Depletion of naive T cells from donor leukapheresis products (LPs) aims at the reduction of alloreactivity, while preserving memory T-cell reactivity (for example, to pathogens). This study established the immunomagnetic depletion procedure under clean room conditions using CD45RA beads and analyzed LPs of six donors for cell composition and functional immune responses. CD45RA depletion resulted in 3.4-4.7 log (median 4.4) reduction of CD45RA þ T cells, thereby eliminating naive and late effector T cells. B cells were also completely removed, whereas significant proportions of NK cells, monocytes and granulocytes persisted. CD45RA-depleted LPs contained effector and central memory CD4 þ and CD8 þ T cells that showed sustained IFN-g secretion to CMV, EBV, Aspergillus and Candida Ags. Alloreactivity was measured in MLRs between donors with complete HLA-mismatch. Alloreactive CD8 þ T cells were strongly reduced (median 41-log) upon CD45RA depletion, whereas alloreactive CD4 þ T cells persisted in significant numbers. In conclusion, clinical grade depletion of CD45RA þ naive T cells from donor LPs is feasible and highly efficient. The depleted products show sustained CD4 þ and CD8 þ T-cell reactivity to pathogens and effectively reduced CD8-mediated alloreactivity. Prophylactic and preemptive infusions after allogeneic SCT may improve T-cell reconstitution and pathogen-specific immunosurveillance, along with lower risk of inducing GVHD.
Allogeneic hematopoietic stem cell transplantation (SCT) regimens incorporating the lymphocytotoxic CD52 antibody alemtuzumab demonstrate efficient engraftment and reduced graft-versus-host disease (GVHD). However, these protocols substantially impair posttransplantation antiviral and antitumor immunity. To accelerate immune reconstitution after alemtuzumab-based reduced-intensity SCT, we administered prophylactic CD8-depleted donor lymphocyte infusions (DLIs) starting on days 60 and 120 after transplantation. DLIs were processed in an immunomagnetic good manufacturing practice depletion procedure resulting in a 2.5-to 6-log reduction in CD8 T cells.
Reactivated varicella-zoster virus (VZV) infection causes herpes zoster and commonly occurs after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Because VZV-specific T cell immunity is essential to prevent virus reactivation, we developed an interferon-gamma enzyme-linked immunosorbent spot (ELISPOT) assay for the sensitive detection of VZV-reactive T cells at the single-cell level ex vivo. We used this assay to monitor the frequency of VZV-reactive T cells in 17 seropositive patients during the first year after T cell-depleted allo-HSCT. The patients did not receive anti-herpesvirus prophylaxis after stem cell engraftment. Independent of the magnitude of transferred donor immunity, VZV-reactive T cell numbers decreased to low levels (median, 2/mL; range, 0 to 35/mL) in peripheral blood early after transplantation. Only patients with subsequent zoster (n = 5) exhibited a dramatic boost in VZV-reactive T cells (median, 366/mL; range, 158 to 756/mL), which was induced by the reactivation event. The postzoster VZV-reactive T cell levels were similar to those seen in healthy virus carriers. In contrast, antiviral T cell levels remained low in patients without VZV disease. Our results demonstrate that VZV-specific T cell immunity recovered efficiently during zoster in T cell-depleted allo-HSCT recipients. It did not reconstitute spontaneously in nonzoster patients, even in the absence of antiviral prophylaxis. Prospective studies should investigate whether VZV vaccination can substitute for natural resensitization by virus disease.
Donor lymphocyte infusions (DLI) are used to resolve mixed T-cell chimerism (TCC) after allo-SCT despite a substantial risk of GVHD. We analyzed the impact of prophylactic CD8-depleted (CD8 depl ) DLI in 20 recipients of anti-CD52 alemtuzumab in vivo T-cell-depleted allografts with declining donor TCC after day þ 60. A total of 13 patients received CD8 depl DLI and 7 patients did not. All but one of the DLI patients converted to complete donor T-cell chimeras, whereas only one non-DLI patient converted spontaneously. DLI induced transient acute GVHD in five and extensive chronic GVHD in two patients. These data suggest the use of CD8 depl DLI as an effective treatment for mixed TCC, particularly in patients at high risk for GVHD. We also observed that the majority of reconstituting donor-derived T cells after alemtuzumab conditioning were CD52-negative. CD8 depl DLI significantly increased the proportion of CD52-positive CD4 T cells, whereby their beneficial effect on reconstituting the post-transplant T-cell repertoire was shown.
93 days (range 15-674) post HSCT. Fourteen (40%) were determined to have invasive ADV disease (7 on tissue biopsy). ADV viral load evaluation over time revealed the following: HVL at presentation in 18 (51.4%) (median 1.1x10 4 ,range 7.4x10 5-6.8x10 9 copies/ ml); 10 (28%) progressed to HVL (median 2 log increase from presentation) at a median of 15 days (range 3-56); and LVL-only at any time point in 7 (20%) pts (median 4000 copies/ml, range 600-8866). Fifteen pts with HVL were treated with cidovofir intravenously or/ and CMX001 a median 7 doses (range 1-38). Despite treatment with antiviral therapy 12 pts (92%) with HVL and 7 pts (87.5%) with LVL-HVL died. Mortality was attributable to ADV in 11 (31.4%) pts. All cause 180 day mortality was 74.3% for pts with ADV. Conclusions: ADV viremia was relatively low (8.7%) in this high risk population and similar to the 5% reported in populations receiving conventional transplants. Determination of viral status in patients with clinical symptoms resulted in a relatively high yield of positivity-40%. The mortality attributable to ADV of 30% suggests the need for development of better treatment modalities. The 180 day all cause mortality of 74% suggests ADV viremia complicates other medical conditions and complications of transplant.
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