Proc. Nat. Acad. Sci. USAVol. 70, No. 12, Part II, pp. 3894-3898, December 1973

Immunological Function of Lymphocytes Fractionated withAntigen-Derivatized Fibers

(cell fractionation/clonal selection theory/adoptive transfer/antigenic specificity)

U. RUTISHAUSER, P. D'EUSTACHIO, AND G. M. EDELMAN

The Rockefeller University, New York, N.Y. 10021

Contributed by Gerald M. Edelman, August 27, 1973

ABSTRACT Specific antigen-binding cells from spleensof immune and nonimmune mice were isolated by themethod of fiber fractionation. After removal from thefibers, these cells were assayed for their viability, theirability to rebind to fibers of the same specificity, and theirin vivo response to the antigen after transfer to syngeneicirradiated recipients. These experiments indicate that thefiber method yields highly enriched populations of specificantigen-binding cells that are viable and include antigen-sensitive bone marrow-derived cells capable of undergoingmitosis and differentiating into antibody-secreting cells.

Despite a number of recent advances, the population dy-namics of the immune response have not been described inquantitative terms, and many of the basic mechanisms ofimmunity are still not well understood. The analysis of thesemechanisms is hindered by the enormous complexity of lym-phocyte populations, and the reduction of this heterogeneityby fractionation would be of obvious value. To carry out thistask, it is necessary to isolate both thymus-derived (T) andbone marrow-derived (B) lymphocytes with restricted rangesof antigen-binding specificities. It is also important that suchfractionated cells remain viable and capable of interactingnormally with antigen and other cells.

Several useful attempts have been made to obtain purifiedpopulations of antigen-binding cells. These include beadcolumns (1, 2), fluorescence-activated cell sorting (3), anddifferential sedimentation of rosettes (4). These fractionationsystems are not entirely adequate, however, either becausethey do not yield specific antigen-binding cells of both the Tand B classes, because they yield cells that are altered intheir response to antigen, or because they yield cell popula-tions that are only partially enriched with respect to theirantigen-binding specificity.

Evidence that the method of fiber fractionation can be usedto isolate specific antigen-binding T and B cells has been pre-sented in several previous publications (5-7). In the presentreport, we provide evidence that these cell populations areviable, highly enriched with respect to their antigen-bindingspecificity, and normally responsive to a specific antigenicstimulus.

MATERIALS AND METHODS

Animals. Specific-pathogen-free Balb/c mice obtainedeither from Jackson Labs (Bar Harbor, Me.) or Charles River

Labs (Wilmington, Mass.), or bred at the Rockefeller Uni-versity from Jackson Labs stock, were used interchangeably inall experiments.

Immunization. Mice were immunized intraperitoneally at8 weeks of age and monthly thereafter with 400 ,ug of Limulushemocyanin (Hcy), or 200 ,ug of hemocyanin conjugated withthe 2,4-dinitrophenyl group (Dnp-Hcy). These antigens wereadsorbed onto bentonite (8) prior to injection. Animals re-ceived 1 to 4 injections; spleens were removed 5 days afterthe last injection.

Preparation of Fibers. Nylon fibers strung in collars werederivatized with Dnp-bovine serum albumin (Dnp-BSA),or p-toluenesulfonyl-bovine serum albumin (tosyl-BSA) asdescribed previously (6). Alternatively, in a new modifica-tion of the method, nylon fibers were coated with Dnp-deriva-tized bovine gelatin (Dnp-Gel). Dnp-Gel was prepared byreacting 20 g of gelatin (Fisher Scientific) with 2 g of 2,4-dinitrobenzenesulfonic acid (Eastman) in 100 ml of 2% K2CO3for 1 hr at 37°. The Dnp-Gel was dialyzed at room tempera-ture against water containing 0.1% merthiolate, and storedat 40 as a 5% gel. Each collar was dipped into a 5% solutionof the Dnp-Gel at 40 50° and immediately centrifuged in aclinical centrifuge (International Equipment, Needham,Mass.) to shake off excess gelatin. Each collar containing thefibers was placed directly in a no. 325 supporting ring of theno. 215 swinging bucket head so that the fibers were horizon-tal and radially aligned, and centrifuged for 10 see at 200 rpm.After centrifugation, the coated fibers were cooled and washedwith cold phosphate-buffered saline.

Preparation and Quantitation of Fiber-Fractionated Cells.Cell suspensions were prepared as described previously (7).Spleen cells in Minimal Essential Medium plus 20 ,ug/ml ofDNase without NaHCO3 (MEM; Microbiological Associates,Bethesda, Md.) were incubated with derivatized or coatedfibers for 1 hr at 40 with gentle shaking (6). Binding to thefibers was 80-95% inhibitable by 300 ,ug/ml of Dnp-BSA orrabbit anti-mouse immunoglobulin G (anti-IgG). Assays forantigen-binding cells were carried out using collars with onerow of 12 fibers each. For preparative fractionation, collarswith two rows of 24 fibers each were used. After removal ofunbound cells by extensive washing, such a collar yielded 2 to5 X 104 fractionated cells.For assay purposes, the number of fiber-binding cells was

determined by a direct count of the cells bound to the edgesof the fibers (5). Specifically bound cells could be released inall cases by plucking the taut fibers with a needle (5). In an

3894

Abbreviations: BSA, bovine serum albumin; FBC, fiber-bindingcells; Gel, bovine gelatin; Hcy, Limulus hemocyanin; MEM,Minimum Essential Medium; PFC, plaque-forming cells; tosyl,p-toluenesulfonyl; T, thymus-derived; B, bone marrow-derived.

Immune Function of Fiber-Fractionated Lymphocytes 3895

alternative procedure, cells bound to Dnp-Gel fibers could bereleased by melting the Dnp-Gel (370, 15 min). The releasedcells were quantitated by a hemocytometer count; viabilityof the fractionated cells was estimated by trypan blue exclu-sion. Specifically, the petri dish containing the suspension ofreleased cells was pressed into a Mallinckrodt no. 8 hollowstopper which in turn was placed in a no. 320 bucket of a no.

269 swinging bucket rotor (International Equipment) andcentrifuged for 10 min at 1000 rpm. The supernate was re-

moved by aspiration and replaced by a small volume of trypanblue solution, and the proportion of cells excluding the dyewas determined in situ.

Rebinding Studies with Fractionated Cells. The efficiencywith which cells removed from Dnp-fibers could rebind thesame (Dnp) or a different (tosyl) antigen was determined bythe fiber-binding assay. Spleen cells were fractionated withpreparative (48-strand) dishes made with Dnp-BSA or Dnp-Gel fibers. The bound cells were removed by plucking intoMEMV plus 10% fetal calf serum (2.5 ml per dish). This cellsuspension was added without dilution to 12-strand dishescontaining fibers derivatized with Dnp-BSA or tosyl-BSA.The specificity of the rebinding was determined by doingduplicate assays in the presence of 300 ,ug/ml of Dnp-BSAor tosyl-BSA. In addition, the binding of various amounts(cells/ml) of unfractionated cells was assayed at the same

time.

Transfer of Cells to Irradiated Recipients. Unimmunizedfemale mice that had been exposed 24 hr previously to 630-730 rad (from a Picker 60Cobalt gamma source) were injectedintravenously with 5 to 10 X 106 splenocytes from mice im-munized with Hcy, plus either 104L105 Dnp- or tosyl-fiber-binding cells (FBC), or 104 106 unfractionated splenocytesfrom mice immunized with Dnp-Hcy. Control mice receivedonly 5 to 10 X 106 cells from mice immunized with Hcy,representing the carrier specificity. The recipient mice were

then injected intraperitoneally with 200 ,ug of Dnp-Hcy ad-sorbed onto bentonite. All materials used in this procedurewere sterile. The sterility of the fractionated cells was main-tained by extensively washing the fiber-bound cells withsterile phosphate-buffered saline. Seven days after transfer,the spleen from each recipient animal was tested for cellssecreting antibodies against Dnp, by a modification of theJerne plaque assay (8). Spleens were teased into Hank'sbalanced salts solution; aggregates were removed by filteringthe cell suspension through nylon mesh; finally the cells were

pelleted by centrifugation and resuspended in the appropriatevolume of medium for the assay. IgM-secreting cells were

detected directly; IgG-secreting cells were detected by theaddition of anti-IgG to duplicate assays (6).

RESULTS

The binding of mouse spleen cells to Dnp-BSA and Dnp-Gelfibers is illustrated in Fig. 1. The appearance and numbers ofcells bound to the two types of fibers did not differ greatly,and in both cases, 75-95% of the binding was inhibitable bysoluble Dnp-BSA, and 85-95% by antibodies against mouse

IgG. Prior to removal from the fibers, over 95% of the cellsexcluded trypan blue (7).

Consistent with previous observations, it was found thatthe viability of the recovered cells varied greatly dependingupon the type of fiber, the removal method, and the mediuminto which the cells were released. Cells could be released from

aM

F

bM

G 2

F_

FIG. 1. Mouse spleen cells bound to (a) Dnp-BSA fibers, and(b) Dnp-Gel fibers. F, fiber; M, medium; G, Dn>-Gel. (X 184magnification)

the fibers either by plucking the taut fibers or, for cells boundto Dnp-Gel fibers, melting the gelatin (Table 1). More cellssurvived removal from Dnp-Gel fibers than from Dnp-BSAfibers, especially if the cells were removed from the Gelfibers by melting the gelatin. In all cases, cell survival was en-hanced by the presence of fetal calf serum in the medium.Furthermore, the proportion of viable plucked cells increasedwhen the cells were incubated for 30 min at 370 in the presenceof serum, as if the cells were repairing lesions in their surfacemembranes. In the absence of serum, however, the viabilityof both fractionated and unfractionated cells decreased. Theseexperiments clearly indicate that the initial high viability ofthe fractionated cells can be maintained by choosing the ap-propriate conditions for cell removal.Although the cells removed from the fibers were viable, it

was not known whether they retained various immunologicalfunctions. As a straightforward test of their antigen-bindingfunction, we determined their ability to rebind specificallyto antigen-coated fibers (Fig. 2 and Table 2). To establish astandard curve, experiments were first done with unfrac-tionated cells. The number of bound cells was a linear functionof the cell concentration over a wide range (Fig. 2), suggesting

TABLE 1. Viability of spleen cells removed from Dnp fibers

Incuba-tiont

Fiber Medium* Removal (min) % Viable:

Dnp-BSA PBS Pluck 30 21 4- 2Dnp-BSA PBS Pluck - 46 i 2Dnp-BSA FCS Pluck 70 + 2Dnp-BSA FCS Pluck 30 81 4± 3Dnp-Gel PBS Pluck 30 21 3Dnp-Gel PBS Pluck - 62 + 4Dnp-Gel PBS Melt 30 70 4 2Dnp-Gel FCS Pluck 83 i 3Dnp-Gel FCS Pluck 30 91 + 2Dn>-Gel FCS Melt 30 97 + 1

Unfractionatedcells PBS 30 63 i 2cells PBS 8-84± 2cells FCS - 30 85 i 1

* PBS, phosphate-buffered saline; FCS, PBS plus 10% fetalcalf serum.

t Incubation was carried out at 370 in the same medium asthe removal process in all cases.

I As determined by trypan blue exclusion. Each number isthe average of three determinations. Standard deviations of themean are shown.

el

Proc. Nat. Acad. Sci. USA 70 (1978)

I

3896 Immunology: Rutishauser et al.

Donor:Dnp-Hcy immunized

1031

10

I/

,L ,L . -L104 i05 10 107 108Cells/4 ml

FIG. 2. Binding of spleen cells to Dnp-BSA fibers. Standardcurves are shown for the binding of previously unfractionatedspleen cells from Dnp-immune (0 0) or unimmunized(O-O) animals as a function of the cell number in the incuba-tion mixture. Standard deviations of the mean are indicated by thebars. The rebinding obtained with 4 X 104 previously fractionatedcells (-) is shown separately. Extrapolation of this binding value(--- -) shows that 62 times as many unfractionated nonimmunecells ( * ) or four times as many unfractionated immunecells (- * -) would be required to produce the same amount ofbinding (see Table 2).

that, under the conditions used, the binding is proportional tothe number of input cells, the binding of one cell is independentof that of another, and the fiber surface is not saturated withcells. As noted previously, a larger proportion of the cellsfrom immunized animals was bound to the fibers than wasthe case with cells from unimmunized animals.The experimental data indicate that Dnp-fractionated cells

did rebind to Dnp-fibers. Cells initially fractionated withtosyl-BSA fibers, however, did not rebind at all to the Dnp-fibers. If binding was expressed as a ratio of the numbers ofcells bound to the numbers of cells incubated, Dnp-fraction-ated cells from both immune and nonimmune mice werefound to bind with the same high efficiency (Table 2). Theconcentration of unfractionated cells required to produce anequivalent level of binding can be estimated as shown in Fig.

TABLE 2. Specific rebinding of fractionated cells toderivatized fibers

SpecificABC inunfrac- Specifictionated binding

Immuniza- popula- Predicted Observed effi-tion tion* enrichmentt enrichment ciencyt

None 1-2 50-100 X 44-78 X 32Primary

(5 day) 3-5 20-33 X 23 X 27Primary

(14 day) 6-8 12-17 X 12-15 X 29Secondary 10-17 6-10 X 6-7 X 39

* Determined by fiber and/or rosette methods with unfrac-tionated spleen cells (6). ABC is antigen-binding cells

t Based on enrichment of cell population for 100% specificantigen-binding cells.

t Defined as the number of cells specifically bound to bothedges of a 10-cm fiber segment under standard conditions at 104fractionated cells per ml (see Fig. 2).

a1T

Unfroctionated spleen cells

105 Dnp-FBC

Fractionation withDnp-BSA fibers

107 unfractionatedspleen cells

Transfer cells i.v. andimmunize with Dnp-Hcy i.p.

Unimmunizedirradiated

recipient

Assoy spleen for cells secretingIgG and IgM ontibody agoinst Dnp

Dnp plaqueassoy

FIG. 3. Protocol for transfer experiments (Tables 3 and 4).The same procedure was used in both sets of experiments, exceptthat fractionation was carried out on Dnp-BSA fibers in one

case (Table 3), and on Dnp-Gel fibers in the other (Table 4).i.v., intravenous; i.p., intraperitoneal.

2. By dividing this cell concentration by the concentrationof fractionated cells used in the rebinding assay, the enrich-ment of antigen-binding cells can be calculated (Table 2).In all cases the observed enrichment was nearly equal to thatexpected for a pure population of antigen-binding cellsspecific for Dnp.The results of experiments designed to test the function of

fractionated cells in vivo are shown in Tables 3 and 4. Frac-tionated Dnp-binding cells from mice immunized with Dnp-Hcy were transferred to syngeneic, irradiated mice and testedfor their ability to produce a specific humoral antibody re-

sponse when stimulated by Dnp-Hcy (Fig. 3). Cells from miceimmunized with Hcy were mixed with the fractionated cellsboth to serve as a carrier and to provide the Hcy-specific Tcells required for a complete response by the Dnp-specificcells. Inasmuch as the unfractionated cells were from micethat were not immunized against Dnp, the frequency of Dnp-specific B cells was relatively low and these cells respondedweakly with the production of both IgM and IgG antibodies.On the other hand, the fractionated cells from mice immunizedwith Dnp responded by the production of IgG antibodies only.

In order to quantitate the enrichment obtained by frac-tionation, it was necessary to determine the response per cellinjected. The number of cells secreting antibody of the IgGclass was not a simple function of the number of transferredcells (Fig. 4). Over the range from 105 to 107 transferred cells,it was found that increasing the cell number by a factor x in-

creased the response by a factor of approximately x2/'.Fractionation by Dnp-BSA (Table 3) or Dnp-Gel (Table 4)

104Donor:

Hcy immunized

Proc. Nat. Acad. Sci. USA 70 (1978)

QuatAr.c

Immune Function of Fiber-Fractionated Lymphocytes 3897

of spleen cells from mice immunized against Dnp-Hcy yieldedcells that produced a Dnp-specific response equivalent'to thatof at least 10 times as many unfractionated cells from thesame animals. In contrast, cells obtained by fractionation ofthese cell populations with tosyl-derivatized fibers did notyield numbers of Dnp plaque-forming cells (PFC) above con-

trol values. There was also no detectable response if the re-

cipient animals were not simultaneously injected with Dnp-Hcy. Inasmuch as these unfractionated cell populations con-

tained 7-15% Dnp-binding cells, the observed enhancement inthe transfer experiments approached the value expected froma pure population of Dnp-specific cells, of which at least theantigen-sensitive B cells are functioning normally. As expectedof cells from immunized mice, enhancements were not ob-served in the IgM response. It is noteworthy that in severalexperiments the Dnp-specific IgM response of 5 X 106 un-

fractionated Hcy cells was partially suppressed in the pres-

ence of 2 X 104 unfractionated Dnp-Hcy cells, but not in thepresence of 2 X 104 Dnp-fractionated cells from the same

Dnp-Hcy population (Table 4).

DISCUSSION

The fractionation of lymphoid cell populations by means ofantigen-derivatized fibers yielded highly enriched populationsof specific antigen-binding cells. The isolated cells were viable,displayed the appropriate antigen-binding specificity, andincluded B cells that responded to stimulation by antigen toproduce specific antibody-secreting cells.Removal of the fractionated cells from the fiber without

damage depends on several factors. In a previous report (5),we noted that cells specifically bound to derivatized fibers or

beads cannot be released by the addition of a competitivebinding inhibitor. It was suggested that, after being specif-ically bound, the cell membrane forms secondary adhesionsto the surface of the fiber or bead, and, therefore, other phys-ical means of removal were adopted. The physical methodsusing taut fibers required shearing of the cell-fiber bond,

TABLE 3. Response of cells isolated with Dnp-BSA fibersafter transfer to irradiated recipients

Transferred cells*

Un- ResponsetUnfrac- frac-

Frac- tionated tionated IgM PFC IgG PFCtionated Dnp-Hcy Hcy per per

Dnp-FBC cells cells spleen spleen

0 0 107 4,163 6,503290 752

0 7X 104 107 5,077 9,5371,583 2,158

0 7X 106 107 4,873 25,533246 2,801

7 X 104 0 107 4,487 30,733354 3,177

* The experimental procedure is summarized in Fig. 3. Thenumbers shown are the numbers of cells injected into eachrecipient mouse.

t Individual recipient spleens were assayed for PFC 7 daysafter transfer. Each number is the average of data from sixmice. The standard deviations of the mean calculated from thedata are shown. Cells from Dnp-immune animals fractionated on

tosyl-BSA fibers did not yield PFC above control values.

27~

9

IgGPFC 3

0 1 3 9 27 81105 Transferred cells

FIG. 4. Efficiency of immune response of unfractionatedspleen cells after transfer to irradiated recipients. The indicatednumber of spleen cells, from mice immunized with Dnp-Hcy, wasmixed with 107 cells from mice immunized with Hcy, and in-jected intravenously into an irradiated syngeneic recipient.The recipient mouse was then immunized with Dnp-Hcy. Thespleen of each recipient was assayed for PFC 7 days later. Eachpoint represents the average of data from three mice; standarddeviations of the mean are indicated by the bars.

which can produce a lesion in the cell surface membrane. Dam-age to the cells is minimized by either (1) using a "soft"gelatin-coated fiber that presumably minimizes the shear re-quired for release, or (2) avoiding shear forces altogether bymelting the cells off a gelatin-coated fiber, and, in all cases,(3) using the appropriate medium and incubation conditionsto allow repair of any lesions produced by the removal pro-cess. With these methods, fractionated cell populations withhigh viabilities can be obtained routinely from either deriva-tized or gelatin-coated fibers.A radical rearrangement or even removal of specific im-

munoglobulin receptors can result from the incubation ofantigen-binding cells with soluble antigen or soluble anti-immunoglobulin (9, 10). It might be expected, therefore, thatthe specific binding of lymphocytes to antigen-derivatizedfibers would cause similar effects. In previous studies (6), wefound that up to 50% of the cells bound to Dnp-fibers werecapable of forming uniform rosettes in situ when incubatedwith cross-reacting 2,4,6-trinitrophenyl-derivatized erythro-cytes. This suggested that, for these cells, the binding eventdid not induce a radical rearrangement of all the receptors.

TABLE 4. Response of cells isolated with Dnp-gelatin fibersafter transfer to irradiated recipients

Transferred cells*

Unfrac- Unfrac- ResponsetFrac- tionated tionated IgM PFC IgG PFC

tionated Dnp-Hcy Hcy per perDnp-FBC cells cells spleen spleen

0 0 5X106 2,240± 1,120±481 171

0 2X104 5X106 1,299± 2,033±4133 444

0 2 X 105 5 X 106 1,390 i 4,182 ±t281 1,188

2X 104 0 5 X 106 2,073 ±t 5,750 ±240 1,285

*Except for the fact that the fractionated cells were preparedwith Dnp-Gel fibers, the transfer procedure was identical to thatdescribed in Table 3.

t Individual spleens were assayed for PFC. Each number isthe average of data from six mice. The standard deviations ofthe mean calculated from the data are shown.

Proc. Nat. Acad. Sci. USA 70 (1978)

3898 Immunology: Rutishauser et al.

The failure of the remaining cells to form rosettes may reflectdifferences in the relative sensitivities of the rosette and fiber-binding assays, or in the antigen-binding properties of T andB cells.Experiments based on the rebinding of fiber-fractionated

cells to fibers of the same specificity circumvent many of theambiguities encountered with the fiber-rosette assay (6). Theresults indicate that the isolated cells, even after plucking,were not grossly impaired in their ability to bind antigen.The conditions of the rebinding assays (incubation at 40 forno more than 2 hr) do not allow time for a significant amountof synthesis or rearrangement of receptors. We therefore con-clude that at least some receptors remain on or in the cellsduring fractionation.

Analysis of the enrichment obtained in the rebinding experi-ments indicates that most of the fractionated cells have hap-ten-specific receptors. Dnp-fractionated cells from both unim-munized and immunized animals were re-bound to antigen-coated fibers with equivalent efficiencies (Table 2), despite thefact that the two populations have been shown to differ mar-kedly in their affinities for the Dnp group (6). These previousstudies also indicated that up to 2% of spleen cells from unim-munized animals can bind to a single haptenic determinant,but that only a small proportion of these cells, those havingreceptors with a relatively high affinity, are stimulated by thatantigen. The lower-affinity cells presumably can bind otherunrelated antigens with a higher affinity and possibly betriggered by them. It is probable, therefore, that cells withlow affinities for Dnp did not function as antigen-sensitivecells in the present experiments. Preliminary experiments ex-amining the relationship between cross-reactivity and affinityin fractionated cell populations from immune and nonimmunemice support this hypothesis.The stimulation by antigen of the fractionated antigen-

binding B cells to differentiate into antibody-secreting cellssuggests that their functional properties have not been alteredby the fractionation procedure. The data are consistent withthe idea that the proportion of Dnp-sensitive B cells in thepopulation of Dnp-binding cells is the same before and afterfractionation. The magnitude of the response obtained withDnp-fractionated cells is well correlated with the ex-pected enrichment and with the results obtained in rebindingexperiments. Although the number of antibody-secreting cellsobtained in vivo for each fractionated cell injected cannot bedetermined directly, an approximate calculation can bemade. The spleen of an animal injected with 104 fractionatedcells yields 2 to 4 X 103 PFC. To calculate the actual yieldof PFC per fractionated B cell, several assumptions and cor-rections are necessary. (1) The number of PFC found on day7 is equal to the total yield of PFC. We have in fact only de-termined that the response is maximal at this time, and areprobably underestimating the total number of PFC. (2) Thehoming efficiency of the transferred cells to the spleen is 3-7%

(11). (3) Sixty percent of the fractionated cells are B cells (7).(4) The viability of the transferred cells is 80-90%. (5) Thefractionated population is 90% Dnp-binding cells, of which,for the highly immune donor cell populations used in thisstudy, about 60-80% have a high enough avidity to be stimu-lated by Dnp-Hcy (6). Applying these corrections to the datain Tables 3 and 4, a range of values of 7 to 50 PFC per frac-tionated antigen-sensitive B cell injected is obtained. Thiscalculation is obviously approximate, but nevertheless doessupport the conclusion that cell division has occurred in re-sponse to the antigen.The unusual exponential relationship between the number

of cells transferred and the response of the recipient (Fig. 4)has not been explained. A number of phenomena could pro-duce such an effect, such as competition for homing sites inthe recipient spleen, or a toxic effect of the transferred cells onthe recipient. Similarly, the apparent suppresive effects of onecell population on the response of another, as observed in theIgM production of unfractionated cells (Table 4), are as yetunexplained. The complexity of the transfer system makesa direct analysis of these problems difficult, and the presenceof complex feedback loops and control steps tends to hinderthe study of more fundamental events in the immune re-sponse. The classical approach to such difficulties is to isolatethe key components of the system to study their propertiesand interactions under more controlled conditions, e.g., intissue culture. Despite these complexities, our present experi-ments indicate that fiber fractionation can be used as a generalmethod for the isolation of specific antigen-binding cells andthat these cells function normally in an in vivo immune re-sponse.

The authors thank Dr. Patricia Spear for her participationduring the initial stages of this work and Miss Barbara McVeetyfor her valuable technical assistance. This work was supportedby USPHS Grants from the National Institutes of Health.

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Proc. Nat. Acad. Sci. USA 70 (1978)