Altered production and renewal of natural killer cells in B-lymphocyte-deficient CBA/N mice

The present study was designed to measure by quantitative and kinetic methods the production and renewal of natural killer (NK) cells in congenitally B-lymphocyte-deficient (CBA/N) mice. The total NK activity (percent specific lysis corrected for changes in whole organ cellularity) of the bone marrow and spleen of immunologically normal (CBA/CaJ) and CBA/N mice was assayed prior to and immediately after 48 h treatment (2 X/day, i.p.) with the cell cycle poison hydroxyurea (HU) and at various intervals throughout the subsequent post-HU recovery period. The total NK activity (TNKA) of untreated CBA/N bone marrow was 154% of that of CBA/CaJ bone marrow while the TNKA of CBA/N spleen was not significantly different (112%) from that of CBA/CaJ spleen. At the conclusion of 48 h HU, bone marrow TNKA of CBA/N and CBA/CaJ mice fell to 60 and 49%, respectively, of their saline-injected (2 X/day, i.p.) control levels, while spleen TNKA fell to 42 and 61%, respectively, of their saline-injected control levels. In the bone marrow, NK cell depletion in response to HU was more rapid in CBA/N mice (day 0.5 after HU) than in CBA/CaJ mice (day 2 after HU). TNKA of the spleen also decreased more rapidly in CBA/N mice (day 2 after HU) than in CBA/CaJ mice (day 3 after HU). The data indicate an enhanced production and turnover of NK cells in CBA/N mice relative to CBA/CaJ mice. Moreover, increased production and renewal of NK cells in CBA/N mice together with virtually unchanged levels of NK activity (112% of CBA/CaJ mice) in CBA/N mouse spleens indicate that mature lytic NK cells in CBA/N spleen but not bone marrow have a significantly shorter post-mitotic life span than do NK cells in the spleens of immunologically normal (CBA/CaJ) mice.     

Production and differentiation of NK lineage cells in long-term bone marrow cultures in the absence of exogenous growth factors.

Neither lytic NK cells nor IL-2-responsive NK precursors were produced in myeloid (Dexter) long-term bone marrow cultures (LTBMC). However, when myeloid LTBMC were switched to lymphoid (Whitlock-Witte) conditions and reseeded ("recharged") with fresh bone marrow cells (BMC), nonadherent cells with NK lytic activity and NK 1.1+ phenotype were produced within 1-2 weeks without the addition of exogenous IL-2 to the cultures. NK- and T cell-depleted BMC proliferated extensively in switched cultures and in 2 weeks generated cells that lysed the NK target YAC-1 but not the LAK target P815. The presence of NK precursors in the cultures was confirmed by reculturing nonadherent cells harvested from recharged LTBMC in fresh medium containing 50 U rIL-2/ml. High levels of NK lytic activity were generated. Sequential expression of NK 1.1 and IL-2 responsiveness followed by lytic activity was demonstrated by harvesting cells early after recharge, prior to the appearance of lytic cells. Elimination of NK 1.1+ cells depleted the ability to respond to IL-2 in secondary culture. Our studies demonstrate that myeloid-to-lymphoid switched LTBMC support the proliferation and differentiation of NK lineage cells from their NK 1.1-, nonlytic progenitors in the absence of an exogenous source of growth factors.     

Protective effect of biological response modifiers on murine cytomegalovirus infection

Pretreatment with two biological response modifiers (BRM), OK-432 and PS-K, protected mice from lethal infection by murine cytomegalovirus (MCMV). This was evidenced by an increase in 50% lethal doses and a decrease in titers of infectious viruses replicated in the liver and spleen. Spleen cells from the BRM-treated mice augmented the natural killer (NK) cell activity and suppressed the replication of MCMV in vitro. During MCMV infection, the NK cell activity of the spleen cells was maintained at a high level in the BRM-treated mice, whereas it was severely impaired in untreated mice. The BRM-induced protection was nullified by concomitant administration of antiasialo GM1 antibody. Interferon was neither induced by BRM treatment nor enhanced in BRM-pretreated and MCMV-infected mice. Thus, the protective effect of OK-432 and PS-K seems to be based on activation of NK cells and prevention of MCMV-induced inhibition of the NK cell activity.     

OK-432 and IL-2-augmental cytotoxicity of human natural killer cells and cytotoxic T lymphocytes at the clonal level

Abstract The biology response modifiers OK-432 and interleukin-2 (IL-2) were found to enhance the lytic capacity of cloned CD3⁻ natural killer (NK) cells and CD3⁺ T cells. With respect to NK cells, only those clones with a high proliferative capacity and cultured without phytohaemagglutinin (PHA) responded with enhaced lytic capacity to OK-432. OK-432, but not IL-2, was found to augment the antibody-dependent cellular cytotoxicity of cloned NK cells. With T-cell clones, OK-432 augmented the cellular cytotoxicity of CD3⁺8⁺ but not that of CD3⁺4⁺ cytotoxic T lymphocytes, while IL-2 augmented the cytotoxicity of both types of clone. Taken together, these results indicate that OK-432-augmented lytic capacity is not restricted to NK cells and its pathway of action may be independent of IL-2.     

Mechanism of NK activation by OK-432 (Streptococcus pyogenes). I. Spontaneous release of NKCF and augmentation of NKCF production following stimulation with NK target cells

The biological response modifier OK-432 (Picibanil) (manufactured in Japan) is produced by lyophilization of cultures of the low virulent Su strain of group A Streptococcus pyogenes of human origin. This preparation has been shown to have multiple effects on the immune system and has been used as an anti-cancer therapeutic agent in man. It has been shown that OK-432 augments the cytotoxic activity of human natural killer (NK) cells. We have proposed that natural killer cytotoxic factors (NKCF) derived from NK cells play a role in the mechanism of NK cell-mediated cytotoxicity (CMC). The present study investigates the underlying mechanism of the OK-432-mediated enhancement of NK activity by determining whether OK-432 has an effect on the induction and activity of NKCF produced by NK cells. Treatment of peripheral blood lymphocytes (PBL) with OK-432 for 20 hr and wash resulted in significant augmentation of NK CMC and this enhancement was dependent on the concentration of OK-432 used. Coculture of the OK-432-treated PBL with U937 resulted in a several-fold enhanced production of NKCF in the supernatant. The NKCF produced were similar to those produced by untreated effector cells in that they had the same NK target specificity for lysis. The time kinetics of stimulation of PBL with OK-432 for optimal production of NKCF was found to be 8-12 hr. It was also observed that culture of OK-432-treated PBL in the absence of stimulator cells spontaneously release significant amounts of NKCF into the supernatant. The supernatant containing NKCF was tested for interleukin 2 (IL-2) activity using an IL-2-dependent HT-2 line. It was found that there was no direct correlation between the levels of NKCF and IL-2 activity. The results of this study demonstrate that OK-432 stimulates NK cells to produce NKCF in the presence or absence of stimulator cells. The optimum concentration of OK-432-induced augmentation of NK CMC paralleled that seen for optimum NKCF production, suggesting that one mode of action of OK432 is to enhance NKCF production in a manner reminiscent of IFN and IL-2. The results also point out that OK-432 acts by a mechanism independent of the action of IL-2.     

Protection of OK-432, a Streptococcus pyogenes preparation, against lethal infection of mice with herpes simplex virus

We have studied the protective effect of OK-432, a biological response modifier (BRM) of Streptococcus pyogenes origin, on the lethal infection of mice with herpes simplex virus (HSV)-1. A single intraperitoneal (i.p.) injection of more than 10 micrograms of OK-432, when given at least two days before the infection, gave a marked effect yielding nearly 100% protection against ordinarily lethal infection. The protection was independent of the amount of infected virus inoculated. When given after the infection, the agent even at the maximal dose (100 micrograms), produced only a marginal effect. A single i.p. administration of OK-432 augmented the natural killer (NK) activity of peritoneal exudate cells and spleen mononuclear cells in mice 2 to 3 days after injection of OK-432, coinciding with the times when it induced a survival effect on HSV-infection. Treating OK-432-treated mice with a combination of an anti-macrophage agent, silica, and an anti-NK cell agent, anti-asialo GM1 serum, before infection diminished the antiviral effect of OK-432. The OK-432 protection against HSV infection was also markedly diminished in athymic nude mice. Thus, the protective effect of OK-432 on lethal HSV infection seems to be based on the activation of NK cells, macrophages, and T lymphocytes.     

Orally administered streptococcal preparation,OK-432 augments the antitumor immunity of patients with gastric or colorectal cancer

A streptococcal preparation, OK-432, was orally administered at a dose of 5 KE to patients with gastric or colorectal cancer for 7–14 days before their operations, and its immunomodulatory effects on peripheral blood lymphocytes (PBL), regional node lymphocytes (RNL) and tumor infiltrating lymphocytes (TIL) were assessed. The group treated with OK-432 included 8 gastric and 6 colorectal cancer patients, and the control group included 8 gastric and 8 colorectal cancer patients. The NK cell activity of PBL was significantly augmented by the oral administration of OK-432, and the proportions of Leu 7+ and Leu 11+ cells in PBL also increased. The responses of PBL and TIL to autologous tumor extracts in the presence of interleukin-2 were enhanced after the oral administration of OK-432. The proportion of OKT8+ cells in PBL increased after treatment with oral OK-432, whereas the proportion in RNL significantly decreased. These results indicate that oral OK-432 affects NK and T cells and may augment the antitumor immunity of patients with gastrointestinal cancer.     

Donor-type activated natural killer cells promote marrow engraftment and B cell development during allogeneic bone marrow transplantation.

Purified NK cells were obtained from mice with severe combined immune deficiency and were activated with human IL-2 (hrIL-2) in vitro to determine if, once activated, these cells could be transferred with compatible bone marrow cells (BMC) and promote marrow engraftment in irradiated allogeneic recipients. After culture with hrIL-2, these cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. These activated NK cells were then adoptively transferred with the donor BMC and rhIL-2 into lethally irradiated allogeneic hosts. The addition of NK cells with the BMC allowed for more rapid hematopoietic engraftment as determined through short term studies, and greater donor-derived chimerism with accelerated reconstitution of the B cell population as determined with long term analysis. No evidence of graft-vs-host disease was detected in the recipients receiving the activated NK cells with allogeneic T cell replete BMC and hrIL-2. The mechanism by which the transferred NK cells improved BMC engraftment was at least partly through the abrogation of the host effector cell's ability to mediate resistance to the marrow graft. Thus, the administration of donor-type activated NK cells with BMC and hrIL-2 may significantly augment hematopoietic engraftment and immune reconstitution in the clinical setting of allogeneic BMT without giving rise to graft-vs-host disease.     

Streptococcal preparation OK-432 augments cytotoxic activity against an erythroleukemic cell line in humans

Summary The effects of OK-432, an inactivated and lyophilized preparation of a low-virulence strain of Streptococcus pyogenes, were evaluated on the cytotoxicity of lymphoid cells against a natural killer (NK)-sensitive erythroleukemic cell line K562 in patients with malignant diseases. When ten patients were treated postoperatively with daily injections of OK-432, a significant degree of augmentation in the cytotoxicity of peripheral blood lymphoid cells was evoked in all the patients, and the maximum level of cytotoxicity was on the third day after the beginning of the treatment. In spite of the successive daily administration, the level of cytotoxicity declined thereafter, but stayed higher than the pretreatment level. When OK-432 was injected IP in three patients with carcinomatous peritonitis, the cytotoxic activity of ascitic lymphoid cells was significantly enhanced. Cytotoxicity of in vitro-cultured lymphoid cells taken from peripheral blood of normal donors was also augmented by the addition of OK-432.     

Changes in immunological parameters in lung cancer patients undergoing immunotherapy with streptococcal preparation OK-432

Summary We have studied the immunological status of patients treated with streptococcal preparation OK-432. Two KE of OK-432 was injected intramuscularly once every week for more than three years unless the patients died. The natural killer (NK) activity in those patients who underwent curative surgery for lung cancer and had no sign of recurrence was significantly increased (P < 0.01) during the OK-432 treatment. However, the NK activity in the patients who had persistent disease (non-resected cases, incompletely resected cases or recurrent cases) was not significantly increased in comparison with that before the immunotherapy. Also, in the cases with no clinical symptoms of recurrence, both the lymphoblastogenetic reactions to the mitogens and the IL-2 production were significantly enhanced(P < 0.01) during the administration of OK-432. Reactions in the SU-PS (polysaccharide taken from the cell-wall fraction of theStreptococcus pyogenes SU strain and containing 7.2% of protein) skin-test appeared to significantly correlate with the immunological status of the patients under OK-432 therapy, but the PHA and PPD skin reactions showed no definitive enhancement. The survival rate of the patients whose SU-PS skin tests were positive during the OK-432 immunotherapy was significantly higher (P < 0.01) than that of the patients with negative reactions.     

Effective Induction of Human NK Cells with OK-432 and Further Augmentation of Their Cytolytic Function by rIL-2

Low concentrations of exogenously added recombinant interleukin 2 (rIL-2) were able to augment OK-432-induced natural killer (NK) cell activity. This kind of augmenting effect depended on the dose of rIL-2 and manifested itself only in PBMC stimulated with OK-432 (OK-MC) followed by rIL-2; augmentation did not happen in the reverse order. The existence of CD16⁺/CD25⁺ (IL-2 receptor positive; IL-2R⁺) and CD57⁺/CD25⁺ double positive cells which possess NK cell surface markers in OK-MC markedly increased in a long-term culture (12 days). A strong positive correlation was observed between the IL-2-dependent augmentation of NK activity and the quantitative changes in cell populations that possessed NK cell phenotypes. Treatment of the day-12-OK-MC with monoclonal anti-CD56 antibody plus complement could almost completely abrogate the augmented NK cytotoxicity. Furthermore, this augmenting effect was detectable within 4 hr after addition of rIL-2 at single cell level, suggesting that the effect did not require NK cell's DNA synthesis. Thus it was suggested that OK-432 could promote and upregulate the expression of IL-2 receptor (CD25) on CD56⁺ NK cell populations. Moreover, it was considered that the interaction of low concentration rIL-2 with IL-2 receptors on OK-432-activated NK cells could augment their lytic function.     

Functional defects of NK cells treated with chloroquine mimic the lytic defects observed in perforin-deficient mice

NK cells are the primary effectors mediating acute rejection of incompatible bone marrow cell grafts. To reduce rejection, we evaluated the ability of chloroquine (CHQ) to prevent perforin-dependent NK cell activity. Perforin is a key cytotoxic component released from the lytic granules of activated NK cells. Generation of functional perforin requires an acidic protease activity that occurs in the secretory, lytic lysosomes. Our hypothesis was that CHQ, a lysosomotropic reagent, would raise the pH of the acidic compartment in which perforin is processed and thereby block perforin maturation and cytotoxicity. We have measured NK cytotoxicity in vivo by clearance of YAC-1 tumor cells from the lungs and by rejection of incompatible bone marrow transplants and in vitro by cytolysis of YAC-1 and Jurkat cells. The engraftment of bone marrow cells was monitored by recolonization of the spleen with hemopoietic cells from transplants of MHC class I-deficient bone marrow cells into lethally irradiated recipient mice. Transplant rejection was compared in two inbred strains of mice: 129, which apparently use perforin-dependent cytotoxicity, and C57BL/6, in which rejection can be perforin-independent. CHQ treatment reduced NK cell activity in 129 mice in which perforin is important for mediating rejection. CHQ affected the fraction of NK cell cytolysis that was Fas independent. In addition, we found that CHQ prevents perforin processing by LAK cells in vitro. These data indicate that CHQ may impair rejection of incompatible bone marrow transplants and other functions mediated by NK and cytotoxic T cells.     

Peri-operative immunotherapy with OK-432

Pre- and postoperative intradermal administration of OK-432 enhanced the SU-PS skin reaction in patients with gastric cancer, but failed to prevent a fall in the NK activity induced by the operation.The change in NK activity was not associated with a change in the proportion of Leu 7-positive cells, but was related to Leu 11a-positive cells. Intradermal injection of OK-432 increased the proportion of Leu 7-positive cells in the patients in whom they accounted for less than 20% of lymphocyte population. The case was the same with Leu 11a-positive cells.Intravenous injection of OK-432 tended to increase suppressor-inducer T cells (CD4⁺2HA⁺ cells), B cells and Leu 7-positive cells. Particularly, the proportions of OK-M₁-positive cells and MHC class II antigen-positive cells increased in all patients. Immunotherapy with OK-432 given intravenously at a dose of 0.1 KE appeared to be safe because no side effects were essentially observed.     

Dual effects of OK-432 on mitogenic response of splenocytes to concanavalin A

OK-432, a streptococcal preparation, was studied for its effect on the concanavalin A (Con A)-induced mitogenesis of the host spleen cells. When mice were given a single intraperitoneal injection of OK-432, there was a substantial increase in the mitogenic response of splenocytes, whereas multiple injections conversely resulted in a marked reduction of the mitogenic response, when the spleen cells were cultured at high cell densities of over than 5 X 10(5) cells/well. The reduced Con A-responsiveness in the latter was not restored by mixing spleen cells from mice given multiple OK-432 injections with those from normal mice. Moreover, splenic macrophages from OK-432-injected mice exhibited marked inhibitory activity against Con A-mitogenesis of normal splenocytes, while normal splenic macrophages failed to show such an effect. Splenic T cells from OK-432-injected mice also showed an inhibitory activity against Con A-mitogenesis of normal splenocytes and similar activity was also noted in normal splenic T cells. Therefore, the OK-432-spleen cells contain two types of suppressor cells; one is a newly elicited suppressor macrophage and the other is a suppressor T cell supposedly resident also in normal spleen cells. In the OK-432-injected spleen cells, accessory cell function for T cell Con A-mitogenesis was markedly reduced. On the other hand, it was noted that the interleukin 2-producing ability of the OK-432-splenocytes was augmented more than that of normal splenocytes, indicating that multiple OK-432 injections also cause an increase in the helper T cell activity of the host spleen cells.     

Unusual T cell populations in adult murine bone marrow. Prevalence of CD3+CD4-CD8- and alpha beta TCR+NK1.1+ cells

The T cell populations present in normal murine bone marrow have not been previously analyzed in detail, mainly because of their relative rarity. In order to permit such analyses, bone marrow T cells were enriched by depleting Mac1-positive cells, which constitute 65 to 90% of bone marrow cells (BMC), and then studied by two-color flow cytometry. Analysis of the remaining cells revealed that the T cell profile of adult murine bone marrow is markedly different from that of other lymphoid organs. A very high proportion of bone marrow CD3+ cells (approximately one-third) are CD4-CD8-. CD3+CD4-CD8- cells are much more concentrated among BMC T cells than among thymocytes or splenic T cells, suggesting that bone marrow may be either a site of extrathymic TCR gene rearrangement, or a major site to which such cells home from the thymus. The expression of NK1.1 was also evaluated on Mac1-depleted BMC populations. Surprisingly, up to 39% of alpha beta TCR+ BMC were found to express NK1.1. Most alpha beta TCR+NK1.1+ BMC also expressed CD4 or CD8. NK1.1+ alpha beta TCR+ cells represented a much greater proportion of BMC T cells than of other lymphoid (splenocyte or thymocyte) T cell populations. Mac1-depleted BMC of nude mice contained very few cells with this phenotype. These results are consistent with the hypothesis that NK1.1+ alpha beta TCR+ cells are generated primarily in the thymus of normal animals and migrate preferentially to bone marrow, where they may function as regulatory elements in hematopoiesis.     

Locoregional immunotherapy of head and neck cancers utilizing allogeneic spleen cells — a report of 2 cases

Frozen-stored human spleen cells (SC) cultured with streptococcus preparation OK-432 acquired direct cytotoxicity to autologous as well as allogeneic tumor cells. The activated cells started to produce cytocidal cytokine TGIF, which is distinct from previously known cytokines.We examined the possibility of allogeneic adoptive immunotherapy (AIT) using these OK-432-stimulated spleen cells (OK-SC) in two cancer patients. Rapid necrosis of cancer tissue and remarkable decreases of tumor markers in tumor effusion were observed. There were no severe side effects.     

Effect of herbal preparation, brahma rasayana, in amelioration of radiation induced damage

Oral administration of brahma rasayana (BR; 10 and 50 mg/dose/animal) for 15 days increased significantly total leukocyte count and percentage of polymorphonuclear cells in irradiated mice. Bone marrow cellularity and alpha-esterase positive cells also increased significantly in radiation-treated animals after BR administration. Number of nodular colonies on the surface of spleen on day seven increased significantly in lethally irradiated recipients receiving bone marrow cells from animals treated with BR. Oral administration of BR also enhanced in serum level of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and granulocyte macrophage-colony stimulating factor(GM-CSF) in normal and irradiated mice. These results indicated that proliferation of stem cells induced by BR in irradiated mice may be related to its stimulation of cytokine production.     

Effects of a low-pathogen environment on natural killer cells of normal and B lymphocyte-deficient mice.

The effect of short-term exposure (1 month) to a low-pathogen environment (LPE) on NK cell levels in the bone marrow and spleen of immunologically normal CBA/CaJ and B-lymphocyte-deficient CBA/N mice was assessed using both functional (51Cr release) and histological methods. The total NK activity (TNKA), i.e. the percentage specific lysis corrected for changes in whole organ cellularity, of the spleens of LPE-exposed CBA/CaJ mice was not significantly different from that of conventionally reared control mice of that strain (112%), while TNKA of the bone marrow of LPE-exposed mice fell to 27% of that of the bone marrow of conventionally reared controls. TNKA of the spleen and bone marrow of LPE-exposed CBA/N mice was reduced (30%) and elevated (120%), respectively, relative to their conventionally reared counterparts. The numbers of asialo-GM-1-positive (ASGM-1+) lymphoid cells, putative NK cells, in CBA/CaJ spleens were more numerous in conventionally reared than in LPE-exposed mice (4.9 X 10(6) vs. 2.7 X 10(6), and were also more numerous in the bone marrow of conventionally reared mice than in LPE-exposed animals (8.0 X 10(4), vs. 3.0 X 10(4) cells). Similarly, in LPE-exposed CBA/N mice, the numbers of ASGM-1+ lymphoid cells in the spleens and bone marrow were lower (2.7 X 10(6) and 5.4 X 10(4), respectively) than those of the spleens and bone marrow of their conventionally reared counterparts (3.8 X 10(6) and 10.0 X 10(4), respectively). The results demonstrate that short-term maintenance in an LPE affected the NK cells of both the spleen and bone marrow of immunologically normal and B-lymphocyte-deficient mice in a strain-specific manner and suggest that the external environment may regulate NK cell production and turnover.     

Myelopoietic Efficacy of Orlistat in Murine Hosts Bearing T Cell Lymphoma: Implication in Macrophage Differentiation and Activation

Orlistat, an inhibitor of fatty acid synthase (FASN), acts as an antitumor agent by blocking de novo fatty acid synthesis of tumor cells. Although, myelopoiesis also depends on de novo fatty acid synthesis, the effect of orlistat on differentiation of macrophages, which play a central role in host’s antitumor defence, remains unexplored in a tumor-bearing host. Therefore, the present investigation was undertaken to examine the effect of orlistat administration on macrophage differentiation in a T cell lymphoma bearing host. Administration of orlistat (240 mg/kg/day/mice) to tumor-bearing mice resulted in a decline of tumor load accompanied by an augmentation of bone marrow cellularity and survival of bone marrow cells (BMC). The expression of apoptosis regulatory caspase-3, Bax and Bcl2 was modulated in the BMC of orlistat-administered tumor-bearing mice. Orlistat administration also resulted in an increase in serum level of IFN-γ along with decreased TGF-β and IL-10. BMC of orlistat-administered tumor-bearing mice showed augmented differentiation into macrophages accompanied by enhanced expression of macrophage colony stimulating factor (M-CSF) and its receptor (M-CSFR). The macrophages differentiated from BMC of orlistat-administered mice showed characteristic features of M₁ macrophage phenotype confirmed by expression of CD11c, TLR-2, generation of reactive oxygen species, phagocytosis, tumor cell cytotoxicity, production of IL-1,TNF-α and nitric oxide. These novel findings indicate that orlistat could be useful to support myelopoesis in a tumor-bearing host.     

Mechanisms of lymphocytic choriomeningitis virus-induced hemopoietic dysfunction.

Results of this study showed that lymphocytic choriomeningitis virus infection causes a marked activation of natural killer (NK) cells not only in the spleen but also in the bone marrow. This activity reached its peak at about day 3 of infection and declined after days 6 to 7. Enhanced NK cell activity was found to correlate with decreased receptivity for syngeneic stem cells in bone marrow and spleen, with the notable exception that decreased receptivity persisted longer in bone marrow. Treatment of infected recipients with anti-asialo GM1 (ganglio-N-tetraosylceramide) significantly increased the receptivity for syngeneic hemopoietic cells. These findings are consistent with the hypothesis that NK cell activation causes rejection of syngeneic stem cells, thus resulting in hemopoietic depression. To understand the mechanisms behind the prolonged decrease in bone marrow receptivity (and bone marrow function in the intact mouse) mentioned above, we followed the changes in the number of pluripotential stem cells (CFU-S) circulating in the peripheral blood and in endogenous spleen colonies in irradiated mice, the limbs of which were partially shielded. It was found that following a marked early decline, both parameters increased to normal or supranormal levels at about day 9 after infection. Because the bone marrow pool of CFU-S is only about 20% of normal at this time after infection, a marked tendency for CFU-S at this stage in the infection to migrate from the bone marrow to the spleen is suggested. It seems, therefore, that as NK cell activity declines, the spleen regains the ability to support growth of hemopoietic cells and the bone marrow resumes an elevated export of stem cells to the spleen. This diversion of hemopoiesis could explain both the long-standing deficiencies of the bone marrow compartment and the prolonged decrease in the receptivity of this organ.