Combinational test of DGKi with PD-1 blocker

scTCR-seq + scRNA-seq

DGKi + Nivolumab combinational therapy

Introduction
- DGKs are physiological regulators of T-cell development,differentiation, and function.
- Inhibition of DGK may restore the T-cell activation program
- Enhanced effects may be seen with combination therapy (Nivolumab + DGKi)


Data

Sample name: P*****
Condition (1 control, 3 test conditions): CONTROL, CND1, CND2, COMBO
Number of cells : ~9000 cells

Initial Processing

## perform default analysis
perform_default_analysis <- function(obj.srt, n_features = 2000, n_pcs = 20, 
                                     dims_for_neighbors = 1:10, 
                                     resolutions = c(0.2, 0.5), 
                                     umap_dims = 1:10) {
  # Step 1: Find variable features
  obj.srt <- FindVariableFeatures(obj.srt, 
                                  selection.method = 'vst', 
                                  nfeatures = n_features)
  
  # Step 2: Scale and normalize data
  all_genes <- rownames(obj.srt)
  obj.srt <- NormalizeData(obj.srt)
  obj.srt <- ScaleData(obj.srt, features = all_genes)
  
  # Step 3: Run PCA
  obj.srt <- RunPCA(obj.srt, 
                    features = VariableFeatures(object = obj.srt), npcs = n_pcs)
  
  # Step 4: Find neighbors
  obj.srt <- FindNeighbors(obj.srt, dims = dims_for_neighbors)
  
  # Step 5: Find clusters
  obj.srt <- FindClusters(obj.srt, resolution = resolutions)
  
  # Step 6: Run UMAP
  obj.srt <- RunUMAP(obj.srt, dims = umap_dims)
  
  # Return the Seurat object with analysis results
  return(obj.srt)
}

# apply
obj.srt <- perform_default_analysis(obj.srt)

Numer of cells

UMAP (sample)

Colored by condition

Colored by each condition

Clustering

Clusters

  1. resolution : 0.2
  2. resolution : 0.5

cluster and condition (res 0.1)

TABLE1 : Number of cells (res 0.2)

HEATMAP1 : Percentage of cells (res 0.2)

cluster and condition (res 0.5)

TABLE2 : Number of cells (res 0.5)

HEATMAP2 : Percentage of cells (res 0.5)



Clonotype Diversity

CTRL

CND1

CND2

COMB

scRepertoire

Clonotype analysis using scRepertoire package

Analysis below represents the basic analysis using the package.

Quantify Clonotypes

Unique clonotypes by TRA chain

Unique clonotypes by TRB chain

Clonotype Abundance

Length of Clonotypes

Compare Clonotypes

Visualize Gene Usage

More Advanced Clonal Analysis

Diversity Analysis




Specific cell type + clonotype

Cytotoxic T Cell Markers and associated clonotypes

Purpose : Try to find the clonotypes with strong cytotoxic T cell marker gene expressions, expecting functional clonotypes to fight the disease.

Cytotoxic T cell markers :

Mostly enzymes that kill target cells.

  • IFNG (Increase when infection)
  • GZMA (Granzyme A)
  • GZMB (Granzyme B)
  • NKG7 (NK cell granule protein 7)



Reordered by clonotype version

Note: No clear pattern across cytotoxic gene expression by clonotypes.