Nonmyeloablative allogeneic stem-cell transplantation can induce sustained regression of metastatic renal-cell carcinoma in patients who have had no response to conventional immunotherapy.

No abstract

Vaccine strategies, such as influenza virus vaccination of the elderly, are highly effective at preventing disease but provide protection for only the responding portion of the vaccinees. Adjuvants improve the magnitude and rates of responses, but their potency must be attenuated to minimize side effects. Topical delivery of strong adjuvants such as heat-labile enterotoxin from Escherichia coli (LT) induces potent immune responses. We hypothesized that LT delivered alone in an immunostimulating (LT-IS) patch placed on the skin at the site of injection could augment the immune response to injected vaccines. This was based on the observation that topically applied LT induces migration of activated antigen-presenting cells (APCs) from the skin to the proximal draining lymph node (DLN), and that APCs loaded with antigen by injection in the same anatomical region also migrate to the same DLN. We observed that when influenza virus vaccine is injected and an LT-IS patch is placed to target the same DLN, the influenza virus antibody response is enhanced. Similarly, influenza virus-specific T cells isolated from the lungs show increased levels of gamma interferon and interleukin-4 production. An LT-IS patch placed near an injected vaccine also leads to increased levels of hemagglutination inhibition titers, enhanced mucosal immunoglobulin A responses, and enhanced antigen presentation. Although the mechanisms by which an LT-IS patch exerts its enhancing effects need further study, the enhanced immune responses, ability to safely use potent adjuvants, and simplicity of LT-IS patch application address an important unmet need and provide a new immune enhancement strategy.

We previously identified the aberrantly expressed cell cycle regulator cyclin B1 as a tumor antigen recognized by antibodies and T cells from patients with breast, lung, and head and neck cancers. Ordinarily expressed only transiently in the G2/M stage of the cell cycle in normal cells, cyclin B1 is constitutively expressed at high levels in the cytoplasm of these and many other tumor types, leading to its recognition by the cancer patient's immune system. We report here an unexpected observation that cyclin B1-specific antibody and memory CD4 and CD8 T cells are also found in many healthy individuals who have no history of cancer. Moreover, young as well as older healthy people have these responses suggesting that events other than cancer, which occur either early in life or throughout life, may lead to aberrant cyclin B1 expression and anti-cyclin B1 immunity. The role, if any, of immunity to this tumor-associated antigen is not known. We wanted to determine specifically whether immunity to cyclin B1 might be important in the immunosurveillance of cyclin B1؉ tumors. We therefore tested in mice the effectiveness of vaccine-elicited anticyclin B1 immunity against a cyclin B1؉ mouse tumor that was chosen based on our published observation that cyclin B1 overexpression is associated with the lack of p53 function. We found that cyclin B1 DNA prime-protein boost vaccine protected mice from a challenge with a tumor cell line that was established from a tumor arising in the p53 ؊/؊ mouse that spontaneously overexpresses cyclin B1.cancer vaccines ͉ human immunology ͉ immunosurveillance ͉ tumor immunology O verexpression of cyclin B1 has been documented in many human cancers, including colorectal, lung, cervical, and head and neck carcinomas (1-5). Additionally, this overexpression has been shown to correlate with worse prognosis in lung, laryngeal, esophageal, and tongue cancers (1,3,(5)(6)(7)(8). Whereas cyclin B1 is expressed transiently in normal cells, in cancer cells, it is expressed constitutively in all stages of the cell cycle (9). Additionally, cancer-associated cyclin B1 overexpression is evident primarily in the cytoplasm where it can be subject to proteasomal processing and presentation in MHC class I. We reported isolation of cyclin B1 peptides from MHC class I molecules of tumor cells that were recognized by tumor-specific memory T cells present in patients with cyclin B1 overexpressing tumors (10). We later showed that patients with cancer and premalignant lesions that overexpress cyclin B1 have cyclin B1-specific antibodies of IgM, IgG and IgA isotypes (11).Cyclin B1 overexpression in many tumors is secondary to the loss of p53 function either through p53 mutations or a deletion (9). Considering that alterations in p53 function happen in many tumors early in the process of transformation, cyclin B1 is a good candidate antigen for both immunotherapy and immunoprevention of a large number of human tumors. Additionally, because cyclin B1 is required for entry into mitosis, it is unlikely that a growing tum...

We have analyzed the mechanism of human endothelial injury in a human peripheral blood lymphocytesevere combined immunodeficient (huPBL-SCID) mouse/human skin graft model of allograft injury and examined the effect of immunosuppressive drugs on this process. In this model , split-thickness human skin containing the superficial dermal microvessels was grafted onto immunodeficient C.B-17 SCID or SCID/beige mice and allowed to heal. Human peripheral blood mononuclear cells (PBMCs) allogeneic to the skin , when subsequently introduced by intraperitoneal injection , caused destruction of the human dermal microvasculature by day 16 , evident as endothelial cell sloughing and thrombosis. In the same specimens , mouse microvessels that invaded the human skin graft were uninjured. Human microvascular cell injury was accompanied by a mononuclear cell infiltrate consisting of approximately equal numbers of human CD4؉ and CD8؉ T cells , some of which contained perforin-positive granules. We found no evidence of human natural killer cells and noted occasional human , but not mouse , macrophages at a frequency indistinguishable from that resident in skin on animals not receiving human PBMCs. These human T cell infiltrates did not extend into adjacent mouse skin. Human immunoglobulin G antibody was detected in the blood and was diffusely present throughout mouse and human tissues in SCID mice receiving PBMCs. Mouse C3 was detected on human dermal vessels in both unreconstituted control animals and those that received PBMCs. Blood and tissues from mice injected with PBMCs depleted of B cells showed no human immunoglobulin, but circulating CD3؉ cells were detected by flow cytometry at levels comparable with those of animals receiving whole PBMCs. Significantly, skin graft infiltration by human T cells and human dermal microvascular injury were equivalent in the B cell-depleted and whole-PBMC-reconstituted mice. Mice inoculated with PBMCs depleted of CD8؉ T cells developed microvascular injury and infiltrates containing perforin-expressing CD4؉ T cells. These data suggested a cytolytic T celldependent mechanism of microvessel injury. We then tested the ability of T cell immunosuppressants, cyclosporine and rapamycin, to attenuate vessel damage.

Intradermal (i.d.) immunization is a promising route of vaccine administration. Suitable i.d. adjuvants are important to increase vaccine efficacy in poorly responding populations such as the elderly or for dose-sparing strategies in the face of vaccine shortages. Bacterial exotoxins, such as Escherichia coli heat-labile enterotoxin (LT), exert strong immunostimulatory effects through binding to monosialoganglioside (GM1) cell surface receptors; however, injection is hampered by local inflammation. We demonstrate that the injection of LT formulations deficient in GM1 binding by mutation (LT(G33D)) or in vitro ligand coupling does not cause localized edema and inflammation in mice, yet these formulations retain potent adjuvant activity by enhancing functional Ab and cellular immune responses to coadministered Ags. Complete protection against in vivo lethal tetanus toxin challenge and the induction of Ag-specific CTL responses capable of killing target cells in vivo indicated in vivo efficacy of the induced immune responses. LT(G33D) proved superior to standard alum adjuvant regarding the magnitude and breadth of the induced immune responses. Immunizations in complex ganglioside knockout mice revealed a GM1-independent pathway of LT adjuvanticity. Immunostimulation by i.d. LT(G33D) is explained by its ability to induce migration of activated APCs to the proximal draining lymph nodes. LT(G33D) is a promising candidate adjuvant for human trials of parenteral vaccines in general and for current i.d. vaccine development in particular.

Summary. To study immune recovery after non-myeloablative, reduced-intensity stem cell allografts (NST) and T-cell-depleted myeloablative transplants (TCD), we measured T-cell subset recovery by flow cytometry, T-cell repertoire by spectratyping and thymic T-cell output using a T-cell receptor excision circle (TREC) assay. We found a rapid and comparable increase in lymphocyte numbers in both NST and TCD, supporting the presence of a powerful drive for lymphocyte recovery after transplant. Spectratyping on d 45 and 100 revealed almost complete normalization of the T-cell repertoire in NST patients by d 45, whereas TCD patients demonstrated marked skewing of the repertoire, persisting to d 100. After NST, there was a significantly higher number of TREC-positive CD4 + and CD8 + cells (P ¼ 0AE02 and P ¼ 0AE01 respectively). However, in both NST and TCD, early T-cell recovery after transplant appeared to result entirely from post-thymic T cells, the expansion pattern of which is most influenced by the starting T-cell dose, but not markedly by graft-versus-host disease (GVHD) or mixed chimaerism. These results define important qualitative differences in the T-cell repertoire according to the type of transplant schedule used.

Summary. Two patients with chronic myeloid leukaemia (CML) received a non-myeloablative preparative regimen of cyclophosphamide and¯udarabine, followed by an unmanipulated, G-CSF-mobilized, peripheral blood stem cell transplant from an HLA-identical sibling. Chimaerism, evaluated in myeloid and T-lymphoid lineages by PCR of minisatellite variable regions, showed day 14 post-transplant haemopoietic recovery to be 90% autologous in both patients. On day 30 the bone marrow showed only 1/20 and 2/18 donor metaphases. By day 100 post transplant both had 100% donor myeloid and lymphoid lineages as assessed by karyotype and minisatellite chimaerism analysis. They subsequently became RT-PCR negative for BCR-ABL. Both survive 7 and 14 months post transplant in molecular remission of CML. In one, donor T cells, stimulated with pre-transplant CML cells, induced 30±50% inhibition of pre-transplant leukaemic CFU-GM, but did not inhibit CFU-GM in the day 60 marrow (46% Ph-negative recipient, 54% donor). These results show that a non-myeloablative allotransplant can induce molecular remissions of CML through a graft-versusleukaemia effect.