Deconvoluting Post-Transplant Immunity: Cell Subset-Specific Mapping Reveals Pathways for Activation and Expansion of Memory T, Monocytes and B Cells

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO+CD62L− effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant

A Pilot Study of IL-2Rα Blockade during Lymphopenia Depletes Regulatory T-cells and Correlates with Enhanced Immunity in Patients with Glioblastoma

Preclinical studies in mice have demonstrated that the prophylactic depletion of immunosuppressive regulatory T-cells (T(Regs)) through targeting the high affinity interleukin-2 (IL-2) receptor (IL-2Rα/CD25) can enhance anti-tumor immunotherapy. However, therapeutic approaches are complicated by the inadvertent inhibition of IL-2Rα expressing anti-tumor effector T-cells.To determine if changes in the cytokine milieu during lymphopenia may engender differential signaling requirements that would enable unarmed anti-IL-2Rα monoclonal antibody (MAbs) to selectively deplete T(Regs) while permitting vaccine-stimulated immune responses.A randomized placebo-controlled pilot study was undertaken to examine the ability of the anti-IL-2Rα MAb daclizumab, given at the time of epidermal growth factor receptor variant III (EGFRvIII) targeted peptide vaccination, to safely and selectively deplete T(Regs) in patients with glioblastoma (GBM) treated with lymphodepleting temozolomide (TMZ).Daclizumab treatment (n = 3) was well-tolerated with no symptoms of autoimmune toxicity and resulted in a significant reduction in the frequency of circulating CD4+Foxp3+ TRegs in comparison to saline controls (n = 3)( p = 0.0464). A significant (p<0.0001) inverse correlation between the frequency of TRegs and the level of EGFRvIII specific humoral responses suggests the depletion of TRegs may be linked to increased vaccine-stimulated humoral immunity. These data suggest this approach deserves further study.ClinicalTrials.gov NCT00626015

Accelerated memory cell homeostasis during T cell depletion and approaches to overcome it.

Partial T cell depletion is used in solid organ transplantation as a valuable strategy of peritransplant induction immunosuppression. Using a murine cardiac allograft model, we recently demonstrated that this led to lymphopenia-induced (homeostatic) proliferation among the residual nondepleted lymphocytes. Rather than promoting tolerance, peritransplant T cell-depleting Abs actually resulted in resistance to tolerance induction by costimulatory blockade. In this study we show that memory T cells predominate shortly after subtotal lymphodepletion due to two distinct mechanisms: relative resistance to depletion and enhanced homeostatic proliferation. In contrast, regulatory cells (CD4+ CD25+ Foxp3+) are depleted as efficiently as nonregulatory cells and exhibit reduced homeostatic expansion compared with memory cells. The resistance to tolerance induction seen with subtotal T cell depletion can be overcome in two different ways: first, by the adoptive transfer of additional unprimed regulatory cells at the time of transplant, and second, by the adjunctive use of nondepleting anti-CD4 and anti-CD8 mAbs, which effectively block homeostatic expansion. We conclude that the resistance to tolerance induction seen after subtotal lymphocyte depletion can be attributed to alterations in the balance of naive, memory, and regulatory T cells. These data have clinically relevant implications related to the development of novel strategies to overcome resistance to tolerance