TCR & CLIC1 Analysis in Sézary Syndrome(Validation Herrara data

Introduction

This analysis validates CLIC1 as a biomarker in Sézary Syndrome using publicly available single-cell RNA-seq data from Herrera et al. (2021). The dataset includes:

• Healthy Controls (HC): 1 donor, blood and skin
• Sézary Syndrome (SS): 6 patients, blood ± skin samples
  
• Mycosis Fungoides (MF): 1 patient, blood and skin

Reference: Herrera et al. (2021) - PMC8532199

https://pmc.ncbi.nlm.nih.gov/articles/PMC8532199/

load libraries

Define File Paths

# Base directory
base_dir <- "/home/bioinfo/1-Thesis_Final_Year_2025/2025-Final_Year_Results/2025-Year3_Analysis/Biomarkers_Validation_with_Public_Data/Herrera_Data/"

# Seurat object
seurat_file <- file.path(base_dir, "Herrara_All_samples.rds")

# TCR data directory
tcr_dir <- file.path(base_dir, "Herrara_TCR_AB")

# Output directory
output_dir <- "output"
dir.create(output_dir, showWarnings = FALSE)

---

Load Seurat Object

cat("Loading Seurat object...\n")

Loading Seurat object...

merged_seurat <- readRDS(seurat_file)

cat("Dataset dimensions:\n")

Dataset dimensions:

cat(sprintf("  - Cells: %d\n", ncol(merged_seurat)))

  - Cells: 30345

cat(sprintf("  - Genes: %d\n", nrow(merged_seurat)))

  - Genes: 33660

cat(sprintf("  - Samples: %s\n", paste(unique(merged_seurat$sample_id), collapse = ", ")))

  - Samples: SS1_Blood, SS2_Blood, SS3_Blood, SS4_Blood, SS5_Blood, SS6_Blood, MF1_Blood, SS1_Skin, SS2_Skin, SS3_Skin, SS4_Skin, MF1_Skin, Healthy_Skin, Healthy_Blood

Load TCR Data

tcr_list <- lapply(names(tcr_files), function(sample_name) {
cat("Reading:", sample_name, "\n")
df <- read.delim(gzfile(tcr_files[[sample_name]]), check.names = FALSE)
colnames(df)[1] <- "clonotype"

df_long <- df %>%
pivot_longer(
cols = -clonotype,
names_to = "cell_barcode",
values_to = "count"
) %>%
filter(count > 0) %>%
mutate(sample_id = sample_name)

return(df_long)
})

Reading: HC1_Blood 
Reading: HC1_Skin 
Reading: SS1_Blood 
Reading: SS1_Skin 
Reading: SS2_Blood 
Reading: SS2_Skin 
Reading: SS3_Blood 
Reading: SS3_Skin 
Reading: SS4_Blood 
Reading: SS4_Skin 
Reading: MFIV1_Blood 
Reading: MFIV1_Skin 
Reading: SS5_Blood 
Reading: SS6_Blood 

combined_tcr <- bind_rows(tcr_list)
cat("Total productive TCR assignments:", nrow(combined_tcr), "\n")

Total productive TCR assignments: 17899 

CLIC1 VALIDATION

Integrate TCR with Seurat

library(stringr)

# Extract alpha and beta chains from clonotype
combined_tcr <- combined_tcr %>%
  mutate(
    TCR_alpha = str_extract(clonotype, "(?<=TRA:)[^|]+"),
    TCR_beta  = str_extract(clonotype, "(?<=TRB:)[^|]+")
  )
# Make cell_barcode match Seurat object
combined_tcr <- combined_tcr %>%
  mutate(cell_barcode = paste0(sample_id, "_", cell_barcode))

# Keep only cells present in Seurat
tcr_in_seurat <- combined_tcr[combined_tcr$cell_barcode %in% colnames(merged_seurat), ]

# Create a metadata table for Seurat
tcr_meta <- tcr_in_seurat %>%
  select(cell_barcode, clonotype, TCR_alpha, TCR_beta) %>%
  column_to_rownames("cell_barcode")

merged_seurat <- AddMetaData(merged_seurat, metadata = tcr_meta)

Identify expanded malignant T cells

# =========================
# Identify expanded malignant T cells per SS sample using scRepertoire
# =========================

ss_samples <- c("SS1_Blood", "SS1_Skin", "SS2_Blood", "SS2_Skin",
                "SS3_Blood", "SS3_Skin", "SS4_Blood", "SS4_Skin",
                "SS5_Blood", "SS6_Blood")

malignant_cells_list <- list()

for (s in ss_samples) {
  sample_cells <- subset(merged_seurat, subset = sample_id == s)
  
  # Skip sample if no TCR_beta
  if (!"TCR_beta" %in% colnames(sample_cells@meta.data)) next
  
  clonotype_counts <- table(sample_cells$TCR_beta)
  expanded_clones <- names(clonotype_counts[clonotype_counts >= 5])
  
  # Skip if no expanded clones
  if (length(expanded_clones) == 0) next
  
  malignant <- subset(sample_cells, subset = TCR_beta %in% expanded_clones)
  
  # Only add if there are cells
  if (ncol(malignant) > 0) malignant_cells_list[[s]] <- malignant
}

# =========================
# Merge all malignant SS cells (robust)
# =========================
malignant_cells_list <- malignant_cells_list[!sapply(malignant_cells_list, is.null)] # remove NULLs
malignant_cells_list <- unname(malignant_cells_list)                                  # remove names
sezary_cells <- Reduce(function(x, y) merge(x, y), malignant_cells_list)             # merge all

highlight top clones

library(scRepertoire)
library(Seurat)

# ================================
# Clonal homeostasis (size distribution)
# ================================
clonalHomeostasis(sezary_cells, cloneCall = "clonotype")

# ================================
#  Clonal proportion / relative abundance
# ================================
clonalProportion(sezary_cells, cloneCall = "clonotype")

NA
NA

highlight top clones

library(scRepertoire)

clonalOverlap(sezary_cells, cloneCall = "clonotype", group.by = "sample_id", method = "morisita")

NA
NA

Separate blood and skin

# ================================
# Merge all Sézary malignant cells with healthy controls
# ================================
# Ensure RNA assay
DefaultAssay(sezary_cells) <- "RNA"

# Normalize and identify variable features
sezary_cells <- NormalizeData(sezary_cells, normalization.method = "LogNormalize", scale.factor = 10000)

Performing log-normalization
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**************************************************|
Performing log-normalization
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**************************************************|
Performing log-normalization
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**************************************************|
Performing log-normalization
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Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
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Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
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Performing log-normalization
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**************************************************|
Performing log-normalization
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**************************************************|
Performing log-normalization
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**************************************************|

sezary_cells <- FindVariableFeatures(sezary_cells, selection.method = "vst", nfeatures = 2000)

Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|

sezary_cells <- ScaleData(sezary_cells)

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sezary_cells <- RunPCA(sezary_cells, npcs = 20)

# Subset healthy control cells
hc_cells <- subset(merged_seurat, subset = sample_id %in% c("Healthy_Blood", "Healthy_Skin"))

# Merge all together
ss_vs_hc <- merge(
  x = sezary_cells,
  y = hc_cells,
  add.cell.ids = c("SS", "HC")
)

# ================================
# Normalize, find variable features, scale, and run PCA & UMAP on merged object
# ================================
DefaultAssay(ss_vs_hc) <- "RNA"
ss_vs_hc <- NormalizeData(ss_vs_hc, normalization.method = "LogNormalize", scale.factor = 10000)

Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
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Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Performing log-normalization
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|

ss_vs_hc <- FindVariableFeatures(ss_vs_hc, selection.method = "vst", nfeatures = 2000)

Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variances
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|

ss_vs_hc <- ScaleData(ss_vs_hc)

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ss_vs_hc <- RunPCA(ss_vs_hc, npcs = 20)
ss_vs_hc <- RunUMAP(ss_vs_hc, dims = 1:20)

Using method 'umap'
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[----|----|----|----|----|----|----|----|----|----|
**************************************************|

# ================================
# Add tissue grouping column
# ================================
ss_vs_hc$tissue_group <- case_when(
  grepl("Blood", ss_vs_hc$sample_id, ignore.case = TRUE) ~ "Blood",
  grepl("Skin", ss_vs_hc$sample_id, ignore.case = TRUE) ~ "Skin",
  TRUE ~ "Other"
)

# ================================
# Visualize CLIC1 expression
# ================================

# FeaturePlot with red gradient
FeaturePlot(ss_vs_hc, features = "CLIC1", reduction = "umap",
            cols = c("grey90", "red"), label = TRUE)

# DotPlot by sample_id with red-grey gradient
DotPlot(ss_vs_hc, features = "CLIC1", group.by = "sample_id") +
  RotatedAxis() +
  scale_color_gradient(low = "grey90", high = "red") +
  ggtitle("CLIC1 Expression by Sample") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot by tissue_group with red-grey gradient
DotPlot(ss_vs_hc, features = "CLIC1", group.by = "tissue_group") +
  RotatedAxis() +
  scale_color_gradient(low = "grey90", high = "red") +
  ggtitle("CLIC1 Expression by Tissue") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# ================================
# Visualize CLIC1
# ================================
# FeaturePlot
FeaturePlot(ss_vs_hc, features = "CLIC1", reduction = "umap")

# DotPlot grouped by sample
DotPlot(ss_vs_hc, features = "CLIC1", group.by = "sample_id") + RotatedAxis()

# DotPlot grouped by tissue
DotPlot(ss_vs_hc, features = "CLIC1", group.by = "tissue_group") + RotatedAxis()

Visualize CLIC1

# DimPlot by sample
DimPlot(ss_vs_hc, group.by = "sample_id", reduction = "umap")

# FeaturePlot for CLIC1
FeaturePlot(ss_vs_hc, features = "CLIC1", reduction = "umap")

# DotPlot for CLIC1 across groups
DotPlot(ss_vs_hc, features = "CLIC1", group.by = "sample_id") + 
  theme(axis.text.x = element_text(angle = 45, hjust = 1))

# FeaturePlot with red gradient
FeaturePlot(ss_vs_hc, features = "CLIC1", reduction = "umap",
            cols = c("grey90", "red"), label = TRUE, repel = T)

FeaturePlots for Top50 UP

top_50_up <- read.csv("top_50_upregulated.csv")        # or read.delim("top_50_up.tsv")
top_50_down <- read.csv("top_50_downregulated.csv")



FeaturePlot(ss_vs_hc, 
             features = top_50_up$gene[1:10], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_up$gene[11:20], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_up$gene[21:30], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_up$gene[31:40], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_up$gene[41:50], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlots for Top50 DOWN

FeaturePlot(ss_vs_hc, 
             features = top_50_down$gene[1:10], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_down$gene[11:20], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_down$gene[21:30], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_down$gene[31:40], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

FeaturePlot(ss_vs_hc, 
             features = top_50_down$gene[41:50], 
             reduction = "umap", 
             cols = c("lightblue", "red"),  # Custom color gradient from light blue to red
             label = TRUE)

  )

Error: unexpected ')' in "  )"

Visualization of Potential biomarkers-Upregulated

# Vector of genes to plot
up_genes <- c("CLIC1", "COX5A","GTSF1", "MAD2L1","MYBL2","MYL6B","NME1","PLK1", "PYCR1", "SLC25A5", "SRI", "TUBA1C", "UBE2T", "YWHAH")

# DotPlot with custom firebrick-red gradient
DotPlot(ss_vs_hc, features = up_genes) +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Upregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot with custom firebrick-red gradient
DotPlot(ss_vs_hc, features = up_genes, group.by = "sample_id") +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Upregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot with custom firebrick-red gradient
DotPlot(ss_vs_hc, features = up_genes, group.by = "tissue") +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Upregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot with custom firebrick-red gradient
DotPlot(ss_vs_hc, features = up_genes, group.by = "condition") +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Upregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot with custom firebrick-red gradient
DotPlot(ss_vs_hc, features = up_genes, group.by = "condition") +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Upregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14))

Visualization of Potential biomarkers-Downregulated

# Downregulated genes
down_genes <- c("TXNIP", "RASA3", "RIPOR2", 
                "ZFP36", "ZFP36L1", "ZFP36L2",
                "PRMT2", "MAX", "PIK3IP1", 
                "BTG1", "CDKN1B")

# DotPlot with firebrick color for high expression
DotPlot(ss_vs_hc, features = down_genes) +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Downregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot with firebrick color for high expression
DotPlot(ss_vs_hc, features = down_genes, group.by = "sample_id") +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Downregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot with firebrick color for high expression
DotPlot(ss_vs_hc, features = down_genes, group.by = "tissue") +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Downregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

# DotPlot with firebrick color for high expression
DotPlot(ss_vs_hc, features = down_genes, group.by = "condition") +
  RotatedAxis() +
  scale_color_gradient2(low = "lightblue", mid = "red", high = "firebrick", midpoint = 1) +
  ggtitle("Expression of Downregulated Genes in Sézary Syndrome") +
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
    axis.text.y = element_text(size = 12),
    plot.title = element_text(hjust = 0.5, face = "bold", size = 14)
  )

Save Seurat object

saveRDS(ss_vs_hc, file = "Sezary_Blood_Skin_vs_HC_TCR_filtered.rds")

saveRDS(ss_vs_hc_harmony, file = "Sezary_Blood_Skin_vs_HC_TCR_filtered.rds")