delta 4 variable region gene segments. V sensitivity preferentially use ...

Immune or Normal y8 T Cells That Assist ap T Cells in Elicitation of Contact Sensitivity Preferentially Use Vy5 and V64 Variable Region Gene Segments' Wlodzimierz Ptak,* Marian Szczepanik,* Rajani Ramabhadrantt and Philip W. Askenase2t In the current study, we confirmed previous findings suggesting that yS T cells were involved in the successful adoptive cell transfer of contact sensitivity (CS) by cup CS-effector T cells. In this study, we used hamsteranti-mouse y6-TCR mAb treatment of CS-effector T cells, followed by enrichment and removal of the yS T cells with goat anti-hamster Ig-linked magnetic beads, or by addition of hemolytic rabbit C. This removal of y6 T cells abrogated adoptive cell transfers of CS, despite the presence of ap T cells that are known to mediate CS. FACS analysis documented enrichment of y6 T cells rising from 1 to 2 % of the starting cells, to 60 to 95% of the magnetic bead adherent cells. Adoptive cell transfer of CS was reconstituted by adding back to the cup cells, highly enriched y6 cells attached to anti-yS-TCR magnetic beads. Not only were @-enriched T cells from sensitized mice able to assist immune CS-effector a p T cells, but y6 T cells from normal nonimmune mice also had CS-assisting activity, and furthermore, neither were MHC-restricted in this function. Thus, CS-assistingy6 T cells were present endogenously in normal mice without priorimmunization, and acted without Ag specificity and without MHCrestriction, to assist CS-effector a$ T cells. Similar studies, with hamster mAbsspecific for Vy and VS portions of yS-TCR, demonstrated that they6 T cells that assisted the CS-effector a@ T cells preferentially expressed Vy5 and V64 in their TCR. PCR analysis on extracted mRNA showed that Vy5 and V64 gene segments indeed were rearranged and expressed in the sensitized and normal lymph nodes; and oneand two-color FACS analysis of magnetic bead-fractionated cells suggested that Vy5 and VS4 were expressed on the same T cells. In summary, these results demonstrated that Vy5+, V64+, y6 T cells were needed to assist cup effector T cells in the adoptive cell transfer of CS.The journal of Immunology, 1996, 156: 976-986.

T

he role of y6 T cells in immune responses is largely unknown. We recently reported that y6 T cells were necessary for optimal adoptive cell transfer of contact sensitivity (CS)3 responses in mice (1). CS reactions are Ag/MHC-specific responses that are elicited in vivo in the skin and are mediated by a@ T cells that probably are specific for hapten conjugated to self peptides and complexed with MHC molecules, on the surface of APCs (2). Our previous work established that CS responses are in fact due to two different Ag-specific Thy-I+, CDS+ cells. The first acting cell, called the CS-initiating cell, is Thy-l+, CD5+, CD4-, CD8-, CD3-, surface Ig-, IL-3Rt, and B220+ (3-5). This Agspecific cell is not a B cell (6), and probably is not an a@or y6 T cell (1, 3, 4, 5 , 7). The CS-initiating cell leads to an early, 2-h, serotonin-dependent local vasoactive response that is required for recruitment of the second acting, classical CS-effector T cell. The CS-initiating cell functions via release ofan Ag-specific factor that *Departmentof Immunology, JagiellonianUniversity College of Medicine, Krakow, Poland; and 'Section of Allergy and Clinical Immunology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06S20 Received for publication January 23, 1995. Accepted for publicatlon November 7, 1995. The costs of publication of thls article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate This fact.

' This work was supported by grants from the National Institutes of Health (Al12211 and AI-26669 to P.W.A.), and the Polish Committee of Scientific Research, and Marie-Curie Fund to W.P.

*

Address correspondence and reprint requests to Dr. Philip W. Askenase, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Abbreviations used in this paper: CS, contact sensitivity; DETC, dendritlc epidermal T cell; MACS, magnetic cell separation system; PCI, picryl chloride; PE, phycoerythrin; OX, oxarolone. Copyright 0 1996 by The Amerlcan Association of Immunologists

acts via mast cells (8, 9) and platelets (10) to cause local release of serotonin to facilitate vascular recruitment of CS-effector T cells (1 1 , 12). These second acting, A g N H C class 11-specific T cells, which depend for local recruitment on the serotonin-mediated CS initiation noted above, induce 24- to 48-h skin-swelling responses by producing Thl cytokines, and are Thy-l+. CDS", CD4', CD8-, CD3+, TCR-aP+, and IL-ZRf (3-15). Our previous studies sought to determine whether early acting CS-initiating cells are yG-TCR+. We found that neither CS-initiating nor CS-effector T cells were yS-TCR+, but that a third population of T cells, called CS-assisting cells, that was necessary for elicitation of the later classical 24- to 48-h phase of CS, was y8TCR+, but was not Ag specific nor MHC restricted ( I , 16). These previous studies did not investigate the Vy and V6 gene segments involved in assistance of the a@CS T effector cells. In addition, some of these studies depleted yS T cells by in vitro coating with hamster anti-yG-TCR mAb, followed by addition of rabbit C, and then i.v. transfer to recipients subsequently skin tested to elicit CS responses. Because mAb + C depletion of y6 T cells probably was completed in vivo (17), we were not able to verify in vitro that the yS T cells were depleted. In addition, even though separation by mAb with immunomagnetic beads was used previously (16), no verification of separation efficiency was provided. In the current study, we again used cell separation by anti-y6 Ab-coated magnetic beads, and also by various anti-Vy and anti-VS mAb. This enabled us to deal with large numbers of cells needed for in vivo transfer experiments, and allowed for efficient recovery of the positively selected cells. In addition, this technique permitted determination by FACS analysis of the surface phenotype of the positively selected cells, and again allowed recovery and adoptive transfer of these selected cell subsets in vivo, to regulate elicitation of CS responses in the recipients. We confirmed 0022-1 767/96/$02.00

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The Journal of immunology that immune or normal yS T cells were required for (YP T cells to adoptively transfer CS, and that V y 5 and Vi34 gene segments were yS CS-assisting cells. Furtherexpressedpreferentiallyinthese more, Vy5 V64 + cells from normal or allogeneic mice mediated CS assistance, and our results suggested that Vy5 and V& were expressed on the same cell.

Materials and Methods Mice Male 6- to 8-wk-old CBA/J and BALB/c mice were obtained from the Jackson Laboratory (Bar Harbor, ME), or from our breeding unit in Krakow, Poland. After arrival, mice were rested at least 1 wk before use.

Reagents Picryl chloride (PCI, trinitrophenyl chloride) was obtained from Chemica Alta (Edmonton. Alberta, Canada), and was recrystallized from methanol/ H,O before use. and protected from light in a desiccator during storage at room temperature. Oxarolone (OX, 4 ethoxymethylene-2-phenyl oxazolone) was obtained from BDH Chemicals Ltd. (Poole. England).

Antibodies and complement The following mAbs were used. Culture supernatants: anti-TCR-yG (clone UC7-13D5, hamster IgG) (18), from Dr. J . Bluestone, University of Chicago (Chicago, IL); anti-TCR-yG (clone GL4, hamster IgG) and anti-Vi34 (clone GL2, hamster IgG) ( I 9), both from Dr. Leo Lefrancois, University of Connecticut (Farmington, CT): anti-Vy5 (clone F536, hamster IgG) (20), from Dr. James Allison, University of California at Berkeley; and anti-V66.3 from Simon Carding (University of Pennsylvania, PA).The yG-TCR V region nomenclature used in this work is that of Heilig and Tonegawa (21). We also used FITC, PE. or biotin-labeled affinity-purified mAbs obtained from PharMingen (San Diego, CA), including UC7, GL4, F536, GL2, and H57 (anti-TCR-P chain).

Immunization for induction o f contact sensitivity-effector cells Mice were contact sensitized by topical painting with 0.15 ml of a 5% solution of PC1 in an ethanohcetone mixture (3:l, v:v) on all four paws and the .;kin of the clipped abdomen and chest.

Adoptive cell transfer of contact sensitivity T cell subpopulation depletion by in vitro treatment with monoclonal antibodies and complement. Four days after CS with PCI, mice were killed, and spleens and lymph nodes were removed. Cell suspensions were prepared and washed three times with PBS. The ability of y6 T cells, and of certain Vy- or VG-expressing subsets, to influence ap T cells in successful adoptive cell transfer of CS was determined by two different protocols. In the experiment shown in Figure 4, immune cells were incubated with anti-yS-TCR mAb. or with specific anti-Vy or anti-VG mAb, for 30 minat 4°C. Usually 7 to 10 X IO7 cells were incubated with 5 ml of mAb-containing supernatant, unless otherwise stated, or with 10 to 20 p g of affinity-purified mAb that was diluted in PBS containing 3% fetal bovine serum, in a volume of 5 ml. After a single wash at 4"C, cells were incubated with rabbit C (Pel Freeze. Brown Deere, WI) (2 X IO7 cells/ml) and then mixed intermittently for an additional 45 min at 37°C. Cells treated with C alone served as controls. mAb- and C-treated cells were washed and resuspended, and cells remaining from 7 to 10 X IO7 starting cells were injected i.v. into naive syngeneic mice via the retro-orbital venous plexus. Magnetic bead cell separation for transfers. A second technique used was separation by mAb-coated magnetic beads ( 1 pm BioMag; Advanced Magnetics, Cambridge, MA). Four-day PCI-immune lymph node cells (4 X 10') were incubated with 50 ml of anti-y6 mAb supernatant at 4°C for 60 min. Then the anti-yG mAb-coated cells were washed three times with PBS. and resuspended in 50 ml PBS + I%FCS, containing goat antihamster Ig-coated magnetic beads at 5 to I O beads per cell. We estimated that yG T cells were 2% of the total, and worked in a slight (SX) bead excess to favor isolation of yS T cells. Then the cell and bead mixture was incubated in a vertical 50-ml tissue culture flask on ice. After 30 min, a flat vertically held 8 X 12-cm magnet (Advanced Magnetics) was applied. After 10 min, medium containing the nonadherent (magnetic bead-negative) cells was removed with a pipette. Then the remaining adherent (magnetic bead-positive) cells were washed in PBS 1 % FCS, recovered by re-attachment to the magnet, and counted. On subsequent FACS analysis, by staining the bead.; and attached cells

+

with FlTC anti"y6, the beads did not stain because the y8-TCR and the anti-yG-TCR were on the cells and not on the beads. Viability was 70 to 80%. Then adherent magnetic bead-positive cells. or the nonadherent magnetic bead-negative cells, or a mixture were injected i.p. into naive recipients in 1.5 ml PBS. In several different experiments, approximately 6 X IOh regulatory adherent yGTCR+ cells and 4 X 1 O7 yG-TCR nonadherent CS-effector cells were injected i.p., per recipient. Microscopy of the magnetic bead-positive cells revealed that many cells had attached beads. No attempt was made to remove the beads from the surface of the cells before injection. It took approximately 15 to 25 donor immune mice to obtain the 2 5 X IO7 yG T cells used in each experiment. Measurement of contact sensitivity responses. Recipients of cells treated in vitro with mAb + C (i.v. transfers; Fig. 3) or with anti-TCR mAb, and then with anti-hamster Ig Ab-coated magnetic beads (i.p. transfers; Figs. I , 2, and 4) were ear challenged. In the experiments shown in Figures I and 4, earchallenge was postponed for 18 h after transfer to allow immune cells to emigrate from the peritoneal cavity, and to optimally detect the 2-h initiating phase of CS (13, 14. 22). Ear-skin challenge consisted of topical application of 1 drop (27-gauge needle) of 0.8% PC1 in olive oil to both sides of both ears. Duplicate measurements of ear thickness were made bilaterally with a micrometer (Mitutoyo, Paramus, NJ) before challenge, and 2,24, and 48 h after challenge, unless stated otherwise. The increase cm. In in ear thickness was expressed as the mean ? SE in units of each separate experiment, there were three or four mice per group. In addition, in each experiment, the ears of nonimmunized controls were challenged and measured 5imilarly. The background-swelling response in this control group usually averaged approximately 1 U at 2 h, 2 U at 24 h, and 3 U at 48 h, and was subtracted from the swelling response of the experimental groups. Thus, the results are presented in the figures as the net increase in ear swelling. ~

Statistics Statistical analysis was performed by using the two-sample, unpaired Student's t test, and p < 0.05 was taken as the level of significance. For each instance in whichp < 0.05 by the Student's t test, the significance also was determined by the nonparametric Mann Whitney U test. In all instances, p < 0.05 also was obtained. Each figure shows the results of a representative experiment that used three or four mice per group, or pooled experiments (Fig. 3).

One-color fluorescence-activated cell sorter analysis Cells harvested as described above for in vivo transfers were kept on ice and diluted to 106/mlin PBS with 2% FCS and 0.01o/o sodium azide (FACS buffer). Then 1 0 0 p1 (10' cells) were added to a groupof wells of a V-bottom 96-well plate kept on ice. Then, either FACS buffer alone (to determine the background fluorescence), or an appropriate dilution of FITC-conjugated anti-TCR-cup (H57), or anti-yG-TCR (UC7) (PharMingen) was added to triplicate wells. In some experiments, aggregated mouse y-globulin or normal human serum was used to block Ig FcRon the assay cell population, but no differences were found, nor was there blocking of staining when we used mAb 2.4G2, i.e., anti-FcyRII (CD32). The plates were covered and incubated on ice for 60 min. Then well5 were washed by addition of FACS buffer and centrifugation at 4°C.Finally, cell pellets were transferred to capped polystyrene Falcon tubes ( 1 2 X 75 mm) after resuspending in 200 pl FACS buffer containing 1 % paraformaldehyde, and stored at 4°C wrapped in aluminum foil. The next morning, FACS analysis was performed on a Becton Dickinson FACStar cell sorter run at approximately 275 mW laser power with an argon laser, for analysis of IO4 cells per sample. For data acquisition and analysis, 21 Lysys II software program was used.

Two-color fluorescence-activated cell sorter analysis Samples were run on the Becton Dickinson FACStar""' flow cytometer, with IO4events per collected sample in list mode for four parameters: SSC, FSC, FLI, and FL2. The laser excitation wave length was at 488 nmat approximately 250 mW. Before analysis, the lymphocyte population was selected in the FSC-SSC dot plot, with dead cells and debris gated o u t , and the resulting gated FlTC dot plot was analyzed for double positive cells.

MiniMACS yS T cell separation and subsequent fluorescence-activated cell sorter analysis To determine by FACS whether both Vy5 and V U were expressed on the same T cells, 4-day PCI-immune lymph node cells were separated using miniMACS immunomagnetic beads (Miltenyi Biotec Inc., Auburn, CA), and the derived column eluate and flow-through fractions were stained with FITC-conjugated mAb antl-Vy5 or with PE-conjugated anti-Vi34.

978 Briefly, cells were washed three times with PBS, and resuspended at IO' per/ml in PBS containing 5 mM EDTA. Then cells were incubated for 20 min on ice with IO p g anti-Vy5, or biotinylated anti-VW mAb per lo7 cells. After incubation, cells were washed twiceand resuspended to 10' cells in 90 p1 of PBS + 5 mM EDTA. Then 10 pl of MACS streptavidin-conjugated microbeads (Miltenyi Biotec Inc.) were added, and cells were incubated for 15 min on ice with vigorous agitation, and then washed twice with PBS + 5 mM EDTA. Pelleted cells were resuspended in 1 ml of 0.5% BSA in PBS, and applied to a miniMACS column fitted to a magnet. The column was washed once with 0.5% BSA-PBS, and flow-through cells were collected as the magnetic nonadherent fraction. Then the column was washed twice with 0.5% BSA-PBS,andremovedfromthemagneticseparator.Themagnetic adherent cells wereflushed out of the column into a fresh tube using the plunger included in the MiniMACS kit. Magnetic adherent and nonadherent fractions were washed and resuspended to lo6 cells/ml in FACSbuffer. Then IO5 cells were incubated either with FACS buffer alone (to determine background fluorescence), or an appropriate dilution of FITC-conjugated anti-Vy5 or PE-conjugated anti-VW for 30 min on ice. During incubation, samples were protected from light. Then cells were washed three times with FACS buffer, resuspended in 200 pl FACS buffer containing 1% paraformaldehyde, and stored at 4 ° C protected from light to be processed for FACS analysis the next day.

Ribonucleic acid isolation Total RNA was isolated from lymphoid organs of mice. The mice were killed by cervical dislocation. Spleens and lymph nodes were removed and were flash frozen by dropping them into separate containers with liquid nitrogen. The frozen tissues were stored at -7OOC until later processing. For RNA isolation, 4 Mguanidinium-thiocyanate solution was added to the frozen tissue at 1 mV0.1 g tissue weight, and the tissue was homogenized using a Virtis homogenizer. Most of the RNA isolation procedure was according to Chomczynski and Sacchi (23). The final RNA preps were stored as aqueous stock at -70°C.The integrity of RNA preps was checked by electrophoresis of an aliquot of 1 to 2 p g RNA in formamideformaldehyde loading buffer containing bromophenol blue. Ethidium bromide was addedto a final concentrationof 40 to 50 pg/ml. After mixingthoroughly, the sample was heated at 65°C for 10 min and electrophoresed through formaldehyde/l% agarose gelin I X MOPS (3-[N-morpholino]propanesulfonic acid) buffer at 1 0 0 V, withconstant circulation.At the end of the run, RNA was visualized using UV light.

Ribonucleic acid polymerase chain reaction RNA-PCR was conducted using the Perkin-Elmer Cetus (Emeryville, CA) RNA-PCR kit. One microgram of total tissue RNA was used per reaction. For reverse-transcriptase reaction to synthesize the first cDNA strand, Cy1 and C6 oligonucleotides were used as 3'-antisense primers for TCR-y and TCR-6 reactions, respectively, at 0.5 p M final concentration (24). For amplification of the TCR-y cDNA, TCR Vy5 and TCR Cy1 oligonucleotides were used as 5'-sense and 3"antisense primers, respectively, at 0.5 p M final concentration (24). Similarly, for amplification of TCR-6 cDNA, TCR V61 or V64, and TCR C6, oligonucleotides were used as 5"sense and 3'-antisense primers, respectively, at 0.5 FM final concentration (25). For the Vy5 and V61 PCR-positive control, RNA was obtained from a Vy5+ V61+ DETCclone that was provided by Dr. Robert Tigelaar, Dermatology Department, Yale Medical School (New Haven, CT). An aliquot of PCR-amplified cDNA was electrophoresed on 1% agarose gel in 1X Tris acetate EDTA buffer at 100 V. Due to low abundance of PCR products, no DNA bands were visualized on ethidium-bromidestaining, except for control DETC cDNA. Then, the gel was Southern transferred onto nitrocellulose membrane using 1OX SSC, followed by baking in a vacuum oven at 80°C for 2 h. The TCR-y and TCR-6 Southern blot was hybridized, at 4 2 T , with 3ZP-labeledTCR Vy4, Cyl, and Js2 probes, respectively. For the TCR-yprobe, V4, Jyl, a 158-bp ClaVXhal fragment, was from V4CUpGEM4 plasmid clone (25). Each probe was labeled with 32Pusing random hexamer primers. The hybridized blots were washed very stringently using 0.1X SSC 0.1% SDS at 60 to 65°C. The washed blots were exposed to Kodak X A R - 5 film at -70°C using intensifying screen. For the V64-CS PCR, the following cDNA primers were used VM,5'-CTGGAGATCTCCGACTCGCAGCTG GGG-3' and C6, 5'-AGGGTAGAAATCmCACCAGACA-3'.

+

Results Magnetic bead separation shows that yS T cells are required for ap T cells to transfer contact sensitivity responses

To verify that yS T cells actually were required for cup T cells to adoptively transfer CS responses, we treated 4-day PCI-immune

V y 5 + A N D VS4+ T CELLS ASSIST

(YO T CELLS

Ear Swellma Response

Group

c

Cell Fraction

Transferred

y6-Enriched

(units x 10-3 +SE) hr

(% positive control)

24 hr

(% posiliva control)

(3'5)

FIGURE 1. Magnetic bead separation shows that y6 T cells are required for ap T cells to transfer CS responsiveness. CBA mice were sensitized actively with PCI. Four days later, lymph node cells were harvested and suspended in hamster anti-murine y6-TCR mAb (UC7) and incubated. Then, after washing, anti-y6-TCR-sensitized cells were resuspended in PBS (with 1% FCS) containingparamagnetic beads coated with goat anti-hamster Ig Ab. Then after incubation on ice, a paramagnet was applied to the flask, and nonadherent and adherent cells were harvested and transferred i.p. to normal mice challenged18 h later on the ears with 0.8% PC1 in olive oil. A negative control ear-swelling group received no transfer, and just were challenged on the ears with 0.8% PCI. This determined the background ear swelling that was subtracted from responses of thevarious cell transfer recipient groups, to obtain the net increase in ear swelling that is shown in the figure. ***Significant vs group A, p < 0.005.

lymph node cells with hamster anti-murine yS-TCR mAb (UC7) and then with goat anti-hamster Ab-coated magnetic beads. After incubation, the magnetic bead adherent cells (the yS-TCR-enriched cells) were isolated with a magnet and transferred to mice i.p., either alone, or mixed with the remaining anti"$ mAb nonadherent cells (Le., containing the remaining TCR-aPf cells) that also were transferred to another group alone, and then recipient mice were challenged on their ears with PC1 18 h later. Untreated PCI-immune lymph node cells transferred 24-h CS reactivity, and also the required, earlier, 2-h, CS-initiating activity (Fig. 1, group A). Transfer of yS-depleted (ap-remaining) cells significantly reduced 24-h CS responsiveness, but not 2-h CSinitiating responsiveness (group B), andanti-yd adherent (78enriched cells) transferred reduced 2-h and 24-h reactions (group C). These results suggest that Ag-specific CS-initiating cells do not have y8-TCR on their surface. In addition, the fact that treatment with anti-yS-TCR mAb did not affect the CS-initiating cells suggests that the inhibition of 24-h transfers in the yS-depleted group B was not due to a nonspecific inhibitory effect of the magnetic bead process. Interestingly, similar to our previous results, 24-h CS was more than restored (125% of positive controls) (Fig. 1, group D) by transferring a mixture of the T remaining cells of group B (i.e., y8depleted) together with the yS-enriched cells of group C. FACS analysis showed that the anti-ya adherent population was highly enriched for yS T cells, i.e., 1 to 2% of the starting cells and 1 to 2% of the anti-yS nonadherent cells were yS-TCR+ (Fig. 2, left and center); while 70% of magnetic beads were adherent, yS-enriched cells that were required for the $3 T cells to transfer CS were yS-TCRf (Fig. 2, right). The cells that were stained with anti-TCR-cup mAb gave reciprocal results, and thus, anti-TCR-cYP mAb served as a control FACS-staining reagent. We concluded that actual addition of y6 T cells was required to assist a@T cells to mediate the adoptive cell transfer of CS responsiveness. Furthermore, biologically active yS CS-assisting cells could be recovered highly enriched via Ab-coated magnetic beads (group C) and

The journal of Immunology

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F I G U R E 2. FACS determination of immunoaffinity magnetic bead enrichment of murine yS T cells using anti-TCR-ys mAb UC7. Murine lymph node cells, obtained 4 days after PC1 contact sensitization, were separated into anti-yG-TCR adherentandnonadherent fractions. Then, unfractionated lymph node cells, cells of the anti-yS-TCR nonadherent population, and also the anti-yS-TCR adherent population were stained with FlTC anti-yS-TCR mAb and analyzed byFACS.

Group -

103

Starting Lymph Node Cells

T Cell Subpopulations Transferred

rs- (aP+) Immune CBA @-

+

Immune CBA $(ap-Enriched)

i-

Immune

$3

Nil

antiky6 TCR Non-adherent Cells (yS Depleted)

24 Hr Ear Swelling Responses (units x 10-3 cm f SE) (yopositive r

l

1

0

2

4

1

6

t

1

1

8 1012

control)

F I G U R E 3.

101

102

103

l~+

anti-yh TCR Adherent Cells (yS Enriched)

76-Enriched T cells from normal

mice assist allogeneic CS-effector T cells. CBA mice were contact sensitized with PCI. On day 4, lymph node and spleen cells were separated into 7 6 ' and 76- fractions with anti-yG-TCR magnetic beads. The yS -depleted fraction (ap T cell-enriched) failed to adoptively transfer CS (group B), but did transfer when supplemented with yS' cells from normal syngeneic CBA spleen (group C), or from allogeneic BALB/c suleen krour,, Di. Statistical significance: "wouu , B vs groups A, C, and D, p < 0.001; group A vs groups C and D, not significant.

I B

Immune CBA $+ Normal CBA y# (SyngenicAssistance)

+

Normal BALB/c $+ Immune CEA $(Ailogenic Assistance)

could be adoptively transferred without intentional detachment of the beads in vivo to express their biologic function. y6 T cells in normal spleen assist ap CS-effector T cells without MHC restriction

The above results confirmed previous findings ( I ) that the y6 T cell-enriched fraction of CS-immune cells was required for immune ap cells to adoptively transfer CS responsiveness. Recently, we showed that unfractionated immune cells were not MHC restricted in this function ( I ) , and in addition, that unfractionated, normal, nonimmune spleen cells also had this CS-assisting function (16). W e now show that y6-enrichedT cells (anti-ys magnetic adherent cells) derived from normal spleen also can assist immune aP T cells in adoptive cell transfer of CS (Fig. 3, group C), and that this y6 CS-assisting activity also is present in normal allogeneic spleen (Fig. 3, groupD). Taken together, these results support y6 T cellsthatarerequiredtoassist aP CStheconceptthat effectorT cells are found in normal mice, and function without classical MHC restriction. Preferential usage of Vy5 and V64 variable region gene segments in yS T cells required for optimal transfer of contact sensitivity

To determine whether there was any preferential usage of particular Vy or V6 gene segments in y6 T cells that wcre required to assist aP T cells in adoptive transfer of CS, 4-day PCI-immune cells were treated in vitro with either of two different pan-antiy6-TCR mAb, or with the four available mAb that are specific for murine V y and V6 gene products, and then were incubated in vitro with C and transferred i.v. As before, significantly reduced transfers were obtained when cells were treated with either UC7 or GL4 (both are pan-anti-y6 mAb) (Fig. 4, groups B and C vs group A). In addition, adoptive cell transfers were inhibited significantly by treatment of the sensitized cells with hamster mAb to either VyS or V64, but treatment with anti-V y2 or V66.3 was not inhibitory.

(100) (22)

(120)