Cellular Immunity and Immunophenotyping

CELLULAR IMMUNITY SUMMARY

  • Phenotype Assessment

    • Utilizing flow cytometry for comprehensive cell morphology, differentiation, and functional state evaluation.

    • Allows for multi-parameter analysis of individual cells.

  • PBMC Isolation

    • Assessing lymphocyte viability from various biological samples (e.g., peripheral blood, bone marrow, lymphoid tissues).

    • Accurate isolation is crucial for downstream assays.

CELL PHENOTYPE

  • Defined by observable morphological features (e.g., cell size, granularity, nuclear-to-cytoplasmic ratio).

  • Influenced by an intricate interplay of genotype (genetic makeup) and environmental factors (e.g., cytokines, growth factors, stress).

CELL IMMUNOPHENOTYPE

  • A distinct set of specific cell surface and intracellular antigens recognized by immune cells.

  • Dynamically affected by the cell's structural integrity, developmental stage (differentiation), and underlying genetic profile (genotype).

  • These antigens are primarily proteins or carbohydrates.

ASSESSMENT OF IMMUNE CELLS

  • Complete Blood Count (CBC)

    • A standard laboratory test including detailed erythrocyte (red blood cell) and leukocyte (white blood cell) evaluations.

    • Provides counts for total WBCs and a differential count of their main types (neutrophils, lymphocytes, monocytes, eosinophils, basophils).

  • Immunophenotyping

    • A sophisticated technique based on the presence or absence of specific cell antigens.

    • Typically identified using fluorescently labeled monoclonal antibodies that bind to these markers.

    • Allows for precise identification and quantification of different immune cell subsets.

CLUSTER OF DIFFERENTIATION (CD) MARKERS

  • A nomenclature system used to identify cell surface molecules (antigens) with specific functions.

  • Crucial for classifying cell types and their differentiation stages.

  • Examples:

    • CD3: Pan-T-cell marker.

    • CD4 and CD8: Distinguish helper and cytotoxic T-cell subsets.

    • CD14: For monocytes/macrophages.

    • CD19/CD20: For B-cells.

    • CD56: For NK cells.

  • Normal CD4/CD8 ratio: Commonly observed between 1.1 to 1.8, with an average of 1.4.

  • Deviations can indicate immune dysregulation or disease.

IMMUNE CELL SUBPOPULATIONS

  • Highly specialized groups of immune cells defined by their unique combination of CD markers, expression of specific transcription factors, and characteristic cytokine production profiles.

  • Examples:

    • nTreg (natural Regulatory T cells): Express CD4, CD25, and FOXP3; suppress immune responses.

    • Tfh (Follicular Helper T cells): Express CD4, CXCR5, PD-1, and secrete IL-21; essential for B-cell help in germinal centers.

    • Th1 (T helper 1 cells): Express CD4, produce IFN-\gamma and IL-2; involved in cellular immunity against intracellular pathogens.

    • Th2 (T helper 2 cells): Express CD4, produce IL-4, IL-5, IL-13; involved in humoral immunity, allergic reactions, and defense against parasites.

    • Th17 (T helper 17 cells): Express CD4, produce IL-17, IL-21, IL-22; involved in host defense against extracellular bacteria and fungi, also implicated in autoimmune diseases.

CELL ISOLATION METHODS

  • Various techniques employed to obtain pure populations of immune cells for research or diagnostic purposes:

    • Density Gradient Centrifugation: Cells separated based on buoyant density (e.g., Ficoll-Paque for PBMC separation from granulocytes and red blood cells).

    • PBMCs, being less dense, remain at the interface after centrifugation.

    • Adherence Methods: Based on the ability of certain cells (e.g., monocytes, macrophages) to adhere to plastic surfaces.

    • Allows for their separation from non-adherent cells.

    • Immunomagnetic Separation (MACS): Uses antibody-coated magnetic beads to positively or negatively select cell populations based on specific surface markers.

    • Cells bound to beads are retained by a magnet.

    • Flow Cytometry (FACS): Provides the highest purity for cell isolation, sorting cells based on their fluorescent properties and light scatter.

FLOW CYTOMETRY (FC)

  • A powerful laser-based technology used to analyze and sort individual cells or particles.

  • Based on their light scattering and fluorescent characteristics as they pass through one or more laser beams.

  • Parameters Collected:

    • Forward Scatter (FSC): Approximately proportional to cell surface area or size.

    • Larger FSC values generally indicate larger cells.

    • Side Scatter (SSC): Provides information about the internal complexity or granularity of a cell.

    • High SSC indicates more internal structures (e.g., granules).

    • Fluorescence: Indirectly indicates the presence and quantity of specific antigens or intracellular components.

    • Identified by reporter molecules (e.g., fluorochromes) attached to antibodies or probes.

  • Data Analysis: Involves sophisticated gating techniques.

    • Specific cell populations are identified and selected (gated) on two-dimensional dot plots or one-dimensional histograms.

    • Allows for quantitative analysis of cell percentages and marker expression.

  • Dual Color Analysis: Utilizes two different fluorescent markers (e.g., FITC and PE) simultaneously.

    • Assesses the co-expression of two distinct antigens on individual cells.

    • Allows for more precise identification and differentiation of complex cell populations.

  • Multi-color Flow Cytometry: Advanced FC setups can use 8, 10, or even more colors (fluorescent antibodies) simultaneously.

    • Enables the identification and characterization of highly complex and rare cell subsets from heterogeneous samples.

APPLICATIONS OF FLOW CYTOMETRY

  • A versatile tool with broad applications in research, diagnostics, and clinical monitoring:

    • Diagnostic tools: For hematological malignancies (e.g., leukemia, lymphoma), identifying aberrant immunophenotypes.

    • Immunodeficiency monitoring: In conditions like HIV/AIDS (e.g., CD4 T-cell counts).

    • T cell cross-matching: For organ transplantation to minimize rejection.

    • Detection: Of autoantibodies in autoimmune diseases.

    • Monitoring: Allergic reactions by analyzing basophil activation.

    • Assessment: Of apoptotic processes and cell cycle analysis.

    • Minimal Residual Disease (MRD): Detection in cancer follow-up.

    • Stem cell enumeration: For transplantation.

PRACTICAL ASPECTS

  • Isolation of PBMCs: Typically involves venipuncture, dilution of blood, layering over Ficoll-Paque, and centrifugation.

    • The mononuclear cell layer is then carefully harvested.

  • Viability assessment: Commonly performed using the trypan blue exclusion method.

    • Cells with compromised membranes (non-viable) absorb the blue dye; viable cells exclude it.

  • Emphasis on understanding histograms, dot plots, and data presentation methods in FC:

    • Interpreting single-parameter histograms for marker intensity.

    • Interpreting two-parameter dot plots for cell population identification and analysis of co-expression.

    • Understanding how to apply gates effectively