDHL5 sgCD70 1", "anti-CD3 + SUDHL5 sgCD70 2", "anti-CD3 + SUDHL5 sgCD70 3")))
+    
+  
+  p <- ggplot(df_plot, aes(x = condition, y = parameter)) +
+    geom_bar(aes(x = condition, y = mean, fill = condition), stat = "identity", position = position_dodge(), color = "grey20") +
+    geom_errorbar(aes(ymin=mean-sd, ymax=mean+sd),
+                  width=0.3,
+                  size=0.3,
+                  color = "grey20") +
+    geom_jitter(aes(x = condition, y = parameter), width = 0.1, color = "grey20", show.legend = F) +
+    scale_fill_manual(values = brewer.pal(11, "RdGy")[c(7,8,9,4,3,2)]) +
+    ylab("% NFkB-activated\nT cells") +
+    xlab("") +
+    scale_y_continuous(expand = c(0,0), limits = c(0,31)) +
+    guides(fill=FALSE) +
+    stat_compare_means(data = df_plot, aes(x = condition, y = PARAMETER, label = ..p.signif..),
+                       inherit.aes = F,
+                       method = "t.test",
+                       comparisons = list(c("anti-CD3", "no stimulation"),
+                                          c("anti-CD3 + SUDHL5 sgControl", "no stimulation"),
+                                          c("anti-CD3 + SUDHL5 sgCD70 1", "anti-CD3 + SUDHL5 sgControl"),
+                                          c("anti-CD3 + SUDHL5 sgCD70 2", "anti-CD3 + SUDHL5 sgControl"),
+                                          c("anti-CD3 + SUDHL5 sgCD70 3", "anti-CD3 + SUDHL5 sgControl")),
+                       label.y = c(21, 23, 25, 27, 29)) +
+    theme_cowplot() +
+    theme(axis.text.x = element_text(angle=45, hjust=1),
+          legend.title=element_blank())
+  
+  pdf("Figure5G_CD70_CRISPR_T_cell_stimulation.pdf", width = 2.75, height = 4.5)
+  p
+  dev.off()
+  
+  
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