Sublethal Dose Of Cyclophosphamide Research Articles

Immune function in cyclophosphamide-treated mice is restored by the T-cell-tropic isoxazole derivative R-13

Reconstitution of the immune function in chemotherapy patients will lead to decreases in post-operative complications. A preliminary investigation showed that an isoxazole derivative R-13 (3,5-dimethyl-isoxazole[5,4-e]8H-triazepin-4-one) hydrochloride, given in a single oral dose to normal mice, induced significant increases in the content of CD4+ cells in the spleens and lymph nodes. That observation prompted the authors to assess the immune reconstituting effects of R-13 in mice pre-treated with cyclophosphamide (CP). Mice were given intraperitoneally (IP) a sublethal dose of CP (200 mg/kg) and then R-13 (as 20 µg IP doses, every 3 days post-CP treatment). Control mice, not treated with CP, received R-13 or the vehicle (DMSO in appropriate dilution). Blood leukocyte and splenocyte numbers, blood cell type levels, splenocyte spontaneous and ConA-induced proliferation, and delayed-type hypersensitivity (DTH) to ovalbumin (OVA) were investigated on day 15 post-CP treatment and five R-13 doses. The humoral immune response (antibody-forming cell development to sheep erythrocytes) was measured 30 days post-CP treatment and 10 R-13 doses. In CP-treated mice, five dosings with R-13 led to increases in numbers of splenocytes and blood leukocytes, as well as in spontaneous and ConA-induced splenocyte proliferation, relative to levels in mice that received only CP 15 days earlier. Blood analysis revealed decreases in neutrophil and eosinophil contents and an increased appearance of lymphocyte immature forms in all mice that received the R-13. Both cell-mediated responses to OVA and humoral immune response to sheep erythrocytes in CP-treated hosts were restored. Based on the data here, it is concluded that R-13 may be of potential value for reconstitution of the immune function of chemotherapy patients.

Abstract 3963: Chemo-resistantregulatory T cells suppress the development of antitumor immunity aftercytotoxic regimens.

Abstract Previous studies demonstrated that naïve T cells transferred into lymphopenic hosts develop into memory like T cells and acquire some effector functions. We and others have shown that sublethal whole body irradiation and transfer of naïve T cells augment antitumor immunity and inhibited tumor progression. Further, we found a significant increase of CD4+CD25+Foxp3+ regulatory T cells (Treg) in irradiated mice. Depletion of those radio-resistant Treg after irradiation and transfer of naïve T cells enhanced generation of antitumor effector T cells and suppressed tumor progression (Baba J, et al. 2012, Blood). The combination of lymphodepletion and transfer of naïve T cells seems to be a promising strategy. However, whole body irradiation has not been routinely used in clinical settings. Previously, we demonstrated that the sublethal doses of cyclophosphamide (CPA) efficiently depleted lymphocytes in mice, and enhanced antitumor effects of transferred naïve T cells. In this study, we examined the effect of lymphodepleting doses of CPA on immune suppressor cells. Similar to the irradiated lymphopenic hosts, a significant increase of Treg was observed in mice treated with CPA. The combination of CPA, transfer of naïve T cells and Treg depletion with anti-CD25 monoclonal antibodies succeeded to cure advanced skin tumors.To evaluate the proliferation and apoptosis of Treg increasing in CPA-treated mice, we performed BrdU incorporation, Ki-67 expression and Annexin V apoptosis assay. The results indicated that the increased percentage of Treg during recovery from lymphopenia is due to the rapid proliferation of Treg that survive CPA treatment. Next, we investigated which subsets of donor T cells are necessary in this combination therapy. CD4+ or CD8+ T cells were depleted from donor T cells before transfer into lymphopenic skin tumor bearing mice. Depletion of CD4+ T cells, but not CD8+ T cells eliminated the antitumor effects. Intracellular cytokine FACS revealed that transfer of CD4+ T cells following CPA treatment induced tumor specific effector CD4+ T cells in lymphopenic hosts. Interestingly, transfer of CD4+ T cells also induced CD8+ effector T cells from CPA treated recipient cells. Depletion of CD8+ recipient cells with anti-CD8 monoclonal antibodies abrogated the antitumor effects of lymphodepletion and CD4+ T cells transfer. Furthermore, Transfer of CD4+ T cells into Rag-2 knockout mice showed no antitumor effects. These results suggested that donor CD4+ T cells induce effector CD8+ T cells originate from recipients after CPA treatment and suppress tumor progression. Our results showed that the combination of CPA treatment, naïve CD4+ T cell transfer, and Treg depletion had potent antitumor efficacy. Both of donor CD4+ T cells and recipient CD8+ T cells were necessary for this augmentation of antitumor immunity. Citation Format: Yu Saida, Satoshi Watanabe, Tomohiro Tanaka, Junko Baba, Koh Satoh, Satoshi Shoji, Natsue Igarashi, Koichirou Nozaki, Masaaki Okajima, Satoru Miura, Junta Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Ichiei Narita. Chemo-resistantregulatory T cells suppress the development of antitumor immunity aftercytotoxic regimens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3963. doi:10.1158/1538-7445.AM2013-3963

Abstract 1550: Successful treatment of advanced tumors with chemo-immunotherapy: a combination of cyclophosphamide and inhibition of chemo-resistant immune suppressor cells

Abstract It is well known that naïve T cells transferred into lymphopenic hosts develop into memory like T cells and acquire some effector functions. We and others have been shown that transfer of naïve T cells following sublethal whole body irradiation augmented antitumor immunity and inhibited tumor progression. Further analyses showed that antitumor effector T cells were primed from transferred naïve T cells. Although the exact mechanism underlying this enhancement of antitumor immune responses remains unclear, possible explanations have been proposed; depletion of suppressor cells, improvement of tumor-antigen presentation, and elimination of lymphocytes competing activation cytokines. Combination of lymphodepletion by cytotoxic regimens, such as chemotherapy or radiotherapy, and transfer of naïve T cells seems to be a promising strategy. Although whole body irradiation induces lymphopenia to tumor-bearing animals and enhances antitumor immunity, it has not been routinely used in clinical settings. To test whether cytotoxic agents deplete lymphocytes and enhance antitumor immune responses, we infused cyclophosphamide (CPA), fludarabine, cisplatin and etoposide at the sublethal doses to mice. CPA treatment depleted 95% of lymphocytes in mice, and enhanced antitumor effects of transferred naïve T cells. Other cytotoxic drugs failed to augment antitumor immunity in combination with transfer of T cells. Previously, we have demonstrated that CD4+CD25+Foxp3+ regulatory T cells (Treg) and CD11b+Gr-1+ myeloid-derived suppressor cells (MDSC) inhibited priming of antitumor effector T cells. Depletion of these suppressor cells increased the number of tumor-specific T cells and augmented antitumor immune responses. To examine the effect of sublethal doses of cyclophosphamide on immune suppressor cells, we injected cyclophosphamide into mice and harvested lymph-nodes and spleens for FACS analyses. Unexpectedly, we found a significant increase in the frequency of Treg and MDSC after CPA treatment. Magnetically isolated Treg and MDSC from cyclophosphamide treated mice suppressed tumor-specific responses of effector T cells in vitro. Depletion of Treg with anti-CD25 monoclonal antibodies following CPA administration and transfer of naïve T cells increased the number of antitumor effector T cells. Further, the combination of Treg depletion, CPA treatment and transfer of naïve T cells succeeded to cure 20-day established skin tumors in mice. We have also been testing whether inhibition of MDSC after CPA treatment augments antitumor immunity. Our results showed that CPA treatment efficiently depletes lymphocytes and enhanced antitumor immune responses despite an increase of immune suppressor cells. Further inhibition of suppressor cells after CPA administration successfully treated advanced tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1550. doi:1538-7445.AM2012-1550

Mobilization with Cyclophosphamide Shifts Hematopoiesis From the Bone Marrow to the Spleen In Mouse.

AbstractAbstract 1590 Introduction:Spleen is an active hematopoietic tissue in adult mice. Spleen niches supporting hematopoiesis were not extensively studied yet. Spleen does not contain osteoblastic niches which are considered to be essential for long-term hematopoiesis, and splenic hematopoiesis is assumed to originate mainly from progenitors with short-term repopulating ability. Normal spleen is primarily a lymphoid organ but it also contributes to myelopoiesis by containing approximately 5% of total body multipotent progenitors CFU-S. We have studied the effect of mobilization with cyclophosphamide on trafficking of hematopoietic stem/progenitor cells (HSPC) from the bone marrow to the spleen, and dependence of normal splenic hematopoiesis on continual supply of HSPC from the bone marrow. Method:Murine congenic model C57Bl/6 Ly5.1/Ly5.2, and C57Bl/6 GFP, was used in the experiments. Kinetics of HSPC in the bone marrow and the spleen after a sublethal dose of cyclophosphamide (135 mg/kg) was measured by clonal assays (CFU-GM and CFU-S – progenitors) and by competitive repopulation assay (short- and long-term repopulating cells, STRC and LTRC). Parabiotic pairs were established from control and cyclophosphamide-treated partners in order to demonstrate HSPC exchange between hematopoietic organs. Selective irradiation of either spleen with bone marrow shielded, or irradiation of bone marrow with spleen shielded, was used to measure significance of HSPC migration from the spleen to the bone marrow during hematopoietic regeneration. Selected cytokine expression in the bone marrow and in the spleen was determined by real-time PCR. Result:Bone marrow HSPC rapidly but transiently regenerated from cyclophosphamide damage. Secondary decrease in HSPC between 5 and 7 days after cyclophosphamide in the bone marrow was accompanied with significant reduction of SDF-1 mRNA expression, mobilization of HSPC into peripheral blood and their rapid accumulation in the spleen. Spleen thus transiently became the main hematopoietic organ in the mouse by containing 69% of total body CFU-S, and also contained significantly more LTRC compared to the femur (Fig). [Display omitted] Surprisingly, there was no increase in SDF-1 mRNA expression during homing of HSPC in the spleen. Spleens of parabiotic partners were rapidly colonized by partner cells including HSPC within two weeks. Bone marrow also contained small but significant amount of partner HSPC one and 2 weeks after parabiosis establishment. In pairs made from control and cyclophosphamide treated mice, mobilized HSPC preferentially engrafted in both own and partner spleens. Spleen cellularity and progenitor content was not affected by selective spleen irradiation and was restored from shielded bone marrow 6 days after irradiation. Expansion of extramedullary splenic hematopoiesis after cyclophosphamide occurred even when HSPC were destroyed in the spleen 1 day after cyclophosphamide, i.e. the spleen expansion was dependent on the bone marrow. Irradiation of bone marrow with spleen shielded led to abruption of splenic hematopoiesis in both control and cyclophosphamide treated mice. Conclusion:There is a quantitatively significant trafficking of HSPC by means of the circulation in mice. The splenic hematopoiesis appears to be almost fully dependent on a continuous supply of HSPC from the bone marrow. This trafficking of HSPC can be enhanced by hematopoietic cell mobilization. In mice, damage caused by cyclophosphamide results in a transient, however quantitatively remarkable relocation of HSPC, including LTRC, from the bone marrow to the spleen. This accumulation of HSPC in spleen is not accompanied with increased expression of SDF-1 mRNA. HSPC circulating in peripheral blood also significantly colonize the bone marrow and actively participate in its hematopoiesis. Support: Projects LC06044 and MSM 0021620806 of the Ministry of Education, Youth and Sports of the Czech Republic. Disclosures:No relevant conflicts of interest to declare.

Oral glutamine protects against cyclophosphamide-induced cardiotoxicity in experimental rats through increase of cardiac glutathione

Objective This study evaluated the effects of supplemental oral glutamine (GLN) on acute cardiotoxicity of cyclophosphamide (CPA) in experimental rats. The dose-related cardiotoxicity of CPA is associated with a rapid decrease in cardiac glutathione (GSH) and oxidative cardiac injury. GLN is a rate-limiting precursor for GSH synthesis during periods of oxidative and other types of stress when it becomes a conditionally essential amino acid. Methods Forty-four male Fischer 344 rats were randomized into two groups to receive 1 g · kg −1 · d −1 of GLN or glycine by gavage. After 2 d of prefeeding, each of these groups was further randomized into three subgroups to receive intraperitoneally a lethal dose of CPA (450 mg/kg), a sublethal dose of CPA (200 mg/kg), or saline (controls). Twenty-four hours later all six groups of rats were sacrificed and blood GLN was measured. Cardiac tissue was examined for histopathologic alterations: GSH and oxidized GSH concentrations. Results The results showed that dietary GLN decreased cardiac necrosis and maintained normal cardiac GSH levels. Elevated cardiac GSH levels in the GLN group correlated with increased arterial GLN levels. GLN protected against the acute cardiotoxic effects of CPA and significantly improved the short-term survival after lethal and sublethal doses of CPA. Conclusion These data suggest that GLN may protect against CPA-related cardiac injury through maintenance of cardiac GSH metabolism.

Lactoferrin Accelerates Reconstitution of the Humoral and Cellular Immune Response During Chemotherapy-induced Immunosuppression and Bone Marrow Transplant in Mice

Experimental evidence from previous studies supports the conclusion that orally administered lactoferrin (LF) restores the immune response in mice treated with a sublethal dose of cyclophosphamide (CP). The aim of this study was to elucidate potential benefit of LF in mice undergoing chemotherapy with busulfan (BU) and CP, followed by intravenous (i.v.) injection of bone marrow cells. CBA mice were treated orally with busulfan (4 mg/kg) for 4 consecutive days, followed by two daily doses of CP delivered intraperitoneally (i.p.) at a dose of 100 mg/kg and reconstituted next day with i.v. injection of 10(7) syngeneic bone marrow cells. One group of these mice was given LF in drinking water (0.5% solution). After treatment, mice were immunized with ovalbumin (OVA) to subsequently measure delayed type hypersensitivity responsiveness and with sheep red blood cells to determine humoral immunity by evaluation of splenic antibody-forming cells. As expected, both humoral and cellular immune responses of mice that were treated with these chemotherapeutic agents was markedly impaired. Here we report that this impairment was remarkably attenuated by oral administration of LF. Humoral immunity fell to levels that were 66-88% lower than that of untreated animals. Humoral immunity of LF-treated animals was equivalent to that of untreated mice within 1 month. Cellular immune responses were inhibited by chemotherapy treatment to a lesser degree, reaching levels that were approximately 50% lower than those of untreated animals. Again, LF mitigated this decrease, resulting in responses that were only slightly lower than those observed in untreated animals. Furthermore, when mice were given a lethal dose of BU (4 x 25 mg daily doses, i.p.) followed by a bone marrow transplant, LF caused enhanced lympho-, erythro-, and myelopoiesis in the bone marrow and appearance of transforming splenic lymphoblasts, similar to effects caused by administration of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). In summary, our study suggests that LF may be a useful agent to accelerate restoration of immune responsiveness induced by chemotherapy in bone marrow transplant recipients.

Enhanced clearance of Escherichia coli and Staphylococcus aureus in mice treated with cyclophosphamide and lactoferrin

Previous studies on cyclophosphamide (CP)-immunocompromised mice showed accelerated reconstitution of immune system function following oral treatment with lactoferrin (LF). The aim of this investigation was to evaluate the ability of mice, treated with a sublethal dose of CP and given LF, to combat bacterial infections. Mice were injected with a single, intraperitoneal dose of CP (350 mg/kg body weight). One group of CP-treated mice was also given LF in drinking water (0.5% solution) for 14 days. Untreated and LF-treated mice served as controls. On day 15 following CP administration, mice were infected intravenously with 10 8 Escherichia coli or 5×10 7 Staphylococcus aureus. Twenty-four hours later, the number of colony-forming units (CFU) in spleens and livers were determined. Phenotypic analysis of blood leukocytes was determined, as well as the ability of splenic and peritoneal cells to produce IL-6 spontaneously and in the presence of lipopolysaccharide (LPS). Treatment with CP, or with CP and LF, led to profound reduction of E. coli CFU in the liver and the spleen; treatment with LF alone had significant inhibitory effects on organ enumerated CFU. S. aureus CFUs were also significantly reduced in spleens of mice treated with CP or CP/LF and, to a lesser degree, after LF alone. These effects were also significantly reduced in the livers. Analysis of blood cellular phenotype revealed total number of peripheral leukocytes was lower in the CP-treated group (52.6%) but not significantly different from control values in CP/LF and LF-treated groups (90.7% and 104.6%, respectively). Conversely, percentage of blood neutrophils was markedly elevated in CP and CP/LF groups—62% and 42.5% vs. 18.4% in controls. These findings were accompanied by production of IL-6 by splenic and peritoneal cells which was significantly increased in CP- and CP/LF-treated groups. It was concluded that the increased clearance of bacteria in the organs of mice treated with CP and CP/LF may result from a rise in the number of neutrophils infiltrating the organs and contributing to accelerated clearance of bacteria. The study also suggests that the ability of cells from CP- and CP/LF-treated mice to produce significantly more IL-6 may also contribute to increased resistance to infections. Lastly, together with our previous data, this study indicates that LF used to reconstitute the antigen-specific immune response in CP-treated mice does not impair their resistance to infection.

Orally administered lactoferrin restores humoral immune response in immunocompromised mice

Cyclophosphamide (CP) is an anti-tumor drug commonly used in the chemotherapy of human cancer and autoimmune diseases. In our previous studies, we have demonstrated that lactoferrin (LF), given orally to CP-immunosuppressed mice, could reconstitute a T cell mediated immune response by the renewal of the T cell population. The aim of this present study was to evaluate the effects of LF on humoral responses in mice treated with cyclophosphamide. We demonstrate that a single, sublethal dose of cyclophosphamide (400 mg/kg body weight) profoundly inhibited the humoral immune response of CBA mice to sheep red blood cells (SRBC), as measured by the number of antibody forming cells (AFC) in the spleen after 5 weeks following CP treatment. Administration of 0.5% bovine LF in drinking water for 5 weeks partially reconstituted the AFC number (30–40% of the control values, but 7–10× more than in CP-treated controls). Determination of T and B cell levels in the spleens by flow cytometry revealed that the content of CD3 + and CD4 + as well as Ig + splenocytes was elevated in the immunocompromised mice treated with LF. In addition, the number of peritoneal macrophages was partially restored following LF treatment. Evaluation of the proliferative response to concanavalin A (ConA) and pokeweed mitogen (PWM) demonstrated that the diminished reactivity of splenocytes from CP-treated mice was significantly enhanced by LF. In summary, we conclude that the prolonged, oral treatment of immunocompromised mice with LF led to partial reconstitution of the humoral response, associated with elevation of T and B cell and macrophage content and the proliferative response of splenocytes to mitogens.

In vivo synergistic effect of the immunomodulator AS101 and the PKC inducer bryostatin

The immunomodulator AS101 has recently been found to have radioprotective properties when injected prior to sublethal and lethal doses of irradiation. In addition, this compound was found to protect mice from hemopoietic damage caused by sublethal doses of cyclophosphamide (CYP) and to increase the rate of survival of mice treated with lethal doses of CYP. AS101 was previously shown to exert a synergistic effect with the PKC-inducer bryostatin in cytokine secretion in vitro. The present studies were designed to evaluate the effects of in vivo combined treatment with AS101 and bryostatin on bone marrow and spleen cellularity and on the number of committed progenitors in the bone marrow at various points of time after their treatment with a sublethal dose of CYP or irradiation. In addition, the combined effect was tested on the survival of mice irradiated with a lethal dose of irradiation. Our data show the presence of synergism which greatly enhances the number of bone marrow and spleen cells 48 hr and 9 days after CYP treatment or irradiation. The combined effect was also demonstrated when bone marrow colony-forming units granulocyte-macrophage (CFU-GM) progenitor cells were evaluated. Moreover, AS101 and bryostatin synergized in their protective effects against lethal damages of irradiation. These results strongly suggest that bryostatin, which lacks tumor-promoting activity, is a particularly good candidate in combination with AS101 for treatment in vivo in counteracting chemotherapy- or radiation-induced hematopoietic suppression or in generally improving the restoration of immune response under conditions involving immune or hemopoietic damage.

The immunomodulator AS101 administered orally as a chemoprotective and radioprotective agent

AS101 [ammonium-trichloro (0,0′ dioxyethylene)tellurate] is a new immunomodulator shown previously to stimulate the production of various cytokines in vitro and in vivo, and to have minimal toxicity. In the present study we explore the possibility of oral administration of AS101 to mice via cannulation in lieu of interperitoneal or intravenous administration reported to date. Our studies show that oral administration of AS101 at a dose ranging between 50 and 100 μg/mouse promotes hemopoietic regeneration after treatment with sublethal doses of cyclophosphamide (CYP) and protects mice from the lethal effects of this compound. In addition, AS101 administered orally confers a strong radioprotective effect upon mice when given before irradiation.

Experimental Escherichia coli peritonitis in immunosuppressed mice: the role of specific and non-specific immunity.

An experimental Escherichia coli septicaemia-peritonitis model was adapted to immunosuppressed mice. The mice were made neutropenic by a sublethal dose of cyclophosphamide, which resulted in a 100-fold increase in their susceptibility to intraperitoneal injection of E. coli O18:K1. A lethal infection could be prevented by passive immunisation with anti-K1 capsular or anti-O18 LPS antibodies but not with anti-J5 bacterial antibodies. The anti-K1 and anti-O18 antisera were able to increase the LD50 of the E. coli challenge by factors of 50 and 5, respectively. The role of non-specific, lipopolysaccharide (LPS)-mediated resistance to infection was also investigated in this model, in which only long-living phagocytic cells such as macrophages are believed to be functional. Pretreatment of mice with LPS was shown to prevent growth of the bacterial challenge in the peritoneal cavity and blood and to result in a five-fold increase in the LD50 of the challenge strain. These findings suggest an important role for macrophages as effector cells in defence against E. coli infection.

In vivo study of lactoferrin and murine rebound myelopoiesis.

Lactoferrin has been reported to inhibit the production of colony-stimulating factor (CSF); thus we sought to study its possible effects on myelopoiesis in vivo. The characteristics of rebound myelopoiesis in C57BL mice injected with a sublethal dose of cyclophosphamide (CY) were used to test lactoferrin for any granulopoietic activity. An experimental group received daily injections of 50 micrograms of human lactoferrin beginning 24 hr after CY injections. By measuring the total nucleated cellularity of the femoral marrow, the peripheral blood count, and the incorporation of tritiated thymidine by the marrow in six replicate experiments, no statistically significant difference was noted between the lactoferrin injected groups and the control groups. Neither the route of lactoferrin administration (i.v. or i.p.) nor the sex of the animal altered the myelopoietic recovery. Lactoferrin had no stimulatory or inhibitory effect on murine rebound myelopoiesis in vivo contrary to the reported in vitro results.

The origin of cytotoxic T lymphocyte precursors (CTL-P): MHC-restricted and alloreactive CTL-P in the spleen during regeneration after a sublethal dose of cyclophosphamide (Cy).

The regenerating spleen 8 days after an injection of a sublethal dose (300 mg/kg) of cyclophosphamide (Cy) had a defective capacity to give rise to cytotoxic T lymphocytes (CTL) in response against allogeneic cells, whereas the cytotoxicity against 2,4,6-trinitrophenyl-(TNP) modified syngeneic cells was at the normal level. Alloresponse was first obtained 2 wk after the Cy treatment. The limiting dilution analysis showed this at the clonal level: the frequency of anti-TNP-specific CTL precursors (CTL-P) in the spleen treated with Cy 8 days previously was the same as the frequency in the normal spleen. The defective alloresponse was due to a decreased number of allospecific CTL-P that was later increasing. The regenerative capacity was not abolished by adult thymectomy or treatment of the mice with a bone marrow-seeking isotope, 89Sr, suggesting that these CTL-P are derived from Cy-resistant splenic precursors rather than from the thymus or bone marrow. These precursors have probably been under thymic education: the dominance of H-2k-restricted CTL over H-2d-restricted CTL in the response of (H-2k X H-2d)F1 mice to TNP-self is known to be influenced by the H-2 genotype of the thymus, and this dominance was also demonstrated with anti-TNP CTL derived from these F1 mice pretreated with Cy. The CTL-P in the regenerating spleen (day 8) were not hydrocortisone sensitive, and nylon wool-purified T cells from this stage had a lactate dehydrogenase (LDH) isoenzyme pattern of the mature T cell type (rather than of the thymocyte type). Thus, in these aspects the T cells of the regenerating spleen resembled normal splenic cells. These data suggest that the Cy-resistant spleen population contains cells that can give rise to CTL-P that have a defective specificity repertoire at the beginning of the regeneration, but later mature to a normally alloreactive population.

Delayed-type hypersensitivity response in mice to Treponema pallidum.

C3H/HeJ mice which had been primed with either virulent or killed T. pallidum were studied for in vivo delayed-type hypersensitivity (DTH) responses to T. pallidum following local footpad challenge. Mice sustaining a chronic infection of 5 months duration failed to develop a DTH to treponemal antigens, whereas priming by a single intravenous injection with killed organisms resulted in a significant DTH response in mice when challenged 5 days later. Treatment of mice prior to priming with a single sublethal dose of cyclophosphamide (100 mg/kg body weight) not only failed to potentiate T. pallidum-DTH, but abrogated the response observed in untreated primed animals.

During lymphatic regeneration, precursors for major histocompatibility complex-restricted cytotoxic T cells appear before alloreactive precursors.

Mice were injected with a sublethal dose of cyclophosphamide (Cy) (300 mg/kg), and the appearance of the capacity of the regenerating spleen to form cytotoxic T lymphocytes (CTL) in response against 2,4,6 trinitrophenyl-coupled syngeneic cells or against allogeneic cells was followed. It was found that 8 da after Cy injection, the spleen contained cells that could give rise to CTL, but only 2,4,6 trinitrophenyl-specific CTL responses could be obtained at this stage. A low alloresponse was first seen 2 wk after Cy injection.

Cytostatic Effect of Spleen Cells of Cyclophosphamide-Treated Mice on Tumor Cells

Spleen cells of mice receiving an i.p. injection of a sublethal dose of cyclophosphamide (CY) have the ability to inhibit tumor growth in vitro. This effect is dose dependent and is maximal at the peak of the spleen regeneration which follows the phase of atrophy due to CY toxicity. This cytostasis is neither tumor-specific nor strain-restricted and the cells responsible for this inhibition of tumor cell multiplication have the characteristics of macrophages: they lack the Thy 1-2 antigenic marker, appear in CY treated nude mice, stick on plastic vessels, and are retained by adherence columns (nylon wool or Sephadex G10); their activity is greatly reduced when a specific macrohpage toxic reagent such as carrageenan is added to the cultures. The effector cells are similar to those which are able to suppress the response of normal splenocytes to T and B mitogens, and which appear in the same conditions of induction, after CY injection.