Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9427-9430, October 1993 Immunology Significant enlargement of a specific subset of CD3+CD8+ peripheral blood leukocytes mediating cytotoxic T- activity during human immunodeficiency virus infection (T lymphocyte subset//AIDS) A. BENSUSSAN*t, C. RABIANt, V. SCHIAVON*, D. BENGOUFAt, G. LECA*, AND L. BOUMSELL* Institut Nationale de la Sant6 et de la Recherche Medicale U93, Association Claude Bernard, and tLaboratoire d'Immunologie et d'Histocompatibilite, H6pital Saint-Louis, 1 Avenue Claude Veliefaux, 75475 Paris cedex 10, France Communicated by Jean Dausset, July 20, 1993

ABSTRACT We have obtained a monoclonal antibody studies have shown that human immunodeficiency virus termed BY55 that defines an 80-kDa cell-surface structure on (HIV) infection significantly changes the phenotype of cir- a subset ofcirculating peripheral blood mononucleocytes. This culating and increases CD8 T lymphocytes (19), structure, which was not detected on most cell lines or activated it was interesting to look for this cytotoxic CD3+CD8+BY55+ lymphocytes, was expressed exclusively on 15-25% of CD2+ circulating cell subset in asymptomatic HIV-positive individ- circulating lymphocytes, including a major subset within the uals with various numbers of CD4 cells. Results indicated CD3- and the T-cell y6+ lymphocytes and a small that peripheral blood CD3+CD8+BY55+ cells significantly percentage of the CD3+CD8+ cells. Moreover, we have shown increased in HIV-positive individuals. that the BY55 molecule delineated the competent killer circu- lating lymphocytes. In the present report, additional two- and MATERIALS AND METHODS three-color immunofluorescence studies of peripheral blood lymphocytes were done to precisely determine BY55 expression mAbs. BY55 mAb was obtained by immunization of within the T-cell population. In normal individuals, peripheral BALB/c mice with the YT2C2 cell line (18). Briefly, spleen blood CD3+CD8+BY55+ cells represented only 5-6% of the cells from immunized mice were fused to the NS1 cell line 5 lymphocytes, and these cells possessed cytolytic activity. Inter- days after the last injection. The initial screening by indirect estingly., we found that the percentage of total BY55+ lympho- immunofluorescence and flow cytometry with a FACStar cytes as well as the percentage of CD3+CD8+BY55+ was microfluorometer (Becton Dickinson) retained all the hybri- significantly increased in peripheral blood lymphocytes of doma-culture supematants reactive with the immunizing human immunodeficiency virus-seropositive individuals. cells but weakly reactive with resting peripheral blood mono- nuclear cells. Cultures containing selected antibodies were cloned twice by limiting dilution. The cloned hybridoma was Use of monoclonal antibodies (mAbs) has allowed the iden- serially passaged by i.p. injection into BALB/c mice primed tification of various cell-membrane glycoproteins that define with pristane. Ascites fluid was collected, ultracentrifuged, phenotypic and functional human lymphocyte subsets (1-3). and affinity-purified, when required. BY55 mAb is an IgM. In particular, it was well established that CD8 and CD4 Other mAbs such as CD3, CD4, CD8, and CD38 were subsets were associated with the major histocompatibility produced locally. These mAbs were used as ascites fluid and, complex (MHC) class I/II restriction ofT-cell recognition (4, when needed, coupled to fluorescein isothiocyanate (FITC) 5). Several cell-surface structures were described as being or biotin. Some antibodies were purchased from Immunotech restricted to a subset of circulating T cells. These included (Marseille, France). dipeptidylpeptidase IV CD26 (6), CD28 (7), CD31 (8), and Isolation of Cell Populations. Human peripheral blood lym- CD38 (9). Except for CD28, the expression of all these phocytes (PBL) were prepared by Ficoll/Hypaque density- molecules increases during cellular activation. Other struc- gradient centrifugation. Cells were washed twice in RPMI tures such as CD25 (10), CD39 (11), CD69 (12), and CD71 (13) 1640 medium (GIBCO) supplemented with 2 mM L-gluta- are characteristic of activated T lymphocytes. In an attempt mine, penicillin/streptomycin (100 units/ml and 100 Mg/ml, to define specific cell subset surface glycoproteins, we char- respectively), and 10% heat-inactivated human serum and acterized various mAbs obtained by repeated immunizations used for immunofluorescence assays. HIV-negative and with functionally defined cell lines (14-16). In particular, we HIV-positive blood samples were obtained from the Labo- had recently reported a mAb, termed BY55 (17), that recog- ratory of Immunology (H6pital Saint-Louis). The HIV- nizes a distinctive 80-kDa structure on the immunizing leu- seropositive population studied was a heterogeneous cohort. kemic line YT2C2 (18). Flow cytometry For isolation of CD8+BY55+ and CD8+BY55- cell popula- analysis revealed that BY55 mAb exclusively stained 15-25% tions, fresh PBL were first depleted in CD4+ cells by passage of the circulating CD2+CD4- lymphocytes of a normal indi- over a column and next were simultaneously labeled with vidual. These cells include a CD2+CD3- competent killer- phycoerythrin (PE)-conjugated CD8 mAb and FITC- cell subset and a minor CD3+ cell subset coexpressing conjugated BY55 mAb. After a final wash, cells were sorted CD8bight molecules. In the present report, we show further into CD8brht+BY55+ (representing 5-7% of the CD4- PBL) antigenic phenotype from studies on 10 normal individuals. and CD8bright+BY55- (representing >60% ofthe CD4- PBL) BY55 mAb stained only 5-6% of circulating CD3+CD8brigh+ fractions using a FACStar microfluorometer (Becton Dick- cells. In addition, we reveal that these cells possess cytolytic Sorted were collected in RPMI 1640 activity, as they mediate CD3-redirected lysis against the Fc inson). populations receptor-bearing tumor cell line K-562. As a number of Abbreviations: mAb, monoclonal antibody; HIV, human immuno- deficiency virus; PBL, peripheral blood lymphocyte(s); CTL, cyto- The publication costs of this article were defrayed in part by page charge toxic T lymphocyte(s); MHC, major histocompatibility complex; PE, payment. This article must therefore be hereby marked "advertisement" whycoerythrin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. TTo whom reprint requests should be addressed. 9427 Downloaded by guest on October 1, 2021 9428 Immunology: Bensussan et al. Proc. Natl. Acad. Sci. USA 90 (1993)

medium/10% heat-inactivated human serum, washed twice, ceptor y8. Although no significant amounts of CD4+ circu- and used as effector cells in cytotoxicity assays. Trypan blue lating cells coexpressed BY55 molecule, a major subset of exclusion was performed on the unsorted population and on T-cell receptor y3+ cells were stained by BY55 mAb (17). sorted populations, and the viability was always >90%. Interestingly, we found that 6% of peripheral blood mono- Immunofluorescence Assays. Phenotypic analyses were nuclear cells coexpressed BY55, CD3, and CD8 molecules, done on whole peripheral blood or Ficoll/Hypaque PBL indicating that only a minor subset of the CD3+CD8+ cells gated on lymphocytes. For two-color immunofluorescence coexpressed BY55 molecule. experiments, cells were simultaneously incubated during 30 Cell-Mediated Cytotoxic Activity of BY55+ Circulating min with a PE-conjugated mAb and FITC-labeled BY55 Cells. We had previously shown that BY55+ circulating cells mAb. Red blood cells were lysed by adding 2 ml of cell contained all the competent MHC-unrestricted killer lym- cytometry lysis solution (Becton Dickinson). The overlap in phocytes (17). However, as the BY55+ lymphocytes consist FITC and PE fluorescence emissions was corrected by using of CD3+CD8+BY55+ cells, corresponding to T lymphocytes an electronic compensation network. For three-color immu- expressing T-cell receptor af3, natural killer CD3-CD8-- nofluorescence assays, 2 x 106 cells or 100 ,l of blood was BY55+, and CD3- or CD3+CD8dim+BY55+ cells, we could simultaneously stained with CD3 Tri-Color (Caltag Labora- not distinguish their respective contribution to the cell- tories, South San Francisco, CA), CD8PE, and FITC-BY55 mediated cytotoxic function. In particular, we could not mAb. Analysis was performed on a FACScan microfluorom- determine whether the MHC-restricted CD3+CD8bright+- eter after compensating spectrum overlaps between FITC BY55+ circulating cell subset exhibited killer activity. There- and PE and between PE and Tri-Color (excitation wavelength fore, to study MHC-restricted killer activity of freshly iso- of Tri-Color was 488 nm; emission wavelength was >650 lated CD3+CD8bright+BY55+ and CD3+CD8bright+BY55- cell nm). populations, CD3-redirected cytotoxic assays were done CD3-Induced Redirected Cytotoxicity Assay. The K-562 with these sorted cells. Moreover, to exclude the natural cells were labeled with 100 ,uCi of 51Cr (1 Ci = 37 GBq) for killer activity ofthe CD3-CD8dim+BY55+ cells against K-562 1 hr, washed, and used as target cells, Anti-CD3 mAb (Leu4, target cells, we separated BY55+ and BY55- cells within IgGl) (final concentration, 10 jig/ml) was added to 51Cr- CD8bnght+ gated cells. A representative cell-mediated cyto- labeled targets 15 min before addition of effector cells. toxicity assay done with unsorted and sorted effector cells is Assays at various effector to target cell (E:T) ratios with 5 x presented in Fig. 1. As expected, a weak but significant CD3 103 labeled target cells per well were done in triplicate in mAb-redirected lysis against the K-562 cells was observed 96-well V-bottom microtiter plates. The final 10% fetal calf with PBL depleted in CD4+ cells. Interestingly, we found that serum/culture medium volume was 200 ,ul in each well. After in contrast to CD3+CD8+BY55- cells, which never exhibited 4 hr of incubation, 100 ,ul was removed from each well, and lytic activity, only the CD3+CD8+BY55+ cell subset medi- radioactivity was counted in a y counter to determine 51Cr ated a strong CD3 mAb-redirected cytotoxicity in a 4-hr 51Cr release. The percentage of lysis was measured according to radioisotope-release assay. the formula: % killing = 100% x [(sample release cpm - CD3+CD8brght+BY55+ Circulating Lymphocytes Increased spontaneous release cpm)/(maximum release cpm - spon- in HIV-Positive Individuals. That HIV infection leads to taneous release cpm)]. changes in phenotype of PBL has been reported; in partic- ular, CD8+ T lymphocytes expressed activation antigens such as HLA-DR and CD38 (20). We examined the blood of RESULTS 20 HIV-positive individuals with different numbers of CD4+ Multicolor Immunofluorescence Analysis of PBL Isolated cells per mm3 (range 902-64 cells per mm3) (Table 2). The from Normal (HIV Seronegative) Individuals with BY55, CD3, mean percentage of CD4+ cells was 18.7%, whereas 62.8% of and CD8 mAbs. To delineate the BY55+ circulating lympho- the lymphocytes were CD8+. Interestingly, we found that cyte subset within the T-cell population, three-color analysis 39.1% of lymphocytes were BY55+. Two-color immunoflu- was done with PBL of 10 different normal individuals. Table orescence revealed that most of these BY55+ cells (76%) 1 shows the values of the mean ± SDs. As previously were also CD8+. As expected, the percentages of reported (17), 21.2% (range 11-32%) of circulating PBL CD8+HLA-DR+ and CD8+CD38+ lymphocytes were en- expressed BY55 mAb-reactive molecule. A little more than hanced. To gain insight into BY55+ PBL subsets in normal 50% of these BY55+ cells were CD3. Half of these CD3 individual and HIV-seropositive individuals, tri-color immu- cells coexpressed low amounts of CD8 molecules. Ten to nofluorescence was used. The results (Table 3) were obtained 15% of BY55+ were CD3+ CD8- cells that may correspond with eight different HIV-positive individuals. Again, BY55+ mainly to CD3+CD4-CD8- cells expressing the T-cell re- circulating lymphocytes were increased in these eight HIV- Table 1. Frequency of BY55+ cells in PBL from normal individuals Donor CD3+ CD3+CD8+ CD3+CD8- CD3-CD8+ CD3-CD8- no. BY55+, % CD8+, % CD3+, % CD8+, % BY55+, % BY55+, % BY55+, % BY55+, % 1 11 41 87 37 4 2 3 3 2 15 19 87 18 5 5 1 9 3 30 48 61 29 6 2 9 8 4 32 37 68 19 7 3 12 10 5 22 45 70 26 3 1 9 6 6 12 29 89 25 4 1 3 4 7 26 35 75 28 5 6 5 14 8 29 29 83 42 13 2 5 8 9 19 38 89 36 9 4 2 3 10 19 41 72 27 4 2 9 7 Mean* 21.2 + 6.6 38.1 ± 8.5 78.2 ± 9.6 28.7 ± 7.3 5.8 ± 2.9 2.7 ± 1.5 5.8 ± 3.5 7.1 ± 3.2 PBL or blood from 10 randomly selected HIV-seronegative adults was labeled for one-, two-, or three-color determination, as described. Samples were analyzed by flow cytometry. *Mean percentage ± SD. Downloaded by guest on October 1, 2021 Immunology: Bensussan et al. Proc. Natl. Acad. Sci. USA 90 (1993) 9429 40) HIV-positive individuals, whereas the percentage of -- CD8 + BY55- CD3+CD8bnrght+BY55+ cells was multiplied by four.

DISCUSSION 30- -A-'fU4 - PB-'1L In this study, we report the delineation of a distinctive CD3+CD8bight+ circulating T-cell subset that mediated CTL CZ activity in normal individuals. This cell subset was individ- ualized within the CD3+CD8+ cell population by the use of 20I a specific mAb termed BY55. This mAb was initially reported to recognize an 80-kDa cell-surface structure on the active natural killer circulating CD3- cells (17). Reactivity of BY55 mAb with these cells was not seen with interleukin 2-depen- dent natural killer or CTL clones and was rapidly lost once the circulating cells were activated in culture. Thus, the BY55 molecule does not seem to correspond to an in vitro cell- surface activation antigen. In HIV-seronegative individuals, circulating cytotoxic 80 40 20 10 1 CD3+CD8bright+BY55+ cells represented 5-6% (range Effector/target ratio 4-13%) ofthe lymphocytes, whereas more BY55+ cells were found within the natural killer CD3- cells whether or not FIG. 1. Determination ofthe CD3-redirected cytotoxicity against dimly expressing CD8 molecules (13% of the PBL). We the -bearing target cells K-562. Freshly isolated periph- demonstrated that sorted CD3+CD8bight+BY55+ cells from eral blood mononuclear cells were depleted in CD4+ cells by passage freshly isolated CD4-PBL mediated potent CD3-redirected over a column and were then simultaneously labeled with FITC- cytotoxicity against Fc receptor-bearing K-562 target cells. conjugated BY55 mAb and PE-conjugated CD8 mAb. Cells were sorted into CD8br8ght+BY55+ and CD8bri&t+BY55- fractions by These results are ofthe utmost interest, particularly because using a FACStar microfluorometer. Sorted populations were col- no CTL activity was observed within the CD8bight+BY55- lected, washed, and tested at various effector/target ratios in cyto- sorted cells. Extensive phenotypic analysis with PBL from toxic T lymphocyte (CTL) assays. Viability ofthe sorted populations HIV-seropositive individuals revealed that the mean percent- was always >90o. age of BY55+ cells increased. This increase was attributable in all cases to an increase in the CD3+CD8bnrght+BY55+ cell subset. In addition, in two individuals an increase in the positive donors. An average of 21% of the circulating lym- natural killer CD3-BY55+ was also seen (see Table 3, donors phocytes were CD3+CD8bright+BY55+, whereas 14.7% ofthe 24 and 25). cells, including 5.7% of CD8dim+ were CD3-BY55+. It is Taken together, these results indicate that BY55 mAb interesting to note that the doubling in BY55+ circulating stained a subset of circulating CD3+CD8+ CTL that was lymphocytes of most HIV-seropositive patients resulted increased in HIV-seropositive individuals. Many studies from an increase in the CD3+CD8brght+BY55+ cell subset. In have focused on the role of CD8 T lymphocytes in the host addition, the increase in the CD3+CD8bri0t+BY55+ cells was response to HIV infection (21-23). In particular, CD8+ not proportional to the rise in the percentage of CD3+CD8+ lymphocytes have been shown to exhibit CTL activity, as cells in HIV-seropositive individuals. Tables 1 and 3 show measured by CD3-induced redirected cytotoxicity (24). This that the percentages of CD3+CD8+ cells was doubled in CTL activity was not correlated with HLA-DR or CD38

Table 2. Frequency of BY55+ cells in PBL from HIV-seropositive individuals Donor CD4+, CD8+ CD8+ DR+, no. no./mm3 CD3+, % CD4+, % CD8+, % BY55+, % BY55+, % % CD8+38+, % 1 902 83 44 41 31 16 24 35 2 624 91 20 71 40 37 40 56 3 515 93 10 82 49 41 57 72 4 455 80 31 43 30 16 18 25 5 458 78 23 58 39 27 38 29 6 394 93 10 80 42 38 49 67 7 368 79 24 55 35 28 30 38 8 340 87 18 70 59 53 49 57 9 328 87 29 56 26 20 39 46 10 293 76 17 65 22 18 48 58 11 258 83 15 68 52 45 50 52 12 233 81 14 69 51 42 45 41 13 230 78 20 54 50 33 29 36 14 198 79 12 66 68 52 42 37 15 191 68 18 59 43 39 43 54 16 186 71 16 59 26 13 38 43 17 130 82 22 64 9 6 33 52 18 128 69 12 62 32 25 45 50 19 110 88 10 81 38 35 70 74 20 64 76 9 54 40 15 36 46 Mean 81.1 + 7.1 18.7 + 10.9 62.8 ± 10.9 39.1 ± 13.3 29.9 + 13.2 41.1 ± 11.4 48.4 ± 13.1 PBL or blood from 20 selected HIV-seropositive adults was labeled for one- or two-color determination. Results are presented as for Table 1. Downloaded by guest on October 1, 2021 9430 Immunology: Bensussan et al. Proc. Natl. Acad. Sci. USA 90 (1993) Table 3. Frequency of BY55+ cells in PBL from HIV-seropositive individuals CD3+CD8+ CD3+CD8- CD3-CD8+ CD3-CD8- Donor BY55+, CD3+CD8+, BY55+, BY55+, BY55+, BY55+, no. CD3+, % CD4+, % CD8+, % t % % % %% 21 80 24 52 43 50 28 3 2 11 22 77 31 45 30 44 16 1 2 11 23 83 15 69 33 66 23 1 2 7 24 90 10 80 52 36 18 0.3 30 4 25 62 12 57 39 51 16 0.5 5 18 26 72 12 60 37 54 22 1 2 8 27 89 5 82 32 80 26 2 0.5 3 28 71 4 69 35 64 20 2 2 10 Mean 78 + 8.9 14.1 + 8.6 64.2 ± 12.2 37.6 + 6.7 55.6 ± 13 21.1 ± 4.2 1.35 ± 0.85 5.7 ± 9.3 9 ± 4 PBL or blood from eight selected HIV-seropositive adults was labeled for one-, two-, or three-color determination. Results are presented as for Table 1. expression on CD8+ lymphocytes. Further experiments will 7. June, C. H., Ledbetter, J. A., Linsley, P. S. & Thompson, determine whether the CD3+CD8brghI+BY55+ cell subset C. B. (1990) Immunol. Today 11, 211-216. 8. Tanaka, Y., Albelda, S. M., Horgan, K. J., van Seventer, exerts CTL activity against HIV-infected cells. As the inter- G. A., Shimizu, Y., Newman, W., Hallam, J., Newman, P. J., action of CD28 with molecules has been described to Buck, C. A. & Shaw, S. (1992) J. Exp. Med. 176, 245-253. augment the generation of CTL in resting T-lymphocyte 9. Funaro, A., Spanoli, G. C., Ausiello, C. M., Alessio, M., populations (25), further multicolor immunofluorescence Roggero, S., Delia, D., Zaccolo, M. & Malavasi, F. (1990) J. studies are needed to determine whether the CD3+CD8brght+- Immunol. 145, 2390-23%. 10. Smith, K. A. (1988) Adv. Immunol. 42, 165-179. BY55+ cytotoxic-cell subset coexpressed CD28 molecules. 11. Gouttefangeas, C., Mansur, I.-G., Schmid, M., Dastot, H., At present, we cannot rule out the possibility that CD28 Gelin, C., Mahouy, G., Boumsell, L. & Bensussan, A. (1992) molecules are not required for CTL function of this later Eur. J. Immunol. 22, 2681-2685.- subset, as Azuma et al. (26) have recently shown that 12. Nakamura, S., Sung, S. J., Bjomdahl, J. M. & Fu, S. M. (1989) CD8bight+CD28- circulating T lymphocytes can mediate J. Exp. Med. 169, 677-689. 13. Trowbridge, I. S. & Lopez, F. (1982) Proc. Natl. Acad. Sci. potent CD3-redirected cytotoxity against Fc receptor- USA 79, 1175-1179. bearing target cells. Finally, until CTL activity against HIV- 14. David, V., Bachelez, H., Leca, G., Degos, L., Boumsell, L. & infected cells is demonstrated with CD3+CD8bfght+BY55+ Bensussan, A. (1990) J. Immunol. 144, 1-6. sorted cells"in HIV-seropositive individuals, it is tempting to 15. Boumsell, L., Schmid, M., Dastot, H., Gouttefangeas, C., speculate that the augmentation of these circulating CTL Mathieu-Mahul, D. & Bensussan, A. (1990) J. Immunol. 145, 2797-2802. corresponds to a fruitful phase of the cellular immune re- 16. Bougeret, C., Mansur, I.-G., Dastot, H., Schmid, M., Mahouy, sponse in these individuals (27). 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