Immunotherapy of EL4 lymphoma with reovirus

Summary EL4 lymphoma was grown as an ascitic tumor in the peritoneal cavity of C57Bl/6 mice. Animals with different tumor burdens (either 10⁷ or 10⁹ cells) were treated with a single intraperitoneal injection of BCNU using doses from 20–40 mg/kg. Response as measured by mean survival time and percent survival was dependent on tumor burden and dose of drug. The objective of chemotherapy was to increase the mean survival time, but not the percent survival, in order to evaluate the therapeutic effect of reovirus. Mice were given 10⁸, 10⁹, or 10¹⁰ Pfu of reovirus at various times with respect to chemotherapy. The number of mice cured after treatment with both BCNU and reovirus was significantly greater compared to mice treated with BCNU only. Mice cured with combination therapy developed tumor-specific immunity as measured by cytotoxic lymphocytes and serum, and resistance to a lethal tumor challenge. The Abbreviations used are: BCNU: 1,3-bis-(2-chloroethyl)-1-nitrosourea; Saline: 0.9% NaCl solution; MEM: minimal essential medium; Pfu: plaque-forming units; FCS: fetal calf serum; BME: basal eagle's medium; SSC: sodium citrate-sodium chloride     

Optimization of reovirus production from mouse L-929 cells in suspension culture

Reovirus serotype 3 Dearing (T3D) has shown potential as a novel cancer therapy. To support the increasing demand for reovirus, a two-stage perfusion mode scheme is proposed for cell growth and reovirus production. Mouse L-929 cells were used as the host for reovirus infection due to their ability to grow well in suspension culture. Several L-929 cell growth and reovirus infection characteristics were investigated and optimized in spinner flask batch cultures. For the growth of L-929 cells, a balanced nutrient-fortification of SMEM medium increased the maximum cell density by 30%, compared to normal SMEM; however, ammonia and lactate accumulations were found to inhibit further cell growth. For the production of reovirus, approximately 90% increase in viral yield resulted when the infection temperature was reduced from 37 to 33 degrees C. Infectious reovirus particles were shown to be stable in conditioned medium at 37 and 33 degrees C. The final virus titer was dependent on the multiplicity of infection (MOI) and the host cell density at the time of infection. A combination of an MOI of 0.1 pfu/cell and an initial host cell density of 1.0 x 10(6) cells/mL in fortified medium resulted in a maximum virus titer of (4.59 +/- 0.16) x 10(9) pfu/mL and a specific yield of (2.34 +/- 0.08) x 10(3) pfu/cell. At an optimal harvest time of the infection process, 99% of the virus was associated with the cellular debris. Finally, the presence of 5.0 mM ammonia in the culture medium was shown to seriously inhibit the reovirus yield, whereas lactate concentrations up to 20 mM had no effect.     

Evaluation of time and dose in treating mammary adenocarcinoma with immunostimulants

Summary BCG, C. parvum, and reovirus were used as immunostimulants in treating murine mammary adenocarcinoma (A-10) after tumor burden had been minimized with BCNU. Immunostimulants were administered at different times with respect to chemotherapy. Different doses were used to determine the optimal response as measured by survival. BCG induced the best response when 6.67×10⁵ organisms were given 2 days after chemotherapy. The optimal response with C. parvum was observed after a dose of 0.35 mg was given 1 or 2 days after chemotherapy. Similarly, reovirus produced the best response when 10¹⁰ plaque-forming units were given 2 days after chemotherapy. These data are consistent with previous findings and support the notion that immunostimulants require an appropriate lymphoid substrate in order to induce an adequate anti-tumor response.Tge abbreviations used are: BCNU: 1,3-bis-(2-chloroethyl)-1-nitrosourea Saline: 0.9% NaCl solution; BCG: Bacillus Calmette-Guerin; C. parvum: Corynebacterium parvum; pfu: plaque-forming unitsThis study was supported, in part, by Contract No. N01-CB-43864 and Grant No. CA 14460 from The National Cancer Institute     

Monocyte chemoattractant protein-1 (MCP-1) gene transduction: an effective tumor vaccine strategy for non-intracranial tumors

Recently, there has been renewed interest in the concept of tumor vaccines using genetically engineered tumor cells expressing a variety of cytokines to increase their immunogenicity. Human MCP-1 (JE) is a potent chemoattractant and activator of monocytes and T lymphocytes and thus a good candidate gene for a tumor vaccine. We therefore evaluated the efficacy of vaccines consisting of irradiated tumor cells transduced with the murine MCP-1 gene in the syngeneic 9L gliosarcoma brain tumor model. 9L cell lines stably expressing murine MCP-1 (9L-JE) and control cell lines expressing neomycin 3 phosphotransferase (9L-Neo) were generated by infection with a Moloney murine leukemia retroviral vector. Fisher 344 rats were immunized with intradermal injections of 5×10⁵ or 2×10⁶ irradiated (5000 cGy) 9L-JE, 9L-Neo, and wild-type 9L (9L-WT) cells. Two weeks later immunized an non-immunized animals were challenged with varyious doses of intradermal (5×10⁶–5×10⁷) or intracerebral (2×10⁴–5×10⁵) 9L-WT cells. Intradermal tumors grew in all non-immunized animals. No tumors grew in animals immunized with irradiated 9L-JE or 9L-Neo cells and challenged with inocula of fewer than 5×10⁵ 9L-WT cells. With higher inocula up to 10⁷ cells, tumors appeared in all the animals. Tumors in animals immunized with 9L-JE were always smaller than tumors in the other groups. In addition, only the 9L-JE vaccine protected against tumor inocula of 5×10⁷ cells. Thus vaccination with MCP-1-expressing cells was able to protect animals against at least a 100-fold larger number of challenge tumor cells than vaccination with control cells. In contrast to studies with intradermal tumors, immunization with 9L-JE and 9L-Neo produced only minimal protection against intracerebral tumors. There was no significant difference between the 9L-JE and 9L-Neo vaccines in intracerebral challenge. This study suggests that tumor vaccines expressing cytokine genes such as MCP-1 can increase the antitumor response. However, the protective effect of these vaccines appears to be largely limited to intradermal tumors rather than intracerebral tumors.     

Activation of p53 by Chemotherapeutic Agents Enhances Reovirus Oncolysis

Mammalian reovirus is a benign virus that possesses the natural ability to preferentially infect and kill cancer cells (reovirus oncolysis). Reovirus exploits aberrant Ras signalling in many human cancers to promote its own replication and spread. In vitro and in vivo studies using reovirus either singly or in combination with anti-cancer drugs have shown very encouraging results. Presently, a number of reovirus combination therapies are undergoing clinical trials for a variety of cancers. Previously we showed that accumulation of the tumor suppressor protein p53 by Nutlin-3a (a specific p53 stabilizer) enhanced reovirus-induced apoptosis, and resulted in significantly higher levels of reovirus dissemination. In this study, we examined the role of p53 in combination therapies involving reovirus and chemotherapeutic drugs. We showed that sub-lethal concentrations of traditional chemotherapy drugs actinomycin D or etoposide, but not doxorubicin, enhanced reovirus-induced apoptosis in a p53-dependent manner. Furthermore, NF-κB activation and expression of p53-target genes (p21 and bax) were important for the p53-dependent enhancement of cell death. Our results show that p53 status affects the efficacy of combination therapy involving reovirus. Choosing the right combination partner for reovirus and a low dosage of the drug may help to both enhance reovirus-induced cancer elimination and reduce drug toxicity.     

Low Dose Influenza Virus Challenge in the Ferret Leads to Increased Virus Shedding and Greater Sensitivity to Oseltamivir

Ferrets are widely used to study human influenza virus infection. Their airway physiology and cell receptor distribution makes them ideal for the analysis of pathogenesis and virus transmission, and for testing the efficacy of anti-influenza interventions and vaccines. The 2009 pandemic influenza virus (H1N1pdm09) induces mild to moderate respiratory disease in infected ferrets, following inoculation with 10⁶ plaque-forming units (pfu) of virus. We have demonstrated that reducing the challenge dose to 10² pfu delays the onset of clinical signs by 1 day, and results in a modest reduction in clinical signs, and a less rapid nasal cavity innate immune response. There was also a delay in virus production in the upper respiratory tract, this was up to 9-fold greater and virus shedding was prolonged. Progression of infection to the lower respiratory tract was not noticeably delayed by the reduction in virus challenge. A dose of 10⁴ pfu gave an infection that was intermediate between those of the 10⁶ pfu and 10² pfu doses. To address the hypothesis that using a more authentic low challenge dose would facilitate a more sensitive model for antiviral efficacy, we used the well-known neuraminidase inhibitor, oseltamivir. Oseltamivir-treated and untreated ferrets were challenged with high (10⁶ pfu) and low (10² pfu) doses of influenza H1N1pdm09 virus. The low dose treated ferrets showed significant delays in innate immune response and virus shedding, delayed onset of pathological changes in the nasal cavity, and reduced pathological changes and viral RNA load in the lung, relative to untreated ferrets. Importantly, these observations were not seen in treated animals when the high dose challenge was used. In summary, low dose challenge gives a disease that more closely parallels the disease parameters of human influenza infection, and provides an improved pre-clinical model for the assessment of influenza therapeutics, and potentially, influenza vaccines.     

Detection of subnanogram amounts of RNA in polyacrylamide gels in the presence and absence of protein by staining with silver

The new ultrasensitive photochemically derived silver stain described for polypeptides in polyacrylamide gels (Merril et al., Science211, 1437–1438 (1981)) also stains nucleic acid in polyacrylamide gels. Reovirus genome double-stranded (ds) RNA segments were clearly detected in gels at about 0.03 ng/mm² with the silver staining technique when either purified virions or isolated, purified dsRNA was analyzed. The silver stain was about 10 to 30 times more sensitive than ethidium bromide for detecting reovirus dsRNA.     

Interferon-inducible Transmembrane Protein 3 (IFITM3) Restricts Reovirus Cell Entry

Reoviruses are double-stranded RNA viruses that infect the mammalian respiratory and gastrointestinal tract. Reovirus infection elicits production of type I interferons (IFNs), which trigger antiviral pathways through the induction of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified, the functions of many of these genes are unknown. The interferon-inducible transmembrane (IFITM) proteins are one class of ISGs that restrict the cell entry of some enveloped viruses, including influenza A virus. One family member, IFITM3, localizes to late endosomes, where reoviruses undergo proteolytic disassembly; therefore, we sought to determine whether IFITM3 also restricts reovirus entry. IFITM3-expressing cell lines were less susceptible to infection by reovirus, as they exhibited significantly lower percentages of infected cells in comparison to control cells. Reovirus replication was also significantly reduced in IFITM3-expressing cells. Additionally, cells expressing an shRNA targeting IFITM3 exhibited a smaller decrease in infection after IFN treatment than the control cells, indicating that endogenous IFITM3 restricts reovirus infection. However, IFITM3 did not restrict entry of reovirus infectious subvirion particles (ISVPs), which do not require endosomal proteolysis, indicating that restriction occurs in the endocytic pathway. Proteolysis of outer capsid protein μ1 was delayed in IFITM3-expressing cells in comparison to control cells, suggesting that IFITM3 modulates the function of late endosomal compartments either by reducing the activity of endosomal proteases or delaying the proteolytic processing of virions. These data provide the first evidence that IFITM3 restricts infection by a nonenveloped virus and suggest that IFITM3 targets an increasing number of viruses through a shared requirement for endosomes during cell entry.     

Increased efficiency of immunotherapy using irradiated tumor cells

Summary The efficacy of irradiated tumor cells combined with chemotherapy or non-specific immunostimulation with complete Freund's adjuvant was tested in a model of minimal residual tumor-bearing syngeneic mice. Male C57BL/6J mice were innoculated in the right rear leg with live tumor cells from a methylcholanthrene induced fibrosarcoma. The tumor was resected when it reached 0.7 cm in diameter and animals were treated with doses of irradiated tumor cells (XTC) from the primary tumor ranging in number from 1×10³ to 9×10³. Best survival was noted using 5×10³ XTC combined with irradiated tumor cells of liver or pulmonary metastases origin, complete Freund's adjuvant or cytoxan. The combination of irradiated tumor cells of metastatic origin did not enhance the therapeutic effect of XTC alone. Freund's adjuvant was not of benefit in enhancing the efficacy of XTC. However, improved survival was noted when chemotherapy in the form of cytoxan was used to supplement XTC. Our data suggests that XTC is more efficacious as a mode of immunotherapy than are live tumor cells. The dose of XTC used is critical in determining its effect. Chemotherapy appears to enhance the benefit of XTC.Research fellow from Zhejiang Medical University, Hongzhou, ChinaSupported by the Brigham Surgical Group, Inc.     

Sensitization to apoptosis underlies KrasD12-dependent oncolysis of murine C26 colorectal carcinoma cells by reovirus T3D

Reovirus T3D is an oncolytic agent that preferentially targets tumor cells expressing an activated Ras oncogene. Ras signaling interferes with the cellular stress response that inhibits translation of reovirus RNAs. Murine C26 colorectal carcinoma cells express a mutant KrasD12 gene. Reovirus T3D efficiently kills C26 cells, but not C26 cells in which the KrasD12 mRNA is stably repressed by expression of KrasD12-directed short-hairpin RNAs. Surprisingly, neither reovirus T3D protein synthesis nor T3D virus yields were suppressed by deletion of KrasD12. Rather, reovirus-induced tumor cell apoptosis was completely abrogated as a result of Kras knockdown. We conclude that sensitization of C26 tumor cells to reovirus-induced apoptosis underlies the Ras dependency of reovirus T3D oncolysis.     

Production of reovirus type-1 and type-3 from Vero cells grown on solid and macroporous microcarriers

Two strains of reovirus were propagated in Vero cells grown in stationary or microcarriers cultures. Vero cells grown as monolayers on T-flasks or in spinner cultures of Cytodex-1 or Cultispher-G microcarriers could be infected with reovirus serotype 1, strain Lang (T1L), and serotype 3, strain Dearing (T3D). A regime of intermittent low speed stirring at reduced culture volume was critical to ensure viral infection of cells in microcarrier cultures. The virus titre increased by 3 to 4 orders of magnitude over a culture period of 150 h. Titres of the T3D reovirus strain were higher (43%) compared to those of the T1L strain in all cultures. Titres were significantly higher in T-flask and Cytodex-1 microcarrier cultures compared to Cultispher-G cultures with respect to either reovirus type. The viral productivity in the microcarrier cultures was dependent upon the multiplicity of infection (MOI) and the cell/bead ratio at the point of infection. A combination of high MOI (5 pfu/cell) and high cell/bead loading (>400 for Cytodex-1 and >1,000 for Cultispher-G) resulted in a low virus productivity per cell. However, at low MOI (0.5 pfu/cell) the virus productivity per cell was significantly higher at high cell/bead loading in cultures of either microcarrier type. The maximum virus titre (8.5 x 10(9) pfu/mL) was obtained in Cytodex-1 cultures with a low MOI (0.5 pfu/cell) and a cell/bead loading of 1,000. The virus productivity per cell in these cultures was 4,000 pfu/cell. The lower viral yield in the Cultispher-G microcarrier cultures is attributed to a decreased accessibility of the entrapped cells to viral infection. The high viral productivity from the Vero cells in Cytodex-1 cultures suggests that this is a suitable system for the development of a vaccine production system for the Reoviridae viruses.     

Isolation of Reovirus T3D Mutants Capable of Infecting Human Tumor Cells Independent of Junction Adhesion Molecule-A

Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D) is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A) on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin) mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA) inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.     

Reovirus and tumor oncolysis

REOviruses (Respiratory Enteric Orphan viruses) are ubiquitous, non-enveloped viruses containing 10 segments of double-stranded RNA (dsRNA) as their genome. They are common isolates of the respiratory and gastrointestinal tract of humans but are not associated with severe disease and are therefore considered relatively benign. An intriguing characteristic of reovirus is its innate oncolytic potential, which is linked to the transformed state of the cell. When immortalized cells are transfected in vitro with activated oncogenes such as Ras, Sos, v-erbB, or c-myc, they became susceptible to reovirus infection and subsequent cellular lysis, indicating that oncogene signaling pathways are exploited by reovirus. This observation has led to the use of the virus in clinical trials as an anti-cancer agent against oncogenic tumors. In addition to the exploitation of oncogene signaling, reovirus may further utilize host immune responses to enhance its antitumor activity in vivo due to its innate interferon induction ability. Reovirus is, however, not entirely benign to immunocompromised animal models. Reovirus causes so-called "black feet syndrome" in immunodeficient mice and can also harm neonatal animals. Because cancer patients often undergo immunosuppression due to heavy chemo/radiation-treatments or advanced tumor progression, this pathogenic response may be a hurdle in virus-based anticancer therapies. However, a genetically attenuated reovirus variant derived from persistent reovirus infection of cells in vitro is able to exert potent anti-tumor activity with significantly reduced viral pathogenesis in immunocompromised animals. Importantly, in this instance the attenuated reovirus maintains its oncolytic potential while significantly reducing viral pathogenesis in vivo.     

Rejection of reovirus-treated L1210 leukemia cells by mice

Summary L1210 leukemia cells were treated in vitro with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and reovirus to determine their interactive effects on rejection of these tumor cells by mice. The cells were treated with BCNU at concentrations of 0, 3, or 10 M, incubated for 48 h, then treated with reovirus at a multiplicity of infection of 0, 10, 30, or 100 for 2, 6, or 12 h. The survival of mice injected with cells treated with any amount of reovirus, regardless of BCNU treatment, was greater than that of mice injected with untreated cells. Exposure of the cells to reovirus for 6 or 12 h increased the survival of mice injected with these cells as compared with that of mice injected with cells exposed to reovirus for 2 h. Of the survivors, 76% were resistant to subsequent challenge with untreated L1210 cells. These results suggest that activities associated with reovirus replication may cause modifications of L1210 cells that enable them to induce an immune response, thus facilitating their rejection. A lack of correlation between differences in DNA synthesis (measured by ³H-thymidine uptake) by treated cells and the ability of those cells to kill recipient mice indicates that rejection of cells treated with reovirus or BCNU is not due to a decrease in their ability to proliferate or, presumably, to generate lethal tumors. The survival of mice injected with treated L1210 cell preparations containing as few as 2.9% reovirus-infected cells was enhanced to the same degree as that of mice injected with those containing as many as 14.6% infected cells, indicating that modification of only a minor component of the tumor cell population is sufficient to alter the ability of the cells to generate a lethal tumor.This work was supported by a research grant from the Miami University Faculty Research Committee and a Sigma Xi Grant-in-Aid of Research     

Interference of anti-tumor and immunosuppressive effects of cyclophosphamide in tumor-bearing rats

Summary Adult rats were given 10⁵ or 10⁶ Yoshida ascites sarcoma (YAS) cells IP and were treated with cyclophosphamide (CY) given IP in single doses of 20 mg/kg or 100 mg/kg, 2 or 5 days after YAS inoculation. Both the curative effect of CY and subsequent resistance to tumor challenge in rats that survived depended on the dose of injected tumor cells and on the dose and time of administration of CY. These three factors determined whether the host's immune response to tumor antigens would develop and contribute to the overall anti-tumor effects of the chemotherapy. The curative effects of CY were significantly less pronounced in T-cell-deficient than in normal rats. Anti-tumor and immunosuppressive activities of CY exerted opposite influences on the ultimate result of the chemotherapy. Adverse immunosuppressive effects prevailed when the drug was administered early (2 days) after YAS inoculation. In this case the chemotherapy was less efficient and the surviving rats were susceptible to a subsequent tumor challenge. Further analysis showed that the injection of CY 2 days after inoculation of YAS antigens induced strong and specific immunologic tolerance to the tumor. In contrast, when a sufficient amount of tumor antigens (higher dose of tumor cells injected and CY injection delayed) elicited an anti-YAS immune response that was not suppressed by early injection of CY (CY administered 5 days after the tumor) effective eradication of tumor cells and anti-YAS resistance in cured animals were observed.Abbreviations YAS Yoshida ascites sarcoma - CY cyclophosphamide - MRBC mouse red blood cells     

Chemoimmunotherapeutic effect of cyclophosphamide on the highly metastatic MAT 13762 tumor

Summary We have observed that cyclophosphamide (CY) treatment of 13762 mammary adenocarcinoma tumor-bearing rats was found to cause tumor regression. Tumor-bearing animals cured with three low doses of CY were partially immune against IV and SC challenge with a high dose of 13762 cells. This immune protection mechanism in CY-cured animals appears to be a T (Ig^–) cell-mediated response. Irradiated rats reconstituted with CY-cured animal spleen cells were also partially protected against IV and SC challenge with 13762 cells, whereas irradiated rats reconstituted with CY-control animal spleen cells were not. In vitro primary and secondary cell-mediated cytotoxic activity of CY-cured spleen cells against target 13762 cells was low. The possible relevance of this tumor-model study is in the understanding of CY-induced tumor immune response and its role in preventing metastases or perhaps recurrent tumor growth.     

Augmentation of immune response to syngeneic fibrosarcoma T241 with in vivo protection

Summary Lewis T241 fibrosarcoma, a syngeneic tumor in C57 BL/6J mice, was found to be poorly immunogenic. When tumor-bearing animals (TBA) were challenged with tumor cells either concomitantly or after excision of a growing tumor no protection was observed. In vivo (Winn) neutralization assays also showed a lack of tumor immunogenicity. However, in vitro studies showed that a significant proliferative response could be elicited from the spleen cells of TBA when these cells were cultured with either mitomycin-C-treated tumor cells or KCl tumor extract. Similarly, macrophage migration inhibition factor (MIF) was produced by TBA spleen cells upon incubation with KCl tumor extract, but no cell-mediated cytotoxicity to T241 target cells was observed with various lymphoid cell populations at any stage of tumor growth. Immunization of syngeneic animals with Vibrio cholerae neuraminidase(VCN)-treated, irradiated tumor cells alone or admixed with Freund's complete adjuvant (FCA) resulted in decreased tumor growth and fewer pulmonary metastases following challenge with 10⁶ tumor cells. No complete tumor rejection was observed. In contrast, 13 of 16 animals immunized with irradiated tumor cells admixed with FCA rejected 10⁵ tumor cells. Animals that grew tumors had significantly reduced tumor growths and pulmonary metastases. Lymph node and peritoneal exudate cells (PEC) of immunized animals showed significant cytotoxicity to T241 cells.     

Selective induction and inhibition of direct and lectin-dependent cell-mediated cytotoxic reactivity

The immune reactivity of mice (C57BL/6, H-2^b) which had been challenged with various numbers (10²–10⁸) of allogeneic tumor cells (P815, H-2^d) was assessed at various times after challenge. Challenge with a high dose (10⁸) of tumor cells resulted in the development of direct cytotoxicity (DCMC), lectin-dependent cytotoxicity (LDCC), delayed-type hypersensitivity (DTH), and antibody production, whereas challenge with lower doses (< 10⁶) of tumor cells favored development of DTH and LDCC with marginal or no DCMC or antibody production. Spleen cells from low-dose alloimmune animals failed to produce DCMC when cultured with P815 cells in vitro and were capable of nonspecifically suppressing the DCMC response of normal spleen cells in MLC. Treatment with cyclophosphamide (100 mg/kg) prior to alloimmunization did not alter the pattern of DTH and cytotoxic reactivity, although treatment after alloimmunization was immunosuppressive for all forms of reactivity. When low-dose challenge was followed by cyclophosphamide treatment and a subsequent high-dose challenge, selective inhibition of DTH, LDCC, and suppressor activity, but not DCMC, was observed. The data suggest that (a) the initial challenge dose plays a significant role in determining which effector and regulatory populations will be activated and what direction the expression of immune reactivity will take; (b) the activated responding populations of DTH, DCMC, and LDCC effector cells are sensitive to cyclophosphamide treatment, whereas the precursors of each are resistant to the effects of the drug; (c) low-dose alloimmunization may be used in combination with cyclophosphamide treatment to modulate DTH, DCMC, and LDCC reactivity in a selective manner; (d) the cytotoxic effector cells responding to highdose challenge and mediating DCMC and those responding to low-dose challenge and mediating LDCC appear to arise from distinct precursor populations.