Cryopreservation of stem cells after collection from peripheral blood or bone marrow for autologous transplantation necessitates protection with dimethyl sulfoxide (DMSO). Unfortunately, DMSO, when infused with the thawed cell suspension, may induce serious complications and side effects. To assess whether depletion of DMSO before autografting affects safety and efficacy, 56 consenting consecutive patients treated with high-dose chemotherapy and autologous blood stem cell transplantation were assigned to obtain either an untreated or DMSO-depleted autograft. On the day of transplantation, the cryopreserved cells were thawed and infused to the patient either immediately or after washing 3 times in normal saline supplemented with 6% anticoagulant citrate dextrose solution. Cell count with viability, clonogenic assay, and phenotyping were performed before and after thawing and after washing. Hematologic recovery, side effects, and complications were recorded. The in vitro and clinical data on 56 patients show that the depletion of DMSO in vitro before autografting does not induce a significant loss of cell number, viability, colony-forming unit-granulocyte-macrophage activity, or number of CD34(+) cells. Furthermore, it leads to a safe and sustained engraftment. The complications and side effects, as recorded by continuous monitoring, were substantially less; however, the procedure takes 3 to 4 hours of laboratory work per patient.

Different "professional" antigen-presenting cells (APC) have unique characteristics that favor or restrict presentation of microbial antigens to T cells, depending on the organism. Cryptococcus neoformans is a pathogenic yeast that presents unique challenges to APC, including its large size, its rigid cell wall, and its ability to stimulate T cells as a mitogen. T-cell proliferation in response to the C. neoformans mitogen (CnM) requires phagocytosis and processing of the organisms by accessory cells prior to presentation of CnM to T cells. Because of the requirement for uptake of the organism and more limited costimulatory requirements of mitogens, macrophages might be the most likely cellular source for the accessory cell. However, the present study demonstrates that a transiently adherent cell that was CD3 ؊ , CD14 ؊ , CD19 ؊ , CD56 ؊ , HLA-DR ؉ , and CD83؉ with a dendritic morphology, rather than monocyte-derived or tissue (alveolar) macrophages, was the most efficient APC for presentation of CnM. A large number of these cells bound and internalized the organism, and only a small number of dendritic cells were required for presentation of the mitogen to T cells. Further, the mannose receptor and Fc␥ receptor II were required for presentation of C. neoformans, as blocking either of these receptors abrogated both uptake of C. neoformans and lymphocyte proliferation in response to CnM. These studies demonstrate the surprising fact that dendritic cells are the most efficient accessory cells for CnM.

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Ciyptococcus neoformans is a pathogenic encapsulated yeast that infects patients that have defective cell-mediated immunity, including AIDS patients. Whole cryptococcal organisms that are killed by heating stimulate normal human lymphocytes to proliferate. However, strains of C. neoformans vary widely in virulence and therefore in their ability to cause disease in humans. To determine the effect of virulence factors such as the cryptococcal capsule, serotype, and the state of the organisms on the lymphocyte response to C. neoformans, human peripheral blood mononuclear cells were stimulated with C. neoformans in vitro and lymphocyte proliferation was determined. The major determinant of the lymphocyte response to C. neoformans was the amount of polysaccharide present. The response was greater after stimulation by minimally encapsulated strains (strains C3D, 68, and 613) than by heavily encapsulated strains (strains 6 and 145). A heavily encapsulated strain (strain 6) did not suppress the response to an acapsular mutant (strain 67). However, the response to an acapsular strain was suppressed by the addition of purified polysaccharide. Human lymphocytes responded to both serotypes of C. neoformans var. neoformans. The antigen responsible for lymphocyte stimulation was preserved despite various techniques of inactivation, including heat, paraformaldehyde fixation, irradiation, and mechanical disruption. Finally, lymphocytes responded equally to live and killed organisms. These results suggest that capsular polysaccharide, a known virulence factor, may suppress the human lymphocyte response to C. neoformans during an infection. Lymphocytes could respond to C. neoformans regardless of the viability of the organism, and they could also respond to disrupted organisms. We speculate that lymphocyte proliferation in vitro could be related to the protective immune response in host defense to C. neoformans and that it is suppressed by virulence factors of C. neoformans.

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