Quantitation of T cell depletion by limiting dilution analysis
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L Moretti1, S Corpuz, L Giuliodori
1Divisione di Ematologia, USL n. 3, Pesaro, Italy.
Background:
We evaluated a culture method for enumeration of residual T cells remaining in marrow after treatment with antibody and complement or with immunotoxin.
Methods:
Marrow cells were cultured at limiting dilutions with phytohemagglutinin in the presence of Epstein Barr virus transformed human lymphoblastoid cells, and supernates were tested three days later for IL-2 by a cell proliferation assay. This method provides a simple, reliable, objective and rapid enumeration of T cells in marrow before and after treatment.
Results:
Approximately 6% of untreated marrow mononuclear cells can produce IL-2 in such clonal cultures. Treatment with antibodies and complement under conditions identical to those used for our previous clinical trials produced a 3.7 log depletion of IL-2 precursors, whereas treatment with a ricin A chain anti-CD3 immunotoxin produced a 3.0 log depletion.
Conclusions:
Clinical correlations are in progress for assessing whether T cell depletion evaluated with the present method equals previous techniques. The extreme depletion of T cells accomplished by these methods may partly account for the high graft failure rate seen in our clinical trials.
Insights
A new culture method accurately counts residual T cells in marrow after antibody or immunotoxin treatment. This technique helps understand T cell depletion and its impact on graft failure rates in clinical trials.
Area of Science:
- Immunology
- Hematology
- Cell Biology
Background:
- Evaluating methods for enumerating residual T cells in marrow post-treatment.
- Assessing T cell depletion after antibody/complement or immunotoxin therapy.
Purpose of the Study:
- To evaluate a novel culture method for quantifying residual T cells in bone marrow.
- To assess the efficacy of T cell depletion strategies in marrow transplantation.
Main Methods:
- Culturing marrow cells at limiting dilutions with phytohemagglutinin and Epstein Barr virus transformed lymphoblastoid cells.
- Measuring interleukin-2 (IL-2) in supernates via a cell proliferation assay to enumerate T cells.
- Utilizing a simple, reliable, objective, and rapid enumeration technique.
Main Results:
- Approximately 6% of untreated marrow mononuclear cells produced IL-2 in clonal cultures.
- Antibody and complement treatment achieved a 3.7 log depletion of IL-2 precursors.
- Ricin A chain anti-CD3 immunotoxin treatment resulted in a 3.0 log depletion of IL-2 precursors.
Conclusions:
- The developed method offers a reliable way to assess T cell depletion in marrow.
- Extreme T cell depletion may contribute to graft failure observed in clinical trials.
- Clinical correlations are ongoing to validate the method against previous techniques.