Mycobacterium Bovis Strain BCG Research Articles

Dynamics of gamma interferon, interleukin-12 (IL-12), IL-10, and transforming growth factor beta mRNA expression in primary Mycobacterium bovis BCG infection in guinea pigs measured by a real-time fluorogenic reverse transcription-PCR assay.

The guinea pig has been utilized as a model for studying infectious diseases because its reactions closely resemble those of humans biologically and immunologically. However, the cytokine responses in this animal remain to be studied. Initially, we established a quantitative assay using a real-time reverse transcription-PCR (RT-PCR) to measure guinea pig gamma interferon (IFN-gamma), interleukin-12 (IL-12), IL-10, and transforming growth factor beta (TGF-beta) mRNA. By preparing primer-fluorogenic probe sets for these cytokines and standard RNA templates corresponding to the target sequence of each cytokine, we obtained linear standard curves essential for quantitative determination. In guinea pigs immunized by intradermal (i.d.) vaccination with the Tokyo strain of Mycobacterium bovis BCG (0.1 mg) or else hyperimmunized with the same vaccine (10 mg) given intravenously (i.v.), peripheral blood mononuclear cells (PBMCs) at 4 weeks showed an increase in IFN-gamma mRNA expression in the latter but not the former animals. However, at week 10, IFN-gamma mRNA expression was markedly elevated in PBMCs, spleen cells, and cells in bronchoalveolar lavage fluid in both the i.d.- and the i.v.-immunized animals, the level of expression being 10 times higher in the latter. In contrast, the expression levels of IL-12 mRNA in PBMCs, spleen cells, and BAL cells were not enhanced in either group at 10 weeks postimmunization. The expression of IL-10 and TGF-beta increased slightly only in PBMCs. Regardless of differences in the levels of cytokine responses, the magnitudes of the purified protein derivative of tuberculin-specific delayed-type hypersensitivity (DTH) skin reactions for the two groups did not differ significantly at 8 weeks postvaccination. In this study, we quantitatively measured IL-10, IL-12, TGF-beta, and IFN-gamma mRNA in BCG-immunized guinea pigs and showed that the level of IFN-gamma mRNA expression does not necessarily reflect the magnitude of the DTH response, suggesting that there may be an intricate relationship between protective immunity, the level of IFN-gamma, and the DTH response. Thus, our quantitative assay would be of use for the development of vaccines using guinea pig models.

Expression of foreign genes in Mycobacterium bovis BCG strains using different promoters reveals instability of the hsp60 promoter for expression of foreign genes in Mycobacterium bovis BCG strains

Setting: Optimization of BCG as a vehicle for live recombinant vaccines requires improved strategies for stable antigen expression. Objectives: To investigate the effects of various combinations of post-translational signals and promoters on expression and stability in different BCG strains. Design: Plasmids were constructed using mycobacterial promoters ( hsp60, 19-kDa antigen, 85A antigen – from the Mycobacterium tuberculosis complex – and the 18-kDa antigen from Mycobacterium leprae) and post-translation signals (85A antigen secretion and 19-kDa antigen acylation signals), coupled with reporter genes. Results: The 19-kDa acylation signal had little effect on expression, while the 85A secretion signal enhanced markedly the levels of cell-associated product. Inclusion of the hsp60 promoter caused plasmid instability; various deletions affecting the promoter region occurred during or soon after transformation, but not during subsequent growth of the transformants, nor with other promoters. BCG Moreau appeared to be more susceptible to deletions than other BCG strains. Conclusions: The 85A signal may prove useful in optimizing gene expression in BCG, irrespective of secretion of the product. Deletions associated with the hsp60 promoter may be due to a transient lethal induction of the hsp60 promoter associated with electroporation. With intact plasmid there was no marked difference in expression between BCG strains.

Plasmidic versus insertional cloning of heterologous genes in Mycobacterium bovis BCG: impact on in vivo antigen persistence and immune responses.

Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag(p26) genes from the simian immunodeficiency virus (SIV) SIVmac251 were engineered so that both genes were cotranscribed from a synthetic operon. The expression cassette was cloned into a multicopy-replicating vector, and the expression levels of both nef and gag in the bivalent rBCG(nef-gag) strain were found to be comparable to those of monovalent rBCG(nef) or rBCG(gag) strains. However, extrachromosomal cloning of the nef-gag operon into a replicative plasmid resulted in strains of low genetic stability that rapidly lost the plasmid in vivo. Thus, the nef-gag operon was inserted site specifically into the BCG chromosome by means of mycobacteriophage Ms6-derived vectors. The resulting integrative rBCG(nef-gag) strains showed very high genetic stability both in vitro and in vivo. The in vivo expression of the heterologous genes was much longer lived when the expression cassette was inserted into the BCG chromosome. In one of the strains obtained, integrative cloning did not reduce the expression levels of the genes even though a single copy was present. Accordingly, this strain induced cellular immune responses of the same magnitude as that of the replicative rBCG strain containing several copies of the genes.

Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG

A recombinant (r-) Salmonella typhimurium aroA vaccine that secretes the naturally secreted protein of Mycobacterium bovis strain BCG, Ag85B, by means of the HlyB/HlyD/TolC export machinery (termed p30 in the following) was constructed. In contrast to r- S. typhimurium control, oral vaccination of mice with the r- S. typhimurium p30 construct induced partial protection against an intravenous challenge with the intracellular pathogen Mycobacterium tuberculosis, resulting in similar vaccine efficacy comparable to that of the systemically administered attenuated M. bovis BCG strain. The immune response induced by r- S. typhimurium p30 was accompanied by augmented interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) levels produced by restimulated splenocytes. These data suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated r- S. typhimurium as carrier is capable of inducing an immune response against mycobacterial antigens.

Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG.

A recombinant (r-) Salmonella typhimurium aroA vaccine that secretes the naturally secreted protein of Mycobacterium bovis strain BCG, Ag85B, by means of the HlyB/HlyD/TolC export machinery (termed p30 in the following) was constructed. In contrast to r-S. typhimurium control, oral vaccination of mice with the r-S. typhimurium p30 construct induced partial protection against an intravenous challenge with the intracellular pathogen Mycobacterium tuberculosis, resulting in similar vaccine efficacy comparable to that of the systemically administered attenuated M. bovis BCG strain. The immune response induced by r-S. typhimurium p30 was accompanied by augmented interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) levels produced by restimulated splenocytes. These data suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated r-S. typhimurium as carrier is capable of inducing an immune response against mycobacterial antigens.

Cloning, expression, and immunological evaluation of two putative secreted serine protease antigens of Mycobacterium tuberculosis.

Culture filtrate proteins (CFP) of Mycobacterium tuberculosis have been shown to contain immunogenic components that elicit at least partial protective immunity against Mycobacterium infection. To clone genes encoding some of the immunogenic proteins, we made a high-titer rabbit anti-CFP serum and used it to screen an M. tuberculosis genomic expression library in Escherichia coli. In this paper, we describe the molecular cloning of two new protein components of CFP and identified them as members of the serine protease gene family. Their open reading frames contain N-terminal hydrophobic secretory signals consistent with their detection in CFP. The predicted molecular masses of the mature, fully processed forms of both antigens are approximately 32 kDa, in agreement with their observed sizes on immunoblots of CFP probed with polyclonal rabbit antisera made to the recombinant proteins. Thus, these proteins have been designated MTB32A and MTB32B. Interestingly, and despite 66% amino acid sequence homology between the two proteins, polyclonal rabbit antisera made to each of the recombinant proteins were found to be specific for the respective immunizing antigens. The recombinant proteins were also evaluated in in vitro assays with donor peripheral blood mononuclear cells (PBMC) from healthy purified protein derivative (PPD)-positive individuals of diverse ethnic backgrounds. MTB32A but not MTB32B stimulated PBMC from healthy PPD-positive donors but not from PPD-negative donors to proliferate and secrete gamma interferon. MTB32A is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M. tuberculosis complex and the BCG strain of Mycobacterium bovis but absent in the environmental mycobacterial species tested. In addition, nucleotide sequence comparison of mtb32a of the avirulent H37Ra strain and the virulent Erdman strain, as well as with the corresponding sequences (identified in the databases) of strain H37Rv and the clinical isolate CSU93, revealed 100% identity. MTB32A, therefore, represents a candidate for inclusion in subunit vaccine development. Finally, the possible role of MTB32 serine proteases as a virulence factor(s) during Mycobacterium spp. infection is discussed.

A dynamic two-dimensional polyacrylamide gel electrophoresis database: the mycobacterial proteome via Internet.

Proteome analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry, in combination with protein chemical methods, is a powerful approach for the analysis of the protein composition of complex biological samples. Data organization is imperative for efficient handling of the vast amount of information generated. Thus we have constructed a 2-D PAGE database to store and compare protein patterns of cell-associated and culture-supernatant proteins of different mycobacterial strains. In accordance with the guidelines for federated 2-DE databases, we developed a program that generates a dynamic 2-D PAGE database for the World-Wide-Web to organise and publish, via the internet, our results from proteome analysis of different Mycobacterium tuberculosis as well as Mycobacterium bovis BCG strains. The uniform resource locator for the database is http://www.mpiib-berlin.mpg.de/2D-PAGE and can be read with a Java compatible browser. The interactive hypertext markup language documents displayed are generated dynamically in each individual session from a rational data file, a 2-D gel image file and a map file describing the protein spots as polygons. The program consists of common gateway interface scripts written in PERL, minimizing the administrative workload of the database. Furthermore, the database facilitates not only interactive use, but also worldwide active participation of other scientific groups with their own data, requiring only minimal computer hardware and knowledge of information technology.

Live antigen carriers as tools for improved anti-tuberculosis vaccines.

Recombinant (r) Mycobacterium bovis BCG strains have been constructed which secrete biologically active listeriolysin (Hly) fusion protein of Listeria monocytogenes. In human and murine macrophage-like cell lines, intracellular persistence of these r-BCG strains was reduced as compared to the parental BCG strain. By immunogold labelling Hly was detected in membrane structures and within the phagosomal space of macrophages. Hly fusions consistently co-localized with a lysosome-associated membrane glycoprotein (LAMP-1) suggesting that membrane attack conformation of Hly was not altered. Although r-BCG microorganisms apparently did not egress into the cytoplasmic compartment of host cells, they both improved major histocompatibility complex class I presentation of co-phagocytosed soluble ovalbumin as compared with wild-type BCG microbes. These data suggest that Hly secretion endows BCG with an improved capacity to stimulate CD8 T cells. Because CD8 T cells play a major role in protection against tuberculosis such Hly-secreting r-BCG constructs are anti-tuberculosis vaccine candidates. In addition, we report on our r-Salmonella typhimurium expression system combined with the HlyB/HlyD/ TolC export machinery for delivering the prominent mycobacterial antigen Ag85B for immune recognition.

Live antigen carriers as tools for improved anti-tuberculosis vaccines

Recombinant (r) Mycobacterium bovis BCG strains have been constructed which secrete biologically active listeriolysin (Hly) fusion protein of Listeria monocytogenes. In human and murine macrophage-like cell lines, intracellular persistence of these r-BCG strains was reduced as compared to the parental BCG strain. By immunogold labelling Hly was detected in membrane structures and within the phagosomal space of macrophages. Hly fusions consistently co-localized with a lysosome-associated membrane glycoprotein (LAMP-1) suggesting that membrane attack conformation of Hly was not altered. Although r-BCG microorganisms apparently did not egress into the cytoplasmic compartment of host cells, they both improved major histocompatibility complex class I presentation of co-phagocytosed soluble ovalbumin as compared with wild-type BCG microbes. These data suggest that Hly secretion endows BCG with an improved capacity to stimulate CD8 T cells. Because CD8 T cells play a major role in protection against tuberculosis such Hly-secreting r-BCG constructs are anti-tuberculosis vaccine candidates. In addition, we report on our r- Salmonella typhimurium expression system combined with the HlyB/HlyD/TolC export machinery for delivering the prominent mycobacterial antigen Ag85B for immune recognition.

Priming of CD8 + CTL effector cells in mice by immunization with a stress protein–influenza virus nucleoprotein fusion molecule

Literature is accumulating which suggests the potential for stress proteins to form the basis of a novel vaccine technology. Immunization with mammalian tumor-derived stress proteins and their associated peptides promote anti-tumor immunity. Vaccination with HIV-1 p24 antigen fused to mycobacterial heat shock protein (Hsp) Hsp71 enhances p24-specific immunity, as measured by p24-specific antibody production and in vitro cell proliferation and cytokine induction. An ovalbumin-Hsp71 fusion protein primes ovalbumin-specific CTL activity and resistance to challenge with an ovalbumin-expressing tumor. We have extended these observations by using a mycobacterial Hsp65 fusion molecule to prime CTL specific for a viral antigen. Gene fusion constructs were generated from DNA encoding Mycobacterium bovis strain BCG Hsp65 and individual fragments of influenza virus nucleoprotein (NP) encompassing H-2K d-and H-2D b-restricted CTL epitopes. The ability of these purified recombinant fusion proteins to prime NP-specific CTL was assessed in mice of appropriate H-2 haplotypes. We observed that adjuvant-free immunization with either fusion protein elicited significant CTL activity when administered at doses of 10–100 μg per mouse. An NP fusion protein made with glutathione- S-transferase failed to elicit NP-specific CTL, indicating that the phenomenon requires Hsp65 sequences. A single immunization with the Hsp65-NP fusion protein elicited CTL activity which persisted for a minimum of 4 months post-immunization, at which time it could be boosted by a second immunization. To our knowledge, this is the first report of a member of the Hsp60 family priming for antigen-specific CTL activity when employed as a fusion protein partner.

A cocktail of Mycobacterium bovis BCG recombinants expressing the SIV Nef, Env, and Gag antigens induces antibody and cytotoxic responses in mice vaccinated by different mucosal routes.

Recombinant live Mycobacterium bovis BCG strains (rBCG) expressing different human immunodeficiency virus (HIV) or simian immunodeficiency (SIV) antigens could be good candidates for the development of vaccines against AIDS. To develop effective HIV/SIV vaccines, humoral and cellular immune responses directed against multiple antigens may be essential for the control of the infection. In this study we immunized BALB/c mice via different mucosal routes (oral, aerogenic, nasal, and rectal) with a mixture of three rBCG strains expressing, respectively, the entire SIVmac251 Nef protein, and large fragments of the Env and Gag proteins. All routes of immunization studied induced immunoglobulin A (IgA) antibodies against mycobacterial PPD, SIV Env, and SIV Gag antigens in feces and bronchial lavages as well as specific immunoglobulin G (IgG) in serum. Strong, specific cytotoxic responses of splenocytes against Nef, Env, and Gag was observed whatever the mucosal route of immunization. Therefore, mucosal vaccination with a cocktail of rBCG strains induces local, specific IgA, systemic IgG, and systemic CTLs against the three SIV antigens expressed. Rectal and oral routes seemed the most appropriate route of vaccination to be used to protect against SIV infection.

Genetic identification of Mycobacterium bovis BCG by restriction fragment length polymorphism analysis of the direct-repeat region.

Restriction fragment length polymorphism (RFLP) analysis was performed on the direct repeat (DR) regions of 14 strains of Mycobacterium bovis BCG. With AluI-digested DNA, BCG Japanese, Russian, and Mexican had differing RFLP patterns but 11 strains, including Pasteur, Glaxo, and Tice, had an identical pattern not detected in over 60 strains of the M. tuberculosis complex. DR analysis can aid in confirming the identification of clinical BCG isolates.

Rapid screening of natural products for antimycobacterial activity by using luciferase-expressing strains of Mycobacterium bovis BCG and Mycobacterium intracellulare

The object of this study was to investigate the ability of a rapid luciferase assay to detect antimycobacterial activity in plant extracts. Recombinant strains of Mycobacterium bovis BCG (rBCG) and Mycobacterium intracellulare expressing firefly luciferase were used as the test organisms. Assays were conducted in a 96-well minitube format under biosafety level 2 conditions. Control and test wells were sampled immediately after inoculation and after 3 (recombinant M. intracellulare) and 5 (rBCG) days of incubation to measure luminescence with a microplate luminometer, and the relative change in luminescence was calculated as a percentage of control values. As an alternative test method, Alamar blue was added after 12 days of incubation, and changes in color were read visually. A total of 480 extracts were tested. Sixteen extracts were active against rBCG, and of those, seven were also active against recombinant M. intracellulare. With activity defined as a relative change in luminescence of < or = 1% (i.e., > or = 99% inhibition) and a persistence of blue color after addition of Alamar blue, there was 99.0% agreement between the two methods. Our results suggest that the luciferase assay is rapid and accurate and has the potential to greatly accelerate the evaluation of antimycobacterial activity in plant extracts in vitro. With this method, it is possible to screen a large number of samples in a short period of time.

Site-directed mutagenesis of the katG gene of Mycobacterium tuberculosis: effects on catalase-peroxidase activities and isoniazid resistance.

Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug-resistant strains are mutated in the katG gene which encodes a catalase-peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug-resistant phenotype have been identified by evaluating INH-resistant clinical isolates and in vitro mutants. In this study, site-directed mutagenesis techniques were utilized to alter the wild-type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG-defective, INH-resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase-peroxidase which reduce catalase activity also decrease peroxidase activity.

Novel insights into the genetics, biochemistry, and immunocytochemistry of the 30-kilodalton major extracellular protein of Mycobacterium tuberculosis.

The 30/32-kDa complex of major secretory proteins are among the most important and intensively studied proteins of Mycobacterium tuberculosis. The proteins have been demonstrated to be immunoprotective and to play a central role in the physiology of the mycobacterium. In this study, we present a series of novel insights into this key protein complex arising out of a combination of genetic, biochemical, and immunocytochemical analyses. Our genetic analyses (i) indicate that the genes are arranged as separate transcription units, (ii) demonstrate that the mature 30-kDa protein of M. tuberculosis differs from the corresponding 30-kDa proteins of two strains of Mycobacterium bovis BCG by only 1 and 5 amino acids, (iii) suggest that expression of the proteins is regulated at the transcriptional level, and (iv) map the transcriptional start site of the 30-kDa protein gene. Our biochemical analyses provide evidence that (i) the 30-kDa protein and the two 32-kDa proteins (i.e., 32A and 32B) are secreted at a ratio of approximately 3:2:1, respectively, (ii) the proteins exist as monomers, (iii) the proteins are not posttranslationally modified by the addition of carbohydrates and lipids, (iv) the 30-kDa and 32A proteins contain one disulfide bridge, and (v) high-level expression and leader peptide processing are achievable in Escherichia coli. Our immunocytochemical analyses demonstrate that the 30/32-kDa complex is expressed in human monocytes and that the proteins are localized to the phagosomal space and the mycobacterial cell wall. These analyses fill important gaps in our knowledge of this critical protein complex of M. tuberculosis and, at the same time, raise new and fundamental questions regarding regulatory mechanisms that control coordinate expression of the proteins at a fixed ratio.

Reporter gene technology to assess activity of antimycobacterial agents in macrophages.

Reporter strains of Mycobacterium tuberculosis and Mycobacterium bovis BCG endogenously expressing firefly luciferase were used in bioluminescence assays to evaluate the activities of isoniazid and rifampin against mycobacteria sequestered in human macrophages. This methodology allowed the efficacy of antibiotics against intracellular mycobacteria to be assessed without the labor-intensive procedures and protracted incubation requirements associated with conventional CFU determinations.

Reactive nitrogen intermediates production from naive and activated monocytes by extracts of Mycobacterium bovis BCG

Extracts of Mycobacterium bovis strain BCG were assessed for in vitro activation of monocytes to produce reactive nitrogen intermediates. The culture filtrate of M. bovis BCG was a strong inducer of nitrite production while live BCG and sonicated antigens were also potent inducers. Other extracts activated monocytes which showed an increase in nitrite production after in vitro BCG infection.

Inositol Phosphate Capping of the Nonreducing Termini of Lipoarabinomannan from Rapidly Growing Strains of Mycobacterium

Previous studies have demonstrated that the nonreducing termini of the lipoarabinomannan (LAM) from Mycobacterium tuberculosis are extensively capped with mannose residues, whereas those from a fast growing Mycobacterium sp., once thought to be an attenuated strain of M. tuberculosis, are not. The noncapped LAM, termed AraLAM, is known to be more potent than the mannose-capped LAM (ManLAM) in inducing functions associated with macrophage activation. Using a combination of chemical and enzymatic approaches coupled with fast atom bombardment-mass spectrometry analysis, we demonstrated that LAMs from all M. tuberculosis strains examined (Erdman, H37Ra, and H37Rv), as well as the attenuated Mycobacterium bovis BCG strain, are mannose-capped with the extent of capping varying between 40 and 70%. The nonreducing termini of LAM from Mycobacterium leprae were also found to be capped with mannoses but at a significantly lower level. A novel inositol phosphate capping motif was identified on a minor portion of the otherwise uncapped arabinan termini of LAMs from the fast growing Mycobacterium sp. and Mycobacterium smegmatis ATCC 14468 and mc(2)155. In addition, an inositol phosphate tetra-arabinoside was isolated from among endoarabinase digestion products of AraLAM and was shown to induce tumor necrosis factor-alpha production. Accordingly, we concluded that AraLAM is characteristic of some rapidly growing Mycobacterium spp. It is distinct from ManLAMs of M. tuberculosis, M. bovis BCG, and Mycobacterium leprae not only in the absence of mannose-capping but also in containing some terminal inositol phosphate substituents which may account for its particular potency in inducing macrophage activation.

BCG-activated NK cells regulate the antibody response to SRBC and restore immune reactivity to PPD in BCG-infected mice.

C57B1/6 mice show a significant increase in the number of natural killer (NK) cells in the peritoneal cavity, four days after intraperitoneal infection with Mycobacterium bovis strain BCG. Cell transfer experiments demonstrated that BCG-induced NK cells are able to depress the induction of antibody response to an unrelated antigen (i.e., sheep red blood cells) in recipient mice. The involvement of macrophages, B and T cells in the phenomenon was ruled out by using different purification steps. In addition, BCG-induced NK cells were shown to be able of partially restoring the DTH response to PPD in recipient mice that were anergic to the latter antigen as a consequence of intravenous infection with large doses of BCG.

Changes of host cell infiltration into Meth A fibrosarcoma tumor during the course of regression induced by injections of a BCG nucleic acid fraction.

MY-1, which consists of DNA and RNA extracted and purified from Mycobacterium bovis strain BCG, causes the regression of various experimental syngeneic tumors when injected intratumorally. In order to identify the host cells involved in the antitumor mechanism(s) of MY-1, we examined Meth A tumors inoculated intradermally to BALB/c mice, which were given multiple injections of MY-1 following tumor inoculation. Histological and immunohistochemical examinations were performed at several time points. On day 4 after inoculation, the MY-1-treated tumors were heavily infiltrated with a heterogeneous population of mononuclear cells with low density nuclei. The MY-1-injected tumors contained asialo-GM1-positive cells and Mac-1-positive cells, which indicated that the infiltrating mononuclear cells were natural killer cells and macrophages. On day 14 after inoculation, the tumors were infiltrated with a large number of L3T4-positive cells and fewer Lyt-2-positive cells, both of which were more abundant in the MY-1-treated tumors than in the control tumors. The observed sequence of host cell infiltration corresponded well with our previous studies which have indicated that the antitumor mechanism of MY-1 is divided into two phases, i.e. the early phase when natural killer cells and macrophages inhibit tumor growth, and the late phase when L3T4-positive cells act to induce tumor regression via a delayed-type hypersensitivity against tumor cells.