Immune Recovery after Timed Sequential Therapy in Acute Myelogenous Leukemia: Discordant Recovery of Naïve T Lymphocytes and Antigen Driven Regulatory T Cells.

Patients who demonstrate a rapid but limited initial lymphocyte recovery following therapy have a significantly better response rate than patients where lymphocyte recovery is markedly delayed. Timed sequential therapy (TST) is a cell cycle dependent based approach for curative therapeutic potential in acute myelogenous leukemia (AML). Recovery of T cells in this setting can be due to thymic dependent T cell development or as a consequence of directed (antigen) expansion of effector T lymphocytes. The present studies evaluated immunological reconstitution of 26 AML patients following induction or consolidation TST. Thirteen patients received cytarabine, daunorubicin and etoposide (AcDVP-16) induction therapy, 3 patients received cytarabine, daunorubicin and cytarabine (AcDAc) and 10 patients received flavopiridol, cytarabine and mitoxantrone (FLAM) either as induction or consolidation therapy. Peripheral blood lymphocytes were collected during the initial phase of white blood cell reconstitution (>200 cells/ cu mm). Lymphocyte Recovery preceded that of myeloid elements (median of day 24 versus day 30) with peak lymphocyte counts ranging from 300–1800 and a median of 650 cells/cu mm. Multi-color flow cytometric analysis revealed recovery of CD4, CD8 and NK cells with the CD3+CD4+:CD3+CD8+ ratio averaging 4.5:1 (range 1:1– 8:1). Assessment of T cell recombinant excision circles (TREC’s) suggested that a significant proportion of the T cells (median 40,000 copies per 100,000 cells; range 100 – 100,000 copies) were recent thymic emigrants. Somewhat surprisingly, there was a significant expansion of the CD4+CD25+Foxp3+ regulatory T cell compartment (median 5.1%; range 2.5 – 12.3%). To examine whether the expansion of this T cell compartment cells was due to endogenous thymic output or developed by homeostatic mechanisms, CD4+CD25+Foxp3+ cells isolated flow cytometrically were assessed for TREC’s and by spectratyping for Vb T cell receptor gene utilization. The results revealed that the majority (>90%) of CD4+CD25+Foxp3+ cells did not express TREC’s and therefore, did not recently emigrate from the thymus. Moreover, there was a marked oligoclonal skewing as defined by a limited Vb T cell receptor repertoire suggesting expansion did not occur by a non-specific, global homeostatic. In contrast, the repertoire of the non-regulatory T cell compartment (CD4, CD8) was unbiased. These results suggest that the expansion of the CD4+CD25+Foxp3+ T cell compartment is either driven by antigen or by restricted homeostatic mechanisms. The lack of significant, correlative levels of typical homeostatic cytokines (i.e., IL-2, IL-7; Bioplex analysis) may provide an environment where CD4+CD25+Foxp3+ T cells can only be driven to expand by antigenic stimulation. The continued presence of antigen driven regulatory T cells may impede the successful deployment of immunologically directed anti-tumor strategies.