Protective immunity induced by an intranasal multivalent vaccine comprising 10 Lactococcus lactis strains expressing highly prevalent M‐protein antigens derived from Group A Streptococcus

Streptococcus pyogenes (group A Streptococcus) causes diseases ranging from mild pharyngitis to severe invasive infections. The N‐terminal fragment of streptococcal M protein elicits protective antibodies and is an attractive vaccine target. However, this N‐ terminal fragment is hypervariable: there are more than 200 different M types. In this study, an intranasal live bacterial vaccine comprising 10 strains of Lactococcus lactis, each expressing one N‐terminal fragment of M protein, has been developed. Live bacterial‐vectored vaccines cost less to manufacture because the processes involved are less complex than those required for production of protein subunit vaccines. Moreover, intranasal administration does not require syringes or specialized personnel. Evaluation of individual vaccine types (M1, M2, M3, M4, M6, M9, M12, M22, M28 and M77) showed that most of them protected mice against challenge with virulent S. pyogenes. All 10 strains combined in a 10‐valent vaccine (M×10) induced serum and bronchoalveolar lavage IgG titers that ranged from three‐ to 10‐fold those of unimmunized mice. After intranasal challenge with M28 streptococci, survival of M×10‐immunized mice was significantly higher than that of unimmunized mice. In contrast, when mice were challenged with M75 streptococci, survival of M×10‐immunized mice did not differ significantly from that of unimmunized mice. Mx‐10 immunized mice had significantly less S. pyogenes in oropharyngeal washes and developed less severe disease symptoms after challenge than did unimmunized mice. Our L. lactis‐based vaccine may provide an alternative solution to development of broadly protective group A streptococcal vaccines.

     

Protective immunity evoked by locally administered group A streptococcal vaccines in mice

The present studies were undertaken to determine the pathogenicity of group A streptococci introduced intranasally (i.n.) into mice in an attempt to mimic mucosal infections in humans and to determine the efficacy of streptococcal vaccines administered via the mucosal route. The LD50 of type 24 streptococci (M24 strep) administered i.n. was 3 x 10(4) CFU. Throat cultures were performed in M24 strep-inoculated mice. Of 11 mice that died, 9 had positive throat cultures 3 or 4 days after i.n. challenge, and of 9 mice that survived, only 1 had a positive throat culture, indicating an association between mucosal infection and death. Postmortem examination performed on 35 mice that died after i.n. challenge showed that all had evidence of disseminated infections, and group A streptococci were recovered from the cervical lymph nodes, blood, spleen, liver, and brain. To determine vaccine efficacy, heat-killed M24 strep or pep M24 were administered i.n. to groups of mice. Whole, heat-killed streptococci and pep M24 administered locally protected mice against death from i.n. challenge infections with homologous M24 strep. The whole cell vaccine also protected against i.n. challenge infections with heterologous type 6 streptococci. Our data suggest that streptococcal vaccines administered locally evoke protective immunity against streptococcal infections.     

Role of group A streptococcal IgG Fc-receptor in induction of anti-IgG by immunization in the rabbit

Previous work has demonstrated that streptococcal IgG Fc-receptors (FcR) may trigger production of anti-IgG after immunization of rabbits with group A streptococci. This effect seemed dependent on in vitro binding of IgG, derived from the growth medium, to the vaccine strains. In the experiments presented here, IgG was eluted from streptococcal strains to be used for immunization of rabbits by 1 M KSCN and washing, a treatment which did not affect the capacity of the strains to bind newly added IgG. Using two IgG FcR-positive group A streptococcal strains (M-types 1 and 22) for intravenous immunization, anti-IgG was found in the sera of 26 out of 28 rabbits, examined 8 weeks after immunization. In contrast, anti-IgG was not induced in 16 rabbits receiving either group A, type T27 or group B, type Ia streptococci both of which lack surface FcR activity. Finally, immunization with purified streptococcal IgG FcR (0.35 mg, given subcutaneously combined with Freund's complete adjuvant and two weeks later intraconjunctivally without adjuvant) also induced anti-IgG. In all rabbits, anti-human rather than anti-rabbit IgG was detected. It is proposed that in vivo interaction between the bacterial FcR and rabbit IgG, resulting in conformation changes in IgG, is a prerequisite for the induction of anti-IgG. Thus, streptococcal triggering of anti-IgG, ascribable to IgG Fc-receptor activity and not requiring presence of foreign IgG, has been demonstrated in the rabbit.     

Role of group A streptococcal IgG Fc-receptor in induction of anti-IgG by immunization in the rabbit

Abstract Previous work has demonstrated that streptococcal IgG Fc-receptors (FcR) may trigger production of anti-IgG after immunization of rabbits with group A streptococci. This effect seemed dependent on in vitro binding of IgG, derived from the growth medium, to the vaccine strains. In the experiments presented here, IgG was eluted from streptococcal strains to be used for immunization of rabbits by 1 M KSCN and washing, a treatment which did not affect the capacity of the strains to bind newly added IgG. Using two IgG FcR-positive group A streptococcal strains (M-types 1 and 22) for intravenous immunization, anti-IgG was found in the sera of 26 out of 28 rabbits, examined 8 weeks after immunization. In contrast, anti-IgG was not induced in 16 rabbits receiving either group A, type T27 or group B, type Ia streptococci both of which lack surface FcR activity. Finally, immunization with purified streptococcal IgG FcR (0.35 mg, given subcutaneously combined with Freund's complete adjuvant and two weeks later intraconjunctivally without adjuvant) also induced anti-IgG. In all rabbits, anti-human rather than anti-rabbit IgG was detected. It is proposed that in vivo interaction between the bacterial FcR and rabbit IgG, resulting in conformation changes in IgG, is a prerequisite for the induction of anti-IgG. Thus, streptococcal triggering of anti-IgG, ascribable to IgG Fc-receptor activity and not requiring presence of foreign IgG, has been demonstrated in the rabbit.     

Antitumor activity of Corynebacterium parvum administered into the pleural cavity of mice

Summary We investigated the efficacy of intrapleurally (IPl) injected 0.25 mg Corynebacterium parvum (CP) against pulmonary tumor deposits of four syngeneic murine tumors, C3Hf/Bu or CBA fibrosarcoma (FSa) or mammary carcinoma (MCa), and compared its efficacy with that of intravenous (IV) CP. A mode of action of CP given by IPl administration was also studied. When given to mice prior to the IV inoculation of tumor cells, IPl and IV CP reduced the number of metastases of C3Hf/Bu-FSa and CBA-MCa equally well. Intrapleural injection of CP within a week after tumor cell inoculation was more effective against metastases in the lung than IV CP. A combination of IPl and IV administration of CP was more effective in the therapy of lung metastases of CBA-FSa and CBA-MCa than either single treatment alone. Intrapleural and IV injections of CP augmented concomitant immunity to artificially produced lung metastases of C3Hf/Bu-FSa. Intrapleural CP was less effective than IV CP in inducing complete regression of the SC growing C3Hf/Bu-FSa.In contrast to IV CP, IPl CP did not markedly influence the spleen weight and cellularity. It was also less effective in increasing the liver weight. However, CP by either route of injection led to a significant increase in the lung weight. CP injected IPl, but not IV, caused a significant increase in the number of nucleated cells in the pleural cavity of mice. More than 90% of these cells were macrophages, and they were found to be cytotoxic for in vitro cultures of CBA-MCa cells. Activated macrophages also mediated in vivo antitumor resistance, as shown by the abolition of this resistance by treatment of the mice with carrageenan.     

Assessment of Human Immune Response to Mutans Streptococcal Glucosyltransferase Peptides Selected by MHC Class II Binding Probability

Dental decay is a major public health challenge, causing substantial social and economic burdens. In animals, vaccination against mutans streptococci, the causative organism, interferes with dental caries. The mutans streptococcal glucosyltransferase (GTF) has been effectively used as a protein antigen in experimental dental caries vaccines. Compared to whole proteins, peptide subunits can focus immune responses on protective epitopes, and not on potentially harmful cross reactive antigens. In the past we selected peptide subunits of GTF for vaccine discovery based on putative functional significance and conservation of GTF primary structure. To focus on the immunogenicity of peptides, we estimated the probability of MHC class II binding. Twenty 20-mer linear GTF peptides were synthesized on this basis and their immunoreactivity explored. Significant human peripheral blood mononuclear cell (PBMC; n = 12) proliferation was observed in response to amino acids (AA) 502–521 (peptide 7), located in the catalytic domain of GTF. Human serum (n = 36) antibody reactivity was observed to AA 438–457 (peptide 5), AA 502–521 (peptide 7), and AA 1376–1395 (peptide 16). Whole saliva mutans streptococcal levels were used as markers of mutans infection, and dental examinations to determine existing and historic caries (DMFS score) were performed. DMFS scores correlated with mutans streptococcal counts, but not with immune responses. We have identified peptides with projected avid MHC-binding activity that reacted with human PBMC and serum antibody, implying that these peptides are immunogenic and may be of significance in a subunit dental caries vaccine.     

Synergistic effects of active specific immunotherapy and chemotherapy in guinea pigs with disseminated cancer

Sewall Wright strain 2 guinea pigs bearing pulmonary metastases of the syngeneic line 10 (L10) hepatocarcinoma were treated with a vaccine composed of 10(7) bacillus Calmette-Guérin admixed with 10(7) x-irradiated L10 tumor cells beginning 10 days after tumor inoculation. Although this treatment failed to cure most of the guinea pigs of their metastatic disease, histologic examination of the pulmonary tumors in the vaccinated guinea pigs provided evidence of a cell-mediated hypersensitivity response that disrupted the normally compact architecture seen in control tumors. When a monoclonal antibody against the L10 tumor was injected i.v. to evaluate the vascular permeability of the tumors, significantly more antibody localized in tumors of vaccinated guinea pigs than in tumors of untreated controls. These results suggested that blood-borne substances could be delivered more efficiently to L10 metastases after the tumor-bearing guinea pigs had been treated with vaccine. To determine whether such increased vascular permeability would enhance the antitumor effects of chemotherapeutic agents, combined immunotherapy and chemotherapy studies were performed. Although cyclophosphamide treatment by itself did not cure L10-bearing guinea pigs, cyclophosphamide used in conjunction with prior immunotherapy increased the survival rate of animals to more than twice that of animals treated with immunotherapy alone (74 vs 33%). These results suggest that one mechanism by which active specific immunotherapy enhances chemotherapy of disseminated tumors is by rendering tumor foci more permeable to subsequently administered cytotoxic drugs.     

Rapid Identification of Mutans Streptococcal Species

Mutans streptococci are considered the predominant pathogens in dental caries. Three methods, i.e. dot blot hybridization analysis, PCR analysis and SDS-blue dextran-PAGE, were examined for identifying mutans streptococcal species. In dot blot hybridization, DNA probe derived from the dextranase gene (dexA) of Streptococcus mutans hybridized with different intensities under the condition of low stringency against each species of mutans streptococci although the dexA probe was specific for S. mutans under the condition of high stringency. Oligonucleotide primers for polymerase chain reaction (PCR) were designed on the basis of the dexA DNA sequence. The primers amplified species-specific PCR products in the reference species (15 strains of 5 species) of mutans streptococci. An electrophoretic profile of dextranases from the mutans streptococci on SDS-blue dextran-PAGE also showed species-specific behavior. These results suggest that the three identification methods examined here are useful for distinguishing the species of mutans streptococci and also indicate that PCR analysis is suitable for simple, rapid and reliable identification of mutans streptococcal species.     

Effect of nonspecific immune stimulation with BCG and polidin on the Ehrlich ascites tumor growth

Investigations were performed to study: 1) the antitumor effect of BCG pretreatment on the development of Ehrlich ascites tumor in mice; 2) the effect of BCG administration in relation to the period of time before tumor inoculation and the dose levels used, and 3) the antitumor effect of an associated pretreatment of BCG and Polidin on the development of Ehrlich ascites tumor. BCG administered prior to Ehrlich ascites tumor inoculation have a protective effect evidenced by a delay in tumor development, a prolonged survival of the tumor host and, in some cases, even inhibition of tumor growth. The effect of BCG was highly dependent on 1) the dose and the time of administration of BCG and) 2 the combined pretreatment of BCG and Polidin.     

Oral immunization of mice with engineered Lactobacillus gasseri NM713 strain expressing Streptococcus pyogenes M6 antigen

The aim of this in vivo study was to evaluate the effects of a recombinant probiotic strain, Lactobacillus gasseri NM713, which expresses the conserved region of streptococcal M6 protein (CRR6), as an oral vaccine against Streptococcus pyogenes. A dose of 10⁹ cells of the recombinant strain in 150 μL PBS buffer was administered orally to a group of mice. One control group received an equivalent dose of Lb. gasseri NM613 (containing the empty plasmid without insert) or and another control group received PBS buffer. Each group contained 30 mice. The immunization protocol was followed on three consecutive days, after which two booster doses were administered at two week intervals. Fecal and serum samples were collected from the mice on Days 18, 32, 46, 58 after the first immunization and Day 0 prior to immunization. Anti‐CRR6 IgA and IgG concentrations were measured by ELISA in fecal and sera samples, respectively, to assess immune responses. Vaccination with the recombinant Lb. gasseri NM713 strain induced significant protection after nasal challenge with S. pyogenes, only a small percentage of this group developing streptococcal infection (10%) or dying of it (3.3%) compared with the NM613 and PBS control groups, high percentages of which developed streptococcal infection (43.3% and 46.7%, respectively) and died of it (46.7% and 53%, respectively). These results indicate that recombinant Lb. gasseri NM713 has potential as an oral delivery vaccine against streptococcus group A.     

Protection, immune responses and side effects in Atlantic salmon (Salmo salarL.) vaccinated against furunculosis by different procedures

In an experimental trial lasting approximately 6 months, 10 different vaccination regimes against furunculosis were studied in Atlantic salmon pre-smolts. Single and repeated administration of vaccine by the intraperitoneal (i.p.), immersion or oral route, and revaccination by combinations of these methods, were tested. In challenge assays initiated 8 and 16 weeks after vaccination, fish injected once with a trivalent vaccine, and fish injected twice with a monovalent vaccine, both containing aluminium phosphate as adjuvant, were moderately protected. Non-injection vaccination protocols consistently failed to protect the fish. Compared with unvaccinated fish, protected groups showed elevated antibody responses toAeromonas salmonicidaantigens throughout the study. Increasedin vitroproliferation of head kidney leucocytes from i.p. vaccinated fish was found 16 weeks after vaccination. The use of a polyvalent vaccine preparation, and revaccination by injection or the oral route improved both immune responses and survival, compared to a single inoculation of monovalent vaccine. In all groups subjected to i.p. administration of vaccine, minor to moderate intraperitoneal lesions were found. In conclusion, i.p. administration of adjuvanted vaccine, preferably in a polyvalent formulation, is the optimal method of inducing anti-furunculosis immunity in Atlantic salmon, and is apparently necessary for an effective immunoprophylaxis of salmonid fish against furunculosis.     

Choice of nutrient media for the laboratory diagnosis of Streptococcus group B

The comparative study of a large assortment of liquid and solid culture media used for the cultivation of streptococci in laboratory practice in the USSR and abroad was carried out with the aim of selecting the optimal media for the laboratory diagnosis of group B. streptococci. Liquid media were tested with the use of 7 streptococcal reference strains, and some of these media, found to yield the best results, were selected for tests on clinical material. The use of liquid accumulation media was shown to permit the isolation of group B. streptococcal strains which could not be detected by the direct inoculation of clinical material into dishes with blood agar. The character of hemolysis induced by group B. streptococci in solid media with 5% of blood added was found to depend on the composition of the medium and the conditions of cultivation.     

Streptococcal L forms and phage. A clinical-epidemiologic study

This study showed that streptococcal L forms could not be isolated from children who were either carriers of group A streptococci or had disease due to this pathogen. It was possible to induce L colony formation in 15 strains of group A. Streptococcal bacteriophages were demonstrated in 20% of group A streptococci isolated from school children who were carriers, but did not have clinical evidence of streptococcal disease, and from 44.9% of children whose physicians considered they had acute streptococcal upper respiratory infections. Lysogeny (bacteriophage) was demonstrated more frequently during 1969-70 when carrier rates were high and from children who had manifest streptococcal disease, suggesting a possible positive relationship between lysogeny, high carrier rates, and infection in the children studied. Lysogeny and erythrogenic toxin production by group A streptococci occurred simultaneously in approximately half of the strains of group A streptococci tested, suggesting that lysogeny is not a sine qua non for erythrogenic toxin production.     

Fluorescent-Antibody Techniques for the Identification of Group D Streptococci: Direct Staining Method

Fluorescent-antibody (FA) techniques were employed in an attempt to develop a rapid test for the identification of group D streptococci. Fresh isolates were obtained from sewege and feces of sheep, cattle, horses, rabbits, chickens, geese, and rats. Identification to species were made by the conventional physiological, biochemical, and serological tests. Both whole and disrupted cells of representative strains of each species were used for the preparation of the group D streptococcus vaccine. Globulin fractions of individual and pooled antisera were labeled with fluorescein isothiocyanate, and the resulting conjugates were tested with homologous and heterologous antigens. The specificity of the conjugates and staining was assessed by adsorption and inhibition tests utilizing controls with homologous and heterologous antigens. Employing the direct staining method and individual and pooled conjugates, it was possible to obtain 84 and 85% positive FA reactions, respectively, with group D streptococcal strains. Trypsinization of the smears prior to staining eliminated all FA cross-reactions observed with non-group D streptococci and staphylococci. These findings suggest that the direct staining method will be of value in the rapid identification of group D streptococci.     

Acceleration of axonal outgrowth in rat sciatic nerve at one week after axotomy

Following injury of sciatic motor axons in the rat, the rate of axonal outgrowth is faster if there has been a prior “conditioning” axotomy. The acceleration of outgrowth is due to an acceleration of SCb, the rate [slow (SC)] component of axonal transport that carries cytomatrix proteins; this occurs throughout the axon by 7 days after the conditioning axotomy (Jacob and McQuarrie, 1991a, J. Neurobiol. 22:570–583). To further characterize the conditioning lesion effect (CLE), it is important to know (1) the minimum effective conditioning interval (time between conditioning and testing lesions), (2) whether the cell body reaction is required, and (3) whether outgrowth accelerates after a single axotomy. Outgrowth distances were measured by radiolabeling all newly synthesized neuronal proteins and detecting those carried to growth cones by fast axonal transport. When the conditioning and testing lesions were made simultaneously (0 day conditioning interval), there was no CLE. With a conditioning interval of 3 days, there was a shortening of the initial delay (before the onset of outgrowth) without a change in outgrowth rate. With conditioning intervals of 7, 14, and 21 days, the rates of outgrowth were increased by 8%, 22%, and 11%, respectively. To determine whether the cell body reaction to axotomy is necessary for the CLE, a nonaxotomizing stimulus to axonal growth (partial denervation) was used in place of a conditioning axotomy. This had no effect on the rate of outgrowth from a testing lesion made 14 days later. Finally, we examined the possibility that outgrowth accelerates after a single lesion. Outgrowth was faster at 6–9 days after axotomy than at 3–6 days (p < 0.001), and accelerated further at 9–12 days (p < 0.001). We conclude that (1) the shortest effective conditioning interval is 3 days; (2) the cell body reaction is necessary for the CLE; (3) axonal outgrowth from a single axotomy accelerates in concert with the anabolic phase of the cell body reaction. The SCb motor is, in turn, upregulated by this reaction. This suggests that the SCb motor responds to a fast-transported signal that is a product of the cell body reaction. © 1993 John Wiley & Sons, Inc.     

Low-dose paclitaxel enhances the anti-tumor efficacy of GM-CSF surface-modified whole-tumor-cell vaccine in mouse model of prostate cancer

Chemotherapy combined with a tumor vaccine is an attractive approach in cancer therapy. This study was designed to investigate the optimal schedule and mechanisms of action of a novel GM-CSF (granulocyte?Cmacrophage colony-stimulating factor) surface-modified tumor-cell vaccine in combination with paclitaxel in the treatment of mouse RM-1 prostate cancer. First, the anti-tumor efficiencies of various dosage of paclitaxel (4, 20, 40?mg/kg) in combination with the vaccine in different administration sequences were examined in the mouse RM-1 prostate cancer model. Then, the in vivo and in vitro effects of various dosage of paclitaxel on RM-1 cells, T cells, and DCs (dendritic cells) were evaluated. The results showed that: (a) the GM-CSF-surface-modified tumor-cell vaccine was more potent at inducing the uptake of tumor antigens by DCs than irradiated tumor cells plus free GM-CSF; (b) 4?mg/kg paclitaxel combined with the GM-CSF-surface-modified tumor-cell vaccine was the most effective at enhancing tumor regression in RM-1 prostate cancer mice when the vaccine was administrated 2?days after paclitaxel; and (c) administration of 4?mg/kg paclitaxel followed by the vaccine induced the highest degree of CD8⁺ T-cell infiltration in tumor tissue, suggesting that the induction of tumor-specific immune response had occurred. These findings suggested that the GM-CSF-surface-modified tumor-cell vaccine may have potential clinical benefit for patients with prostate cancer when it is combined with paclitaxel. Furthermore, the effect of immunochemotherapy depends on careful selection of paclitaxel dosage and the sequence of paclitaxel/vaccine administration.     

Cadmium,copper, and zinc levels in the rice and rice field soil of Houston,Texas

Fifty-one pairs of hulled rice samples and the soil from which each rice sample was grown were analyzed for heavy metals in August, 1979, in order to estimate the background contamination of cadmium (Cd), copper (Cu), and zinc (Zn) in rice grown in the Houston, Texas area. Both samples were divided into three groups by soil types and colors. The cadmium concentration in Texas rice was only one-half to one-quarter lower than that of Asian rice. However, the levels of Cu and Zn in rice in Texas were similar to those reported. Soil heavy metals were lower than ever reported, but these values were consistent with the geochemical characteristics of the Texas Houston area. No particular relationship was found between the three metals in rice and the metals in soil where the sampled rice was grown.     

Antitumor effect of the streptococcal preparation OK-432 in a murine model of ovarian cancer

Summary The antitumor effects of the streptococcal preparation OK-432 were analyzed in a murine ovarian teratocarcinoma (MOT) model. Administration of OK-432 i.p. prevented tumor outgrowth in 75% of mice challenged with 10³ MOT cells i.p. 24 h previously. Treatment was less successful in mice challenged with 10⁴ or 10⁵ cells, preventing tumor growth in 25% of the former and only 5% of the latter group. Tumor-challenged mice cured by injections of OK-432 were not rendered resistant to a subsequent challenge with 10³ MOT cells 75 days after initial treatment. Only the i.p. route of administration was effective as i.v. OK-432 did not prolong survival of tumor-challenged mice. An antitumor response was detected as early as 24 h after i.p. treatment. This correlated temporally with an influx of neutrophils into the peritoneal cavity. Peritoneal cells obtained between 6 and 24 h after treatment were capable of lysing MOT targets in vitro. A single cell cytotoxicity assay demonstrated that peritoneal neutrophils, elicited by i.p. injection of OK-432, could bind to and lyse MOT targets. These data indicate that OK-432 is effective against small tumor cell inocula in this murine model of ovarian cancer and, furthermore, that the neutrophilic response into the peritoneal cavity plays a role in tumor rejection.     

Septicaemic infections in pigs,caused by haemolytic streptococci of new Lancefield groups designated R,S and T

Pyogenic streptococci isolated from outbreaks and from sporadic infections in pigs and piglets were characterized by the almost unique combination of the properties of -haemolysis on horse blood agar and acid production from inulin. Three new serological groups were recognized, each with a single antigen different from those of any of the Lancefield groups. These antigens are polysaccharides located in the cell wall. About half the number of haemolytic streptococcal strains isolated from pigs were group R or group S streptococci, a few strains were group T streptococci, and the remaining strains were group L streptococci,S. equisimilis, group E streptococci, or group P streptococci, in this order of frequency. Only one out of about 150 haemolytic strains could not be identified serologically. Group R and group T streptococci differ from group S streptococci by acid production from raffinose. Infections with group R streptococci appeared to occur independently of age, whereas infections with group S streptococci were almost entirely confined to piglets.     

Effects of antithymocyte serum on lymph node cells participating in the graft-vs-host reaction.

BALB/c mice were injected with 0.1 ml of antithymocyte serum (ATS) and tested at various times thereafter for graft-versus-host (GVH) reactivity of lymph node cells (LNC) and spleen cells (SC). Splenomegaly produced by LNC or SC injected alone was used as a measure of precursor T cell function and the capacity of such cells to produce synergy when combined with normal thymocytes was used to evaluate amplifier T cell activity. In lymph node, both precursor and amplifier function were strikingly depressed 2 days after ATS administration; by day 11, precursor function showed slight recovery while amplifier activity had recovered to supranormal levels. In spleen, precursor activity recovered two fold between 2 and 11 days after ATS inoculation but no amplifier activity could be detected at day 11. Since donors had not been deliberately stimulated with alloantigens prior to testing of LN and SC GVH acivity, these studies demonstrate (a) that precursors and amplifiers are disinct T cell populations that are committed to express unique functional activities before antigen exposure and (b) that, following depletion with ATS, these two populations recover independently at different rates in separate lymphoid compartments.