BATF3 Human

What is BATF3 and what are its primary functions in human immune cells?

BATF3 (Basic Leucine Zipper ATF-Like Transcription Factor 3) is a 14.5-20 kDa nuclear member of the bZIP family of proteins. It functions as a transcription factor that controls the differentiation of CD8+ conventional dendritic cells (cDCs) in the immune system. BATF3 acts via formation of heterodimers with JUN family proteins, recognizing and binding DNA sequence 5'-TGA[CG]TCA-3' to regulate target gene expression. In humans, BATF3 is preferentially expressed in effector CD4 T cells and conventional dendritic cells, where it plays critical roles in immune response regulation and cell fate determination .

How does BATF3 expression differ across human immune cell populations?

BATF3 shows differential expression across immune cell populations. It is predominantly expressed in:

• Effector CD4 T cells but not in regulatory T (Treg) cells

• CD141+ Myeloid Dendritic Cells

• CD8α+ Thymic Conventional Dendritic Cells

• Conventional dendritic cells (CD11c+)

• Th1 cells

The expression is typically transient in T cells, appearing within the first days after T cell priming and having long-lasting effects. Detection methods such as flow cytometry have shown BATF3 expression in human PBMC monocytes, particularly in CD11c+ populations, and Jurkat T cell leukemia cell lines .

What is the role of BATF3 in dendritic cell development and how can researchers study this function?

BATF3 is essential for the development of conventional type 1 dendritic cells (cDC1), specifically controlling the differentiation of CD8α+ and CD103+ DCs. These BATF3-dependent cDC1s are critical for priming CD8+ T cell-mediated immunity against intracellular pathogens and malignancies.

To study this function, researchers can:

• Use Batf3-knockout mouse models that specifically lack cDC1 populations

• Perform lineage-tracing experiments using cell-specific markers (CD103, CD8α, XCR1)

• Implement conditional knockout systems to temporally control BATF3 deletion

• Conduct bone marrow chimera experiments to assess cell-intrinsic effects

• Analyze human BATF3 polymorphisms and correlate with dendritic cell phenotypes .

How does BATF3 influence CD8+ T cell memory formation and what are the molecular mechanisms involved?

BATF3 programs CD8+ T cell memory through multiple mechanisms:

• When overexpressed in CD8+ T cells, BATF3 enhances their survival and transition to memory state

• BATF3 regulates T cell apoptosis and longevity via the proapoptotic factor BIM

• BATF3-expressing T cells show normal expansion and differentiation but reduced contraction phase

• Gene Ontology analyses revealed that BATF3-induced genes are enriched for DNA and mRNA metabolic processing, ribosomal biogenesis and metabolic pathways such as glycolysis

BATF3 overexpression increases expression of IL7R, a surface marker associated with T cell survival, long-term persistence, and positive clinical response to adoptive cell therapy. Mechanistically, BATF3 overexpression increases chromatin accessibility at regions near memory-associated genes (TCF7, MYB, IL7R, CCR7, SELL) while reducing accessibility at exhaustion-associated loci (TIGIT, CTLA4, LAG3) .

What is the role of BATF3 in regulatory T cell (Treg) differentiation and function?

BATF3 acts as a fate-decision factor in T cell differentiation by inhibiting regulatory T cell development. Key findings include:

• BATF3 is preferentially expressed in effector CD4 T cells but not in Treg cells

• Ectopic expression of BATF3 inhibits Foxp3 induction, the master transcription factor for Treg differentiation

• BATF3-deficient CD4 T cells preferentially differentiate into Treg cells in vitro and in vivo

• Mechanistically, BATF3 binds to the CNS1 region of the Foxp3 locus and reduces its expression

• BATF3 knockout mice show enhanced Treg function in gut-associated immune disease models

This inhibitory role positions BATF3 as a critical regulator of the balance between effector and regulatory T cell populations .

What are the most reliable methods for detecting and quantifying BATF3 expression in human samples?

For detecting and quantifying BATF3 in human samples, researchers can use several complementary approaches:

For optimal results, intracellular staining protocols with proper fixation and permeabilization are essential when using flow cytometry. Both polyclonal and monoclonal antibodies are available, with monoclonals offering better specificity for human BATF3 .

How can researchers effectively manipulate BATF3 expression in primary human T cells for functional studies?

Several approaches are available for manipulating BATF3 expression in primary human T cells:

• Lentiviral/Retroviral Expression Systems:

  • Compact size of BATF3 (381 bp) makes it amenable to viral delivery

  • Can be incorporated into the same lentivirus delivering CAR or TCR constructs

  • Consider including tracking markers (GFP) to identify transduced cells

• CRISPR-Based Methods:

  • CRISPRa for gene activation: dSaCas9-based systems can upregulate endogenous BATF3

  • CRISPRi for gene repression: Target BATF3 promoter regions

  • CRISPRko for complete knockout: Multiple guide RNAs targeting different exons

• Transfection Approaches:

  • Electroporation of BATF3 mRNA for transient expression

  • Nucleofection of plasmid DNA for longer expression

• Inducible Systems:

  • Tet-On/Off systems for temporal control of BATF3 expression

  • Destabilization domain-based systems for rapid protein depletion

For optimal results, researchers should consider timing of expression manipulation relative to T cell activation state, as BATF3 effects are context-dependent .

What are the key considerations when using BATF3 knockout models for immunological research?

When using BATF3 knockout models, researchers should consider:

• Developmental vs. Acute Effects:

  • Constitutional knockouts affect development of cDC1s, causing broad immunological defects

  • Consider conditional/inducible knockouts to separate developmental from functional effects

• Cell Type-Specific Consequences:

  • Primary defect is in CD8α+ and CD103+ dendritic cell development

  • Secondary effects on CD8+ T cell priming and memory formation

  • Altered IgA-coating of bacteria and microbial dysbiosis

• Compensatory Mechanisms:

  • Alternative transcription factors may partially compensate for BATF3 deficiency

  • Assess expression of related factors (BATF, BATF2, AP-1 family members)

• Strain and Environmental Considerations:

  • Different mouse strains may show variable phenotype severity

  • Microbiome composition significantly impacts phenotype (consider co-housing controls)

  • Diet interactions are particularly important (high-fat diet enhances metabolic phenotypes)

• Experimental Design Controls:

  • Include bone marrow chimeras to distinguish cell-intrinsic from environmental effects

  • Use mixed bone marrow chimeras with WT and knockout cells to control for systemic effects .

How does BATF3 contribute to anti-tumor immunity and cancer immunotherapy?

BATF3 contributes to anti-tumor immunity through multiple mechanisms:

• Development of Critical Dendritic Cell Populations:

  • BATF3 is essential for the development of cDC1s (CD103+ DCs) that cross-present tumor antigens to CD8+ T cells

  • BATF3-dependent DCs are required within the tumor microenvironment (TME) for PD-1/PD-L1 blockade efficacy

• T Cell Programming Effects:

  • BATF3 overexpression in CD8+ T cells enhances their survival and transition to memory state

  • Decreases expression of exhaustion-associated genes like TIGIT, CTLA4, and LAG3

  • Programs a transcriptional profile that correlates with positive clinical response to adoptive T cell therapy

• Costimulatory Functions:

  • BATF3+ DCs provide critical 4-1BB/4-1BBL costimulatory signals within the tumor microenvironment

  • These signals are required for the efficacy of checkpoint blockade therapy

For therapeutic applications, BATF3 overexpression markedly enhanced the potency of CAR T cells in both in vitro and in vivo tumor models. Its compact size makes it particularly amenable to integration into current adoptive cell therapy manufacturing processes .

What is the role of BATF3 in metabolic disorders and intestinal homeostasis?

BATF3 plays a protective role against metabolic syndrome and maintains intestinal epithelial homeostasis:

• Metabolic Regulation:

  • BATF3-deficient mice develop metabolic syndrome characterized by insulin resistance, elevated blood glucose and serum insulin levels, increased body weight and white adipocyte size

  • Hyperinsulinemia and hypercholesterolemia are the earliest metabolic changes observed in lean BATF3-deficient mice

• Intestinal Barrier Function:

  • BATF3 deficiency leads to altered localization of tight junction proteins (occludin-1, ZO-1, claudin-2) in intestinal epithelial cells

  • This results in increased intestinal permeability, which contributes to low-grade inflammation

  • Treatment with glycolysis inhibitor 2-deoxy-D-glucose reduced intestinal inflammation and restored barrier function

• Microbiome Effects:

  • BATF3-deficient mice show decreased IgA-coating of fecal bacteria

  • Display microbial dysbiosis with decreased levels of beneficial bacteria like Akkermansia muciniphila and Bifidobacterium

  • Antibiotic treatment prevents metabolic syndrome development in these mice

These findings suggest that BATF3-dependent dendritic cells play a critical role in maintaining intestinal barrier function and preventing metabolic dysregulation through microbiome-dependent mechanisms .

How does BATF3 interact with other transcription factors to regulate gene expression?

BATF3 functions through a complex network of interactions with other transcription factors:

• JUN Family Interactions:

  • BATF3 heterodimerizes with JUN family proteins to bind DNA

  • Acts as a transcriptional repressor when heterodimerizing with JUN

  • May function in repression of interleukin-2 and matrix metalloproteinase-1 transcription

• IRF4 Cooperative Activity:

  • Forms a BATF3/IRF4 complex that can bind the IL-9 promoter to induce Th9 cell differentiation

  • CRISPRko screens identified IRF4 as a cofactor of BATF3 in T cells

• Glucocorticoid Receptor Interactions:

  • BATF3 acts as a gene-specific coactivator of the Glucocorticoid Receptor (GR)

  • This coactivator potency is influenced by the sequence of the GR binding site

  • Interaction with GR is modulated by the GR "lever arm" domain

• JUNB Cooperation:

  • CRISPRko screens revealed that BATF3 heterodimerizes with JUNB in CD8+ T cells

  • This interaction is essential for driving transcriptional programs

Understanding these interactions is crucial for developing strategies to modulate BATF3 activity in therapeutic contexts .

What are the chromatin-level effects of BATF3 and how can they be studied?

BATF3 exerts significant effects on chromatin accessibility and epigenetic regulation:

• Chromatin Accessibility Changes:

  • BATF3 overexpression increases accessibility at regions near memory-associated genes (TCF7, MYB, IL7R, CCR7, SELL)

  • Decreases accessibility at exhaustion-associated loci (TIGIT, CTLA4, LAG3)

  • Approximately 25% of genes that change expression with BATF3 overexpression show corresponding changes in local chromatin accessibility

• DNA Binding Patterns:

  • BATF3 binds to the CNS1 region of the Foxp3 locus to reduce its expression

  • Acts by recognizing and binding the DNA sequence 5'-TGA[CG]TCA-3'

• Methodological Approaches:

  Technique	Application	Key Considerations
  ATAC-seq	Maps genome-wide chromatin accessibility	50,000 cells minimum; sort for viable cells
  ChIP-seq	Identifies direct BATF3 binding sites	Requires high-quality ChIP-grade antibodies
  CUT&RUN	Alternative to ChIP with lower cell numbers	Better signal-to-noise ratio than ChIP
  HiChIP	Identifies long-range chromatin interactions	Maps 3D chromatin organization at BATF3 loci
  CRISPRi-seq	Screens for functional impact of BATF3 binding	Requires dCas9-KRAB targeting to BATF3 sites

HOMER motif analysis can be used to find transcription factor binding motifs that contribute to changes in chromatin accessibility with BATF3 overexpression compared to control cells .

What are the current challenges and future directions in human BATF3 research?

Current challenges and future directions in BATF3 research include:

• Safety Considerations for Therapeutic Applications:

  • Sustained expression of BATF3 combined with other genetic alterations could potentially lead to antigen-independent clonal T cell expansion

  • Need to develop transient delivery methods, modulate transgene expression, or integrate suicide switches

• Cell Type-Specific Functions:

  • Better understanding of BATF3 roles beyond dendritic cells and T cells

  • Context-dependent functions in different tissue microenvironments

• Temporal Dynamics:

  • Elucidating the temporal regulation of BATF3 expression during immune responses

  • Understanding how transient expression yields long-lasting effects

• Tissue-Specific Effects:

  • Exploring BATF3 function in specialized tissue niches (e.g., tumor microenvironment, intestinal mucosa)

  • Differences between circulating and tissue-resident immune cells

• Systems Biology Approaches:

  • Integration of multi-omics data to build comprehensive models of BATF3 regulatory networks

  • Single-cell approaches to address cellular heterogeneity

• Therapeutic Engineering:

  • Developing optimized BATF3 variants for enhanced T cell persistence in adoptive therapies

  • Combining BATF3 with other transcription factors for synergistic effects

• Metabolic Integration:

  • Further understanding how BATF3 connects immune function with metabolic regulation

  • Potential for targeting BATF3 in metabolic diseases .