Autologous dendritic cells pulsed with HPV E7 protein can induce T cell responses in a portion of late stage cervical cancer patients. Boosting of immune responses by adjuvants and vaccination of tumor HLA-positive patients will be mandatory in future trials.

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IntroductionFor many patients with leukemia lacking a matched hematopoietic stem cell donor, transplantation of HLA-incompatible stem cells remains the only curative treatment option. 1 However, donorpatient disparities at single or multiple HLA alleles increase the risks of graft rejection or severe graft-versus-host disease (GVHD). [2][3][4][5][6][7] Both complications are mainly mediated by alloreactive T cells that recognize oligopeptides in association with foreign HLA molecules. 8,9 T-cell depletion (TCD) is an efficient means for reducing alloreactivity 10 and is mandatory if donors share only one HLA haplotype with the recipients. 11 In this haploidentical setting, however, rigorous TCD abrogates specific immunity against pathogens and leukemia cells resulting in higher mortality from infections and disease relapse. 12,13 The precursor frequency of alloreactive T cells is much higher than that of T cells recognizing infectious agents or malignant cells. [14][15][16] Thus, depletion strategies need to substantially reduce alloreactive T cells, while sparing antipathogen and antitumor specificities. Various selective allodepletion (SAD) approaches have been developed for this purpose. Most of them rely on the ex vivo stimulation of donor lymphocytes derived from peripheral blood or bone marrow samples against recipient alloantigenpresenting cells over 1 to 7 days. Activated alloreactive T cells are subsequently depleted either by concomitant costimulatory blockade 17 ; by photodynamic purging 18,19 ; by fluorescence-activated cell sorting 20,21 ; by CD95-mediated apoptosis 22 ; or by targeting the activation-induced antigens CD25, [23][24][25][26][27][28][29][30][31] CD71, and HLA-DR. 24 Two of these approaches already demonstrated in vivo efficacy in terms of GVHD reduction and graft survival in haploidentical transplantation. 17,32,33 However, disease relapse remained the major complication in patients receiving allodepleted grafts. This might be explained by the inadequately low precursor frequencies of leukemia-reactive T cells within infused lymphocytes.We describe herein an alternative approach that is based on the in vitro expansion of donor T-cell lines through repeated stimulations with allogeneic leukemia or tumor cells prior to SAD. Because responding T cells durably expressed high levels of CD25, CD69, and CD95, we searched for alternative activation-induced antigens suitable for the allodepletion step. Among several candidates tested, the tumor necrosis factor receptor superfamily member CD137 (4-1BB) 34,35 demonstrated the most favorable expression kinetic in allostimulated T-cell cultures. Using immunomagnetic CD137 depletion reagents we obtained a 1-log reduction of anti-HLA mismatch specificities in T-cell lines stimulated with HLA-incompatible leukemia or tumor cells. Allodepleted cultures showed considerable residual antitumor and antiviral CD8 T-cell responses. We also introduce HLA-negative K562 cells 36 transfected with single HLA-class I molecules as an efficient tool to detect and d...

Selective depletion of alloreactive T cells from stem-cell allografts should abrogate graft-versus-host disease while preserving beneficial T cell specificities to facilitate engraftment and immune reconstitution. We therefore explored a refined immunomagnetic separation strategy to effectively deplete alloreactive donor lymphocytes expressing the activation antigen CD69 upon stimulation, and examined the retainment of antiviral, antileukemic, and immunoregulatory T cells. In addition to the CD69 high T cell fraction, our studies retrieved two T cell subsets based on residual CD69 expression. Whereas, truly CD69 neg cells were devoid of detectable alloresponses to original stimulators, CD69-low (CD69 low )-expressing T cells elicited significant residual alloreactivity upon restimulation. In interferon-c enzyme linked immunospot assays, anti-cytomegalovirus and anti-Epstein-Barr virus responses were preserved at significant numbers among CD69 neg T lymphocytes. Accordingly, T cells recognizing the leukemia-associated Wilm's tumor-1 antigen were still detectable in the CD69 neg subset. However, antiviral and antileukemic specificities were also consistently found within CD69 low T cells, suggesting that memory-type donor T cells were partially captured due to residual CD69 expression. Finally, CD4 þ CD25 þ Foxp3 þ immunoregulatory T cells did not upregulate CD69 upon allogeneic stimulation. Our data suggest that CD69-mediated removal of alloreactivity can result in efficient allodepletion, but may partially affect the persistence of antiviral and antileukemic donor memory specificities captured among CD69 low -expressing lymphocytes.

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