Application of HSPCs in Cell Therapy - Session 2

Main ideas, concepts, and lessons

Purpose of the session

• Introduces the basics of HLA (human leukocyte antigen) relevant to transplantation.
• Explains how to identify immunologic matches for donor cells.
• Covers common laboratory tests used before and after transplantation, especially:
  • Crossmatch
  • Chimerism analysis
  • Measurable residual disease (MRD) testing

What HLA is and why it matters

• The HLA (human leukocyte antigen) complex is a cluster of tightly linked genes on chromosome 6.
• HLA genes encode cell-surface proteins that act as identification tags so the immune system can distinguish:
  • Self vs. non-self (endogenous vs. foreign/exogenous proteins)

Two main HLA classes

• Class I (genes HLA-A, HLA-B, HLA-C)
  • Present on all nucleated cells
  • Present endogenous peptides to CD8+ cytotoxic T cells
• Class II (genes HLA-DR, DP, DQ)
  • Found on antigen-presenting cells
  • Present exogenous peptides to CD4+ helper T cells
  • Activates an immune response

HLA polymorphism (why matching is hard)

• The HLA region is highly polymorphic, meaning many alleles exist per gene.
• Example: HLA-A has >7,500 known variants.
• Result:
  • It is very unlikely that unrelated individuals share the same HLA allele combinations.
  • This creates millions of possible HLA combinations, making matching challenging.

HLA matching in transplantation types

Allogeneic HSCT (hematopoietic stem cell transplantation)

• Includes replacing the recipient’s diseased/deficient immune and hematopoietic systems with donor stem cells.
• HLA compatibility is crucial because HLA drives self vs non-self recognition.
• Matching is typically defined at the allelic level (specific HLA protein variants).

Unrelated donor “gold standard”:

• A full match at A, B, C, and DRB1 loci (sharing the same alleles).

Additional targets used in many centers:

• High-resolution matching at HLA-DQB1, aiming for a 10/10 match.

Consequences of mismatch may include:

• Immune rejection
• Graft-versus-host disease (GVHD)
• Relapse
• Graft failure

Solid organ transplantation

• Requires less strict HLA matching than HSCT.
• The major concern highlighted is donor-specific antibodies in the recipient.

T-cell roles in HSCT (benefit and harm)

Graft-versus-leukemia (GVL)

• Donor T cells can recognize and eliminate residual malignant cells via an HLA-mediated mechanism
• Helps reduce relapse

Graft-versus-host disease (GVHD)

• Donor T cells attack healthy recipient tissues
• Also HLA-mediated

GVHD management approaches

• T-cell depletion from the graft
• Immunosuppressive drugs
• Close donor-recipient HLA matching

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Donor types for allogeneic HSCT (detailed list)

• Matched related donor (about 20% in the US)

  • Typically a sibling
  • Donor and patient are fully HLA matched at the allelic level

• Unrelated donor (most common currently in the US)

  • Sourced from the general population
  • Used when there is no HLA-matched sibling

• Haploidentical transplant (about 20%)

  • Used when:
    • No HLA-matched sibling exists, and
    • No suitable HLA-matched unrelated donor can be found
  • Typically a partially matched family member, often a parent
  • Mismatched for at least two HLA antigens

• HLA mismatched unrelated donor (used in ~10% of transplants)

  • Used when no suitable family member is available

• Umbilical cord blood

  • Donor cells from a newborn’s umbilical cord
  • Often uses multiple cord units due to lower cell counts than adult grafts
  • Because cord cells are immature, HLA matching requirements are less strict

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Laboratory testing and assays (detailed bullet points)

Core goals

• Labs test samples from patient and donor to determine compatibility, which is essential for:
  • HSCT
  • Solid organ transplant

1) HLA typing (foundational test)

• Most important first test
• HSCT: typically high-resolution molecular typing to the allelic level
• Solid organ: often antigen-level typing unless molecular typing is available

2) HLA antibody screening (solid organ emphasis)

• Performed for solid organ transplantation
• Purpose:
  • Detect all patient anti-HLA antibodies
  • Identify donor-specific antibodies that could harm the graft

3) Crossmatch assays (solid organ emphasis)

• Purpose:
  • Detect whether the recipient has anti-HLA antibodies that could react against donor cells and cause graft rejection
• Described as:
  • A miniature solid organ transplant in a dish

Two crossmatch formats described

• Cellular crossmatch

  • Prospective donor lymphocytes mixed with recipient serum
  • If anti-HLA antibodies are present, they bind donor cells
  • Detection methods:
    • Flow cytometry crossmatch
      • Detection via anti-human globulin antibody + flow cytometry
    • Complement-dependent crossmatch
      • Uses a cell killing assay read out via microscopy

• Prerequisite: isolating donor lymphocytes from donor blood

Automation / workflow support (company methods mentioned)

• STEMCELL EasySep Direct

  • Immunomagnetic isolation of T cells, B cells, or total lymphocytes directly from whole blood
  • Uses antibody + magnetic particle targeting to remove unwanted leukocytes/RBCs
  • Uses an EasySep magnet (e.g., EasyEights magnet mentioned)

• STEMCELL RoboSep automation

  • RoboSep S: runs 4 samples
  • RoboSep 16: runs 16 samples
  • Cells can be isolated in about 25 minutes and used immediately in crossmatch assays

4) Chimerism analysis / engraftment monitoring (mostly HSCT)

• Primary use: HSCT, but increasingly used in some solid organ cases
• Purpose:
  • Determine the proportion of hematopoietic cells derived from donor vs. recipient

Methods mentioned

• Informative polymorphic genetic markers
• Molecular techniques including:
  • STR analysis
  • PCR (including quantitative PCR and digital droplet PCR)
  • NGS (next-generation sequencing)

Clinical interpretation

• If recipient-derived cells persist, it may indicate:
  • Potential graft failure or relapse
• Therefore, patients need close monitoring

Best practice emphasized

• Analyze lineage-specific cell populations
• Requires high purity cells
  • Positive selection techniques are preferred
• T cells and myeloid cells are common lineages

Technical challenge

• Early post-transplant (first ~6 weeks):
  • Patient lymphocytes may be below detection
• Automation with RoboSep recommended for:
  • Consistent performance
  • Saving technologist time

Chimerism types and detection

• Complete donor chimerism

  • Assumes no recipient-derived hematopoietic cells
  • Detection limit typically ~5% recipient cells in lymphoid and myeloid lineages

• Mixed chimerism

  • Both donor and recipient-derived cells present
  • Historically associated with:
    • graft rejection
    • and in malignancy contexts: relapse
  • Updated nuance:
    • If recipient-derived cells are not malignant, stable mixed chimerism can occur with normal hematopoiesis/immune function

Why combine with MRD testing

• The combination of chimerism + measurable residual disease is gaining popularity
• Enables earlier intervention for possible relapse

5) Measurable residual disease (MRD) testing (malignancy monitoring)

• Definition:
  • MRD = a small number of cancer cells remaining after treatment/eradication during conditioning
  • These residual cells can cause relapse
• Key lesson:
  • Even one residual cell can potentially lead to relapse
  • Therefore, curing requires eliminating all malignant cells

Why MRD is hard

• Post-transplant cell counts can be too low for morphology
• So more sensitive molecular assays are used

MRD detection methods described (with sensitivity ranges)

• Multi-parameter flow cytometry

  • Sensitivity: about 1 in 10,000 to 1 in 100,000
  • Advantages:
    • fast
    • applicable to all patients
  • Disadvantages:
    • interpretation can be challenging and needs expertise
    • relies on distinguishing neoplastic vs normal antigen expression
    • depends on proper antibody stain selection

• Quantitative PCR (including digital droplet PCR)

  • Sensitivity: about 1 in 100,000
  • Widely used for tracking genetic aberrations
  • Limitations:
    • examines a limited number of targets
    • still requires technical expertise for interpretation

• NGS (next-generation sequencing)

  • Sensitivity: about 1 in a million (highest sensitivity)
  • Like qPCR, can track genetic aberrations
  • Advantage:
    • can evaluate many genetic targets simultaneously
  • Disadvantages:
    • interpretation complexity
    • high technical expertise needed

Key takeaway

• Higher assay sensitivity → better MRD detection → lower chance of relapse
  • Earlier detection supports intervention

Improving sensitivity via cell population selection

• As with chimerism:
  • sensitivity can improve by analyzing specific cell populations
• Positive selection recommended to ensure high purity testing cells

Examples of enrichment products/approaches mentioned

• B-cell malignancies (e.g., CLL): CD19 or CD19-20 EasySep kits for MRD analysis
• Multiple myeloma: plasma cell enrichment using EasySep CD138 positive kits

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Overall summary / session wrap-up

• HLA enables immune recognition of self vs. non-self, mainly via T cells.
• HLA is highly polymorphic, making matching difficult.
• In HSCT, HLA matching helps prevent:
  • acute tissue rejection-related complications
  • and is closely linked to GVHD
• MRD is the remaining small number of malignant cells that can cause relapse.
• The session prepares viewers to:
  • recall HLA fundamentals
  • recognize donor types
  • identify methods for chimerism and MRD measurement.

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Speakers / sources featured

• STEMCELL (company)
  • Mentioned products/systems: EasySep Direct, EasyEights magnet, RoboSep S, RoboSep 16, EasySep kits
  • Including CD19/CD19-20 and CD138 positive enrichment