Administration of herpes simplex-thymidine kinase-expressing donor T cells with a T-cell-depleted allogeneic marrow graft.

Administration of donor T cells expressing the herpes simplex-thymidine kinase (HS-tk) with a hematopoietic stem cell (HSC) transplantation could allow, if graft-versus-host disease (GVHD) was to occur, a selective in vivo depletion of these T cells by the use of ganciclovir (GCV). The study evaluates the feasibility of such an approach. Escalating numbers of donor HS-tk-expressing CD3(+) gene-modified cells (GMCs) are infused with a T-cell-depleted bone marrow transplantation (BMT). Twelve patients with hematological malignancies received 2 x 10(5) (n = 5), 6 x 10(5) (n = 5), or 20 x 10(5) (n = 2) donor CD3(+) GMCs/kg with a BMT from a human leukocyte antigen (HLA)-identical sibling. No acute toxicity was associated with GMC administration. An early increase of circulating GMCs followed by a progressive decrease and long-lasting circulation of GMCs was documented. GCV treatment resulted in significant rapid decrease in circulating GMCs. Three patients developed acute GVHD, with a grade of at least II, while one patient developed chronic GVHD. Treatment with GCV alone was associated with a complete remission (CR) in 2 patients with acute GVHD, while the addition of glucocorticoids was necessary to achieve a CR in the last case. Long-lasting CR occurred with GCV treatment in the patient with chronic GVHD. Unfortunately, Epstein-Barr virus-lymphoproliferative disease occurred in 3 patients. Overall, the administration of low numbers of HS-tk-expressing T cells early following an HLA-identical BMT is associated with no acute toxicity, persistent circulation of the GMCs, and GCV-sensitive GVHD. Such findings open the way to the infusion of higher numbers of gene-modified donor T cells to enhance post-BMT immune competence while preserving GCV-sensitive alloreactivity.

The safety and efficacy of zotepine in the treatment of schizophrenia: Results of a one-year naturalistic clinical trial.

INTRODUCTION AND METHOD: The safety and efficacy of zotepine,75 - 450 mg/day, were evaluated in an open multicentre one-year study in patients suffering from acute exacerbation of schizophrenia; total exposure amounted to 152.78 years. RESULTS: Mean BPRS total score was reduced from 51.7 at baseline to 40.8 at end-point (P<0.05). Similar significant reductions at all study time-points were recorded for BPRS total and subscores, CGI severity and improvement, BAS total scores and SANS total and global scores. Significant improvements in EPMS and AIMS were recorded from week 12 to end-point. Clinically significant improvements in acute symptoms, detected early in the study, were maintained to end-point. CONCLUSION: Zotepine was well tolerated: weight gain, reduced serum uric acid, raised liver enzymes and increased heart rate were associated with chronic zotepine treatment. Seven patients experienced seizures during the study, although concomitant medications and a known historical predisposition to seizure are factors likely to have contributed to these events. The improvements in negative symptoms and low propensity to cause further extrapyramidal side-effects support the importance of zotepine in maintenance treatment.

Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma.

Lymphomas express a tumor-specific antigen which can be targeted by cancer vaccination. We evaluated the ability of a new idiotype protein vaccine formulation to eradicate residual t(14;18)+ lymphoma cells in 20 patients in a homogeneous, chemotherapy-induced first clinical complete remission. All 11 patients with detectable translocations in their primary tumors had cells from the malignant clone detectable in their blood by PCR both at diagnosis and after chemotherapy, despite being in complete remission. However, 8 of 11 patients converted to lacking cells in their blood from the malignant clone detectable by PCR after vaccination and sustained their molecular remissions. Tumor-specific cytotoxic CD8+ and CD4+ T cells were uniformly found (19 of 20 patients), whereas antibodies were detected, but apparently were not required for molecular remission. Vaccination was thus associated with clearance of residual tumor cells from blood and long-term disease-free survival. The demonstration of molecular remissions, analysis of cytotoxic T lymphocytes against autologous tumor targets, and addition of granulocyte-monocyte colony-stimulating factor to the vaccine formulation provide principles relevant to the design of future clinical trials of other cancer vaccines administered in a minimal residual disease setting.

A closed culture system for the ex vivo transduction and expansion of human T lymphocytes.

A phase I clinical trial is currently being performed at our institution, with the aim of evaluating the feasibility and toxicity related to the administration of herpes simplex thymidine kinase gene-expressing human primary T lymphocytes following allogeneic hematopoietic stem cell transplantation. The need for safe and standardized preparation conditions for gene-modified cells is crucial. We describe the closed culture system used in the current trial for ex vivo retroviral-mediated gene transfer and transduced cell selection. Cell handling is performed in closed systems using a sterile connection device that avoids opening the culture system. Cell numbers during the production process increased from 93 +/- 16 on day 0 to 440 +/- 92 x 10(6) on day 12 (7.2 +/- 1.4-fold increase) (n = 11). Transduction efficiency before and after G418 resistance-based selection was 13.5 +/- 3.8% and 90.0 +/- 1.4%, respectively. Safety and efficacy testing included a search for replication-competent retrovirus, endotoxins, Mycoplasma, and bacterial contamination (n = 0/9), PCR-DNA, % CD3+ cells (91 +/- 2%), and viability after thawing (82 +/- 3%). Effective working time from day 0 to day 12 is approximately 20 h. The closed system we developed allows for safe and reproducible ex vivo preparation of gene-modified primary T lymphocytes for clinical use.

In vivo alloreactive potential of ex vivo-expanded primary T lymphocytes.

BACKGROUND: We are presently investigating the therapeutic potential of herpes simplex-thymidine kinase-expressing donor T cells in the setting of a T cell-depleted allogeneic bone marrow transplantation. The generation, expansion, and selection of the gene-modified T cells require a 12-day ex vivo culture period in high-dose interleukin (IL)-2 that could significantly alter their in vivo alloreactivity. METHODS: We evaluated the alloreactive potential of such cultured cells in a murine allogeneic bone marrow transplantation model. RESULTS: The present studies demonstrate that ex vivo-expanded cultured T cells are capable of strong alloreactivity as evidenced by the occurrence of lethal acute graft-versus-host disease (GVHD). However, GVHD mortality after administration of the cultured T cells occurred later than after the administration of a same number of fresh T cells. Similar kinetics of GVHD-induced mortality between cultured and fresh T cells required a 10-fold increase in the number of cultured T cells, indicating a reduced alloreactive potential of these cells. The addition of a 2-day "resting" period in low-dose IL-2 resulted in T cells with enhanced alloreactive potential identical to the alloreactivity observed with fresh T cells. CONCLUSION: Ex vivo IL-2-expanded T cells are capable of significant in vivo alloreactivity. However, an increase in the number of cultured T cells administered or the introduction of a short resting culture period prior to infusion is necessary in order to achieve in vivo alloreactivity identical to the alloreactivity observed with fresh T cells.

Retrovirus-mediated transfer of the herpes simplex type I thymidine kinase gene in alloreactive T lymphocytes.

We have demonstrated in previous studies that retrovirus-mediated transfer of the herpes simplex thymidine kinase (HS-tk) and neomycin phosphotransferase (neo) genes in CD3/IL-2 stimulated primary T lymphocytes followed by G418 selection resulted in T cells retaining both interleukin-2 (IL-2) and alloresponsiveness and specifically inhibited by ganciclovir (GCV). A clinical trial examining the therapeutic potential of such gene-modified donor T cells after allogeneic bone marrow transplantation is presently underway. In the present study, we have investigated the feasibility and consequences of replacing polyclonal stimulation of T cells by an allogeneic stimulation prior to retrovirus-mediated gene transfer. Exposure of allostimulated primary donor T lymphocytes to retrovirus-containing supernatant resulted in T cells resistant to G418 while maintaining a strong, GCV-sensitive, allogeneic response when subsequently restimulated with the initial allogeneic cells. Control nontransduced cells identically stimulated exhibited a weaker, GCV-insensitive, allogeneic proliferative response. The transduced T cells were also capable of GCV-sensitive alloreactivity when exposed to third-party cells with, however, a lower proliferative response than that seen with the allogeneic cells used for stimulation at the time of transduction. Importantly, this difference in the proliferative responses was not observed with control nontransduced cells identically stimulated. A similar response pattern was observed with respect to pre-cytotoxic T lymphocyte (CTL) frequencies. Overall, retrovirus-mediated gene transfer after an allogeneic stimulation can lead to efficient transduction and the pattern of alloreactivity of the HS-tk-expressing cells is consistent with the preferential transduction of alloantigen-specific dividing T cells. Such an approach could be used to generate cells both strongly alloreactive and GCV-sensitive for in vivo therapeutic use.

Gene transfer applied to the modulation of alloreactivity.

Allogenic hematopoietic stem cell transplantation is associated with a severe complication induced by the T-cells present in the graft: graft-vs-host disease (GVHD). While effectively preventing GVHD, ex vivo T-lymphocyte depletion of the graft unfortunately increases graft rejection and reduces the graft-vs-leukemia (GVL) effect. The ex vivo transfer to the herpes simplex thymidine kinase (HS-tk) suicide gene into T-cells before their infusion with the hematopoietic stem cells should allow for selective in vivo depletion of these T-cells with ganciclovir (GCV) if subsequent GVHD was to occur. In patients not experiencing GVHD, and therefore at a higher risk of relapse, one could preserve the beneficial effects of the donor T-cells on tumor control. Lastly, the early presence of donor T-cells in all patients should contribute to successful engraftment. We have demonstrated that retroviral-mediated transfer of HS-tk and Neomycine resistance genes in T-lymphocytes, followed by G418 selection, results in T-cells specifically inhibited by GCV with no bystander effect. In a phase I study, escalating amounts of HS-tk expressing T-cells will be infused in conjunction with a T-cell depleted marrow graft to allogenic HLA identical recipients. Toxicity, survival, alloreactivity and GCV-sensitivity of the gene-modified cells will be monitored. If successful, such an approach could significantly contribute to expanding the use of alloreactivity as a treatment modality.

Transfer of myeloma idiotype-specific immunity from an actively immunised marrow donor.

The idiotype of myeloma immunoglobulin can be used as a unique tumour-specific antigen. We tested the hypothesis that tumour antigen-specific immunity can be transferred from bone-marrow-transplant donor to recipient. We immunised a healthy sibling donor with myeloma immunoglobulin from the plasma of the recipient, conjugated to an immunogenic carrier protein and emulsified in an adjuvant, before marrow transplantation. Detection of a lymphoproliferative response, a parallel response in the carrier protein, recovery of a recipient CD4+ T-cell line with unique specificity for myeloma idiotype, and demonstration by in-situ hybridisation that the cell line was of donor origin, proved that a myeloma idiotype-specific T-cell response was successfully transferred to the recipient. Donor immunisation with myeloma idiotype may represent a new strategy for enhancing the specific anti-tumour effect of allogeneic marrow grafts.

The kinetics of cytoplasmic granule secretion in natural killer cytotoxicity.

Antiserum against purified cytoplasmic granules from rat LGL tumor cells, and protein A-gold immunoelectron microscopy were used to study the secretory events in lysis of YAC-1 tumor cells by rat LGL tumor cells or by isolated LGL from normal rats. After 30 min incubation of effector and target cells together, gold-labeled cytoplasmic granules were often seen concentrated in the area of the LGL adjacent to the bound YAC-1 target. Within 60 min, the granules were observed to move to the cell border near the conjugated site. At this point, the granules were fused with the cell membrane, and subsequently released the gold-labeled contents into the junction between the LGL and the target cell. Gold particles could be seen at the E-T interface, on the LGL surface, or sometimes on the target cell surface. These data provide direct evidence for the hypothesis that under conditions of active cytotoxicity, natural killer cells secrete their cytoplasmic granule contents leading to the deposition of granule material on the target cell surface and the eventual lysis of the cell.

Ganciclovir treatment of herpes simplex thymidine kinase-transduced primary T lymphocytes: an approach for specific in vivo donor T-cell depletion after bone marrow transplantation?

Allogeneic bone marrow transplantation (BMT) is associated with a severe complication--graft-versus-host disease (GVHD). Although effectively preventing GVHD, ex vivo T-lymphocyte marrow depletion unfortunately increases graft rejection and reduces the graft-versus-leukemia (GVL) effect. The ex vivo transfer of the herpes simplex thymidine kinase (HS-tk) suicide gene into T cells before their infusion with hematopoietic stem cells could allow for selective in vivo depletion of these T cells with ganciclovir (GCV) if subsequent GVHD was to occur. Thus, one could preserve the beneficial effects of the T cells on engraftment and tumor control in patients not experiencing severe GVHD. To obtain T cells specifically depleted by GCV, we transduced primary T cells with a retroviral vector containing the HS-tk and neomycin resistance (NeoR) genes. Gene transfer was performed by coculturing PHA +/- CD3- or alloantigen-stimulated purified T cells on an irradiated retroviral vector producer cell line or by incubating the T cells in supernatant from the producer. Subsequent culture in G418 for 1 week allowed for the selection of transduced cells. GCV treatment of interleukin-2-responding transduced and selected cells resulted in greater than 80% growth inhibition, whereas GCV treatment of control cells had no effect. Similarly, the allogeneic reactivity of HS-tk-transduced cells was specifically inhibited by GCV. Combining transduced and nontransduced T cells did not show a bystander effect, thus implying that all of the cells inhibited by GCV were indeed transduced. Lastly, studies involving the transduction of the HUT-78 (T-lymphoma) cell line suggest that stable expression of HS-tk can be maintained over 3 months in vitro in the absence of G418. In summary, we have established the feasibility of generating HS-tk-transduced T cells for subsequent in vivo transfer with hematopoietic stem cells and, if GVHD occurs, specific in vivo GCV-induced T-cell depletion in allogeneic BMT recipients.

Activated natural killer cells and interleukin-2 promote granulocytic and megakaryocytic reconstitution after syngeneic bone marrow transplantation in mice.

Purified populations of natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID). SCID spleen cells were cultured and activated with recombinant human interleukin-2 (rhIL-2) in vitro. The activated NK cells were then transferred with syngeneic BALB/c bone marrow cells (BMC) and rhIL-2 into lethally irradiated syngeneic recipients to determine their effect on long-term hematopoietic reconstitution. On analysis, the transfer of rhIL-2-activated NK cells along with BMC resulted in significant increases in splenic and BM hematopoietic progenitor cells when compared with those for mice not receiving NK cells. Histologic and flow cytometric analysis showed a marked increase in granulocytic and megakaryocytic lineage cells present in the spleens of the mice receiving activated NK cells. Analysis of the peripheral blood indicated that the transfer of activated NK cells with BMC also significantly improved platelet and total white blood cell counts, with increases in segmented neutrophils. Erythroid recovery was not affected. Finally, lethally irradiated mice receiving activated NK cells and rhIL-2 along with limiting numbers of syngeneic BMC showed a marked increase in survival rate. These results show that the use of populations enriched for activated NK cells after syngeneic BM transplantation (BMT) has a profound enhancing effect on engraftment primarily affecting megakaryocytic and granulocytic cell reconstitution. Therefore, the transfer of activated NK cells and rhIL-2 may be of clinical use to promote hematopoietic reconstitution after BMT.

Natural killer cells and bone marrow transplantation.

Bone marrow transplantation is currently used in the treatment of a variety of neoplastic and nonneoplastic diseases. However, significant obstacles still limit the efficacy of this procedure. These include the occurrence of graft-versus-host disease, the failure of the marrow to engraft, the susceptibility of patients to opportunistic infections during the period of immunodeficiency after transplantation before full recovery of immune function, and finally, the recurrence of the cancer. Natural killer (NK) cells are lymphoid cells responsible for mediating a variety of immunologic and homeostatic functions. Initially described almost 20 years ago, the full range of functions carried out by these enigmatic cells continues to unfold. NK cells may be both beneficial and deleterious in bone marrow transplantation, depending on their genotype and activation status. Resting host-derived NK cells appear capable of mediating resistance to both autologous and allogeneic bone marrow cell grafts. At the other end of the spectrum, the transfer of activated NK cells of donor type appears to produce multiple beneficial effects during both syngeneic and allogeneic bone marrow transplantation. Here, we review and attempt to reconcile the literature concerning the basic biology of NK cells and their effects on hematopoiesis, both in vitro and in vivo. We also discuss the current issues in bone marrow transplantation and the potential role NK cells may play in determining the outcome of the marrow graft, the occurrence of graft-versus-host disease, and the generation of a graft-versus-tumor response when bone marrow transplantation is used for the treatment of cancer.

Interleukin-1 administration before lethal irradiation and allogeneic bone marrow transplantation: early transient increase of peripheral granulocytes and successful engraftment with accelerated leukocyte, erythrocyte, and platelet recovery.

Administration of interleukin-1 beta (IL-1 beta) before a lethal irradiation with or without allogeneic bone marrow transplantation (BMT) protects greater than 90% of the irradiated mice. To approach the mechanisms responsible for the radioprotective effect of IL-1, we examined the effects of IL-1 pretreatment on engraftment and kinetics of peripheral blood, spleen, and marrow cell reconstitution after irradiation and BMT. Although the BMT was not necessary for the survival of the IL-1-pretreated lethally irradiated mice, allogeneic marrow did engraft in these mice as evaluated in the spleen and marrow 2 months after BMT. IL-1 pretreatment significantly accelerated hematopoietic recovery versus transplanted saline-treated controls with a pronounced enhancement of peripheral leukocyte, platelet, and erythrocyte recovery. Leukocyte recovery in IL-1-pretreated mice was unique in that IL-1 first induced an early transient (maximum at day 7) increase of peripheral granulocytes before accelerating leukocyte recovery after day 11. IL-1 pretreatment also significantly enhanced marrow cell recovery after allogeneic BMT with an eightfold increase in marrow cellularity from day 4 to 11 versus control transplanted mice. When lethal irradiation was not followed by allogeneic BMT. IL-1 pretreatment also affected the peripheral reconstitution of leukocytes, platelets, and erythrocytes. Interestingly, in the absence of BMT, IL-1 also induced an early circulation of peripheral granulocytes. Overall, our data demonstrate that a single administration of IL-1 before lethal irradiation and allogeneic BMT can induce an early transient increase of circulating granulocytes, followed by an accelerated multilineage recovery and long-term allogeneic engraftment.

Characterization of non-lytic cytolysin-membrane intermediates.

In order to understand the nature of cytolysin-membrane interactions, the characteristics of stable, non-lytic cytolysin-target cell intermediates formed at low ionic strength, neutral pH, and at physiological ionic strength, pH 6.0, were examined. Protease treatment of cytolysin-RBC intermediates formed at low ionic strength inhibited subsequent hemolysis when the intermediates were exposed to physiological ionic strength and pH. Similarly, when such intermediates were treated with anti-granule and anti-cytolysin antibodies a significant dose-dependent inhibition of hemolysis was observed. These results suggested that in this non-lytic state the cytolysin molecule was exposed on the RBC surface. If low ionic strength or pH 6.0 generated intermediates were washed in 0.5 M NaCl, hemolytic activity was greatly reduced and cytolysin activity could be recovered from the medium. In addition to RBC, both murine (Yac-1 and Lettre ascites) and human (K562) tumor targets formed cytolysin-target cell intermediates at low ionic strength and at low pH. Multilamellar vesicles composed of either phosphatidylcholine, sphingomyelin or phosphatidylserine inhibited the binding of cytolysin to RBC at both low ionic strength and pH 6.0 indicating a lack of polar head group specificity for cytolysin binding.

Islet cells but not thyrocytes are susceptible to lysis by NK cells.

BB rats develop both pancreatic insulitis and lymphocytic thyroiditis, but whereas spontaneous autoimmune diabetes is common, hypothyroidism is rare. Splenic natural killer (NK) cells from acutely diabetic (AD) BB rats and from athymic nude rats are known to be cytotoxic to rat islet cells in vitro. To investigate possible differential tissue susceptibility to lysis by NK cells or their cytokines such as cytolysin (perforin) or NK cytotoxic factor (NKCF), we used an in vitro 51Cr-release assay to measure the cytotoxicity of splenocytes, cytolysin or NKCF against Wistar Furth (WF) and Fischer 344 (F-344) rat islet cells, and FRTL-5 F-344-derived and WRT Wistar-derived rat thyrocytes. The results demonstrated that spleen cells from AD-BB (RT1u) rats and athymic F-344 nude (RT11) rats are cytotoxic to WF (RT1u) islets and F-344 (RT11) islets, but not to FRTL-5 (RT11) or WRT (class I RT11) thyrocytes. WF and F-344 rat spleen cells were not cytotoxic to any of these cells. Thyrocytes are known to express class II molecules on their surface in chronic thyroiditis. We found that treatment of thyrocytes with interferon-gamma (IFN-gamma) induced class II expression but did not increase the cytotoxicity of splenocytes against these cells. Cytolysin and NKCF were both cytotoxic to islets in a dose dependent manner, but FRTL-5 thyrocytes were resistant to killing by these cytokines. These findings suggest that islet cells and thyrocytes in vitro are differentially susceptible to lysis by NK cells.

An improved in vitro assay to quantitate chemotaxis of rat peripheral blood large granular lymphocytes (LGL).

We have developed an improved method to study the directed migration, or chemotaxis, of rat peripheral blood large granular lymphocytes (LGL) in vitro. A modified Boyden chamber technique was used to measure chemotaxis of LGL through polycarbonate filters that had been coated with different basement membrane components. LGL were found to adhere to collagen types I and IV, laminin and fibronectin. However, only collagen type IV was not in itself chemotactic for LGL. Migrated cells could be identified both morphologically and phenotypically as LGL on collagen type IV-coated filters after incubation with a chemotactic stimulus. LGL were found to display chemotaxis to a number of different stimuli, including the classical chemoattractant agents N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and complement fragments present in activated sera. However, the degree of response to these stimuli was much less than that of isolated peripheral blood neutrophils or monocytes. In contrast, all three cell types showed increased chemotaxis to the diacyl glycerol analog 1-oleoyl 2-acetyl glycerol (OAG), which induced a 4-14 fold stimulation of migration. Induction of chemotaxis of LGL by OAG was time and dose-dependent, as confirmed using checkerboard assays. In summary, we have developed a rapid, quantitative method to measure chemotaxis of LGL in vitro. This technique may now be utilized to identify naturally occurring chemoattractants for LGL and to study the intracellular and regulatory events associated with LGL migration.

Differential regulation of interleukin-1 gene expression in human CD3- large granular lymphocytes.

Our laboratory analyzed the expression of lymphokine and cytokine mRNA in CD3- peripheral blood large granular lymphocytes (LGL). Herein we present evidence that this subset of lymphocytes can synthesize IL-1 beta mRNA constitutively and that the cytoplasmic mRNA levels of IL-1 beta can be increased rapidly by interleukin (IL)-2. IL-1 alpha mRNA is expressed constitutively very infrequently and increases in IL-1 alpha mRNA are seen only after prolonged incubation with IL-2. Furthermore, IL-1 activity could not be detected in LGL culture supernatants, indicating that other processes may be involved in releasing biologically active IL-1 from LGL. In addition, MAb to the p75 IL-2 receptor on LGL abrogated IL-2 induction of IL-1 beta mRNA, suggesting that IL-2 signaling via the p75 IL-2 receptor induced IL-1 beta gene expression in LGL. Since, in contrast to T cells, LGL are capable of mediating effector functions without prior stimulation, they are said to be already "primed" for response. Overall, these data suggest that constitutive lymphokine gene expression may be involved in the in vivo priming of LGL.

Anti-asialo GM1 antiserum treatment of lethally irradiated recipients before bone marrow transplantation: evidence that recipient natural killer depletion enhances survival, engraftment, and hematopoietic recovery.

Natural killer (NK) cells are reported to have an important role in the resistance of lethally irradiated recipients to bone marrow transplantation (BMT). Therefore, we investigated the effects of recipient NK depletion on survival, chimerism, and hematopoietic reconstitution after lethal irradiation and the transplantation of limiting amounts of T-cell-deficient bone marrow (BM). When administered before BMT, anti-asialo GM1 (ASGM1) antiserum treatment, effective in depleting in vivo NK activity, was associated with a marked increase in survival in 3 of 3 allogeneic combinations (BALB/c into C3H/HeN, C57B1/6, or C3B6F1). This enhanced survival was independent of the susceptibility of each recipient strain to accept BALB/c BM. Moreover, recipient anti-ASGM1 treatment was also effective in increasing survival in recipients of syngeneic BM, suggesting that NK cells can adversely affect engraftment independent of genetically controlled polymorphic cell surface determinants. Analysis of chimerism in surviving animals 2 months post-BMT showed that recipient NK depletion significantly increased the level of donor engraftment when high doses of BM were transplanted. These studies also demonstrated that anti-ASGM1 pretreatment mainly resulted in an increase in extramedullary hematopoiesis in the second and third week after irradiation. Anti-ASGM1 treatment also dramatically accelerated the rate of appearance of donor-derived cells with a higher level of donor-cell engraftment apparent at a time when the differences in survival between NK-depleted and control BMT recipients became significant. Peripheral cell counts were also affected by NK depletion, with significantly enhanced platelet and red blood cell recovery and a moderate increase in granulocyte recovery. The overall favorable influence of anti-ASGM1 recipient treatment on hematopoietic events post-BMT suggests that, in humans, pretransplant regimens aimed toward NK depletion should be evaluated.

Cytoplasmic components of natural killer cells limit the growth of Cryptococcus neoformans.

Murine natural killer (NK) cell-mediated inhibition of growth of a yeast-like target cell, Cryptococcus neoformans, was completely abrogated by blocking the effector cell secretory process with monensin. Therefore, further studies were performed to determine the ability of various cytoplasmic fractions of NK cells to mediate inhibition of cryptococcal growth. Percoll-fractionated homogenates of rat LGL tumor cells demonstrated that the granule-containing fractions plus three additional sets of less dense cytoplasmic fractions displayed anti-cryptococcal activity; whereas only the cytoplasmic granule-containing fractions had cytotoxic activity against YAC-1 tumor cell and sheep erythrocyte targets. Maximal cryptococcal growth inhibition induced by LGL granules occurred after a 1 h incubation, required the presence of Ca2+ (1.0 mM) or Mg2+ (0.5 mM or 5.0 mM), and was completely abrogated in the presence of rabbit anti-LGL granule IgG. Cytolysin, the granule component which mediates tumor cell and sheep erythrocyte lysis, effectively limited the growth of cryptococci. Since Percoll gradient fractionation of the LGL homogenates demonstrated three separate peaks of anti-cryptococcal activity other than the granule peak, it is possible that the cytolysin-containing granules are not the only subcellular component of NK cells playing a role in inhibition of C. neoformans growth.