of February 8, 2018.This information is current as Mice

Vaccination against Tuberculosis Infection in Effective Bacillus Calmette-Guérinthe Site of Stimulus: Tardiness Critical for B Cells Delay Neutrophil Migration toward

Alexander S. AptVladimir V. Evstifeev, Konstantin B. Majorov and Tatiana K. Kondratieva, Elvira I. Rubakova, Irina A. Linge,

http://www.jimmunol.org/content/184/3/1227doi: 10.4049/jimmunol.0902011December 2009;

2010; 184:1227-1234; Prepublished online 18J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2010 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

B Cells Delay Neutrophil Migration toward the Site ofStimulus: Tardiness Critical for Effective BacillusCalmette-Guerin Vaccination against TuberculosisInfection in Mice

Tatiana K. Kondratieva,1 Elvira I. Rubakova,1 Irina A. Linge, Vladimir V. Evstifeev,

Konstantin B. Majorov, and Alexander S. Apt

Mutations in the btk gene encoding Bruton’s tyrosine kinase cause X-linked immune deficiency, with impaired B lymphocyte

function as themajor phenotype. Earlier, we demonstrated that CBA/N-xidmice, unlike the wild-type CBAmice, were not protected

by bacillus Calmette-Guerin (BCG) vaccination against tuberculosis infection. Because IFN-g–producing T cells and activated

macrophages are key elements of antituberculosis protection, it remained unclear how the mutation predominantly affecting B cell

functions interferes with responses along the T cell–macrophage axis. In this study, we show that B cell deficiency leads to an

abnormally rapid neutrophil migration toward the site of external stimulus. Using adoptive cell transfers and B cell genetic

knockout, we demonstrate a previously unappreciated capacity of B cells to downregulate neutrophil motility. In our system, an

advanced capture of BCG by neutrophils instead of macrophages leads to a significant decrease in numbers of IFN-g–producing

T cells and impairs BCG performance in X-linked immune-deficient mice. The defect is readily compensated for by the in vivo

neutrophil depletion. The Journal of Immunology, 2010, 184: 1227–1234.

Mutations in the gene encoding Bruton’s tyrosine kinase(Btk) cause severe X-linked agammaglobulinemia inhumans (1, 2), and a more mild X-linked immune

deficiency (XID) in mice (3, 4). The XID phenotype in mice is dueto a partial block of B lymphocyte development that is caused bya missense mutation (R28C) in the N-terminal pleckstrin homol-ogy domain of Btk, leading to a total lack of B1 lymphocytes anda substantial decrease in numbers of conventional B2 lymphocytes(5, 6). Because the major phenotype of Btk deficiency is impairedB lymphocyte development and function, interest in Btk immunefunctions was focused on B cell responses (7–10). However, Btk isalso expressed and functions in myeloid lineage cells (11–14), andB1a cells are essential not only for T-independent innate hostresponses but also for adaptive immunity (15). These observationshave stimulated an investigation into a possible role for Btk ina broader range of immune responses.In our early studies, we demonstrated that CBA/LacN-xid mice

were much less efficiently protected by bacillus Calmette-Guerin(BCG) vaccination against subsequent infection with virulentMycobacterium tuberculosis compared with their CBA/Lac coi-sogenic counterparts. This was an intriguing observation, given

the paucity of knowledge regarding genetic control of vaccineefficacy against any infection. The defect in CBA/N mice provedto be X-linked and was accompanied by a marked decrease in theT cell-proliferative activity in response to mycobacterial Agscompared with the wild-type mice (16). Combining the generallyaccepted concept that effector and memory CD4+ T cells are thekey elements of vaccine-induced protection against mycobacteria(17) with the apparent defect in B cell functions associated withthe xid mutation, we hypothesized that the Ag-presenting capacityof B cells was impaired in XID mice, leading to insufficient T cellactivation. After demonstrating equal capacities of CBA- andCBA/N-purified splenic B cells to present mycobacterial and ir-relevant Ags to T cell clones of appropriate specificities (16), wetemporarily put away the discovered phenomenon, lacking rea-sonable hypotheses and research tools to study it further. Therenaissance of our interest was stimulated by the demonstrationthat btk gene mutations are expressed not only in B lymphocytesbut also in many other cells of immune system (11–14), althoughnot in mature T cells (18). The latter suggests that alterations inthe interactions between cell types other than T cells per se mightaccount for the diminished BCG vaccine efficacy in XID mice.In this study, we show that extremely rapid neutrophil migration

toward the site of BCG injection in XID animals is a key feature oftheir response toBCG.Identicalphenotype isexpressedinCD192/2,B cell-deficient mice. Using adoptive cell transfer, we demonstratea previously unappreciated capacity of B cells to inhibit earlyneutrophil migration to the inflammatory site. In our system, thisleads to alterations in BCG distribution among different types ofphagocytes. As a consequence, after BCG vaccination, the numbersof CD4+ T cells producing IFN-g in the spleen are a significantlylower in XID compared with thewild-typemice. In the lung, similardifferences are observed exclusively following vaccination andsubsequent infection but not after vaccination alone. Using in vivoneutrophil depletion shortly before BCG injection, we demonstraterestoration of the vaccine performance in B cell-deficient animals.

Laboratory for Immunogenetics, Central Institute for Tuberculosis, Moscow, Russia

1T.K.K. and E.I.R. contributed equally to this work.

Received for publication June 24, 2009. Accepted for publication November 6, 2009.

This work has been supported by the National Institutes of Health Grant AI078864(to A.S.A.), the Russian Foundation for Basic Research Grant 07-04-00447 (to T.K.K.), and the International Science and Technology Center Grant 3626.

Address correspondence and reprint requests to Dr. Alexander S. Apt, Laboratory forImmunogenetics, Central Institute for Tuberculosis, Yauza Alley, 2, Moscow 107564,Russia. E-mail address: asapt@aha.ru

Abbreviations used in this paper: BCG, bacillus Calmette-Guerin; Btk, Bruton’styrosine kinase; PMN, polymorphonuclear neutrophil; TB, tuberculosis; XID, X-linked immune deficiency.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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Materials and MethodsMice, vaccination, and infection

CBA/LacStoCit (hereafter, CBA), CBA/NCit (CBA/N), C57BL/6JCit (B6),and B6.CD192/2 mice containing homozygous cre insertion in the cd19coding sequence (19) were bred and maintained under conventional con-ditions with water and food provided ad libitum at the Animal Facilities ofthe Central Institute for Tuberculosis (Moscow, Russia), according to theguidelines of the Russian Ministry of Health, National Institutes of HealthOffice of Laboratory Animal Welfare (OLAW) Assurance number A5502-11. Breeding pairs of CD192/2 mice were a gift from D. Kuprash (En-gelhardt Institute, Moscow, Russia). Mice of both sexes were used at 2–3mo of age; no sex-related phenotypic differences were noticed throughoutthe study.

Mice were vaccinated with 2 3 107 CFU of Mycobacterium bovis BCG(substrain Pasteur) in 0.3 ml saline s.c. in the dorsum; a group of mice,which received 2 3 105 BCG CFU, was included in the experiment de-scribed in Fig. 1C. Mice were infected either i.v. with 5 3 105 CFU orintratracheally with 104 CFU of midlog-phase M. tuberculosis strainH37Rv (original stock was a gift from Dr. G. Marchal, Institute Pasteur,Paris, France), as described earlier (20, 21). Mortality of the mice wasmonitored daily starting day 14 following infection. To assess mycobac-terial multiplication in spleens and lungs, 0.1 ml serial 10-fold dilutions ofsterile whole-organ 2-ml homogenates were plated onto Dubos agar, andcolonies were counted after 18–20 d of incubation at 37˚C. All experi-mental procedures were approved by the Institutional Animal Care andUse Committee.

ELISPOT assay

Single cell suspensions were obtained individually from lungs and spleensof mice exactly as described previously (20). Sterile filter Millipore plateswere coated with rat Ab against murine IFN-g (BD Pharmingen, SanDiego, CA), washed, and blocked with RPMI 1640 containing 10% FCS(HyClone, Logan, UT). Cells from three individual animals per group wereadded to the wells with 4 doubling dilutions, starting 1 3 106 cells/welland cultured for 48 h in medium alone or in the presence of 10 mg/mlmycobacterial sonicate. Following staining with biotin-labeled rat Abagainst murine IFN-g (BD Pharmingen), spots were counted using ELI-SPOT Bioreader 4000 Pro-X (BioSys, Karben, Germany), calculated, andnormalized for the bulk individual samples. The results are displayed asthe mean 6 SD per organ.

Adoptive transfers

To prepare radiation bone marrow chimera, CBA/N recipients were irra-diated at 9.5 Gy from a 60Co source and within 6 h restored by the i.v.injection of bone marrow cells freshly isolated from femurs of CBA donors(one-donor-to-one-recipient transfer; 15–20 3 106 cells/mouse). Fourcontrol mice, which did not receive protective cell transfer, died at days9–11 following irradiation with signs of acute bone marrow radiationdisease. Recipients were rested for 6 wk and used for vaccination/infectionexperiments. Fetal liver cells were obtained from 17–18-d CBA embryos,and 10 3 106/mouse were injected i.v. into adult CBA/N recipients 4 wkbefore starting vaccination procedures. Peritoneal cavity cells from CBAdonors, either nonseparated or depleted of plastic-adherent cells, weretransferred into the peritoneal cavities ofCBA/N recipients (53 106/mouse),and BCG was injected i.p. after a 15-h interval.

T cell lines and proliferative response

A total of 2 3 106 immune cells isolated from popliteal lymph nodes ofCBA and CBA/N mice, immunized in the footpads with mycobacterialsonicate in incomplete Freund’s adjuvant 12 d before, were cultured in 1ml RPMI 1640 containing 10% FCS, 10 mM HEPES, 4 mM L-glutamine,5 3 1025 M 2-ME, vitamins, piruvate, nonessential amino acids, andantibiotics (all components from HyClone) in 24-well plates (CostarCorning, Badhoevedorp, The Netherlands) for 14 d in the presence of 10mg/ml sonicate. Live immune cells (.93% viability by trypan blue ex-clusion) were isolated by centrifugation at 25003 g for 20 min at 23˚C, ona 1.088 g/ml Lympholyte M gradient (Cedarlane Laboratories, Hornby,Ontario, Canada), washed twice, and counted. The next stimulation cyclewas accomplished by coculturing 2 3 105 isolated cells with irradiated (12Gy) 1.5 3 106 splenic APCs in the presence of sonicate for another 2–3-wk period. These cycles were repeated for four to five times, until the cellsstarted to grow as a sonicate-specific T cell line (.99% of CD4+ T lym-phocytes). Cell samples were kept at 2150˚C until used. To assess pro-liferative response, T cells were thawed and washed, and 5 3 103 cells/well were cultured for a total of 48 h at 37˚C, 5% CO2, in the presence of

indicated numbers of APCs obtained from peritoneal cavities (Fig. 2A). Allcultures were performed in triplicate. For the last 18 h, cultures werepulsed with 0.5 mCi/well methyl-[3H]thymidine. Cultures were harvestedonto glass microfiber filters using a cell harvester (Scatron, Oslo, Norway)for liquid scintillation counting. Results are expressed as counts per minute(cpm 6 SD).

Transmigration assay

Chemotaxis was evaluated in 24-well Transwell plates with 3-mm pore-sizefilters (Costar Corning). Lower chambers were filled with either culturemedium (supplemented RPMI 1640) alone, or medium containing 50 3103 peritoneal plastic-adherent cells (.90% F4/80+, ,2% Ly-6G+, Mph inFig. 2E), or 50 3 103 nonadherent cells (4% F4/80+, 21% CD3+, 75%CD19+, ,2% Ly-6G+, Lym in Fig. 2E), or a 1:1 mixture of these pop-ulations (Mph + Lym in Fig. 2E), with or without addition of 106 BCGCFU/well. A total of 5 3 105 cells isolated from peritoneal cavities 4 hafter peptone stimulation (.96% Ly-6G+ polymorphonuclear neutrophils[PMNs]) in 100 ml medium were added to the upper chambers and allowedto migrate through the membrane for 1 h at 37˚C. The cells were collectedtotally from the lower chambers, stained with anti–Ly-6G mAbs, andquantified by flow cytometry.

Chemokine levels in peritoneal exsudates

Peritoneal cavities of individual CBA and CBA/N mice (four per group)were washed with 3 ml heparin-containing saline at 30 and 90 min afterinjection of either 1.0 ml saline containing 108 BCG CFU or saline alone(control). Peritoneal cells were removed by centrifugation, and the contentof KC, MIP-2, and G-CSF in supernatants was assessed in ELISA formatusing commercially available kits (R&D Systems, Minneapolis, MN) ac-cording to the manufacturer’s instructions.

Gene expression evaluation

Total RNA from peritoneal cavity cells of individual micewas isolated usingthe commercial SV Total RNA Isolation System (Promega, Madison, WI).Reverse transcription of mRNA was carried out exactly as described pre-viously (22). Quantitative real-time RT-PCR with cDNA was performedusing iCycler iQ Multicolor Real-Time PCR Detection System (Bio-Rad,Hercules, CA). The following specific primers and TaqMan probes werepurchased from DNA Synthesis (Moscow, Russia): csf3, F 59-GCTGCT-GCTGTGGCAAAGT-39, R 59-AGCGCTGACAGTGACCAGG-39; probe,FAM-59-CACTATGGTCAGGACGAGAGGCCGTT-39-BHQ1;mip2, F 59-CACTCTCAAGGGCGGTCAAA-39, R 59-CAGGTCAGTTAGCCTTGC-CTTT-39; probe, FAM-59-CCCTGGTTCAGAAAATCATCCAAAAGA-39-BHQ1; kc, F 59-TGTCAGTGCCTGCAGACCAT-39, R 59-GTGGCTATG-ACTTCGGTTTGG-39; and probe, FAM-59-CATCCAGAGCTTGA-AGGTGTTGCCCTC-39-BHQ1. The PCR reaction was performed in a 25-ml final volume of water containing 2 ml cDNA, 2.5 ml 103 TaqPol buffer(Promega), 1 ml 5 mM 29-deoxynucleoside 59-triphosphates, 1 ml of 10 mMforward and reverse primer mix, 0.5 ml of 10 mM TaqMan probe, 0.5 mlTaqDNA polymerase (5 U/ml; Promega). PCR amplifications were per-formed in triplicate using an identical PCR program for all genes: 5 min at94˚C, followed by 50 cycles alternating 15 s at 94˚C and 1min at 60˚C. Geneexpression levels in the lung tissue of individual mice were normalized tothose of b-actin (and confirmed against GAPDH). To quantify the resultsobtained by real-time PCR, the comparative threshold method was usedexactly as previously described (23), with the expression of the results asmean fold increase 6 SEM for groups of three mice in each of three in-dependent experiments.

Neutrophil depletion in vivo and cell staining

CBA/N mice were injected i.p. with 100 mg/mouse anti–Ly-6G, RB6-8C5mAbs provided by S. Kaufmann (Max Planck Institute, Berlin, Germany).Control CBA/N mice were injected with either 100 mg/mouse of irrelevant,isotype-matched, IgG2b mAbs against a potato virus, provided by A.Avdienko (Central Institute for Tuberculosis), or left untreated. Fifteenhours later, the efficacy of neutrophil depletion (∼99%) was assessed byflow cytometry of peripheral blood cells using FITC-conjugated anti–Ly-6G mAb 1A8 (BD Pharmingen). Mice were vaccinated s.c. with 2 3 107

CFU of BCG and challenged 5 wk later by the i.v. route with 5 3 105 CFUof M. tuberculosis H37Rv. Abs PE-anti-F4/80 (clone CI:A3-1; CaltagLaboratories, Burlingame, CA), PE-anti-CD19 (clone 1D3; BD Pharmin-gen), and PE-anti-CD3 (clone 145-2C11; BD Pharmingen) were used toenumerate peritoneal macrophages, B cells, and T cells, respectively.

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ResultsAdoptive transfer of the wild-type immunocytes restores BCGperformance in XID mice

Impaired performance of BCG vaccine in CBA/N-xid mice wasoriginally discovered in the acute tuberculosis (TB) model usinghigh-dose i.v. challenge, which caused rapid mortality and cannotbe considered as adequately mimicking chronic TB infection (16).Before further studying the phenomenon, we tested BCG vaccineefficacy under more physiological conditions (i.e., in mice vacci-nated with lower doses of BCG and infected with lower doses ofM.tuberculosis delivered via i.v. and i.t. routes, as described earlier[20, 21]). As shown in Fig. 1A–C, BCG vaccination provideda significantly stronger protection against subsequent TB challengein the wild-type CBA, compared with CBA/N-xid mice, irre-spective of the route and dose of infection, or the dose of vaccine.Under different experimental conditions, BCG vaccination efficacyin CBA/N mice varied between 0 and 35% of that in CBA mice.To find out whether it is possible to compensate for the defect of

XID mice by the adoptive transfer of immunocytes from the wild-type coisogenic mice, we performed two types of experiments.First, we showed that the total replacement of immune system ofCBA/N recipients with that of CBA donors in CBA → CBA/Nradiation bone marrow chimeras restores the capacity of recipientsto respond to BCG vaccination by decreasing the load of myco-bacteria in their lungs (Fig. 1D) and increasing the survival time(Fig. 1E). Transfer of syngenic CBA/N cells to CBA/N recipientshad no effect (data not shown), and a somewhat shorter survivaltime of all animals compared with other experiments was likelydue to a general adverse effect of irradiation. Second, it was ofinterest to test whether a biased restoration of the B cell pool inXID mice is sufficient to compensate for the defect in response toBCG, given that the main difference between XID and wild-typemice is a marked B cell deficiency of the former. Thus, wetransferred fetal liver cells from CBA donors into intact CBA/Nrecipients, being aware that the fetal liver is a good source of Bcell precursors, especially of B-1a lymphocytes (5) and that therecipient’s T cells will continue normal functioning in non-irradiated recipients. Our results indicate that this type of transferfully restored the B cell pool in lymphoid organs of CBA/N re-cipients to the wild-type level (Fig. 1F). Evaluation of BCG-induced protection in the recipient mice demonstrated a significantdecrease in the lung CFU counts (data not shown) and an increaseof survival time (Fig. 1G) compared with control animals. Thus,a biased restoration of B cell pool in XID mice was sufficient tonormalize the protective effect of BCG vaccination.

In the absence of B cells neutrophils display increasedmigration toward the site of external stimulus

Because the capacities of purified CBA and CBA/N B cells topresent mycobacterial and irrelevant Ags to T cell clones wereshown to be equal (16), we hypothesized that the defective BCGperformance in XID animals could be due to an impaired pro-cessing of BCG and/or presentation of mycobacterial Ags toT cells by phagocytes. To address this issue, we compared thecapacity of CBA and CBA/N peritoneal exudate cells loaded in vivowith live BCG to induce proliferative responses of mycobacteria-specific T cell lines. Mice were inoculated with BCG i.p., and aftera 2-h interval, peritoneal exudate cells were isolated, washed, andcocultured with T cells. Immediately after isolation, the viabilityof peritoneal cells did not differ between the two mouse strains, asassessed by the trypan blue exclusion. As shown in Fig. 2A, APCsfrom CBA mice induced proliferation of both CBA and CBA/NT cells in a dose-dependent manner, whereas there was no re-

sponse in the presence of CBA/N APC. This total lack of activityprompted us to re-evaluate the viability of the APCs at the timewhen cells were put into coculture with the T cells after washingand suspension procedures. It was found that the majority ofperitoneal exsudate cells extracted from XID mice died within 20–40 min after isolation, so very small numbers of live APC of CBA/N origin were present in cocultures.Neutrophils are the cells that have a very short life span and are

extremely sensitive to physical manipulations. Therefore, we eval-uated theneutrophil contentamongthecells,which infiltrated thesiteof BCG injection in CBA and CBA/Nmice. As shown in Fig. 2B, at1.5–2.0 h post-BCG injection, neutrophils (myeloperoxidase-posi-tive PMN cells) represented ∼70% of the total cell population inCBA/Nmice,whereas inCBAanimals their influxwas substantiallypostponed, and the proportion of neutrophils remained,20% at 2 hpost-BCG injection (i.e., when APCs were isolated in previousexperiments).Moreover, 1.5 h after BCG injection, the total numberof infiltrating neutrophils was ∼1 log higher in CBA/N comparedwith CBA mice (Fig. 2C), despite the fact that the total cell countswere ∼4-fold higher in CBA animals because of the presence ofintact B cell population (data not shown). The difference was notspecific for BCG injection, because inoculation of zymosan par-ticles resulted in similar interstrain differences at the 1.5–2.0-h timepoint (data not shown). On the other hand, the difference totallydepended upon an external stimulus, because there was no differ-ence between naive CBA and CBA/N mice regarding the ratio ofPMN cells in bone marrow (40.8 6 2.1 and 40.5 6 1.8%, re-spectively) or in peripheral blood (20.3 6 1.6 and 18.3 6 1.9%,respectively).In the next series of experiments, we investigated whether

adoptive transfers of cells from CBA to CBA/N mice, which wereeffective in restoration of protective response following BCG vac-cination, changed the pattern of neutrophil influx to the site of BCGinjection. We transferred into peritoneal cavities of CBA/N miceeither a nonseparated population (containing ∼18% F4/80+ mono-cytes, ∼17% CD3+ T cells, and ∼60% CD19+ B cells) or plasticadherent-depleted population (,4% F4/80+, ∼21% CD3+, and∼75% CD19+) of cells from the peritoneal cavities of CBA donors.Fifteen hours later, recipients and naive CBA control mice receivedi.p. 108 CFU of BCG, and the dynamics of neutrophil influx wascompared between groups. As shown in Fig. 2C, 2D, both types oftransfers substantially decreased the speed of neutrophil migrationin CBA/N recipients, making them indistinguishable from CBAcontrol mice. Because adherence to plastic removed ∼90% of theF4/80-positive monocytes from the transferred population, leavingthe effect of transfer unaltered, and CBA/N mice are not defectiveregarding T cell numbers and function, our results suggested that Bcells play an important regulatory role in this system by decreasingthe mobility of neutrophils triggered by an external stimulus. Toconfirm this function of B cells, we compared cell migration after i.p. BCG injection between B6.CD192/2 mice, which display a se-vere deficiency of B cells (19) and the wild-type B6 mice. It wasfound that at 1.5 h post-BCG injection, the neutrophil content inperitoneal cavities was 54 6 6% and , 2% (p , 1026), re-spectively. This provides direct genetic evidence that B cells neg-atively regulate neutrophil migration.

Xid neutrophils have an increased motility

To further investigate the role of B cells in regulation of neutrophilmobility, cells were isolated from peritoneal cavities of CBA/N andCBA mice 3.5 h after peptone injection (.90% Ly-6G+ PMN) andput into the upper chambers of Transwell plates. The lowerchambers were supplied either with culture medium alone (con-trol), or with different populations of peritoneal exudate cells from

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CBA mice, with or without BCG, as indicated in Fig. 2E. After 1 hof migration, the total number of Ly-6G+ cells that migrated intolower chambers was assessed by flow cytometry. As shown in Fig.2E, neither combination of cells, or cells plus BCG, in the lowerchamber affected migration of neutrophils compared with themedium alone control. However, the migratory capacity of CBA/Nneutrophils appeared to be 3- to 5-fold higher than that of their CBAcounterparts, irrespective of the cell combinations in cocultures.Analogous experiments performed with peritoneal cavity PMNobtained at 3.5 h after BCG injection provided identical results(data not shown). Thus, we confirmed the results obtained in vivoregarding substantial differences in the mobility of neutrophils

between the wild-type and XID mice, but, unlike the adoptivetransfer experiments (Fig. 2C, 2D), we were unable to inhibitneutrophil migration in vitro. The most likely explanation for thisdiscrepancy is that the inhibitory effect of B cells on neutrophilmigration is not direct but mediated via microenvironmental ele-ments lacking in the in vitro setting (see Discussion).Differences in neutrophil migration are not due to differences in

the level of chemokine production immediately after BCG injection.It is well established that an orchestrated production of two CXC

chemokines, KC and MIP-2, as well as the PMN growth factor G-CSF, is required tomobilize neutrophils into inflammatory sites (24,25). Therefore, we assessed the levels of these mediators in peri-toneal cavities of CBA and CBA/N mice shortly after BCG in-jection. Mice injected with PBS served as controls. The levels of allthree factors in saline-injected control mice were below sensitivityof corresponding ELISA kits. However, as early as 30 min post-BCG injection the contents of KC and MIP-2 grew dramatically inperitoneal cavities of CBA and CBA/N mice (KC, 14136 304 and847 6 352 pg/ml, respectively, p . 0.05; MIP-2, 865 6 204 and705 6 169 pg/ml, respectively, p . 0.05). By the 1.5-h time point,similarly assessed KC contents rapidly increased and reached veryhigh (.2 ng/ml) levels in mice of both strains (data not shown).Such instant responses cannot be based on de novo protein syn-theses and clearly point at the release of presynthesized factorsfrom host cells. The lack of intrastrain differences is not surprising,because CXCL1 chemokines, such as KC and MIP-2, are producedpredominantly by epithelial and endothelial cells, which do notexpress Btk and are functionally identical in the wild-type and XIDmice. Thus, a delay of neutrophil influx in the wild-type CBA micein our system is likely due to its active inhibition rather thandownregulation of CXCL1 chemokine production by B cells.Regarding G-CSF, the factor produced by bone marrow-derived

cells, we were able to demonstrate interstrain differences that fol-lowed the pattern of neutrophil influx.Whereas the level ofG-CSF inperitoneal cavities of intact or saline-injected mice of either strainwas substantially lower than sensitivity of the test, after BCG in-jection, significantly more G-CSF was present in CBA/N comparedwith CBA mice (485 6 28 and 279 6 29 pg/ml, respectively, p =0.023, unpaired t test) at a 1.5-h time point (i.e., when substantiallymore neutrophils arrived in peritoneal cavities of the former mice).

The expression of genes encoding neutrophil chemoattractantsby peritoneal exsudate cells

Besides paracrine regulation of neutrophil trafficking by chemo-kines provided by several stromal sources, this response clearlydisplays an autocrine component (24). Therefore, we assessed thelevel of expression of genes encoding these molecules in the cellsof peritoneal cavity before and after BCG injection. The results ofquantitative RT-PCR were normalized at two sequential steps ofexperiment: total RNA was extracted from equal numbers ofperitoneal cells (4 3 106 in each group), and equal quantities ofRNAwere used for the cDNA synthesis. As shown in Fig. 3, in theabsence of BCG (time 0), cells washed out of the peritoneal cavityof the wild-type CBA mice expressed higher levels of mRNA forG-SCF (Fig. 3A) and KC (Fig. 3B) than their CBA/N counterparts.The levels of expression of MIP-2 (Fig. 3C), and IL-6 and IL-17(data not shown) mRNAs were not different between mice of thetwo strains. After BCG injection, rapid changes in gene expressionoccurred that paralleled the cellular phenotypes characteristic ofthe wild-type and XID mice. The level of gene expression for allthree key chemokines, G-CSF (Fig. 3A), KC (Fig. 3B), and MIP-2(Fig. 3C), increased dramatically within 45 min postinoculation ofBCG in XID mice, whereas in wild-type animals, it was elevatedonly for MIP-2, but dropped for G-CSF, or remained unchanged

FIGURE 1. Defective phenotypes expressed in CBA/N mice and res-

toration of BCG performance using adoptive cell transfer approaches.

Compared with CBA mice, protection against TB challenge after BCG

vaccination was significantly (p , 0.01, Gohan’s criterion for survival

curves) weaker in CBA/N-xid after injection of 5 3 105 M. tuberculosis

CFU i.v. (A), or 104 CFU i.t. (B); n = 9 for each group, results presented as

mean 6 SEM. The defect did not depend on vaccination dose (C, N,

nonvaccinated control; n, 2 3 105 CFU of BCG s.c.; hatched bars, 23 107

CFU of BCG s.c., challenge, 53 105 CFU ofM. tuberculosis i.v., n = 8 for

each group, mean 6 SEM). In CBA → CBA/N radiation bone marrow

chimera (9.5 Gy irradiation, 2 3 107 donor BMC per recipient), protective

effect of BCG was restored in terms of both mycobacterial lung CFU

counts (D, n = 4 per group, 3-wk postchallenge, p , 0.01, one of two

similar experiments), and prolongation of survival time (E, n = 8 per group,

23 107 CFU of BCG, challenge, 53 106 CFU ofM. tuberculosis i.v., p,0.05, Gohan’s criterion). Adoptive transfer of 2 3 107/mouse fetal liver

(FL) cells from CBA donors to nonirradiated adult CBA/N recipients re-

stored the numbers of B cells in their lymphoid organs to the levels

characteristic for the wild-type mice (F) and was sufficient for restoration

of BCG-provided protection (G).

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for KC. At 1.5–2.0 h post-BCG injection, the level of expressionof all genes in wild-type and XID mice equalized (Fig. 3A–C),exactly as did the neutrophil content in their peritoneal cavities ata later time point (∼3.5 h; Fig. 2B).

An impaired IFN-g production by CD4 T in XID mice

Interstrain differences in the dynamics of neutrophil arrival to thesite of BCG injection resulted in a distinct distribution of cell-associated bacilli in CBA and CBA/Nmice. Thus, after 2 h post-i.p.injection, the vast majority of BCG was associated with mono-nuclear cells in the wild-type mice (Fig. 4A) but with PMN in theXID animals (Fig. 4B). This difference may influence the de-velopment of adaptive immunity against BCG, thus we examinedhow the capacity of CD4+ T cells to produce IFN-g in response tomycobacterial Ags differed between CBA and CBA/N mice, giventhat this immune function is considered a critical component ofanti-TB defense (17).At week 5 after BCG vaccination, prior to TB challenge, mice of

the two strains displayed low and equal numbers of IFN-g–producing CD4+ T cells in their lungs (Fig. 4C). However, shortlyafter infection with virulent M. tuberculosis, a substantial 15-foldincrease in numbers of IFN-g–positive cells was observed in CBAmice, whereas their CBA/N counterparts displayed only a 3-foldincrease (Fig. 4D). Interestingly, although infection itself wasa more powerful stimulus for IFN-g production in the lungs thanBCG vaccination (∼5000 and ∼1500 of IFN-g–positive cells perlung, respectively), in the absence of BCG vaccination there wasno difference between mice of the two strains. Thus, in the organpredominantly affected by infection (lung), the difference betweenthe wild-type and XID mice became evident only after the sec-

ondary contact with mycobacteria. However, in the lymphoid or-gan responsible for the initiation of acquired immunity (spleen)the number of IFN-g–positive cells grew 1 log higher in the wild-type compared with XID mice after BCG vaccination in the ab-sence of challenge (Fig. 4E). Moreover, adoptive transfer of CBAfetal liver cells in CBA/N recipients resulted in a 3-fold increase innumbers of IFN-g–positive cells in the lungs following vaccina-tion and infection (data not shown). Thus, a biased restoration of Bcell pool in XID mice was sufficient to normalize both the num-bers of IFN-g–producing cells and the protective effect of BCGvaccination (see above). Thus, a direct link between capacities to

FIGURE 2. Neutrophil migration toward the

site of BCG injection in XID andwild-typemice.

A, CBA but not CBA/N peritoneal APCs loaded

with BCG in vivo for 2 h can present mycobac-

terial Ags to specific T cell lines (one of two

similar experiments). B and C, After i.p. BCG

injection, inXIDmice, neutrophilsmigrate to the

peritoneal cavity much more rapidly than in

wild-type mice (summary of eight independent

experiments; mean 6 SD). The difference in

migration speed is abrogated by the adoptive

transfer of either whole (D) or nonadherent (E)

population of CBA peritoneal exsudate cells

(summary of two independent experiments, three

mice in each group, n = 6). F, In the Transwell

system, CBA/N neutrophils migrate much faster,

compared with CBA neutrophils, irrespective of

migration stimuli (two independent experiments

with mixtures of cells from three mice in each,

p , 0.01, unpaired t test).

FIGURE 3. Differences in chemokine gene expression between XID and

wild-type mice. Early after BCG injection the expression of mRNA en-

coding key neutrophil-attracting factors G-CSF (A) and KC (B) increases

in CBA/N but decreases in CBA mice (summary of three independent

experiments, mixtures of RNA isolated from three mice in each, n = 9).

There was no difference in the dynamics of expression of mRNA for some

other factors involved in neutrophil migration control, MIP-2 (C), IL-6,

and IL-17 (data not shown). Results are expressed as fold increase 6 SEM

compared with naive CBA animals. Samples of RNAwere analyzed by the

quantitative real-time PCR assay, and gene expression levels in peritoneal

cells were normalized to those of b-actin.

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react normally to the vaccine by enhancing the numbers of IFN-g–producing T cells and to be protected against TB challenge wasestablished in our model.

Neutrophil depletion in vivo restores BCG performance in XIDmice

A major confounding variable between the sets of data presentedabove is the use of two different routes of BCG injection. Indeed,s.c. vaccination provides an adequate tool to demonstrate differ-ences in BCG vaccination efficacy and to evaluate shifts in immuneresponses after adoptive transfers but does not allow isolation oflocal cell populations before and after vaccination. On the otherhand, i.p. injection is perfect for the analyses at the cellular level butpoorly mimics a normal vaccination procedure. A straightforwardapproach to link the data on defective BCG performance in CBA/N-xid mice with an uncontrolled neutrophil influx is to removeneutrophils from XID animals at the very moment of vaccineinjection and to assess shifts in vaccination efficacy. To this end,we administered i.p. anti–Ly-6G, RB6-8C5, and irrelevant, iso-type-matched Abs to groups of CBA/N mice at day 21, vacci-nated these as well as intact animals with 2 3 107 CFU of BCG s.c. at day 0, infected all animals i.v. with virulent M. tuberculosis5 wk later, and compared CFU counts in lungs and spleens, as wellas the content of IFN-g–producing CD4+ T cells in the lungs, atweek 3 postchallenge.

A single administration of RB6-8C5 Abs appeared to be quiteefficient for short-term neutrophil depletion, because 15 h after theadministration the neutrophil counts in the peripheral blood oftreated mice dropped ∼99% compared with isotype controls, withtotal restoration of the neutrophil population 4 d afterward (datano shown). As shown in Fig. 4F, 4G, neutrophil-depleted, BCG-vaccinated XID mice showed highly significant (p = 0.012, Mann-Whitney U test) ∼8-fold reductions in CFU counts in lungs andspleens compared with control groups. Moreover, the number ofIFN-g–producing CD4+ T cells per lung was ∼3-fold higher inneutrophil-depleted animals (data not shown). These results pro-vides direct in vivo evidence that in mice defective in B cell-dependent neutrophil motility control, depletion of neutrophilssubstantially improves the efficacy of BCG vaccination.

DiscussionThe differences between XID and the wild-type mice in thenumbers of CD4+ T cells producing IFN-g provide rational ex-planation of the difference in the vaccine performance but nomechanistic underpinning of the phenomenon, the search forwhich was the essence of this work.While studying whether reduced T cell response in XID animals

could be due to an impaired processing of BCG and/or presentationof mycobacterial Ags to T cells by phagocytes, we established thatthe two mouse strains differ profoundly regarding the speed of

FIGURE 4. BCG phagocytosis, T cell re-

sponses, and restoration of BCG performance

by the in vivo neutrophil depletion. The vast

majority of i.p.-injected BCG is associated with

mononuclear cells in CBA (A), but with PMN in

CBA/N (B) mice, as demonstrated by auramine

staining (original magnification 3400) for my-

cobacteria with Giemsa counterstaining of cy-

tospin preparations. Before challenge, BCG-

vaccinated mice of the two strains did not differ

by numbers of IFN-g–producing CD4+ T cells

in their lungs (C), but at week 3 postinfection,

the number of these cells in the lungs of the

wild-type CBA mice was approximately four

times (p , 0.01, Mann-Whitney U test) higher

(D). In the spleen, the numbers of IFN-g–

producing T cells differed ∼10-fold (p, 0.001)

after BCG vaccination in the absence of chal-

lenge (E). Magnetically sorted CD4+ lung

T cells (C and D) or bulk spleen cells (E) from

three individual mice in each group were ana-

lyzed using the ELISPOT assay; the results of

one of two similar experiments are presented as

mean 6 SD. Neutrophil depletion prior to BCG

vaccination resulted in significant (p , 0.01;

Mann-Whitney U test) ∼8-fold reductions in

CFU counts in lungs (F) and spleens (G) com-

pared with control groups.

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neutrophil influx at the site of BCG injection (Fig. 2). We dem-onstrated that adoptive transfers of peritoneal cells and, impor-tantly, macrophage-depleted cells from CBA to CBA/N mice weresufficient to restore the control of neutrophil migration in XIDmice(Fig. 2). Moreover, B cell-deficient CD192/2 mice showed alter-ations in neutrophil migration identical to those in XID mice,providing an independent genetic evidence of the inhibitory role ofB cells in neutrophil migration. In addition, results from the ex-periments with fetal liver cells and B-enriched peritoneal cells (Fig.2) should be evaluated in the context of 1) competitive disadvantagefor other Btk-sufficient cell types transferred from CBA donors and2) unimpaired function of Btk-negative T cells in CBA/Nmice (18).Taken together, these data strongly suggest that B cells play a pre-viously underscored regulatory role in neutrophil locomotion.Neutrophils migrate in response to mycobacteria, engulf the

bacilli and undergo apoptosis (26–29). Interstrain differences inthe dynamics of their arrival to the site of BCG injection resultedin a distinct pattern of distribution of bacilli: shortly after in-jection, the vast majority of BCG was associated with mono-nuclear cells in the wild type, but with polymorphonuclear cells inthe XID animals (Fig. 4). This difference may influence the de-velopment of immune responses against BCG. Although there isevidence that neutrophils effectively transport intradermally in-jected BCG into lymph nodes (30, 31) and that the interactions ofBCG-infected neutrophils with dendritic cells and macrophagesresult in T cell cross-priming and proinflammatory reactions (32),immunological consequences of these events are not necessarilybeneficial for the host. Mycobacteria-induced neutrophil activa-tion leads to the acceleration of their apoptosis through mecha-nisms dependent on TLR2 and p38 MAPK (27). It was shownrecently that, after ingestion of BCG-containing apoptotic neu-trophils, macrophages intensively formed lipid bodies and pro-duced large quantities of PGE2 and TGF-b, which activelysuppress T cell activation (33). Similar conclusions on inhibitionof immune responses as a consequence of interaction betweenmycobacteria-activated apoptotic neutrophils and dendritic cellswere drawn from another recent study (36). Reasoning along theselines, we studied in more detail the features of neutrophil migra-tion in our system.In vitro experiments demonstrated that the migratory capacity of

CBA/N neutrophils is 3- to 5-fold higher than that of their CBAcounterparts, irrespective of the cell combinations in cocultures.Thus, we confirmed the results obtained in vivo regarding sub-stantial differences in the mobility of neutrophils between the wild-type and XID mice, but unlike the adoptive transfer experiments,we were unable to inhibit neutrophil migration in vitro (Fig. 2). Themost likely explanation for this discrepancy is that the inhibitoryeffect of B cells on neutrophil migration is not direct but mediatedvia microenvironmental elements lacking in the in vitro setting.Neutrophil migration from the bloodstream depends on a complexnetwork of chemokines, cytokines, integrins, and selectins, someautocrine, and some provided by endothelial and epithelial cells(35, 36). Thus, the conditions of neutrophil migration through themembrane in vitro differ profoundly from those during in vivoextravasation. However, a higher level of spontaneous migrationof CBA/N neutrophils in our experiments is in good agreementwith recent data demonstrating significantly enhanced PMN lo-comotion through membranes in the presence of the selective Btkinhibitor LFM-A13 (37). Taken together, these findings demon-strate that an enhanced motility of PMN is another phenotypicexpression of xid mutation. It is not known why functionallycompetent Btk is needed for normal PMN locomotion; however,there is evidence that this kinase is involved in actin polymeri-zation and cytoskeleton dynamics in B cells (38).

An orchestrated production of two CXC chemokines, KC andMIP-2, as well as the PMN growth factor G-CSF, is required tomobilize neutrophils into inflammatory sites (25). In response toBCG injection, high amounts of CXC chemokines were instantlyreleased into peritoneal cavities of both XID and wild-type mice,indicating that an inhibitory action of B cells onto neutrophiltrafficking has features of active suppression rather than inhibitionof chemokine production. An instant chemokine release in re-sponse to mycobacterial stimulus clearly indicates that there arerich depots of these molecules (one, very likely, is the liver; A.Gleiberman, personal communication) able to release pre-existingfactors in response to different stimuli, including those providedby intracellular pathogens. This aspect of an early response tomycobacteria deserves further investigation.Animal models have failed to demonstrate a clear role of neu-

trophils in response against mycobacteria, due primarily to con-flicting data (39–41). However, more recently we and others usinggenetic approaches demonstrated deleterious rather than beneficialeffects of these early inflammatory cells in the course of chronicmycobacterial infections (28, 42, 43). Similar results were obtainedin the mouse model of leishmaniasis (44, 45). It is difficult to judgewhich feature of a strong neutrophil response is more deleteriousfor the host: a temporary shelter granted to a parasite (the “Trojanhorse” concept, see Refs. 28 and 44), or T cell response inhibition(33, 34), or both. Nevertheless, it is likely that an exaggeratedneutrophil response is a kind of “biological mistake” when it occursin the context of chronic infections caused by sophisticated in-tracellular pathogens. Data presented herein add the phenomenonof BCG capture by neutrophils to this line of evidence.A few recent studies demonstrated that B cells have a suppressive

effect on the inflammatory response (46, 47), in particular, CD5+

B1 cells specifically downregulated T cell-mediated inflammation(47). Our results indicate that inhibition of neutrophil migrationmay be yet another regulatory B cell function, adding evidence tothe negative association between B cells and neutrophils in thehost response to infection (48)—the phenomenon awaiting de-tailed mechanistic dissection.

AcknowledgmentsWe thank T. Radaeva and V. Sosunov for their expert help with quantitative

real-time RT-PCR evaluations.

DisclosuresThe authors have no financial conflicts of interest.

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