What are the cells of acquired immunity, their cytokines, and associated Immunohistochemistry (IHC) markers?

Cells of Acquired Immunity: Cytokines and IHC Markers

Primary Cellular Components

The acquired immune system consists of two fundamental lymphocyte populations: T cells mediating cellular immunity and B cells mediating humoral immunity, both originating from common precursors but functionally distinct in their cytokine production patterns and effector mechanisms. 1, 2

T Lymphocytes

T cells are the predominant cytokine producers upon activation, with distinct functional subsets characterized by specific markers and cytokine profiles 3:

Key IHC Markers for T Cell Identification:

• CD3: Pan-T cell marker 3
• CD4: Helper T cell subset marker 4
• CD8: Cytotoxic T cell subset marker 3, 4
• CD69: Early activation marker 3
• PD-1: Activation and exhaustion marker 3
• LAMP1 (CD107a): Degranulation marker for cytotoxic function 3

T Cell Cytokine Production:

• IFN-γ (Interferon-gamma): Primary effector cytokine measured by intracellular staining or ELISPOT 3
• IL-2 (Interleukin-2): T cell growth factor, marker of polyfunctional responses 3
• TNF (Tumor Necrosis Factor): Pro-inflammatory cytokine, component of polyfunctional T cell responses 3
• Perforin 1 (PRF1): Cytotoxic effector molecule 3
• Granzyme B (GZMB): Cytotoxic effector molecule 3

Polyfunctional T cells producing multiple cytokines simultaneously (IFN-γ, IL-2, TNF) represent higher quality immune responses and correlate with better clinical outcomes in immunotherapy trials 3.

B Lymphocytes

B cells require specific differentiation and activation conditions to produce cytokines, unlike T cells that produce multiple cytokines immediately upon activation 5, 6:

B Cell Cytokine Production Patterns:

• IL-6 (Interleukin-6): Influences CD4+ T cell development 6
• IL-10 (Interleukin-10): Regulatory cytokine, predominantly from marginal zone and B1 cell subsets 6, 7
• IL-35: Regulatory function 6
• IFN-γ: Produced by follicular B cells under specific stimulation (combined TLR or phorbol ester activation) 6, 7
• TNF: Influences T cell responses 6
• Lymphotoxin: Essential for lymphoid organ development 6

B Cell IHC Markers:

• CD19/CD20: Pan-B cell markers 3
• Surface immunoglobulin: B cell receptor 2
• CD80, CD83, CD86: Costimulatory molecules when B cells function as antigen-presenting cells 3

Critical distinction: B cell cytokine production depends on their differentiation state and the nature of activating stimulus (TLR2/4/9 for IL-10, combined TLR stimulation for IFN-γ), contrasting sharply with dendritic cells due to differential TLR signaling molecule expression 7.

Antigen-Presenting Cells (APCs)

Dendritic Cells and Macrophages

APCs bridge innate and adaptive immunity through antigen presentation and cytokine secretion 3, 2:

APC Maturation Markers (IHC):

• MHC Class II molecules: Antigen presentation capacity 3
• CD80: Costimulatory molecule, upregulated during maturation 3
• CD83: Maturation marker 3
• CD86: Costimulatory molecule, upregulated during maturation 3

APC Cytokine Secretion:

• IL-1β (Interleukin-1 beta): Pro-inflammatory, requires mature form detection 3
• IL-6: Immunostimulatory phenotype 3
• IL-12: Critical for Th1 differentiation, standardized potency assay for DC vaccines 3
• IL-23: Immunostimulatory function 3

IL-12p70 production by dendritic cells represents a validated functional potency assay that correlates with clinical outcomes in DC-based vaccine trials 3.

Regulatory Cell Populations

Regulatory T Cells (Tregs)

Regulatory T cells suppress inflammatory responses through anti-inflammatory cytokine production 8, 1:

Treg Markers:

• CD4: Surface marker 1
• CD25: IL-2 receptor alpha chain 1
• Foxp3: Transcription factor, definitive Treg marker 3

Treg Cytokines:

• IL-10: Anti-inflammatory, limits excessive inflammation 8
• TGF-β: Regulatory function 8

High frequencies of disease-specific CD4+CD25+Foxp3+ T cells with low IFN-γ production correlate with vaccine failure in therapeutic settings 3.

Natural Killer (NK) Cells

NK cells represent innate lymphocytes that bridge innate and adaptive immunity 8:

NK Cell Markers:

• CD56: NK cell marker 8
• CD16: Fc receptor for antibody-dependent cellular cytotoxicity 8
• CD107a: Degranulation marker for cytotoxic function 3

NK Cell Function:

• MHC-I recognition: NK cells spare healthy cells expressing normal MHC-I levels but target cells with downregulated MHC-I 8
• Cytotoxicity measurement: Flow cytometry-based assays measuring target cell death and NK phenotype simultaneously 3

Standardized Detection Methods

Flow Cytometry-Based Approaches

Multiparametric flow cytometry remains the gold standard for simultaneous assessment of cell surface markers, intracellular cytokines, and functional status 3:

• Intracellular cytokine staining: Detects IFN-γ, IL-2, TNF, perforin, granzyme B with surface phenotyping 3
• Activation marker assessment: CD69, LAMP1, PD-1 expression 3
• Proliferation tracking: CFSE dilution assays 3

Functional Assays

ELISPOT represents the most thoroughly standardized functional assay for quantifying antigen-specific cytokine-producing cells 3:

• IFN-γ ELISPOT: Correlates with clinical outcome in multi-center trials 3
• Granzyme B ELISPOT: Measures cytotoxic function, correlates better with chromium release than tetramer staining 3

Critical caveat: MHC tetramer frequencies alone do not correlate with clinical outcome, but IFN-γ ELISPOT responses do 3, emphasizing the importance of functional over phenotypic assessment.

Cross-Priming Assessment

The definitive test of adaptive immunity involves measuring APC ability to cross-prime cytotoxic T lymphocytes through sequential coculture assays 3:

1. Phagocytosis: CFSE or PKH26 dual-labeling of APCs and target cells 3
2. APC maturation: CD80/CD83/CD86 upregulation by flow cytometry 3
3. T cell activation: CD69, LAMP1, PD-1 expression 3
4. Cytotoxic function: Target cell lysis measurement 3

Pattern Recognition and DAMP Detection

Acquired immunity initiation requires innate recognition through pattern recognition receptors (PRRs) detecting pathogen-associated molecular patterns and damage-associated molecular patterns (DAMPs) 8:

Key DAMPs Recognized:

• ATP: Recruits APCs to cell death sites 8
• HMGB1: Nuclear protein released upon damage 8
• Calreticulin: ER chaperone exposed on damaged cells 8
• Heat shock proteins (HSP70, HSP90): Stress-induced proteins 8

These DAMPs activate APCs to initiate adaptive immune responses, bridging innate recognition to acquired immunity 8.