Mechanisms of Specific Immunological Unresponsiveness to Bacterial Lipopolysaccharides

Transcription

1 INFECTION AND IMMUNITY, Dec. 1987, P /87/ $02.00/0 Copyright C) 1987, Americn Society for Microbiology Vol. 55, No. 12 Mechnisms of Specific Immunologicl Unresponsiveness to Bcteril Lipopolyscchrides KAREN L. ELKINS,* PHILIP W. STASHAK, AND PHILLIP J. BAKER Lbortory of Microbil Immunity, Ntionl Institute of Allergy nd Infectious Diseses, Bethesd, Mrylnd Received 20 July 1987/Accepted 15 September 1987 Low-dose priming of mice with Escherichi coli 0113 lipopolyscchride (LPS) results in the development of immunologicl memory, wheres low-dose priming with E. coli 055 LPS or Serrti mrcescens LPS induces significnt ntigen-specific unresponsiveness. All three preprtions of LPS induced prolifertion of mouse splenocytes with similr time course nd [3H]thymidine uptke. There ws no correltion between the smll mounts of serum ntibody detected by enzyme-linked immunosorbent ssy fter low-dose priming nd the subsequent genertion of either memory or unresponsiveness. Further, the pssive trnsfer of smll mounts of LPS-specific ntibody hd no significnt effect on the mgnitude of the plque-forming cell (PFC) response elicited fter subsequent immuniztion. Reduction of the PFC response to E. coli 055 LPS occurred fter low-dose priming of nulnu (s well s nul+) mice; however, unresponsiveness could not be generted in nu/nu mice by low-dose priming with S. mrcescens LPS. Thus, lthough the development of low-dose unresponsiveness to S. mrcescens LPS ppers to involve T cells, the response to E. coli 055 LPS does not. Enhncement of the primry PFC response to S. mrcescens LPS could be trnsferred with low-dose-primed spleen cells depleted of Lyt-2+ T cells; this suggests tht the mgnitude of the PFC response to this preprtion of LPS is negtively influenced by Lyt-2+ T cells nd positively influenced by Lyt-2- spleen cells (i.e., L3T4+ T cells). These findings indicte tht T cells pper to be involved in regulting the mgnitude of the ntibody response to some types of bcteril LPS. Previous studies hve shown tht the mgnitude of the specific ntibody response to some types of bcteril lipopolyscchride (LPS) cn be incresed or decresed by prior exposure (priming) with single injection of subimmunogenic dose of LPS (4, 14, 37, 39, 50, 51). These effects re not due to chnges in the isotype of the ntibody produced fter immuniztion or to ltertions in the time course of the plque-forming cell (PFC) response (14). The genertion of memory or unresponsiveness is specific for the serotype of the LPS used for priming (14), finding tht is consistent with erlier work showing tht immunologicl memory could be induced by priming with the nonmitogenic, 0-polyscchride portion of the Escherichi coli 0113 LPS molecule (ntive protoplsmic polyscchride or NPP; 50). In experiments using mice tht were low-dose primed nd then immunized with suboptiml, optiml, or suproptiml mounts of E. coli 055 or Serrti mrcescens LPS, the mgnitude of the specific PFC response to ll immunizing doses of homologous LPS tested ws reduced by low-dose priming (14). This suggested tht unresponsiveness is not due merely to the removl of injected LPS by circulting ntibody, since it could thus not be brogted by immuniztion with lrge mounts of LPS. We further considered it unlikely tht unresponsiveness ws due to ntibodymedited feedbck, becuse (i) few or no specific PFC could be detected in the spleen fter low-dose priming with ny LPS used, suggesting tht little circulting LPS-specific serum ntibody is produced upon priming, wheres reltively lrge mounts re involved in ntibody-medited feedbck (7, 47); nd (ii) unresponsiveness could be elicited by immuniztion only 2 dys fter priming with subimmunogenic dose of LPS, i.e., t time when little serum ntibody could be present. Becuse preexisting specific serum ntibody hs been reported to both increse nd * Corresponding uthor decrese the mgnitude of the ntibody response upon immuniztion with ntigen (reviewed in reference 47), prticulrly in the cse of E. coli 055 LPS (7), we exmined the reltionship between ny LPS-specific serum ntibody produced in response to low-dose priming nd the expression of unresponsiveness or memory. We further evluted the contributions of T-cell popultions. The results indicte tht specific ntibody-medited feedbck does not contribute to the genertion of memory or unresponsiveness. Antigenspecific unresponsiveness to S. mrcescens LPS my be regulted by T cells, wheres regultion of the mgnitude of the PFC response to E. coli 0113 or E. coli 055 LPS is not nd ppers to be more complex. MATERIALS AND METHODS Mice. Femle BALB/cByJ mice were purchsed from Jckson Lbortories (Br Hrbor, Mine); they were 8 to 12 weeks of ge t the time of use. Femle BALB/c nulnu nd nul+ mice (mtched littermtes) were obtined from the Ntionl Institutes of Helth Smll Animl Section nd were 8 to 10 weeks of ge t the time of use. Antigens. All preprtions of LPS used were extrcted from cell wlls of bcteri by the phenol-wter procedure (27, 38). A preprtion of E. coli 0113 LPS, used throughout, ws gift of J. A. Rudbch, Ribi ImmunoChem (Hmilton, Mont.); its immunologicl properties hve been previously described (4, 22, 23, 38, 39, 50, 51). E. coli 055 LPS (lots RIR-326, RIR-342, nd ) nd S. mrcescens LPS (lots RIR-322 nd RIR-332) were purchsed from Ribi ImmunoChem. These preprtions of LPS ppered to be devoid of lipid A-ssocited protein, since they were not mitogenic in vitro for C3H/HeJ lymphocytes nd were not immunogenic in vivo for C3H/HeJ mice; no protein could be detected by UV spectroscopy or by chemicl (Lowry) nlysis (dt not shown). All solutions of LPS nd other regents used in vivo in this study were prepred in sterile,

2 3094 ELKINS ET AL. pyrogen-free sline (Abbott Lbortories, Chicgo, Ill.). Mice were immunized intrperitonelly (i.p.) with the doses of LPS indicted in the text. Detection of ntibody-producing cells. Antibody-producing PFC secreting immunoglobulin M (IgM) ntibody specific for LPS were detected by slide version of the technique of loclized hemolysis-in-gel (5, 6). Sheep erythrocytes were sensitized with the pproprite LPS by the chromic chloride coupling method (6), using 1 mg of LPS to sensitize 0.5 ml of pcked erythrocytes. The results obtined for individul mice (PFC per spleen) were corrected by subtrction for smll numbers of sheep erythrocyte-specific PFC detected, so tht only vlues for LPS-specific PFC re considered in this report. Since the vlues for PFC per spleen re log normlly distributed (18), geometric mens of the vlues for groups of similrly treted mice were clculted. The dt re expressed s the men log1o LPS-specific PFC per spleen ± stndrd error of the men (SEM) for groups of 5 to 10 mice or s the ntilog (ctul PFC) of the men. Student's t test ws used to evlute the significnce of the differences observed (44). Differences were considered to be significnt when probbility (P) vlues of <0.05 were obtined. Lymphocyte stimultion. Prolifertive responses to LPS, B-cell mitogen, were mesured by [3H]thymidine incorportion (49). Splenocytes obtined from norml BALB/cByJ femle mice were cultured in 96-well flt-bottomed microtiter pltes (Costr, Cmbridge, Mss.) t 8 x 105 cells per well in serum-free RPMI 1640 (Meditech/Sybron, Wshington, D.C.; <10 pg of endotoxin per ml) contining 100 U of penicillin per ml, 100,ug of streptomycin per ml, 2 mm glutmine, nd 5 x 10-5 M 2-mercptoethnol. After 2 dys, the cells were pulsed for 6 h with 0.5,uCi of [3H]thymidine (6.7 Ci/nmol; New Englnd Nucler Corp., Boston, Mss.) per well. The smples were hrvested onto glss-fiber filter ppers nd counted in Econofluor-2 scintilltion fluid (New Englnd Nucler Corp.) in Beckmn LS-8100T liquid scintilltion counter. ELISA. Ser obtined from mice bled vi the lterl til vein were ssyed for nti-lps ntibody by serotypespecific enzyme-linked immunosorbent ssy (ELISA). Immulon I microtiter pltes (Dyntech, Alexndri, V.) were coted with 1 p,g of E. coli 0113 LPS, E. coli 055 LPS, or S. mrcescens LPS, diluted in phosphte-buffered sline (ph 7.4), per well nd incubted overnight t 4 C. Pltes were wshed with solution of 0.15 M NCl-0.05% Tween 20 nd blocked for 30 min t 37 C with solution of 10% newborn clf serum in phosphte-buffered sline. Seril twofold dilutions of test nd control ser (50 RI per well) were dded, nd the pltes were incubted for 90 min t 37 C nd then wshed. Horserdish peroxidse-lbeled got nti-mouse IgM, got nti-mouse IgG, got nti-mouse IgA, or got nti-mouse immunoglobulin (100,ul per well; Southern Biotechnology, Birminghm, Al.; specificity nd optiml dilutions, determined in preliminry experiments, rnging from 1:500 to 1:4,000) ws dded, nd pltes were incubted for 90 min t 37 C. Color ws developed by the ddition of substrte of ABTS [2,2'-zinobis(3-ethylbenzthizolinesulfonic cid); Sigm Chemicl Co., St. Louis, Mo.] in 0.05 M citric cid-0.05 M N2HPO4 buffer (ph 4.0) with 0.06% hydrogen peroxide. The enzymtic rection ws stopped fter bout 15 min by the ddition of 50,ul of 1.5% KFl per well, nd pltes were red on MR600 plte reder (Dyntech) with 410-nm test wvelength nd 490-nm reference wvelength. Becuse purified preprtions of nti-lps ntibody were unvilble for use s stndrd, the ELISA titer ws INFECT. IMMUN. estblished by compring the opticl density redings of the test serum smple with those of norml mouse serum; in ll cses, norml mouse ser were prepred from individul mice before immuniztion with LPS. In the present work, the titer is designted s the lst seril dilution tht is.2 times the opticl density of the mtched dilution of norml mouse serum, s well s reding of opticl density unit fter subtrcting bckground vlues (the verge of eight control wells contining developing regents but no serum smple). The dt re expressed s the men log2 titer ± SEM for groups of five or six mice or s the ntilog (titer) of the men. No binding greter thn bckground, or greter thn mtched dilutions of norml mouse serum, of immune mouse ser ws observed on uncoted pltes or pltes coted with n irrelevnt ntigen, type III pneumococcl polyscchride (SSS-111) (dt not shown). Cell trnsfer studies. Single-cell suspensions were prepred from spleens of BALB/cByJ mice tht were low-dose primed with S. mrcescens LPS (5 ng i.p.) 5 dys previously. Cell popultions were depleted by two rounds of tretment with the indicted monoclonl ntibodies for 30 min on ice, followed by tretment with 1:10 rbbit complement (Low- Tox; Cedrlne, Hornby, Ontrio, Cnd) for 45 min t 37 C. All tretments were performed t densities of 108 cells per ml in RPMI 1640 contining 2% fetl bovine serum. Monoclonl ntibodies nd their concentrtions used for depletion were: RL172.4, or nti-l3t4 (10), tissue culture superntnt diluted 1:2; 19/178, or nti-lyt-2.2 (21), 25-fold concentrte of tissue culture superntnt diluted 1:10; nd nti-thy-1.2 (New Englnd Nucler Corp.), diluted 1:500. Optiml concentrtions of ll ntibodies nd complement for tretment were determined in preliminry experiments. Depletion ws confirmed in ech experiment by fluorescencectivted cell sorter nlysis using fluorescein-conjugted monoclonl ntibodies (nti-lyt-2, nti-thy-1.2, nd nti- IgM; Becton-Dickinson, Mountin View, Clif.) or ffinitypurified nti-l3t4 monoclonl ntibody (GK1.5; 13) nd fluorescein-conjugted got nti-rt IgG ntibody (Southern Biotechnology). Contmintion with residul L3T4+, Lyt- 2+, or Thy-1.2+ cells, s pproprite, ws routinely less thn 2%. Cells were injected intrvenously (i.v.) through lterl til vein into recipient BALB/cByJ mice, which were immunized (i.p.) with 0.1 or 1,ug of S. mrcescens LPS, s indicted, 1 h fter cell trnsfer. RESULTS Mitogenic response to E. coli 0113 LPS, E. coli 055 LPS, nd S. mrcescens LPS. The prolifertive response of murine B cells to LPS is primrily result of the recognition of lipid A (25-27, 31, 45), whose chemicl structure is similr between different types of LPS from members of the fmily Enterobctericee (27). The mgnitude of the prolifertive response is lso influenced by the composition of the polyscchride portion of the molecule (17, 52). Therefore, we compred the mitogenic response of BALB/cByJ spleen cells to these preprtions of LPS to determine whether differences in their mitogenicity exist which might relte to their bility to induce unresponsiveness or memory. Spleen cells were cultured in stndrd prolifertion ssy (49) with different mounts of LPS nd pulsed with [3H]thymidine fter 2 dys. The results showed tht ll preprtions of LPS tested induced moderte prolifertive response, with comprble dose-response reltionships (Fig. 1). Pek responses were obtined with 20 to 100,ug of E. coli 0113 LPS, E. coli 055 LPS, or S. mrcescens LPS per ml, nd the mgnitude

3 VOL. 55, 1987 REGULATION OF UNRESPONSIVENESS TO LPS ,000 40,000 36,000' ~20,000 15,000 10,00 5,00 I I I ) E. c ON US cells per well, with purified B cells, or with cells pulsed 1, 3, or 4 dys fter the strt of cultures (dt not shown). Role of LPS-specific serum ntibody in the induction of memory or unresponsiveness fter low-dose priming. Previous studies indicted tht few, if ny, LPS-specific splenic PFC could be detected t ny time point fter low-dose priming (14), suggesting tht little serum ntibody is produced upon low-dose priming. However, since it hs been reported tht serum ntibody cn modulte the ntibody response pro- 0 E. =W 016 LPS duced upon subsequent immuniztion with E. coli 055 LPS (7), we sought to determine whether smll mounts of specific serum ntibody produced fter low-dose priming might contribute to the development of either LPS-specific S. & M LPS memory or unresponsiveness. Two pproches were used to exmine this issue. First, serum smples were collected from low-dose-primed mice t vrious times fter priming; these mice were then immunized with n optiml dose of LPS, nd 4 dys fter immuniztion, the mgnitude of the serum ntibody nd PFC responses ws determined. This permitted the correltion of the mgnitude of the PFC response produced with serum ntibody levels t the time of immuniztion. Second, pools of serum were obtined from mice given low doses of LPS; smples of serum from these pools were pssively trnsferred to recipient mice which were simultneously immunized with n optiml dose of LPS. To determine the reltionship between serum ntibody nd the genertion of LPS-specific memory in situ, groups of mice were low-dose primed.(dy 0) with 50 ng of E. coli 0113 LPS or with sline. Individul serum smples were collected , 4, nd 8 dys fter priming; the mice were then immunized pg/mi LPS with 1,ug of E. coli 0113 LPS (or sline) 8 dys fter priming (when memory cn be redily elicited; 4). Ser were col- of spleen lected nd PFC responses were determined 4 dys fter FIG. 1. Dose-response reltionships for stimulltion cell prolifertion by E. coli 0113, E. coli 055, nd. S. mrcescens immuniztion (dy 12). Low-dose priming with 50 ng of E. LPS. BALB/cByJ spleen cells were cultured s descarbed in Mterils nd Methods with the indicted mounts of th4 coli 0113 LPS resulted in the production of very smll, but e vrious LPS detectble, mounts of E LPS-specific coli preprtions. After 2 dys of incubtion, the cells were pulsed for 6 h with 0.5,uCi of [3H]thymidine per well, hrvested ntibody (Tble 1). Men IgM serum titers of E. coli 0113 SEM ws less thn 10% in ll cses, but is omittedi for clrity of LPS-specific ntibody rnged from 1:40 to 1:60 4 dys fter presenttion. Symbols: 0, E. coli 0113 LPS; 0, E. coli 055 LPS; X, low-dose priming, nd ntibody ws still present 8 dys fter S. mrcescens LPS. priming, i.e., t the time of immuniztion; "priming" with sline did not result in the formtion of detectble LPSspecific serum ntibody. No LPS-specific IgM serum ntibody could be detected 2 dys fter low-dose priming, nd of the response t different doses ws similr for different no LPS-specific serum IgG or IgA ws detected t 4 nd 8 preprtions of LPS. The reltively greter pelk response to dys fter low-dose priming (dt not shown). Immuniztion E. coli 055 LPS ws consistently observed in ( other ssys, of the sline-primed group with 1,ug of E. coli 0113 LPS lthough the mgnitude of the difference ws uisully not s resulted in men PFC response of 18,535 PFC per spleen lrge s tht noted in this prticulr experimernt. The sme nd men IgM serum ntibody titer of 1:320; smll mounts trends were found when the ssy ws perform ed in medium of LPS-specific serum IgG could lso be detected. Immuninumbers of ztion of the low-dose primed group with 1,ug of E. contining fetl bovine serum, with different coli Priming TABLE 1. Dy 4 IgM titer Low-dose priming with E. coli 0113 LPS genertes incresed ntibody responses' Dy 8 Dy 12 IgM titer Immuniztion PFC/spleen IgM titer IgG titer LPS 1:60 1:80 Sline 55 1:53 1:61 (1.783 ± 0.153) (1.903 ± 0.165) (1.736 ± 0.710) ± 0.153) (1.783 ± 0.120) Sline <1:20b <1:20 LPS 18,535 1:320 1:85 (4.268 ± 0.047) (2.505 ± 0.135) (1.929 ± 0.214) LPS 1:40 1:46 LPS 61,281 1:1,114 1:279 (1.602 ± 0.135) (1.662 ± 0.176) (4.787 ± 0.055) ( ) (2.445 ± 0.140) BALB/cByJ femle mice were injected (i.p.) with 50 ng of E. coli 0113 LPS or sline on dy 0. They were individully bled 2, 4, nd 8 dys lter nd immunized (i.p.) with 1,Lg of E. coli 0113 LPS on dy 8. All mice were individully bled nd LPS-specific PFC responses were determined 4 dys fter immuniztion (dy 12). IgM titer dt re geometric mens of individul serum titers; men log2 titer ± SEM for groups of five mice is shown in prentheses. PFC/spleen dt re geometric mens of individul PFC per spleen; men log10 PFC per spleen + SEM for groups of five mice is shown in b prentheses. Below the limit of detection (1:20 serum dilution).

4 3096 ELKINS ET AL LPS resulted in typicl memory response (4, 37, 39, 50, 51) with men of 61,281 PFC per spleen nd men IgM serum ntibody titer of 1:1,114; these vlues were significntly greter thn the corresponding responses of unprimed, immunized mice (P < nd P < 0.01, respectively). The LPS-specific IgG serum ntibody response lso incresed, to men of 1:279, lthough this difference ws not significnt (P > 0.05). Very few PFC (55 PFC per spleen) were detected 12 dys fter low-dose priming lone. However, smll mounts of LPS-specific serum IgM (1:53) were still detected 12 dys fter priming, when smll mounts of LPS-specific serum IgG lso could be detected (1:61). Therefore, the development of memory to E. coli 0113 LPS occurs in the presence of smll but detectble mounts of E. coli 0113 LPS-specific IgM serum ntibody. In ddition to the LPS-specific IgM ntibody response, smll but detectble mounts of LPS-specific IgG serum ntibody could be detected fter both primry nd secondry immuniztions. No LPS-specific IgA ntibody ws detected throughout the course of the study (dt not shown). Studies of the serum ntibody responses to E. coli 055 LPS (Tble 2) nd S. mrcescens LPS (dt not shown) lso indicted tht smll mounts of LPS-specific serum IgM could be detected fter low-dose priming, even though subsequent immuniztion with these LPS preprtions resulted in the genertion of immunologicl unresponsiveness. Groups of mice were low-dose primed (dy 0) with 2 ng of E. coli 055 LPS (or sline), nd individul serum smples were collected 2 nd 4 dys lter. Groups of mice were immunized with 1,ug of E. coli 055 LPS (or sline) 6 dys fter low-dose priming; ser were collected nd PFC responses were determined 4 dys fter immuniztion (dy 10). No E. coli 055 LPS-specific serum ntibody could be detected within 2 dys fter low-dose priming (Tble 2). Low-dose priming (with 2 ng of E. coli 055 LPS) resulted in the development of detectble mounts of LPS-specific IgM serum ntibody 4 dys fter priming (men IgM ntibody titers, 1:121 to 1:211); no ntibody ws detected in mice given only sline. No LPS-specific IgG or IgA serum ntibody could be detected t either time point fter low-dose priming (dt not shown). Immuniztion of low-dose-primed mice with 1,ug of E. coli 055 LPS on dy 6 resulted in significntly reduced PFC responses (68,507 PFC per spleen) s compred with sline-primed, immunized control mice (298,299 PFC per spleen) (P < 0.01). The LPS-specific IgM serum ntibody responses of low-dose-primed, immunized mice (men IgM titer of 1:4,457) were lso significntly reduced compred with those of sline-primed mice (men IgM serum ntibody titer of 1:13,512; P < 0.05), lthough the mgnitude of the reduction of the serum ntibody response ws less thn tht INFECT. IMMUN. of the PFC response. Very smll mounts of LPS-specific serum IgG lso were detected fter immuniztion; the mgnitude of these titers did not pper to be ffected by low-dose priming (P > 0.05). No LPS-specific IgA serum ntibody ws detected fter priming or immuniztion (dt not shown). Low-dose priming lone resulted in smll numbers of detectble LPS-specific PFC nd smll mounts of LPS-specific IgM serum ntibody. Experiments following the sme formt were conducted using S. mrcescens LPS, with comprble results. Smll mounts of LPS-specific IgM were detected 4 (but not 2) dys fter low-dose priming, nd subsequent immuniztion with S. mrcescens LPS resulted in reduced IgM serum ntibody titers in primed mice s compred with unprimed control mice (dt not shown). Tken together, the results indicte tht low-dose priming with ny of the LPS preprtions used results in the development of smll mounts of LPS-specific IgM serum ntibody (Tbles 1 nd 2 nd dt not shown), even though few LPS-specific PFC re detected in the spleen t ny time fter low-dose priming (14). LPS-specific IgM serum ntibody could be detected within 4 dys fter low-dose priming, but not within 2 dys fter low-dose priming. Immuniztion with n optiml dose of ny of the LPS preprtions used lso resulted in the development of smll mounts of LPS-specific IgG serum ntibody (even though no LPS-specific IgG PFC could be detected in the spleen; 14), in ddition to reltively lrge mounts of LPS-specific IgM serum ntibody, within 4 dys fter immuniztion. Low-dose priming with either E. coli 055 LPS or S. mrcescens LPS significntly reduced the mgnitude of the serum ntibody response to the sme preprtion of LPS, lthough pprently not to the sme degree s the PFC response ws reduced. It should be noted tht no direct correltion ws observed between the mount of LPSspecific IgM serum ntibody detected in the serum of n individul low-dose-primed mouse nd the degree of memory or unresponsiveness expressed upon subsequent immuniztion (dt not shown). Thus, there do not pper to be mjor qulittive differences between ny of these LPS preprtions with regrd to the genertion of serum ntibody fter low-dose priming, despite their functionl differences in the genertion of memory or unresponsiveness. The influence of these smll mounts of LPS-specific serum ntibody on the mgnitude of the response to immuniztion with n optiml dose of LPS lso ws studied by pssively trnsferring serum contining smll mounts of nti-lps ntibody into recipient mice t the time of immuniztion. The mounts used were chosen to pproximte those detected fter low-dose priming. Groups of BALB/ cbyj mice were injected with 50 ng of E. coli 0113 LPS; TABLE 2. Low-dose priming with E. coli 055 LPS genertes reduced ntibody responses' Dy 2 Priming IgM titer Dy 4 IgM titer Dy6Dy Dy 6 PFC/spleeniIgMotiternIgG_titer 10 immuniztion IgG LPS <1: 20b 1:211 Sline 2,671 1:368 <1: ob (2.325 ± 0.181) (3.427 ± 0.138) (2.565 ± 0.176) Sline < 1: 20b < 1: 20b LPS 298,299 1:13,512 1:17 (5.475 ± 0.045) (4.131 ± 0.074) (1.241 ± 0.241) LPS <1:20b 1:121 LPS 68,507 1:4,457 1:15 (2.084 ± 0.399) (4.836 ± 0.174) (3.659 ± 0.241) (1.181 ± 0.181) BALB/cByJ femle mice were injected (i.p.) with 2 ng of E. coli 055 LPS or sline on dy 0. They were individully bled 2 nd 4 dys lter nd immunized (i.p.) with 1 p.g of E. coli 055 LPS or sline on dy 6. All mice were individully bled nd LPS-specific PFC responses were determined 4 dys fter immuniztion (dy 10). IgM nd IgG titers re geometric mens of individul serum titers; men log2 titer ± SEM for groups of five mice is shown in prentheses. PFC/spleen dt re geometric mens of individul PFC per spleen; men loglo PFC per spleen ± SEM for groups of five mice is shown in prentheses. I Below limit of detection (1:20 serum dilution for IgM ssy, 1:10 serum dilution for IgG ssy).

5 VOL. 55, 1987 their ser were collected nd pooled 8 dys fter priming, i.e., t time when memory is demonstrble (see Tble 1). For comprison, nother group of mice ws immunized with n optiml dose (10 jig of E. coli 0113 LPS), nd their ser were collected 5 dys fter immuniztion, t the pek of the serum ntibody response (37). The E. coli 0113 LPS-specific titers were determined by ELISA; the titers of these prticulr pools were found to be similr to, but slightly greter thn, the men of groups of individul mice (Tble 1). Thus, the mount of LPS-specific ntibody trnsferred (i.v.) in 0.2 ml of these serum pools (bout one-fifth of the totl munrne blood volume) pproximtes the mounts found in situ fter low-dose priming. The trnsfer of norml mouse serum hd no significnt effect (P > 0.05) on the mgnitude of the PFC response to n optiml dose of E. coli 0113 LPS (Tble 3). The trnsfer of ser from mice given n optiml dose of LPS significntly decresed (P < 0.01) the mgnitude of the PFC response to n optiml dose of E. coli 0113 LPS; this observtion is similr to tht previously reported for the effect of reltively lrge mounts of LPS-specific serum ntibody on the response to E. coli 055 LPS (7). However, the trnsfer of ser from low-dose-primed mice only slightly decresed the mgnitude of the response to E. coli 0113 LPS, nd this slight decrese ws not significnt (P > 0.05). Certinly the trnsfer of this smll mount of E. coli 0113 LPS-specific serum ntibody did not produce memory response s ws observed fter low-dose priming nd immuniztion with E. coli 0113 LPS. The effect of pssive trnsfer of LPS-specific serum ntibody on the responses to E. coli 055 LPS nd S. mrcescens LPS ws lso studied. As documented previously (7), the trnsfer of reltively lrge mounts of E. coli 055 LPSspecific serum ntibody reduced the mgnitude of the LPSspecific PFC response; however, the trnsfer of ser from low-dose-primed mice hd no significnt effect on the mgnitude of the LPS-specific PFC response (dt not shown). Similrly, pools of ser were collected from mice given low dose (5 ng) or n optiml dose (1 jig) of S. mrcescens LPS; these were ssyed for LPS-specific ntibody nd pssively TABLE 3. Effect of pssive trnsfer of nti-e. coli 0113 immune serum on the primry PFC response to E. coli 0113 LPS0 Mteril trnsferred PFC/spleenb % Responsec Medium (BSS) ± (14,198) Optiml-dose serum ± (4,716) Low-dose serum (8,824) NMS pool ± (13,999) First, ser were obtined s follows. BALB/cByJ femle mice were bled before immuniztion or priming to obtin pool of norml mouse serum (NMS). One group ws immunized (i.p.) with 10 Fg of E. coli 0113 LPS per mouse nd bled 5 dys lter (optiml-dose serum pool); nother ws given (i.p.) 50 ng of E. coli 0113 LPS per mouse nd bled 8 dys lter (low-dose serum pool). Anti-E. coli 0113 IgM, IgG, nd IgA titers of pooled ser, determined in n isotype-specific ELISA, were 1:1,280, 1:160, nd <1:10, respectively, for optiml-dose ser nd 1:320, 1:10, nd <1:10, respectively, for low-dose ser. Then BALB/cByJ femle mice were injected (i.v.) with 0.2 ml of blnced slt solution (BSS), 0.2 ml of the optiml-dose serum pool, 0.2 ml of the low-dose serum pool, or 0.2 ml of norml mouse serum ( 1:1 pool of prebleed ser from optiml-dose nd low-dose mice). Mice were immunized (i.p.) with 10 jlg of E. coli 0113 LPS immeditely fter serum trnsfer, nd LPS-specific PFC responses were determined 4 dys lter. b Men logl0 LPS-specific PFC per spleen ± SEM for groups of eight mice; geometric mens re shown in prentheses. c Reltive to the medium-injected control group. REGULATION OF UNRESPONSIVENESS TO LPS 3097 TABLE 4. Effect of pssive trnsfer of nti-s. mrcescens immune serum on the primry PFC response to S. mrcescens LPS Mteril trnsferred PFC/spleenb % Responsec Medium (25,432) Optiml-dose serum (13,802) Low-dose serum ± (21,470) NMS pool ± (26,066) First, ser were obtined s described in Tble 3, footnote, except tht optiml-dose ser were obtined from mice immunized with 1,zg of S. mrcescens LPS per mouse nd bled 5 dys lter nd the low-dose ser were from mice given 5 ng of S. mrcescens LPS per mouse nd bled 5 dys lter. Anti-S. mrcescens IgM, IgG, nd IgA titers, determined in n isotypespecific ELISA, were 1:5,120, 1:160, nd <1:10, respectively, for optimldose ser nd 1:640, <1:10, nd <1:10, respectively, for low-dose ser. Then, BALB/cByJ femle mice were injected (i.v.) with medium or serum s described in Tble 3, footnote. They were immunized (i.p.) with 1 p.g of S. mrcescens LPS immeditely fter serum trnsfer, nd LPS-specific PFC responses were determined 4 dys lter. b Men loglo LPS-specific PFC per spleen ± SEM for groups of seven mice; geometric mens re shown in prentheses. c Reltive to the medium-injected control group. trnsferred into recipients which were then immunized with n optiml dose of S. mrcescens LPS. Wheres the titers of the pools pproximted those found in situ fter immuniztion (Tble 4 nd dt not shown), only trnsfer of the ser from mice immunized with the optiml dose of LPS significntly ffected the mgnitude of the LPS-specific PFC response to n optiml dose of S. mrcescens LPS (Tble 4; P < 0.01). Repeted experiments of similr design for ech preprtion of LPS gve comprble results. Therefore, the elbortion of smll mounts of LPS-specific serum ntibody fter low-dose priming does not significntly influence the development of memory or unresponsiveness upon immuniztion with these preprtions of LPS. Cellulr components of the development of memory or unresponsiveness. Previous work hs shown tht memory to E. coli 0113 LPS cn be generted in T-cell-deficient nulnu mice, suggesting tht functionl T cells re not required in the genertion of memory (50). However, it is possible tht the unresponsiveness observed fter low-dose priming with E. coli 055 LPS or S. mrcescens LPS my be medited by T cells nd tht this would preclude the development of memory. To test this possibility, groups of BALB/c nulnu nd nul+ mice were primed with 500 pg of E. coli 055 LPS ( different lot from tht used for Tble 2); they were then immunized with n optiml dose (1,ug) of E. coli 055 LPS 6 dys lter, nd PFC responses were ssessed 4 dys fter immuniztion. The response to E. coli 055 LPS ws significntly (P < 0.01) reduced by low-dose priming with E. coli 055 LPS in both nulnu nd nul+ mice (Tble 5); the degree of unresponsiveness observed ws ctully greter in the nulnu thn in the nul+ mice in this prticulr experiment. This indictes tht T cells re not involved in generting the observed unresponsiveness to E. coli 055 LPS. However, this ws not the cse when similr experiment ws performed using S. mrcescens LPS (Tble 6). Although lowdose priming significntly -(P < 0.01) reduced the response to n optiml dose (1 jg) of S. mrcescens LPS in BALB/c nul+ mice, low-dose priming hd no effect on the mgnitude of the response to n optiml dose in BALB/c nulnu mice. Repeted experiments of ech type gve comprble results.

6 3098 ELKINS ET AL. TABLE 5. Low-dose priming with E. coli 055 LPS reduces the mgnitude of the PFC response to n optiml dose in nulnu mice' Tretment Mice PFC/spleenb % Response' Priming Immuniztion BALB/c nu/+ LPS Sline ± (334) Sline LPS ± (41,759) LPS LPS ± (19,404) BALB/c nulnu LPS Sline ± (271) Sline LPS ± (84,524) LPS LPS ± (12,373) BALB/c femle mice, s indicted, were primed (i.p.) with 500 pg of E. coli 055 LPS (LPS) or sline (Sline); 6 dys lter, they were immunized (i.p.) with 1 plg of E. coli 055 LPS (LPS) or sline (Sline). PFC responses specific for E. coli 055 LPS were determined 4 dys fter immuniztion. b Men loglo LPS-specific PFC per spleen ± SEM for groups of five mice; geometric mens re shown in prentheses. c Reltive to the sline-primed, immunized controls. Thus, T cells my ply centrl role in the development of unresponsiveness to S. mrcescens LPS, nd regultion of the ntibody response to S. mrcescens LPS my be qulittively different from tht observed for E. coli 055 LPS. Since the inbility to induce unresponsiveness to S. mrcescens LPS in nulnu mice suggested tht the ntibody response to this ntigen might be regulted by T cells, cell trnsfer experiments were conducted to obtin direct informtion in this regrd. The trnsfer of low-dose-primed whole spleen cell popultions gve vrible results, which suggested the presence of competing positive or negtive influences in the popultions of primed spleen cells trnsferred (dt not shown). Therefore, experiments using primed spleen cells depleted of vrious subpopultions of T cells were conducted. Groups of mice were primed with low dose (5 ng) of S. mrcescens LPS. Five dys lter, one group ws scrificed nd spleen cell suspensions were prepred. TABLE 6. Low-dose priming with S. mrcescens LPS does not reduce the mgnitude of the PFC response to n optiml dose in nulnu mice Tretment Mice PFC/spleenb % Response' Priming Immuniztion BALB/c nul + LPS Sline (2,518) Sline LPS ± (23,543) LPS LPS ± (9,314) BALB/c nulnu LPS Sline ± (1,546) Sline LPS ± (15,893) LPS LPS ± (16,671) BALB/c femle mice, s indicted, were primed (i.p.) with 5 ng of S. mrcescens LPS (LPS) or sline (Sline); 5 dys lter, they were immunized (i.p.) with 1,ug of S. mrcescens LPS (LPS) or sline (Sline). PFC responses specific for S. mrcescens LPS were determined 4 dys fter immuniztion. b Men loglo LPS-specific PFC per spleen ± SEM for groups of six mice; geometric mens re shown in prentheses. C Reltive to the sline-primed, immunized controls. The cells were treted with (i) complement lone, (ii) nti- L3T4 monoclonl ntibody (RL172.4)' plus complement, (iii) nti-lyt-2.2 monoclonl ntibody (19/178) plus complement, or (iv) mixture of these ntibodies (nti-thy-1.2, RL172.4, nd 19/178) plus complement. The'efficcy of T-cell depletion ws confirmed by fluorescence-ctivted cell sorter nlysis (dt not shown). After tretment with monoclonl ntibody, 2 x 107 remining vible cells were trnsferred (i.v.) into recipient mice, which were immunized 1 h lter with 0.1,ug of S. mrcescens LPS (i.p.). Here, we elected to use suboptiml dose of LPS for immuniztion to detect positive s well s negtive effects tht might be produced by trnsferred cells; the results of other studies (14) showed tht the mgnitude of the ntibody response to suboptiml dose of LPS ws more redily reduced by low-dose priming thn tht elicited by n optiml dose. Comprble results were obtined in other experiments using mice immunized with n optiml mount (1 jig) of S. mrcescens LPS, however (dt not shown). The trnsfer of complement-treted spleen c'ells from low-dose-primed mice enhnced the mgnitude of the nti-s. mrcescens LPS PFC response (P c 0.05) (Tble 7). Tretment of primed spleen cells with nti-l3t4 ntibody plus complement removed the bility of the popultion to enhnce the mgnitude of the PFC response upon trnsfer, indicting tht L3T4+ T cells positively influenced th'e response. The remining cells (which re Lyt-2+ T cells, B cells, nd mcrophges) ppered to hve no effect (P > 0.05) on the mgnitude of the ntibody response. However, tretment of primed cells with nti-lyt-2 ntibody nd complement before trnsfer reveled n bility of the remining cells (L3T4+ T cells, B cells, nd mcrophges) to increse the PFC response (P < 0.05). Tht this effect is not due to B cells or mcrophges is shown by the fct tht the trnsfer of cells from which ll T cells were depleted did not lter the mgnitude of the ntibody response (P > 0.05). DISCUSSION INFECT. IMMUN. LPS, which is polymer composed of 0-ntigen polyscchrides, centrl core polyscchride, nd lipid A sec- TABLE 7. Enhncement of the nti-s. mrcescens LPS PFC response trnsferred with low-dose-primed, Lyt-2- spleen cells Mteril trnsferred PFC/spleenb % Responsec Medium ± (12,448) Complement-treted ± primed spleen cells (26,069) Anti-L3T4 + C'-treted ± primed spleen cells (13,983) Anti-Lyt-2 + C'-treted ± primned spleen cells (40,711) Anti-T cell + C'-treted ± primed spleen cells (13,190) S. mrcescens LPS ± (1,256) Spleen cells were obtined from BALB/cByJ femle mice 5 dys fter priming with 5 ng of S. mrcescens LPS (i.p.) nd were treted with the indicted ntibodies plus complement s described in Mterils nd Methods. Smples of 2 x 107 cells were injected (i.v.) into recipient BALB/cByJ mice. (Other mice primed simultneously were primed 5 dys lter [S. mrcescens LPS] s control for efficcy of priming.) Recipient mice were then immunized (i.p.) with 0.1,ug of S. mrcescens LPS 1 h fter cell trnsfer. bmen loglo LPS-specific PFC per spleen ± SEM for groups of seven mice; geometric mens re shown in prentheses. c Reltive to the medium-injected, immunized controls.

7 VOL. 55, 1987 tion, is n integrl component of grm-negtive bcteril membrnes (26, 27, 31). Anti-LPS ntibody, directed primrily ginst the' 0 ntigens, is produced in both humns nd mice during the course of bcteril infections, or s result of deliberte immuniztion with either whole bcteri or purified LPS. This ntibody my hve role in protection ginst disese, since pssively trnsferred polyclonl or monoclonl nti-lps ntibodies cn pro'tect mice ginst lethl infection with Pseudomons eruginos (40), E. coli Kl (36), nd severl strins of Slmonell typhimurium (12, 24, 33). LPS hs lso been widely studied s model thymus-independent ntigen (11). It is likely tht regultion of the ntibody response to LPS is complex nd qulittively different from tht for conventionl protein ntigens. In contrst to most protein ntigens, LPS is metbolized reltively slowly nd remins present in tissues for long periods of time (8, 9). A prticulrly unusul feture of ll LPS molecules is the presence of lipid A, which hs wide rnge of phrmcologicl nd djuvnt ctivities (25-27, 31, 38, 45); these ctivities could esily influence the production of nti-0-ntigen ntibody. Priming of mice with subimmunogenic doses of E. coli 0113 LPS, followed by immuniztion with n optiml dose of LPS week or more lter, results in IgM PFC responses tht re much greter thn those for the primry ntibody response (4, 23, 37, 39, 50, 51). Whole LPS must be used for immuniztion to elicit memory (50), nd memory cnnot be generted in LPS-defective C3H/HeJ mice (39). This suggests tht B-cell recognition of lipid A is importnt for the expression of memory. We therefore considered the possibility tht the genertion of B-cell memory to 0 polyscchrides is generl property of LPS preprtions nd is relted to the ctivtion of B cells by the lipid A portion of the LPS molecule. However, efforts to generte memory in mice to E. coli 055 LPS nd S. mrcescens LPS gve different results from those previously obtined with E. coli 0113 LPS: priming with subimmunogenic dose of these types of LPS resulted' in PFC responses tht were significntly reduced upon immuniztion with the sme LPS (14). The mounts of E. coli 055 LPS or S. mrcescens LPS required to prime for the development of reduced PFC responses were quite low (500 pg to 5 ng), nd priming reduced PFC responses to ll doses of LPS used to immunize. These effects were not due to ltertions in the time course of the ppernce of PFC or to chnge in the isotype of ntibody produced fter priming nd immuniztion (14). Differences between'the time required for the development of physiologicl tolernce (19, 20) to LPS (hours or weeks) nd the time required for the induction of low-dose unresponsiveness (dys), s well s differences in dose requirements, suggest tht physiologicl tolernce does not contribute to the development of ntigen-specific lowdose unresponsiveness to LPS. To understnd how priming with some preprtions of LPS cn result in the genertion of either immunologicl memory or unresponsiveness, the three preprtions of LPS used in our work were compred in severl wys. All were mitogenic for unseprted mouse spleen cells (Fig. 1), s well s for purified B cells (dt not shown), nd induced comprble mounts of [3H]thymidine incorportion. The polyscchride side chin composition of S. mrcescens LPS is more heterogeneous thn tht of either E. coli 0113 LPS or E. coli 055 LPS by sodium dodecyl sulfte-polycrylmide gel electrophoresis nlysis nd silver stining (dt not shown; 16, 35, 46). The physicl stte of isolted (or membrne-bound) LPS could influence the qulity of the REGULATION OF UNRESPONSIVENESS TO LPS 3099 LPS ntibody produced fter immuniztion. Others hve shown tht polyscchride-rich (rther thn lipid A-rich) hybrid mcromolecules stimulte less [3H]thymidine incorportion by murine spleen cells thn might be predicted on the b'sis of lipid A content; this suggests tht the 0-ntigen components of the LPS molecules hve negtive influence on nonspecific mitogenicity (17, 52). Although the mechnism for such n effect is unknown, it hs been proposed tht polyscchrides my msk the lipid A moiety, thereby interfering with the 'physicl- interction of the lipid with B-cell membrnes nd subsequent signl trnsduction (52).' Similrly, it is possible tht quntittive differences in polyscchride or lipid A content of n LPS preprtion could influence the genertion of specific ntibody responses to LPS. Antibody-medited feedbck inhibition is n importnt component of the ntibody response to LPS (7, 47), nd the ntibody mde fter immuniztion with n optiml dose of E. coli 055 LPS is thought to be responsible for the cyclic ppernce of PFC nd serum ntibody; LPS complexed with ntibody my be- removed from the circultion nd then relesed to stimulte further ntibody synthesis (7). Since the infusion of lrge mounts of IgM nti-e. coli 055 ntibody cn reduce the bility to mke n ntibody response (7), we crefully exmined the possibility tht smll mounts of ntibody produced s result of low-dose priming might exert negtive influence on the ntibody response elicited upon s'ubsequent immuniztion, even though no LPS-specific PFC could be detected fter priming (14). Smll mounts of IgM nti-lps ntibody were indeed detected by ELISA 4 dys fter low-dose priming (Tbles 1 nd 2 nd dt not shown); it is uncler whether this is due to sensitivity differences between the ELISA nd the PFC ssy or to production of specific ntibody outside the spleen. However, no reltionship ws noted between the mount of LPS-specific IgM ntibody detected in the serum of individul primed mice nd the degree of memory or unresponsiveness expressed upon subsequent immuniztion (dt not shown). Although serum ntibody responses lso were suppressed or enhnced by low-dose priming, the degree of difference in serum responses between primed nd unprimed mice did not seem to be s gret s the difference in PFC responses; it is possible'tht the less-ffected serum responses re the result of compenstory chnges following low-dose priming, such s extrsplenic ntibody production or chnges in the secretion rte of individul PFC. In the second series of experiments, ser obtined from low-dose-primed mice were trnsferred to recipient mice to ssess the effect of the smll mounts of ntibody present on the primry response to n optiml immunizing dose; lrger mounts of ntibody, obtined from mice immunized with n optiml dose of LPS, were lso trnsferred for comprison. Although nti-lps PFC responses could be reduced by the trnsfer of lrge mounts of homologous nti-lps ntibody (Tbles 3 nd 4 nd dt not shown; 7), smller mounts (comprble to tht present in situ fter low-dose priming) hd no effect on the mgnitude of the homologous nti-lps PFC response. Similr results were obtined with ech of the three types of LPS considered. It is therefore most unlikely tht the smll mounts of serum ntibody produced fter low-dose priming re responsible for the development of memory or unresponsiveness, becuse (i) only smll mounts of serum ntibody could be detected fter low-dose priming (Tbles 1 nd 2 nd dt not shown), wheres lrge mounts re required to demonstrte ntibody-medited feedbck inhibition (Tbles 3 nd 4 nd dt not shown; 7,

8 3100 ELKINS ET AL. 47); nd (ii) reduced responses were demonstrble in mice s erly s 2 dys fter priming with subimmunogenic dose of LPS, i.e., t time when no serum ntibody could be detected (Tbles 1 nd 2 nd dt not shown; 14). In other studies, we hve observed tht immuniztion of geneticlly defective CBA/N mice with E. coli 0113 LPS resulted in PFC response comprble to tht of norml CBA/CJ mice, wheres very few PFC could be detected in spleens of CBA/N mice fter immuniztion with E. coli 055 LPS or S. mrcescens LPS (K. L. Elkins, P. W. Stshk, nd P. J. Bker, mnuscript in preprtion). CBA/N mice hve n X-linked immunodeficiency tht results in poor ntibody responses to some ntigens nd incresed susceptibility to some pthogens (1, 32, 33, 42). The nture of the CBA/N defect remins unknown, but it hs been ttributed to incomplete mturtion in the B-cell comprtment, lck of B-cell subpopultion designted Lyb-5 (34, 42, 43), or both. A norml ntibody response to E. coli 0113 LPS is elicited in CBA/N mice (23); it is therefore possible tht the B-cell defect which results in bsence of ntibody responses to E. coli 055 LPS nd S. mrcescens LPS is relted to the bility of these preprtions of LPS to induce unresponsiveness in norml mice, in tht susceptibility to unresponsiveness my involve B-cell subpopultion bsent in CBA/N mice. Conversely, the development of memory to E. coli 0113 LPS, which my be function of the B-cell subpopultion or mturtion stte present in CBA/N mice, my be regulted in different fshion from tht of the other preprtions of LPS. Unresponsiveness ws consistently obtined in BALB/c nulnu mice fter low-dose priming with E. coli 055 LPS, but not with S. mrcescens LPS (Tbles 5 nd 6). This implies tht two different mechnisms exist for the genertion of unresponsiveness; one is T-cell dependent (in the cse of S. mrcescens LPS), wheres the other is not (in the cse of E. coli 055 LPS). We cnnot exclude the possibility tht some T-cell precursors known to be present in nulnu mice (15, 41) my hve differentited to functionlly ctive T cells upon priming with E. coli 055 LPS; however, priming of nulnu mice with either preprtion of LPS did not ugment the mgnitude of the ntibody response to sheep erythrocytes, helper-t-cell-dependent ntigen (dt not shown). The development of unresponsiveness fter low-dose priming with S. mrcescens LPS, nd its pprent T-cell dependence, is similr in mny respects to tht observed with nother helper-t-cell-independent bcteril ntigen, type III pneumococcl polyscchride (SSS-III), which is clerly influenced by both T suppressor cells nd T mplifier cells (3). Consequently, we exmined the effect of trnsferring lymphocytes from low-dose-primed mice on the ntibody response to S. mrcescens LPS. The trnsfer of low-doseprimed whole spleen cell popultions gve vrible results, which suggested the presence of competing positive or negtive influences in popultions of primed spleen cells (dt not shown). The trnsfer of untreted or complementtreted spleen cells from low-dose-primed mice usully enhnced the mgnitude of the nti-s. mrcescens LPS PFC response (Tble 7), nd the removl of L3T4+ T cells brogted the bility of these cells to enhnce the mgnitude of the PFC response upon trnsfer; this indictes tht L3T4+ T cells positively influence the response. Further, the removl of Lyt-2+ T cells before trnsfer permitted the remining cells (i.e., L3T4+ T cells) to cuse mrked increse in the PFC response; this ws consistent finding in severl experiments. These experiments indicte tht L3T4+ T cells cn increse the mgnitude of the PFC response to S. mrcescens LPS. This is consistent with other studies in which in vivo removl of L3T4+ T cells before immuniztion with LPS results in decresed primry nti-lps PFC responses (Elkins et l., in preprtion). The demonstrtion of this increse upon trnsfer of Lyt-2- popultions further suggests tht Lyt-2+ T cells cn negtively regulte the mgnitude of the response when present nd, therefore, my hve role in generting unresponsiveness. To dte, we hve not been ble to reproducibly demonstrte suppression by trnsfer of L3T4- (Lyt-2+) popultions; however, mny vribles, such s the number of cells trnsferred, the time of immuniztion reltive to cell trnsfer, nd the time nd dose of priming reltive to cell trnsfer, remin to be ssessed in systemtic mnner. Experiments to evlute the effect of trnsferring primed, positively selected T-cell subpopultions re lso in progress. There is much evidence for T-cell recognition of LPS, lthough the prt(s) of the LPS molecule recognized by T cells is unknown (2, 29, 30, 41, 48). The serotype specificity of the effects of low-dose priming observed in the present work indictes tht T-cell recognition of the 0-ntigen (polyscchride) portion of the LPS molecule, rther thn the lipid A portion, my be importnt in regulting nti-lps ntibody responses, especilly S. mrcescens-specific responses. This suggestion is supported by previous studies in which oscilltion of the nti-e. coli 0113 LPS PFC response fter primry immuniztion ws observed in C3H/HeJ mice, which cnnot recognize lipid A, but ws not observed in nulnu mice, which lck functionl T cells (22). Other studies hve shown tht the nonspecific mitogenic response, s well s the nti-trinitrophenyl ntibody response, to E. coli K235 LPS (conjugted to trinitrophenyl) is greter in mgnitude in germfree BALB/c mice thn in conventionlly housed mice; both of these responses could be suppressed by T cells from conventionl mice (28). Our studies further implicte T cells in the regultion of nti-lps ntibody responses nd stress the importnce of prior exposure in determining responsiveness. Clerly, the mechnisms responsible for regulting nti- LPS ntibody responses re multifctoril. Although we do not believe tht endogenous ntibody plys mjor role in the genertion of memory or low-dose unresponsiveness, lrger quntities of nti-lps ntibody cn clerly medite feedbck inhibition. The responses to vrious LPS preprtions my be function of different B-cell subpopultions or their mturtionl sttes. Finlly, T-cell regultion of the mgnitude of ntibody responses, t lest to some LPS preprtions, is evident despite their conventionl clssifiction s "T-independent" ntigens. Studies re in progress to further chrcterize the fctors tht influence nti-lps ntibody responses nd, thus, tht my be importnt considertions in the control of grm-negtive bcteril disese. ACKNOWLEDGMENTS INFECT. IMMUN. We thnk Jose Ayl for excellent nd enthusistic technicl help, Mtthew Pollck nd Mry Alice Miller for ssistnce with the sodium dodecyl sulfte-polycrylmide gel electrophoresis nlysis of the LPS preprtions, nd Brend Mrshll for preprtion of the mnuscript. LITERATURE CITED 1. Amsbugh, D. F., C. T. Hnsen, B. Prescott, P. W. Stshk, D. R. Brthold, nd P. J. Bker Genetic control of the ntibody response to type III pneumococcl polyscchride in mice. I. Evidence tht n X-linked gene plys decisive role in determining responsiveness. J. Exp. Med. 136:

9 VOL. 55, Armerding, D., nd D. H. Ktz Activtion of T nd B lymphocytes in vitro. I. Regultory influence of bcteril lipopolyscchride (LPS) on specific T-cell helper function. J. Exp. Med. 139: Bker, P. J., D. F. Amsbugh, P. W. Stshk, G. Cldes, nd B. Prescott Regultion of the ntibody response to pneumococcl polyscchride by thymus-derived cells. Rev. Infect. Dis. 3: Bker, P. J., J. R. Hiernux, P. W. Stshk, nd J. A. Rudbch Cyclic development of immunologicl memory to bcteril lipopolyscchride. Infect. Immun. 48: Bker, P. J., nd P. W. Stshk Quntittive nd qulittive studies on the primry ntibody response to pneumococcl polyscchride t the cellulr level. J. Immunol. 103: Bker, P. J., P. W. Stshk, nd B. Prescott The use of erythrocytes sensitized with purified pneumococcl polyscchrides for the ssy of ntibody nd ntibody-producing cells. Appl. Microbiol. 17: Britton, S., nd G. Moller Regultion of ntibody synthesis ginst Escherichi coli endotoxin. I. 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