INFECTION AND IMMUNITY, Dec. 1983, p. 1136-1143 Vol. 42, No. 30019-9567/83/121136-08$02.00/0Copyright C) 1983, American Society for Microbiology

Infection of Mouse Lymphoblastoid Cell Lines withMycoplasma hyorhinis: Complex Nature of Mycoplasma-Host

Cell InteractionsGARY H. BUTLER* AND ERIC J. STANBRIDGE

Department of Microbiology, College of Medicine, University of California, Irvine, California 92717

Received 23 May 1983/Accepted 2 September 1983

Mycoplasma hyorhinis infection of lymphoid cells is a complex process.Mycoplasmas adsorb to cell surface receptors and undergo lateral redistributionon the cell membrane. This process culminates in the formation of co-caps ofmycoplasmas and specific cell surface antigens. One or more of these antigensmay be a M. hyorhinis receptor(s) or may bear a receptor moiety(s). We show thatthe cell surface antigens Thy-1.2 and Thy-1.1, and to a lesser extent H-2 and gp7O,but not T200, are co-capped with M. hyorhinis on the membranes of acutelyinfected mouse thymic lymphoblastoid cell lines. These antigens may representmultiple receptor(s) for M. hyorhinis since there is no correlation between theexpression of any individual antigen and the susceptibility of these cell lines toinfection.

Mycoplasmas, the smallest free-living organ-isms isolated to date (15), are also associatedwith animal cells as extracellular parasites onthe external surfaces of plasma membranes. Theclose association of mycoplasmas and the hostcell plasma membranes has pathological conse-quences ranging from the development of chro-mosomal aberrations (16) to host cell damageresulting from recognition of adsorbed myco-plasmas by the host's immune system. The highdegree of species and tissue specificity of myco-plasmas in vivo (17) implies the existence ofspecific host cell plasma membrane receptorsfor mycoplasmas. Analysis of several in vitrocell systems has shown that mycoplasma recep-tors may be protease (13) or neuraminidase(4,13) sensitive. In addition, glycophorin, a ma-jor sialoglycoprotein of erythrocytes, mediatesthe adherence of mycoplasmas to erythrocytes(1). The important binding component appearsto be sialic acid residues (2). Little more, howev-er, is known regarding the nature of host cellreceptors for mycoplasmas.To define the nature of cellular receptors for

mycoplasmas, we have developed an in vitrosystem using a highly cytoadsorptive strain ofMycoplasma hyorhinis and a set of well-charac-terized murine lymphoblastoid cell lines. Wehave previously shown the unique phenomenonof the adherence of M. hyorhinis to lymphoblas-toid cells followed by their lateral redistributionon the cell membrane to form caps at one end ofinfected cells (18). In this report, we show thatspecific lymphoblastoid cell surface glycopro-

teins co-cap with M. hyorhinis. We further showthat the expression of no single lymphoblastoidcell surface antigen which co-caps with myco-plasmas correlates with the degree of mycoplas-ma infection. The significance of these observa-tions with respect to the identification of M.hyorhinis receptors on lymphoblastoid cell linesis discussed.

[Preliminary data on infection of S49 (Thy-1+)and S49 (Thy-1-a) cells have been published(17a).]

MATERIALS AND METHODS

Mycoplasmas. A highly cytoadsorptive strain of M.hyorhinis was used throughout these experiments. M.hyorhinis was maintained by passage in HeLa cellcultures in minimal essential medium (GIBCO Labora-tories, Grand Island, N.Y.) with 10% calf serum(Irvine Scientific, Irvine, Calif.). Supernatants of thesecultures were centrifuged at 450 x g for 5 min and usedfor infection of lymphoblastoid cell lines. The concen-tration of viable mycoplasmas in these supernatantswas evaluated by serial dilution and growth of colonieson modified Hayflick medium (18).

Cell lines. All lymphoblastoid cell lines used in thestudy were obtained from R. Hyman, Salk Institute,San Diego, Calif. The designations and surface pheno-types of these cell lines are summarized in Table 1.These lines were maintained in Eagles minimal essen-tial medium with 10% calf serum and 2% horse serum.The hybridoma cell line 30-H12 (ATCC TIB 107),which produces monoclonal antibody to Thy-1.2, waspurchased from the American Type Culture Collec-tion, Rockville, Md., and was maintained in RPMImedium containing 10% fetal bovine serum. The hybri-doma cell line TD11e7, which produces monoclonal

1136

CO-CAPPING OF M. HYORHINIS AND CELL ANTIGENS 1137

TABLE 1. Phenotypes of cell lines

PhenotvpeLine Strain MHC" Reference

Thy-i H-2 T2O0 gp7O TL

S49 (Thy-1+) BALB/c D 1.2 Low + Low + 5. 7S49 (Thy-1-a) BALB/c D - Low + Low + 5. 7. 19BW5147 (Thy-1+) AKR K 1.1 + + + - 7. 8, 11BW5147 (Thy-1-a) AKR K - + + + ND" 7, 19BW5147 (Thy-1-e) AKR K - ND + + ND 20BW5147 (T200-a) AKR K Low + - + ND 11AKR1 (Thy-1+) AKR K 1.1 ND ND ND ND 6AKR1 (Thy-I-d) AKR K T25-' ND ND ND ND R. Hyman"R1 (TL+) C58 K 1.2 + + + + 8-10Rl (TL ) C58 K 1.2 - + + + 8-10Ri (Thy-1-a) C58 K - ND ND ND ND R. HymanTlMi (Thy-I+) C57BL/6 B 1.2 + ND ND - 5. 7TlMi (Thy-1-c) C57BL/6 B - + ND ND - 5. 7. 19

'MHC, Major histocompatibility complex.^ ND, Not determined.These cells also lack T25, the intracellular precursor to Thy-i.

"Personal communication.

antibody to Thy-1.1, was obtained from the SalkInstitute and maintained in Dulbecco modified Eaglemedium (GIBCO) with 10% fetal bovine serum. Allcell lines used in these studies were regularly screenedfor mycoplasma contamination before experimentaluse and were always negative.

Antisera. Monoclonal antibodies to Thy-1.2 andThy-1.1 were obtained from ascites fluids of Nu/Numice. Monospecific antiserum to Rauscher murineleukemia virus gp7O was obtained from J. Gruber,Office of Programs and Logistics, National Institutesof Health, Bethesda, Md. Culture supernatant contain-ing 1312.3 monoclonal antibody to T200 was a gift of I.Trowbridge, Salk Institute. Rhodamine-conjugatedrabbit anti-rat immunoglobulin G and rhodamine-con-jugated goat anti-mouse immunoglobulin G were pur-chased from Cappel Laboratories, West Chester, Pa.Mycoplasma-infected cells were identified by usingfluorescein-conjugated burro anti-M. hyorhinis.Mouse anti-H-2k antiserum (C3H.SW x B10.D2) Flanti-BP8 absorbed with EL4 cells was a gift of P.Frost, University of California, Irvine.

Co-capping of M. hyorhinis and lymphoblastoid cellsurface antigens. Lymphoblastoid cells were infectedwith M. livorhinis at a multiplicity of 1 to 5 CFU percell. Cultures were incubated at 37°C in 4% CO, forvarious time intervals. Cells were harvested by centrif-ugation at 450 x g for 2 min and washed twice in coldisotonic phosphate-buffered saline containing i02 MNaN, (PBS-azide). This buffer was used throughoutthe procedure to prevent antibody-mediated redistri-bution of antigens. Approximately 5 x 10' cells wereresuspended in 50 1d of antibody diluted in PBS-azideand incubated at 0°C for 30 min. Cells were washedtwice in cold PBS-azide and incubated with rhoda-mine-conjugated antiglobulin, washed, and reactedwith fluoresceinated burro anti-M. livorhinis antibody.The distributions of antigens and mycoplasmas oncells were evaluated by epifluorescence with a Leitzfluorescence microscope. The distribution of anti-mycoplasma antibody on cell surfaces was classifiedinto three categories: pinpoints, less than three isolat-ed areas of fluorescence: patches. multiple areas of

fluorescence randomly distributed over the cell sur-faces; and caps, concentrations of intense fluores-cence at one pole of cells. Co-caps of cell surfaceantigens and M. hilorhinis were identified as coincid-ing rhodamine-stained and fluorescein-stained caps.To determine the degree of co-capping of M. livorhinisand cell surface antigens, 30 to 50 mycoplasma cappedcells were evaluated for co-capping. Uninfected cells,as well as cells that did not express the relevantspecific antigens, were processed with all experimentsto determine the normal distribution of cell surfaceantigens in the absence of mycoplasmas.Capping of killed mycoplasmas. M. hyorhinis cul-

tures were inactivated by exposure to tit irradiationfor 3 min. The organisms were then pelleted, resus-pended in growth medium, and added to S49 cultures.No attempt at quantification was made, since allorganisms were killed. At several periods after theaddition of the killed mycoplasmas, the S49 cells wereassayed for the presence of pinpoints, patches, andcaps by using fluoresceinated anti-M. hyorhinis anti-serum. Cultures were tested at time zero and whenev-er cells were assayed for the presence of viablemycoplasmas.

RESULTS

Stages of acute M. hyorhinis infection. Evi-dence of acute M. hvorhinis infection of lym-phoblastoid cell lines first became apparent by 4to 8 h postinfection. The initial stages of infec-tion were characterized by host cells havingisolated pinpoints of fluorescence when stainedwith fluoresceinated anti-M. hyorhinis (Fig. 1).As infection progressed, pinpoint staining pat-terns were replaced by patch infections, whichdeveloped as large numbers of mycoplasmasadsorbed to cells and began to move laterally onthe cell surface. Acute infection culminated at 30to 36 h with extensive capping of mycoplasmasat one pole of the cell. Lateral redistribution of

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1138 BUTLER AND STANBRIDGE

X e ________________t

FIG. 1. Illustration of the pinpoint, patch, and cap distribution of mycoplasmas on the surface of S49(Thy-l) cells infected with M. hyorhinis. (a) S49 (Thy-l) cell stained with fluoresceinated anti-M. hyorhinisantibody showing a pinpoint of infection. (b) A representative cell showing typical patchy distribution ofmycoplasmas. (c) Representative cell with a classical mycoplasma cap. Bar, 5 p.m.

mycoplasmas on the cell membrane is shown bythe progressive replacement of pinpoints withpatches and patches with single caps (Fig. 2A).At 4 h postinfection, 95% of infected S49 (Thy-1+) cells were pinpoint infections. By 24 h, only4% of infected cells were pinpoints; the rest ofthe infected cells exhibited patches or caps ofmycoplasmas. Previous observations with elec-tron microscopy (18) showed that pinpoints arecomposed of single or a few mycoplasmas;patches are composed of greater numbers, andcaps may contain as many as 50 mycoplasmas.Previous studies showing the detection of patch-es and caps in the presence of 10-2 M NaN3 at0°C, as well as on paraformaldehyde-fixed cells,showed that redistribution of mycoplasmas isnot due to an antibody-mediated event (18).Furthermore, these studies also indicated thatthe same kinetics of patching and capping oc-curred even when infective doses as high as 20mycoplasmas per cell were used (18).Capping of killed mycoplasmas. The clear

demonstration of nonviable mycoplasma capson the surface of S49 cells (Fig. 3) lends furthercredence to the fact that the caps are not arti-facts due to the formation of microcoloniesgenerated by mycoplasma replication. The samechronological progression of pinpoints, patches,and caps was seen with the killed mycoplasmas(data not shown). Thus, the appearance of capsis not due to the attachment of single largeaggregates of mycoplasmas to the surface of S49cells. At no time during the entire cappingprocess were viable mycoplasmas detected.

Infection of Thy-l+ and Thy-1-a cell lines.Acute M. hyorhinis infection of S49 (Thy-i+)and S49 (Thy-1-a) cell lines with identical dosesof mycoplasmas showed that the former alwaysbecame infected to a significantly higher levelthan the latter when evaluated by direct immu-nofluorescence (Fig. 4 and Table 2). This wasapparent throughout the course of infection (Fig.

5). During the early stages of infection five- tosixfold more S49 (Thy-i+) cells were infectedthan S49 (Thy-1-a) cells. At 24 h postinfection,more than 50% of the S49 (Thy-1+) cells were

C-

0U

0

0-

Bi

(a

0

4-

E2

Time [hours post infectionlA-A-A._._ . _

rime lhours post infectionl

FIG. 2. Kinetics of infection of (A) S49 (Thy-I')and (B) S49 (Thy-1-a) cell lines with M. hyorhinis.Symbols: A, pinpoints; 0, patches; 0, caps.

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00opop

I I.- -El--- --

CO-CAPPING OF M. HYORHINIS AND CELL ANTIGENS 1139

FIG. 3. Demonstration of capping by nonviable mycoplasmas which had been killed by exposure to UVirradiation. At 24 h after the addition of killed mycoplasmas, S49 cells were stained for M. hyorhinis antigens asdescribed in the text. (a) Phase contrast; (b) cap of UV-killed M. hyorhinis on the same cell. Bar, 5 p.m.

infected, with the vast majority exhibiting patch-es or caps. In contrast, only 15% of the S49(Thy-1-a) cells were infected by 24 h, and themajority of cells exhibited pinpoints (Fig. 2B).The higher level of infection of S49 (Thy-i+)cells was also apparent by evaluation of thenumbers of mycoplasmas present in the culturesupernatants. At 24 h postinfection S49 (Thy-1 +)

rrr r-w-_-' 'e 44%f ,

IC.Ws

culture supernatants had three times more M.hyorhinis CFU than S49 (Thy-1-a) superna-tants. The threefold difference represents a mini-mal estimate, since mycoplasmas accumulate insupernatants by the shedding of caps whichremain intact (G. H. Butler and E. J. Stan-bridge, manuscript in preparation) and, as notedabove, may contain as many as 50 organisms.

9,'

v~ ~v t04,l..

,a

* .W~ *sw~Ce*fS_FIG. 4. M. hyorhinis-infected S49 (Thy-i+) and S49 (Thy-i-a) cells at 24 h postinfection stained for M.

hyorhinis as described in the text. (a) Phase contrast of S49 (Thy-1+) cells; (b) distribution of M. hyorhinisorganisms on cells in the same field; (c) phase contrast of S49 (Thy-1-a) cells; (d) distribution of M. hyorhinisorganisms on cells in the same field as in (c). Note the significantly higher level of infection and higher proportionof caps in the Thy-1.2+ cell line. Bar, 10 p.m.

-

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1140 BUTLER AND STANBRIDGE

Comparisons of levels of infection of BW5147(Thy-i+) and BW5147 (Thy-i-a) cell lines alsoshowed that the Thy-i+ line was consistentlyinfected more heavily than the Thy-i-a line(Table 2).

Co-capping of Thy-1 and M. hyorhinis. Weevaluated co-capping of Thy-i and M. hyorhinisto determine whether Thy-1 expressed on wildtype, S49 (Thy-l+), and BW5147 (Thy-l+) cellswas associated with adsorbed mycoplasmas.Double-label immunofluorescence staining ofmycoplasma-infected Thy-i+ cells for Thy-1.1or Thy-1.2 and M. hyorhinis showed that both ofthese antigens were co-capped with M. hyor-hinis. At 24 h postinfection, Thy-1.2 was co-capped with approximately 76% of M. hyorhiniscaps on S49 (Thy-i +) cells (Table 3 and Fig. 6),and Thy-1 co-capped with 45% of M. hyorhiniscaps on BW5147 (Thy-l+) cells (Table 3). Con-trol experiments in which 103 uninfected cellswere examined showed less than 0.5% redistri-bution of Thy-1.2 at the poles of cells.

Examination of the distribution of anothermajor surface protein, the 200-kilodalton glyco-protein T200, after infection of S49 (Thy-l+),S49 (Thy-1-a), BW5147 (Thy-I'), and BW5147(Thy-1-a) cell lines with M. hyorhinis showedthat no co-capping of T200 and mycoplasmasoccurred (Table 3 and Fig. 7). Therefore, co-capping of Thy-1 with M. hyorhinis appeared tobe relatively selective.

Co-capping of gp7O and H-2 antigens with M.hyorhinis. To determine whether other cell sur-face antigens were co-capped with M. hyorhinis,we conducted double-label immunofluorescenceassays to evaluate additional antigens. The gp7Oand H-2k antigens were expressed on BW5147(Thy-l') and BW5147 (Thy-1-a) lines, butH-2d and gp7O were not detected on S49 (Thy-I1) or S49 (thy-1-a) lines by immunofluores-cence assays, although very low levels havebeen detected by other investigators (5) with themore sensitive cytotoxicity assays (Table 1).The gp7O antigen was co-capped with the major-ity of the mycoplasma caps on BW5147 (Thy-i +and BW5147 (Thy-1-a) cells (Table 3). H-2k wasco-capped with 100% of the mycoplasma capson BW5147 (Thy-l+) cells and with the majority

TABLE 2. Infection of lymphoblastoid cell lineswith M. hyorhinis

% of Degree of infection at 24Cell line cells h (% of infected cells)

infected Pinpoint Patch Cap

S49 (Thy-1+) 85 53 20 27S49 (Thy-i -a) 32 84 12 4BW5147 (Thy-1+) 35 60 28 12BW5147 (Thy-1-a) 9 93 5 2

Time (hours post infectionIFIG. 5. Comparison of level of infection of S49

(Thy-1l) (U) and S49 (Thy-1-a) (*) cell lines with M.hyorhinis. Infection was measured by direct immuno-fluorescence as described in the text.

of mycoplasma caps on BW5147 (Thy-1-a)cells.

Complexity of M. hyorhinis association with cellsurface antigens. In the experiments describedabove, Thy-l+ cells became infected with M.hyorhinis to significantly higher levels than Thy-1-a cell lines, and both Thy-1.2 and Thy-1.1were among cell surface antigens selectivelyassociated with M. hyorhinis caps. Thus, wewanted to determine whether the correlationbetween M. hyorhinis infection and Thy-1expression was a generally occurring phenome-non. To accomplish this, we examined the infec-tion of several additional cell lines with M.hyorhinis (Tables 1 and 4). These data showedthat three Thy-1.2+ cell lines, Ri (TL+),Rl(TL-), and TIMI (Thy-i+) as well as oneThy-1.1+ cell line, AKR (Thy-l+), did not be-come heavily infected with M. hyorhinis. At 24 hpostinfection, the AKR (Thy-l+) cell line re-mained virtually uninfected. The RI (TL+), Ri(TL-), and TlMl (Thy-i+) lines had many in-fected cells; however, few infected cells werecapped. S49 (Thy-1 +) cells infected with thesame sample of mycoplasmas were heavily in-fected, with 66% of the cells infected and 42% ofinfected cells capped. Furthermore, two Thy-1.2- cell lines, Ri (Thy-1-a) and TlMi (Thy-1-c), could be infected to high levels, with asignificant percentage of the infected cells show-ing caps. The Ri (Thy-1-a) cell line was infectedto an extremely high level, with 100% of the cells

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CO-CAPPING OF M. HYORHINIS AND CELL ANTIGENS 1141

TABLE 3. Selective co-capping of cell surfaceantigens with M. hyorhinis

% of caps co-capped (% of cells with caps)aAntigen S49 S49 BW5147 BW5147

(Thy-l+) (Thy-l-a) (Thy-l+) (Thy-l-a)Thy-1.2 76 (27) bThy-1.1 - 45 (6)gp70 - - 68 (6.5) 83 (3)H-2k 74 (6.5) 100 (3.5)T200 0 (23) 0 (3) 0 (8) 0 (2)

a Percentage of mycoplasma caps co-capped withantigen at 24 h postinfection. Numbers in parenthesisare the percentages of total cells with mycoplasmacaps.

b , Antigen was not detected by indirect immuno-fluorescence.

infected and approximately half of the infectedcells having mycoplasma caps. In addition, only2% of these cells were pinpoint infections. Thus,the expression of Thy-1 only correlated to highinfection in comparisons of S49 (Thy-1 +) cells toS49 (Thy-1-a) cells and BW5147 (Thy-1+) cellsto BW5147 (Thy-P-a) cells. Since RI (Thy-1-a)cells infected to high levels, the complementa-tion class A glycosylation defect did not conferprotection against M. hyorhinis infection.M. hvorhlinis infection of the various cell lines

did not correlate with the expression of othercell surface antigens on any cell lines evaluated.The TL antigens TL1, TLI, and TL3 were notrelated to infection, since Ri (TL+) and Ri(TL-) cells showed that the latter could beinfected slightly better (Table 4). Similarly,expression of gp7O, H-2, and T200 could not becorrelated with levels of M. hyorhinis infection(Table 4).

DISCUSSIONThese data clearly demonstrate that acute M.

hvorhinis infection of the highly susceptible S49(Thy-1 +) cell line is a complex process involvingadsorption, lateral redistribution, capping, andshedding of mycoplasmas. Unlike adsorption ofsome soluble ligands and viruses to cellularreceptors, adsorption of M. hyorhinis to hostcells is a very slow process. Although individualmycoplasmas may be detected on cells by immu-nofluorescence and electron microscopy, pin-point infections do not become evident until 4 to8 h postinfection. Thus, mycoplasma adsorptionprobably does not follow first-order kinetics,and multiple contacts between mycoplasmasand host cells may be required for adsorption.Furthermore, the association of multiple hostcell surface antigens with M. hyorhinis capssuggests that multiple ligand receptor interac-tions may be required for effective adsorption.In contrast, the adsorption of solubilized M.

hvorhinis components to S49 (Thy-I') cells is amuch more rapid process because it may involveonly single ligand-receptor interactions (G. H.Butler and E. J. Stanbridge, Abstr. Annu. Meet.Am. Soc. Microbiol. 1983, G23, p. 100). Onceadsorbed to cellular receptors, M. hvorhinisorganisms undergo patching and capping on thecell membrane. This is also a slow process,which culminates at 24 to 36 h postinfection withshedding of caps. After these events steady-state infection occurs and is characterized by thesimultaneous presence of pinpoints, patches,and caps in approximately equal numbers.We have demonstrated that the process of

patching and capping is a naturally occurringphenomenon by several criteria. First, detectionof patches and caps with antibody in the pres-ence of sodium azide at 0°C, as well as onparaformaldehyde-prefixed cells, shows thatthese events are not mediated by antibody cap-ping. In addition, caps can be detected in theabsence of antibody by Giemsa staining or elec-tron microscopy (18). Second, capping of killedmycoplasmas and mycoplasma membrane prep-arations indicates that the caps are not devel-oped as a consequence of replication of myco-plasmas on the cell membrane.

Infection of resistant cell lines such as S49(Thy-1-a) occurs by the same process as infec-tion of susceptible lines. The numbers of infect-ed cells, however, are significantly fewer, andmost infected cells have only single pinpoints offluorescence when stained for M. hvorhinis.We have used the phenomenon of mycoplas-

FIG. 6. M. hvorhinis-infected S49 (Thy-l+) cell at24 h postinfection stained for Thy-1.2 and M. hyorhinisas described in the text. (a) Phase contrast; (b) cap ofM. hyorhinis on the same cell; (c) co-cap of Thy-1.2 onthe same cell; (d) distribution of Thy-1.2 on an unin-fected S49 (Thy-1+) cell. Bar, 10 ,.m.

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1142 BUTLER AND STANBRIDGE

ma capping on the plasma membrane of hostcells to demonstrate that cell surface glycopro-teins selectively associate and redistribute withmycoplasmas on the cell membrane. Double-label immunofluorescence studies showed thatThy-1, H-2, and gp7O co-capped with M. hyor-hinis on cells which expressed levels of theseantigens detectable by immunofluorescence. OnS49 (Thy-l+) cells, most mycoplasma capsshowed co-capping of Thy-1.2. On BW5147(Thy-l+) cells, Thy-1.1 was co-capped with 45%of M. hyorhinis caps, and H-2k and gp7O wereco-capped on virtually all of the mycoplasmacapped BW5147 (Thy-1+) and BW5147 (Thy-1-a) cells. In contrast, T200, a major cell sur-face glycoprotein expressed on all of these cellsas well as on S49 (Thy-1-a) cells, was not co-capped on any cell line. The selective associa-tion of Thy-1, H-2, and gp7O with M. hyorhinison the cell membrane supports the observationsof Wise et al. (21) that M. hyorhinis recoveredfrom the supernatants of infected BW5147 (Thy-1 +) cells could adsorb out the cytotoxic antibod-ies toward uninfected cells from antisera to Thy-1.1 and H-2k. These supernatants, however,were unable to adsorb the activity from anti-gp7O sera. We suggest that shed mycoplasmacaps retain some of the cell surface antigens withwhich they were associated on the surfaces ofcells. The lack of association of gp7O with shedmycoplasmas may simply reflect the fact thatgp7Os are moderately hydrophobic molecules(14) and may not be easily removed from the cellmembrane when caps are shed into culture su-

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FIG. 7. M. hyorhinis-infected S49 (Thy-1+) cell at24 h postinfection stained for T200 and M. hyorhinis asdescribed in the text. (a) Phase contrast; (b) cap of M.hyorhinis on the same cell; (c) distribution of T200 onthe same cell; (d) distribution of T200 on an uninfectedS49 (Thy-1 +) cell. Bar, 10 p.m.

TABLE 4. Infection of lymphoblastoid cells with M.hvor-hinis

% of Degree of infection at 24Cell line cells h (% of infected cells)

infected Pinpoint Patch Cap

AKR1 (Thy-1) 22 100 0 0AKR1 (Thy-1-d) 6 100 0 0Rl (TL+) 70 69 31 0RI (TL-) 53 72 17 11Rl (Thy-1-a) 100 2 44 54BW5147 (Thy-1-e) 30 100 0 0BW5147 (Thy-1-a) 12 93 5 2BW5147 (T200-a) 40 100 0 0BW5147 (Thy-1+) 35 60 28 12TlMl (Thy-1+) 43 75 17 8TiM1 (Thy-1-c) 94 18 37 45S49 (Thy-1+) 66 25 23 42S49 (Thy-l-a) 32 84 12 4

pernatants. In contrast, Thy-1 is more looselyassociated with cell membranes and may evenbe released spontaneously from cells (3).To determine which cell surface antigens were

involved in the adsorption of M. hyorhinis tocells, we measured the degree of infection ofseveral well-characterized sets of thymic lym-phoblastoid cell lines. We first compared infec-tion of Thy-1+ and Thy-1- cell lines, since initialstudies (18) showed that S49 (Thy-1+) cells werehighly susceptible to M. hyorhinis infection, andsince co-capping studies indicated a selectiveassociation between Thy-1 and mycoplasmas onthe cell surface. These studies showed that S49(Thy-1+) cells were more heavily infected thanS49 (Thy-1-a) cells and that BW5147 (Thy-1+)cells were more susceptible to infection thanBW5147 (Thy-1-a) cells. S49 (Thy-1-a) cellswere derived from S49 (Thy-1+) cells by immu-noselection of Thy-i-negative subpopulationswith anti-Thy-1.2; BW5147 (Thy-1-a) cells weresimilarly derived from BW5147 (Thy-1+) cellswith anti-Thy-1.1 serum (5,7).Our studies indicate that both of these theta-

negative cell lines were similar to their respec-tive parental cell lines for those cell surfaceantigens we evaluated by immunofluorescence.These observations led us to the hypothesis thatThy-1 was important for adsorption of M. hyor-hinis to thymic lymphoblastoid cell lines. To testthis hypothesis, we evaluated the infection ofthe additional Thy-i-positive cell lines TlMl(Thy-l+), Ri (TL+), Ri (TL-), AKR1 (Thy-lf),and BW5147 (T200-a) and of the additional Thy-1-negative cell lines TlMi (Thy-1-c), Ri (Thy-1-a), AKR1 (Thy-1-d), and BW5147 (Thy-i-e).These studies revealed the complexity of inter-actions between M. hyorhinis and thymic lym-phoblastoid cells. The Thy-i-positive cell linesAKR (Thy-f+), TlMl (Thy-f+), BW5147

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CO-CAPPING OF M. HYORHINIS AND CELL ANTIGENS

(T200-a), and Ri (TL+) infected poorly, where-as the Thy-i-negative cell lines Ri (Thy-i-a)and TlMi (Thy-1-c) infected well. Ri (Thy-1-a)cells infected better than S49 (Thy-l+) cells.Thus, expression of Thy-1 did not generallycorrelate with the susceptibility of thymic lym-phoblastoid cells to M. hyorhinis infection. Inaddition, since Ri (Thy-i-a) cells became veryheavily infected with M. hyorhinis, the class Aglycosylation defect did not confer protectionagainst infection. Similar comparisons of othercell lines studied showed that gp7O, H-2, TL, orT200 expression did not correlate with the levelof M. hyorhinis infection.These data suggest that infection of mouse

thymic lymphoblastoid cells is a complex phe-nomenon involving at least two types of interac-tions. First, M. hyorhinis surface molecules mayinteract with many lymphoblastoid cell surfaceantigens by binding to some common elementsuch as carbohydrate moieties shared by theseantigens. This is supported by the association ofM. hyorhinis with Thy-1.2, Thy-1.1, gp7O, andH-2k. The failure of T200 to co-cap with myco-plasmas suggests that this molecule may lack thecommon receptor moiety. We speculate that thecommon element may be carbohydrate becauseassociations between other mycoplasmas, i.e.,Mycoplasma pnelimoniae, and host cell surfaceglycoproteins have been well documented (12).Furthermore, Blenk et al. (H. Blenk, R. Arndt,and B. Blenk, Abstr. 4th Int. Congr. Mycoplas-mology 1982, p. 96) isolated carbohydrate-bind-ing proteins from mycoplasmas. Second, addi-tional interactions may be required for effectiveinfection of lymphoblastoid cells by M. hyor-hinis. This is apparent beause expression ofnone of the cell surface glycoproteins we studiedcorrelated with infection. Preliminary observa-tions (Butler and Stanbridge, abstract G23) sug-gest that as yet unidentified low-molecular-weight cell surface antigens are involved in theadsorption of intact M. hyorhinis to cellularproteins. Identification of these proteins, as wellas identification of the common element sharedby co-capped glycoproteins, will allow us todetermine their importance relative to infectionof cells and co-capping of cellular antigens.

ACKNOWLEDGMENTS

We are grateful to the various investigators noted fordonations of cell lines and antisera. We are especially gratefulto Robert Hyman for his discussion and comments.These studies were supported by Public Health Service

grant Al 15702 from the National Institutes of Health. E.J.S. isthe recipient of a Research Career Development Award fromthe National Cancer Institute (CA00271).

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