INFECTION AND IMMUNITY, Dec. 1977, p. 646-63 Vol. 18, No. 3Copyright@ 1977 American Society for Microbiology Printed in U.S.A.

Induction and Expression of Immunity AfterBCG Immunization

M. J. LEFFORD

Trudeau Institute, Inc., Saranac Lake, New York 12983

Received for publication 11 July 1977

The induction and expression of immunity to Mycobacterium tuberculosisafter BCG immunization by intravenous, subcutaneous, and pulmonary routeshas been investigated in mice. The speed with which protective immunity wasengendered was a function of inoculum size; the immunization route was a lessinfluential factor. Tuberculin hypersensitivity varied both with the inoculum sizeand immunization route, being least after pulmonary immunization. Once im-munity was established, a steady state ensued in which the number of sensitizedlymphocytes in the spleen was similar, regardless of the route or dose of vacci-nation. Actively immunized animals controlled intravenous and subcutaneouschallenge infections, regardless ofthe method ofvaccination. However, pulmonarychallenge was resisted most efficiently by mice immunized by the pulmonaryroute. Adoptive immunity was well expressed in the spleen only, but the lungswere no more deficient in this regard than the footpad. It is suggested thatenhanced immunity in the lungs depends on a population of resident sensitizedlymphocytes.

There is ample published evidence that theroute of immunization with BCG determines thelevel of delayed tuberculin hypersensitivity(DTH) and resistance to reinfection with tuber-cle bacilli. Subcutaneous/intradermal inocula-tion favors the development of tuberculin DTH,compared with pulmonary (P) and intravenous(i.v.) immunization (14, 16). On the other hand,although animals infected by the P, i.v., or sub-cutaneous routes are resistant to i.v. challengewith virulent tubercle bacilli, resistance to pul-monary challenge is observed only after P ori.v. immunization (1, 3, 4, 17, 24).The latter observations in particular are dis-

turbing because they do not conform to thedemonstrated efficacy of intradermal immuni-zation with BCG in protecting humans againstpulmonary tuberculosis (22). Moreover, sincethe cells that confer long-term antituberculosisimmunity are long-lived, recirculating, smalllymphocytes (21), it is puzzling that these cellsshould be less available to combat pulmonaryinfection than infection at any other site.

Earlier studies have been limited to the re-sponse of actively immunized animals to i.v. orP challenge. The use of actively immunized an-imals incurs the problem of nonspecific resist-ance that is found to a greater or lesser extentin animals immunized with BCG (5, 18). In suchanimals, it is impossible to determine how muchof the observed resistance to reinfection is at-tributable to specific or nonspecific effects. The

comparison of the lungs and spleen as sites oftuberculosis infection also creates some difficul-ties. The spleen is the major central lymphoidorgan through which the recirculating lympho-cytes traverse (13, 15). The lung parenchymacontain small foci of lymphoid tissue (26) butcannot be regarded as lymphoid organs to whichlymphocytes "home." Consequently, it could beargued that the lungs are at a disadvantage tothe spleen in their content ofimmunocompetentcells.The purpose of this study was to critically

reevaluate certain variables that may influencethe induction and expression of anti-tuberculosisimmunity. These factors include the dose ofBCG and its route of administration, and theexpression of immunity in the spleen, the lungs,and one other peripheral site, the hind footpad(FP). The problem of nonspecific resistance hasbeen circumvented by testing the ability ofspleen cells from immunized mice to confer anti-tuberculosis immunity upon nonimmune recipi-ents (19).

MATERIALS AND METHODSAnimals. Female (C57B1/6 x DBA/2) F1 inbred

mice (B6D2) were introduced into experiments when6 weeks old.Mycobacteria. Strains of mycobacteria were ob-

tained from the Trudeau Mycobacterial Culture Col-lection, Saranac Lake, N.Y. The organisms were grownin Proskauer and Beck liquid medium (Difco Labora-tories, Detroit, Mich.), to which were added Tween

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VOL 18, 1977

80 and glycerol. Culture vials were stored at -70°C,thawed before use, and diluted appropriately in salineto contain the required number of viable bacilli permilliliter. BCG Pasteur (TMC 1011) and Mycobacte-rium tuberculosis RlRv (TMC 205) were used forimmunization and challenge, respectively.Immunizaon. The required numbers of viable

BCG in appropriate volumes of saline were injectedeither i.v. in 0.2 ml or into the left hind footpad(LHFP) in 0.04 ml. For P infection, 10 ml of BCGculture containing approximately 2 x 108 viable bac-teria per ml was nebulized in a Middlebrook AirborneInfection Apparatus (Tri-R Instruments, RockvilleCentre, N.Y.) in which the mice were held. Thisprocedure implanted approximately 104 viable BCGinto the lungs. The implantation inoculum waschecked by making viable counts of BCG in the lungsof mice immediately after infection.

Challenge. Mice were challenged with RlRv atdoses of l05 i.v., 104 into the right hind footpad(RHFP), or 104 into the lungs as described for BCGimmunization above.DTH. Lyophilized human tuberculin-purified pro-

tein derivative was obtained from the National Insti-tute of Allergy and Infectious Diseases, Bethesda, Md.The material was brought to a concentration of 125ug by solution in sterile saline containing 0.05% Tween80. The thickness of the RHFP was measured withdial-gauge calipers before injection of 5 yg of purifiedprotein derivative (0.04 ml) and was remeasured 24 hlater. The difference in FP thickness was expressedin 0.1-mm units. An increase of 2 units (0.2 mm) ormore was accepted as a positive result, denoting thepresence of DTH.

Irradiation. Before challenge with RlRv andtransfer of spleen cells, all recipient mice and theircontrols were exposed to 500 rads of total body irra-diation from a "Cs source (19).Spleen cels. Spleens were removed from donor

mice and teased apart, and the dissociated cells werewashed in ammonium chloride/tris(hydroxy-methyl)aminomethane buffer to lyse erythrocytes (6).The remauiing cells were washed in Dulbecco phos-phate-buffered saline and suspended in the same so-lution to a density of 2.5 x 108 leukocytes per ml.Each recipient received 0.2 ml of this suspension,containing 5 x 107 leukocytes, injected i.v. The viabil-ity of each spleen cell suspension was checked beforeinjection by trypan blue exclusion. For the entireseries of experiments, the cell viabilities were in therange of 60 to 80%.

Viable counts of mycobacteria. Spleens andlungs were homogenized in glass tubes with matchingmotor-driven Teflon pestles. Feet were severed at theankle and allowed to soak for 30 min in 0.1% benzal-konium chloride to kill skin surface contaminants.Each foot was then cut into four to five coarse piecesand homogenized in sterile saline with a Virtis grinder(Virtis Co., Gardiner, N.Y.). Appropriate dilutions ofhomogenate were plated on Middlebrook 7H-10 agar(Difco) and incubated for 14 to 21 days at 370C; thencolony counts were made. The counts were expressedeither as geometric means or as an index of immunitythat was estimated by subtracting the geometric meancount of a test group from that of the control group.

IMMUNITY INDUCED BY BCG 647

Statists. Comparison of group means was madeafter analysis of variance and application of the Qtest (32). All DTH and viable-count means were basedon groups of five mice.

RESULTSIndUction of immunity after immuniza-

tion with BCG by the i.v. and LEOFP routes.Mice were immunized with either 106 BCG i.v.,103 BCG i.v., or 106 BCG into the LHFP. Atintervals thereafter, mice of each group wereused for tuberculin tests and as spleen cell do-nors. Mice immunized with 106 BCG i.v. orLHFP developed unequivocal tuberculin hyper-sensitivity at 3 to 4 weeks, whereas the groupreceiving 103 BCG i.v. never exhibited DTH(Fig. 1). Repeated experiments revealed consid-erable inter-experimental variation in the devel-opment and decay of DTH, but there were cer-tain consistent features (Fig. 1 and 4). Only thelarger doses of BCG resulted in perceptibleDTH, which was maximal at 3 to 4 weeks andthen waned rapidly. LHFP immunization re-sulted in a biphasic response, and the state ofhypersensitivity was sustained for a longer pe-riod of time. In all experiments, the level ofDTH 3 months after immunization was trivial,regardless of the route and dose of BCG.The ability of spleen cells of vaccinated mice

to confer adoptive immunity upon syngeneicrecipients is illustrated in Fig. 2. There was asmall but highly significant (P< 0.01) transfer ofimmunity by spleen cells of FP-immunized miceas early as week 1. By week 2, spleen cells ofdonors given 106BCG i.v. were also effective (P< 0.01). Sensitized lymphocytes were detected

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a.400U.

4

3

2

1

WooksFIG. 1. DTH after BCG immunization by i.v. and

subcutaneous routes. Symbols: D, 108 BCG i.v.; *,103 BCG i.v.; A, 10a BCG LHFP.

648 LEFFORD IFC.IMN

a-ft-er a longer interval in mice vaccinated with103 BOG i.v., but by week 6 the spleen cells ofeach group conveyed immunity to the sameextent, and this capability did not diminish dur-ing the remaining 6 weeks of the experiment.Development of immunity after i.v.,

LHFP, and P vaccination with BCG. Thenext point of interest was whether immunizationby the P route was more or less effective thanthe i.v. or LHFP routes. Groups of mice wereimmunized with either 106 BOG LHFP, 103 BOGi.v., or iO' BOG P. At 2-week intervals, viablecounts of BOG were made from organs intowhich the inocula had been directly implanted,i.e., the LHFP, spleen, and lungs, respectively(Fig. 3). No apparent growth of BOG occurredin the LHFP, probably because the large inocu-lum induced a rapid immune response that pre-vented multiplication (18). As will become evi-dent later, smaller inocula of BOG did multiplyin the FP (Fig. 6 to 8). After i.v. infection, BOGmultiplied in the spleen for 4 weeks and thenslowly decreased in number. Growth of BOG inthe lungs after aerosol infection was substantial,and large numbers of viable orgaism werepresent in this organ throughout the course ofthe experiment.The development of tuberculin DTH in these

mice is shown in Fig. 4. In this experiment, eachgroup of immunized mice exhibited DTH atweek 4, but its level varied with the route ofimmunization. Mice imunzd in the LHFPwere significantly more sensitive (P <0.01) than

2

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*0£

0

P-infected mice, with the remaining group, i.v.,occupying an intermediate position.

Transfer of adoptive immunity was notachieved until 4 weeks after immunization, atwhich point all groups of vaccinated mice wereeffective donors (Fig. 5). Between 4 and 6 weeks,cells from LHFP-immnizd donors were moreeffective than those from P-immunized mice (P<0.01), but subsequently the difference betweenthe three groups of cell recipients was insignifi-cant.Expression of active and adoptive im-

munity at different sites. The above experi-ments suggested that, regardless of the dose ofBOG or its route of administration, the spleencells of vaccinated mice eventually developed a

7

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. 4.0a

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4 KAm '

ftC-.--'

0 1 2 3 4 5 6 ' 8 10 12-

WeeksFIG. 2. Transfer of adoptive immunity to M. tuber-

culosis with 5 X 1(1' spleen cells at intervals afterimmunization with either 10r, BCG i.v. (U), 103 BCGi.v. (0), or 10 BCG LHFP (A).

0 2 4 6 8 10 12

WeeksFIG. 3. BCG was inoculated either i.v., subcuta-

neously, or into the lungs, and viable counts weremade fr-om the 8pleen (40), FP (A), and lungs (U),respectively.

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02

0O. A

Weeks

FIG. 4. Induction of DTH after BCG immuniza-tion by PP (A), i.v. (@), and P (U) routes.

INFECT. IMMUN.

I

VOL. 18, 1977

uniform capacity to transfer anti-tuberculosisimmunity within 8 weeks of immunization. Inthe next series of experiments, mice were vacci-nated with BCG, l03 i.v. (Fig. 6), 106 LHFP (Fig.7), or 1iO P (Fig. 8). Eight weeks later, whenthe ability of spleen cells to transfer immunity

2

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~~ ~ ~ ~ ~ ~ ~ ~ ~

2 4 6 8 10 12

WeeksFIG. 5. Adoptive immunity in recipients of spleen

cells from mice immunized by FP (A), i.v. (@), andP (U) routes.

IMMUNITY INDUCED BY BCG 649

7

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FIG. 7. Adoptive and active immunity after FPimmunization with BCG. Symbols: See legend ofFig.6.

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6

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WeeksFIG. 6. Adoptive and active immunity after i.v. im-

munization with BCG. Symbols: *, normal controls;A, recipients of 5 x iO7 spleen cels from immunizeddonors; U, actively immunized donors; x, chaUengeinoculum; 0, residual BCG in donor organs.

6

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FIG. 8. Adoptive and active immunity after P im-munization with BCG. Symbols: See legend ofFig. 6.

I1

650 LEFFORD

was established, either the actively immunizedmice were challenged with RlRv, or their spleencells were transferred to syngeneic recipientsthat were also challenged, together with irradi-ated controls. Three types of challenge wereevaluated: 105 i.v., a procedure that depositedapproximately 104 bacilli in the spleen; 104RHFP; and 104 P. Viable counts were madefrom the appropriate organs (spleen, RHFP, andlungs, respectively) 1 h after challenge and 2and 4 weeks later (Fig. 6 to 8).The effects of spleen-cell transfer will be con-

sidered first. As in earlier experiments, spleencells from the different groups of immunizeddonors provided excellent protection (P < 0.01)against i.v. infection with RlRv, and immunitywas manifest within 2 weeks of challenge. Theextent of adoptive immunity in the spleen wasusually less than that in actively immunizeddonors (P < 0.01) at 2 weeks (Fig. 6 and 7), butat 4 weeks there was no difference between thecounts in the two types of mice (Fig. 6 to 8).The expression of adoptive immunity in the

RHFP and lungs was defective as comparedwith the spleen. Spleen cells from i.v.- and P-vaccinated mice did not confer resistance ineither of these sites at 2 weeks after challengeor in the RHFP at 4 weeks (Fig. 6 and 8). Amodest degree of protection, 0.41 loglo unit (P <0.05), was expressed in the lungs at 4 weeks inrecipients of cells from i.v.-immunized mice (Fig.6). Spleen cells from LHFP-immunized micewere more effective. They conferred resistancein the RHFP at 2 weeks (P < 0.05) and 4 weeks(P < 0.01) and in the lungs at 4 weeks (P < 0.01)(Fig. 7). It is notable that this particular batchof cells also conferred the highest level of adop-tive immunity in the spleen (Fig. 6 to 8).Before reinfection with RlRv, donor mice of

each group were killed, and viable counts ofresidual BCG from the immunizing inoculumwere made in the spleen, RHFP, and lungs (Fig.6 to 8). Each group of donor spleens containedapproximately 103 viable BCG at the time ofchallenge and an undetectably low (<102) num-ber of organisms in the RHFP. Less than 102viable BCG were isolated from the lungs ofLHFP- or i.v.-immunized mice, but approxi-mately 105 BCG were present in the lungs of P-immunized mice. This population of BCG wasapproximately 10-fold greater than the challengeinoculum of RlRv (Fig. 8).Turning now to the expression of active im-

munity, the anti-mycobacterial mechanism wasmost effective in the RHFP, where growth ofthe organisms was completely suppressed andthere was a net decrease of viable organisms by2 weeks (Fig. 6 to 8). There was limited growthof RlRv in the spleens during the first 2 weeks

after challenge, but the inhibition of growth washighly significant (P < 0.01) relative to normalmice. The control of challenge infection in theRHFP and spleen was closely similar in allgroups of vaccinated mice. On the other hand,the ability to control P infection varied with theroute of immunization. Only mice vaccinated bythe P route inhibited the growth of RlRv inthe lungs (P < 0.01) within 2 weeks of reinfection(Fig. 8), but all donor mice controlled the infec-tion (P < 0.01) by 4 weeks (Fig. 6 to 8).

DISCUSSIONAn examination of the induction of immunity

after BCG vaccination has been made by themethod of adoptive transfer of resistance withlymphoid cells. This system has been shown todepend on an immunologically specific popula-tion of T-lymphocytes and circumvents theproblem of nonspecific resistance that exists inthe actively immunized donors (19, 20). It wasfound that the rapidity of onset of immunitywas a function of the size of the immunizinginoculum, a conclusion that had been inferredin earlier work (18). The influence of inoculationroute was less apparent, but it appeared thatimmunity appeared first after FP, then i.v., andfinally P vaccination. More impressive than therate of induction ofimmunity was the consistent,stable level at which the numbers of sensitizedlymphocytes in the spleen achieved equilibrium.Within 2 months of immunization, this equilib-rium was such that the transfer of adoptiveimmunity with 5 x 107 spleen cells was closelysimilar regardless of the dose and route of vac-cination. This steady state reflects a homeostaticmechanism that compensates for a 1,000-foldvariation in inoculum size, and is the more re-markable because the initial lymphoprolifera-tive response to living BCG is very much de-pendent on the dose (21).Tuberculin hypersensitivity after immuniza-

tion with BCG was a function of dose and routeof administration. The highest levels of DTHwere obtained with 106 LHFP, followed by 106i.v., 103 i.v., and lastly 104 P. The levels of DTHvaried considerably between experiments. It isworth noting that the fluctuation in DTH is inmarked contrast to the consistency with whichspleen cells conferred immunity.This study has confirmed the observations of

others that P immunization with living BCGengenders a greater resistance to subsequent Preinfection than does i.v. or subcutaneous vac-cination (1, 3, 4, 17, 24). Admittedly, the i.v.immunization procedure of Ribi et al. (30) alsoproduces strong resistance to P challenge, butthis is due to the vehicle in which the antigen

INFECT. IMMUN.

VOL. 18, 1977

is injected, an oil-in-water emulsion. The oildroplets, into which antigen is incorporated, lo-calize in the lungs due to embolism of the pul-monary capillaries and induce extensive granu-

lomatosis (1, 29). Similarly, the system of Myr-vik (25) also induces chronic pulmonary granu-

lomatosis. By contrast, i.v. injection of livingBCG in saline deposits 90% of the inoculum inthe liver, 10% in the spleen, and only 1% in thelungs (10, 11). Hence, the i.v. injection of myco-bacterial antigens in oil and aerosol infectionwith BCG both constitute vaccination by the Proute.A number of explanations for the association

between P immunization and pulmonary resist-ance will now be considered. Ribi and co-work-ers have shown that the protective action of hisnonliving vaccines is a function of the granulo-matosis that is produced, measured as increasein lung weight (2, 23). The implication of thisobservation is that the increased resistance toP infection is due to nonspecific resistance ex-

erted by the macrophages participating in thepulmonary granulomas. An analogous situationexists in animal that have been immunized withliving BCG, in whom resistance to reinfectionat a particular site is associated with the persist-ence of live vaccinating organisms at that site(17). These observations are supported by thisstudy, in which mimal resistance to reinfectionin the lung was expressed in mice carrying a

substantial number ofBCG (>105) in that organat the time of challenge. The association be-tween resistance at a given site and persistenceof immuniing organisms is not invariable. Inthis study, resistance to reinfection was highestin the RHFP, from which BCG was never iso-lated. Whereas it is not contended that activatedmacrophages in the lungs or other organs donot restrain the growth of challenge organism,it is doubtful whether such action fully accountsfor the experimental results. The growth ofRlRv in the lungs of i.v.- and FP-immunizedmice is unimpeded for at least 14 days (Fig. 6and 7), whereas the effective duration ofnonspe-cific resistance is measured in as many hours(28). It is therefore probable that some othercomponent of the granulomata, namely lympho-cytes, makes a potent contribution.

Clearly, if aerosol immunization resulted in a

larger or more efficient population of specificallysensitized lymphocytes than i.v. or FP inocula-tion, increased resistance to reinfection at everysite would logically ensue. This conjecture isuntenable, because resistance to reinfection inthe spleen and the RHFP was no better after Pthan i.v. or FP immunization, and the same

holds true for adoptive immunity. Another pos-sibility is that, due to the adjuvant properties

IMMUNITY INDUCED BY BCG 651

of mycobacteria, immunization with BCG in-duces not only an immune response to mycobac-terial antigens but an autoimmune response totissues at the site of infection (7, 9). If this wereso, P and FP infection might be expected togenerate lymphocytes that produced better pro-tection at the homologous than the heterologoussite. The results of the cell transfer experimentsprovide no support for this notion. Cells fromP-immunized mice did not confer preferentialprotection upon the lungs of recipients. Para-doxically, cells from i.v.- and FP-immunizedmice were more effective in this regard. More-over, actively immunized mice combated i.v. andFP challenge with equal facility, regardless ofthe immunization route.

It is proposed that the sheer proximity ofsensitized lymphocytes to the implanted, ho-mologous, challenge organisms results in abrisker anamnestic response, because time isgained that would otherwise be spent in recruit-ing a sufficient number of sensitized lympho-cytes from the circulation into the site of chal-lenge. Such tissue lymphocytes may correspondto the sessile memory cells ofhumoral immunity(12, 31) and may share identity with the lungcells that can produce migration-inhibiting fac-tor after immunization via the respiratory tract(8, 14, 27, 33). The rapid expression of cell-me-diated immunity in the spleen is attributable inpart to the migration through that organ ofrecirculating lymphocytes in which anti-tuber-culosis immunity is vested (21), obviating theneed for further recruitment. Moreover, regard-less of the route of immunization, a significantnumber of living BCG eventually reach thespleen, where they generate tuberculoid granu-lomas.

It has been argued that adoptive immunitycannot be expressed in the lungs (34), the impli-cation being that there is defective migration ofsensitized lymphocytes into this organ. The datain Fig. 7 refute this contention. Admittedly,adoptive immunity is poorly expressed in thelungs as compared with the spleen. But thelungs are not unique in this respect, because theFP is milarly affected. Furthermore, in someexperiments (Fig. 6 and 7) there was significantadoptive immunity in the lungs. It is pertinentthat the number of cells transferred, 5 x 107,was relatively modest, and there is a strongpossibility that more substantial effects wouldresult from the use of larger doses of cells. Al-though adoptive immunity is expressed to asmilar extent in the lungs and FP, active im-munity is inexplicably more effective at the lat-ter site.

In conclusion, it appears that there is a widevariation in intinsic resistance to tuberculosis

652 LEFFORD

TABLE 1. Expression of adoptive and activeimmunity to tuberculosis at different sites

Type of Level of resistance in:immunity Spleen FP Lungs

Adoptive Moderate Low Low

Active Moderate High Variablea

a Low after i.v. or LHFP immunization; moderate after Pimmunization.

in different organs (Table 1). There appears tobe a rational explanation for the relatively goodresistance of the spleen. The intrinsic suscepti-bility of the lungs and FP is high and low,respectively, and currently there is no good rea-son for this discrepancy. The resistance of asusceptible organ like the lung can be increasedby appropriate immunization, but only at thecost of inducing substantial ganulomatous dis-ease. There was no association between anti-tuberculosis immunity, active or adoptive, andthe level of tuberculin hypersensitivity.

ACKNOWLEDGMENTSI thank Robert J. Emerson and Dwight Milne for excellent

technical assistance.This work was supported by Public Health Service grants

AI-12404 and AI-07015 and contract NO1 AI-52529 from theNational Institute of Allergy and Infectious Diseases, and byPublic Health Service grant 5S07 RR05705 from the Divisionof Research Resources.

LITERATURE CITED

1. Anacker, R. L., W. R. Barclay, W. Brehmer, G.Goode, R. H. List, E. Ribi, and D. F. Tarmina. 1969.Effectiveness of cell walls of Mycobacterium bovisstrain BCG administered by various routes and in dif-ferent adjuvants in protecting mice against airborneinfection with Mycobacterium tuberculosis strainH37Rv. Am. Rev. Respir. Dis. 99:242-248.

2. Anacker, R. L, W. R. Barclay, W. Brehmer, C. L.Larson, and E. Ribi. 1967. Duration of immunity totuberculosis in mice vaccinated intravenously with oil-treated cell walls of Mycobacterium bovis strain BCG.J. Immunol. 98:1265-1273.

3. Anacker, R. L., E. Ribi, D. F. Tarmina, L. Fadness,and R. E. Mann. 1969. Relationship of footpad sensi-tivity to purified protein derivatives and resistance toairborne infection with Mycobacterium tuberculosis ofmice vaccinated with mycobacterial cell walls. J. Bac-teriol. 100:51-57.

4. Barclay, W. R., W. M. Busey, D. W. Dalgard, R. C.Good, B. W. Janicki, J. E. Kasick, E. Ribi, C. E.Ulrich, and E. Wolinsky. 1973. Protection ofmonkeysagainst airborne tuberculosis by aerosol vaccinationwith Bacillus Calmette-Guerin. Am. Rev. Respir. Dis.107:351-358.

5. Blanden, R. V., M. J. Lefford, and G. B. Mackaness.1969. The host response to Calmette-Guerin Bacillusinfection in mice. J. Exp. Med. 129:1079-1107.

6. Boyle, W. 1968. An extension of the 5"Cr-release assayfor the estimation of mouse cytotoxins. Transplantation6:761-764.

7. Burell, R. G., and M. S. Rheins. 1958. Production ofanti-lung substances in rabbits by homologous tuber-culous tissue antigens. Am. Rev. Respir. Dis. 78:259-267.

INFECT. IMMUN.

8. Cantey, J. R., and W. L. Hand. 1974. Cell-mediatedimmunity after bacterial infection of the lower respira-tory tract. J. Clin. Invest. 54:1125-1134.

9. Cate, C. C., and R. Burrell. 1974. Lung antigen inducedcell-mediated immune injury in chronic respiratory dis-ease. Am. Rev. Respir. Dis. 109:114-123.

10. Colfins, F. M., and T. E. Miller. 1969. Growth of a drug-resistant strain of Mycobacterium bovis (BCG) in nor-mal and immunized mice. J. Infect. Dis. 120:517-533.

11. Collins, F. M., L. G. Wayne, and V. Montalbine. 1974.The effect of cultural conditions on the distribution ofMycobacterium tuberculosis in the spleens and lungsof specific pathogen-free mice. Am. Rev. Respir. Dis.110:147-156.

12. Ellis, S. T., and J. L. Gowans. 1973. The role of lym-phocytes in antibody formation. V. Transfer of immu-nological memory to tetanus toxoid: the origin of plasmacells from small lymphocytes, stimulation of memorycells in vitro and persistence of memory. Proc. R. Soc.London Ser. B 183:125-139.

13. Ford, W. L. 1969. The kinetics of lymphocyte recircula-tion within the rat spleen. Cell Tissue Kinet. 2:171-191.

14. Galindo, B., and Q. N. Myrvik. 1970. Migratory re-sponse of granulomatous alveolar cells from BCG-sen-sitized rabbits. J. Immunol. 105:227-237.

15. Gowans, J. L., and E. J. Knight. 1964. The route ofrecirculation of lymphocytes in the rat. Proc. R. Soc.London Ser. B 159:257-282.

16. Kawata, H., Q. N. Myrvik, and E. S. Leake. 1964.Dissociation of tuberculin hypersensitivity as mediatorfor an accelerated pulmonary granulomatous responsein rabbits. J. Immunol. 93:433-438.

17. Larson, C. L, and W. C. Wicht. 1962. Studies of resist-ance to experimental tuberculosis in mice vaccinatedwith living attenuated tubercle bacilli and challengedwith virulent organisms. Am. Rev. Respir. Dis.85:833-846.

18. Lefford, M. J. 1971. The effect of inoculum size on theimmune response to BCG infection in mice. Immunol-ogy 21:369-381.

19. Lefford, M. J. 1975. Transfer of adoptive immunity totuberculosis in mice. Infect. Immun. 11:1174-1181.

20. Lefford, M. J., D. D. McGregor, and G. B. Mackaness.1973. Immune response to Mycobacterium tuberculosisin rats. Infect. Immun. 8:182-189.

21. Lefford, M. J., D. D. McGregor, and G. B. Mackaness.1973. Properties of lymphocytes which confer adoptiveimmunity to tuberculosis in rats. Immunology25:703-715.

22. Medical Research Council. 1972. BCG and vole bacillusvaccines in the prevention of tuberculosis in adolescenceand early adult life. Bull. W.H.O. 46:371-385.

23. Meyer, T. J., E. Ribi, and I. Azuma. 1975. Biologicallyactive components from mycobacterial cell walls. V.Granuloma formation in mouse lungs and guinea pigskin. Cell. Immunol. 16:11-24.

24. Middlebrook, G. 1961. Immunological aspects of airborneinfection: reactions to inhaled antigens. Bacteriol. Rev.25:331-346.

25. Myrvik, Q. N., E. S. Leake, and S. Oshima. 1962. Astudy of macrophages and epithelioid-like cells fromgranulomatous (BCG-induced) lungs of rabbits. J. Im-munol. 89:745-751.

26. Nagaishi, C. 1972. Functional anatomy and histology ofthe lung. University Park Press, Baltimore.

27. Nash, D. R., and B. Holle. 1973. Local and systemiccellular immune responses in guinea-pigs given antigenparenterally or directly into the lower respiratory tract.Clin. Exp. Immunol. 13:573-583.

28. North, R. J. 1974. T cell dependence of macrophageactivation and mobilization during infection with My-cobacterium tuberculosis. Infect. Immun. 10:66-71.

29. Ribi, E., R. L. Anacker, W. R. Barclay, W. Brehmer,G. Middlebrook, K. C. Milner, and D. F. Tarmina.

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1968. Structure and biological functions of mycobacte-ria. Ann. N.Y. Acad. Sci. 154:41-57.

30. Ribi, E., C. Larson, W. Wicht, R. List, and G. Goode.1966. Effective nonliving vaccine against experimentaltuberculosis in mice. J. Bacteriol 91:975-983.

31. Smith, J. B., A. J. Cunningham, K. J. Lafferty, andB. Morris. 1970. The role of the lymphatic system andlymphoid cells in the establishment of immunologicalmemory. Aust. J. Exp. Biol. Med. Sci. 48:57-70.

32. Snedecor, G. W., and W. G. Cochran. 1967. Statistical

IMMUNITY INDUCED BY BCG 653

methods, 6th ed. The Iowa State University Press,Ames.

33. Spencer, J. C., R. H. Waldmani, and J. E. Johnson.1974. Local and systemic cell-mediated immunity afterimmunization of guinea pigs with live or killed M.tuberculosis by various routes. J. Immunol.112:1322-1328.

34. Truitt, G. L, and G. B. Mackaness. 1971. Cell-mediatedresistance to aerogenic infection of the lung. Am. Rev.Respir. Dis. 104:829-843.