Robust polyfunctional T-helper 1 responses to multiple fungal antigens from a cell population generated using an environmental strain of Aspergillus fumigatus.

BACKGROUND AND AIMS: Aspergillus fumigatus infections are the leading cause of invasive fungal infection-related deaths in stem cell transplant patients, and may be amenable to correction with adoptive immunotherapy providing T lymphocytes specific for A. fumigatus. However, a clinically usable source of antigen and a reliable procedure for the generation of large numbers of Aspergillus-specific T lymphocytes to clinical-grade standards is not available. METHODS: An environmental strain of A. fumigatus (WMAfES) was isolated and cultured using materials and reagents suitable for clinical manufacture. Water-soluble lysate from germinated conidia of WMAfES was used as the antigen source. Peripheral blood mononuclear cells were stimulated with antigen-pulsed autologous dendritic cells on days 0 and 7. Cells were expanded with a cocktail of interleukin (IL)-2, IL-7 and IL-15 from days 7 to 21. RESULTS: We obtained a mean 32.8-fold increase in cell numbers over 21 days of culture (n = 8). Resultant cultures were predominantly effector and central memory CD4(+) T cells, which produced T-helper (h)1 and Th17 cytokines when restimulated with A. fumigatus antigen derived from environmental or clinically isolated A. fumigatus. Cultured cells exhibited a high level of specific expansion and chemokine production when restimulated. Moreover, cultured cells cross-reacted with antigens from other fungi, including Penicillium, Candida albicans and other non-fumigatus Aspergillus species. CONCLUSIONS: We describe a simple, robust, reproducible and clinically applicable procedure using a clinically appropriate antigen preparation for the expansion of polyfunctional A. fumigatus-specific T cells from normal donors of varying HLA types.

Clinical-grade varicella zoster virus-specific T cells produced for adoptive immunotherapy in hemopoietic stem cell transplant recipients.

BACKGROUND AIMS: Varicella zoster virus (VZV) causes life-long latent infection in healthy individuals, which reactivates in 10-68% of stem cell transplant patients. Reconstituting immunity through adoptive transfer of T cells specific for VZV may aid in the prophylaxis and treatment of VZV infections. The potential for generating T cells specific for VZV using a clinically approved VZV vaccine strain was investigated. METHODS: The Varivax® vaccine was used to stimulate peripheral blood mononuclear cells from healthy donors. Only reagents approved for clinical manufacture were used. Monocyte-derived dendritic cells pulsed with Varivax (R) were used to stimulate autologous mononuclear cells at a responder to stimulator ratio of 10:1. On day 7, a second stimulation was performed; 20 U/mL interleukin (IL)-2 were added from day 7 and 50 U/mL IL-2 from day 14 onwards. Cell phenotype and functionality were assessed after 21 days of culture. RESULTS: A mean increase of 11-fold in cell number was observed (n= 18). Cultures were mainly T cells (mean CD3 (+) 89.7%, CD4 (+) 54.2%, CD8 (+) 28.7%) with effector and central memory phenotypes. Cells produced one or more T helper (Th)1 cytokine (interferon-γ, tumor necrosis factor-α and IL-2), and CD4 (+) (but not CD8 (+) ) cells expressed the cytoxicity marker CD107 when restimulated with VZV antigens. CONCLUSIONS: We have demonstrated a clinically applicable method that yields high numbers of highly reactive T cells specific for VZV. We propose that reconstructing host immunity through adoptive transfer of VZV-specific T cells will reduce the frequency of clinical VZV infection in the period of severe immune suppression that follows allogeneic stem cell transplantation.

In vitro generation of influenza-specific polyfunctional CD4+ T cells suitable for adoptive immunotherapy.

BACKGROUND AIMS: Influenza viruses cause potentially fatal respiratory infections in stem cell transplant patients. Specific T cells provide long-lived host adaptive immunity to influenza viruses, and the potential for generating such cells for clinical use was investigated. METHODS: The inactivated influenza vaccine (Fluvax) approved for human use was used as the antigen source. Monocyte-derived dendritic cells pulsed with Fluvax were used to stimulate autologous peripheral blood mononuclear cells (PBMC) on days 0 and 7. Cells were expanded with interleukin (IL)-2 from day 7 onwards. Cell numbers and phenotype were assessed on day 21. The presence of influenza virus-specific cells was assessed by cytokine production and proliferative responses following restimulation with influenza antigens. RESULTS: Over 21 days of culture, a mean fold increase of 26.3 in cell number was observed (n = 7). Cultures were predominantly effector and central memory CD4+ cells, and expressed a phenotype characteristic of activated antigen-specific cells capable of B-cell helper function. Cytotoxic CD4+ and CD8+ cells specific for influenza and a high percentage of CD4+ cells specific for each of three influenza viruses targeted by Fluvax (H1N1, H3N2 and Brisbane viruses) were generated. In addition, T cells expanded when restimulated with antigens derived from influenza viruses. CONCLUSIONS: We have demonstrated a clinically usable method for producing influenza virus-specific T cells that yield high numbers of highly reactive CD4+ cells suitable for adoptive immunotherapy. We propose that reconstructing host immunity through adoptive transfer of influenza virus-specific T cells will reduce the frequency of influenza-related deaths in the period of severe immune suppression that follows stem cell transplantation.

BK virus-specific T cells for use in cellular therapy show specificity to multiple antigens and polyfunctional cytokine responses.

BACKGROUND: BK virus (BKV) infection causes hemorrhagic cystitis posthemopoietic stem-cell transplant and graft loss in renal transplant recipients. Reactivation occurs in up to 60% of patients in both groups. Treatment-related cellular immunosuppression is a major contributor to the development of BKV-related disease, but the targets of the immune response are not well characterized. Immunotherapy by adoptive transfer of cellular effectors has been shown to be effective in controlling and preventing some virus-related diseases in transplant recipients, particularly Epstein-Barr virus and cytomegalovirus. Infusion of BKV-specific T cells may potentially reconstitute functional BKV immunity and reduce clinical complications of BKV infection. METHODS: BKV-specific T cells for clinical use in adoptive immunotherapy were generated using monocyte-derived dendritic cells pulsed with overlapping peptide mixes spanning the five BKV proteins VP1, VP2, VP3, large T antigen, and small T antigen. Phenotypic and functional characteristics of the cells were investigated as well as their antigen specificity. RESULTS: Expanded CD4(+) and CD8(+) cells responded to restimulation with BKV peptides principally from VP1, large T, or small T antigens; produced multiple cytokines; and showed cytotoxic activity against antigen-coated targets. CONCLUSIONS: Possible clinical uses for BKV-specific T cells generated using this method include immune reconstitution posthemopoietic stem-cell transplantation or prophylaxis and treatment of immune deficiency in renal transplant recipients, fulfilling the need for effective therapy for BKV-related hemorrhagic cystitis and renal dysfunction.

The potential and limitations of p38MAPK as a drug target for the treatment of hematological malignancies.

The classical role of p38MAPK involves the transmission of death signals from stress stimuli such as ionizing radiation, inflammation and chemotherapy in normal and malignant cells. However emerging evidence point to it having pleiotropic functions, ranging from cellular proliferation and survival, to cell cycle arrest and regulation of the tumor microenvironment. The mechanisms responsible for these diverse and rather contradictory functions of p38MAPK are only now being unraveled. In some hematological malignancies, p38MAPK is constitutively activated. The reasons for this are not fully understood, but can result from underlying genetic lesions such as the bcr/abl translocation. The involvement of p38MAPK in mediating cell cycle arrest in the face of cytotoxic damage suggests that p38MAPK may be a therapeutic target. The role of p38MAPK in cytokine production by the stromal compartment adds another level of complexity to the regulation of hematological malignancies that exhibit a degree of stromal dependence, at least some of which is cytokine driven. p38MAPK dependent cytokine production by malignant hematopoietic cells has also been observed, and promotes tumor cell adhesion, angiogenesis and osteoclastogenesis. This suggests a mutual relationship between tumor and stromal cells. In contrast, p38MAPK is required for the effects of cytotoxic agents in some hematological malignancies. These conflicting qualities point to the importance of careful consideration of the use of p38MAPK inhibitors as a therapeutic strategy.

p38MAPK inhibitors attenuate cytokine production by bone marrow stromal cells and reduce stroma-mediated proliferation of acute lymphoblastic leukemia cells.

OBJECTIVE: The bone marrow microenvironment provides critical support for the growth and survival of acute lymphoblastic leukemia cells and protection against the effects of chemotherapeutic agents. Although the mechanisms are not fully understood, it is likely that they are mediated at least in part by stromal derived cytokines and chemokines. RESULTS: We have demonstrated that inhibition of p38(MAPK) in bone marrow stromal cells reduced the production of IL-6, VEGF, PDGF and CXCL12. In addition to the known role of CXCL12 in ALL cell stromal-dependent proliferation, we have shown that VEGF and PDGF also provide important proliferative cues for ALL cells, both as exogenous single agents and as bone marrow stromal culture-derived factors. In contrast we could not detect a significant role for IL-6 in ALL stromal-dependent proliferation. Consistent with these findings inhibition of p38(MAPK) significantly reduced stromal-dependent proliferation of ALL cells. METHODS: Cell proliferation was measured by (3)H-thymidine assays, survival by Annexin V/PI staining, gene expression by microarray, cytokine protein levels by antibody microarrays and/or ELISA and cellular signaling by western blotting. CONCLUSION: These findings suggest that inhibition of p38(MAPK) may provide a useful adjunct to current treatment strategies by retarding ALL cell growth.

Interaction of interleukin-7 and interleukin-3 with the CXCL12-induced proliferation of B-cell progenitor acute lymphoblastic leukemia.

BACKGROUND AND OBJECTIVES: The chemokine stroma-derived factor 1a (SDF-1a or CXCL12) is essential for proliferation of B lineage acute lymphoblastic leukemia (ALL) cells in their physiological microenvironment, bone marrow stroma. CXCL12 synergizes with cytokines that stimulate myeloid cells, but its interaction with cytokines affecting lymphoid cells has not been examined. We investigated whether interleukin (IL)-7 and IL-3 interact with CXCL12 to regulate ALL proliferation. DESIGN AND METHODS: The survival of ALL cells in serum-free cultures, with or without stromal support and cytokines, was assessed by flow cytometry, and proliferation by 3H-thymidine incorporation. Signaling mechanisms were assessed by western blotting of phosphorylated forms of signaling molecules and by the use of specific inhibitors. RESULTS: CXCL12, IL-3, and IL-7 had only marginal effects on ALL cell survival under serum-free conditions. However, these molecules individually induced significant proliferative responses in stromal cultures of 11 cases of ALL. The combination of CXCL12 with IL-7 or IL-3 produced a variety of responses, with clear synergistic or additive interactions observed in four cases. Synergistic proliferation in response to CXCL12 plus IL-7 was associated with enhanced phosphorylation of the mitogen-activated protein kinases, ERK-1/2 and p38, and AKT, and was partially inhibited by pretreatment of cells with inhibitors for p38 MAPK and phosphatidylinositol 3-kinase, implicating these pathways in the proliferation in response to IL-7 plus CXCL12. INTERPRETATION AND CONCLUSIONS: These findings indicate a complex interaction between signaling from the CXCR4 receptor on ALL cells with those initiated by the cytokines IL-7 and IL-3, suggesting that CXCL12 may facilitate ALL proliferation by enhancing cytokine-signaling pathways in responsive cases.