Fine specificity analysis of human influenza-specific cloned cell lines

CELLULAR

IMMUNOLOGY

94, 394-405 (1985)

Fine Specificity

Analysis of Human Influenza-Specific Cloned Cell Lines

GHISLAINE STERKERS, *,I JEAN MICHON,* YVETTE HENIN,* ELISABETH GOMARD,* CLAUDE HAmouN,-f AND JEAN PAUL LEVY* *Laboratoire d’lmmunologie et Virologie des Tumeurs, INSERM U 152, HGpital &chin, 27 rue du Faubourg Saint Jacques, 75014, Paris, and flrniti d’Ecologie Virale (Centre National de la grippe), Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France Received January 22, 1985; accepted April 16, I985 Influenza-specific human-T-cell clones, proliferating in the presence of virus-infected cells with restriction by class II molecules and displaying class II-restricted CTL activity or specific helper activity in antibody synthesis, have been analyzed for antigenic specificities. All of them were obtained by in vitro stimulation against influenza A/Texas virus. In all cases the virus specificity appeared identical in cytolytic and proliferative responses.Three of the clones were broadly cross-reactive, recognizing all or almost all type A influenza strains. The three remaining clones were subtype specific when tested with human strains and recognized the surface glycoproteins of influenza virus. One of these lines reacted with an epitope of the neuraminidase N2 while the other two recognized the hemagglutinin H3. By using a large panel of mammalian and avian influenza strains, it can be demonstrated that hemagglutinin-specific human T cells can recognize a cross-reacting determinant shared by H3 and H4 subtypes of hemagglutinin which has never been detected with antibodies. Q 1985 Academic FWS, IIIC. INTRODUCTION

The specificities of anti-influenza antibodies have been extensively studied. Most of them recognize strain-specific epitopes of the viral surface glycoproteins, hemagglutinin (HA)’ and neuraminidase (NA) (1, 2). In contrast, most cytolytic T lymphocytes (CTL) usually show a high degree of cross-reactivity for target cells infected with serologically distinct type A influenza strains when whole populations of murine and human T cells are used as effecters (3-5). This could be explained by recent results that have indicated that most mouse influenza-specific CTL recognize internal virus components shared by almost all influenza strains (6). However, by using target cells transfected with the HA gene, it was demonstrated that the HA surface molecule can serve as a target antigen for both subtype-specific and cross-reactive subpopulations of mouse influenza CTL (7). Recently, it has been ’ To whom correspondence should be sent. * Abbreviations used: HA, hemagglutinin antigen of influenza virus; NA, neuraminidase antigen of influenza virus; M, matrix antigen of influenza virus; NP, nucleoprotein antigen of influenza virus; P, polymerase antigen of influenza virus; CTL, cytolytic T lymphocytes; %RR, relative response percent; HAU, hemagglutinin units; [3H]Tdr, tritiated thymidine; HLA, major histocompatibility complex in human; PBM, peripheral blood mononuclear cells. 394 000%8749185$3.00 Copyright 0 1985 by Academic Press,Inc. All rights of reproduction in any form -cd.

SPECIFICITY OF ANTI-INFLUENZA

HUMAN

395

T CELLS

possible to obtain and maintain for several months influenza-specific cloned cell lines that have very precise antigenic specificities (8-15). Such clones are of great help in determining the fine specificities of CTL or helper cells. By such a method, it has been possible to demonstrate that T cells recognize HA in human and mouse (8, 9), polymerase P3 (10) and nucleoprotein NP (11) in mouse, or other crossreactive determinants ( 12- 15) in both species. However, the antigenic repertoire of influenza-specific T cells has yet to be well characterized in the human system, and this is of particular interest since in addition to B cells ( 16, 17), T cells are important in the immune defense of mammalian against influenza infection (5, 18-23). CTL play a role in recovery from infection (18, 20, 22) and helper T cells may play a role in cross-protection against different influenza virus strains (21, 23). Here we report the specificities of six long-term-cultured influenza-specific humanT-cell lines. Five of them are cloned cell lines having CTL activity, the remaining one having a specific helper activity for B cells in antibody synthesis. Four different types of specificities are described for these six lines. It is especially noteworthy that two of them react with a cross-reactive HA determinant of H3 and H4 subtypes which has never been detected by antibodies. TABLE 1 Hemagglutinin and Neuraminidase Subtypes of the Influenza Viruses Used in This Study Subtype

Species of origin b

Hemagglutinin ’

Neuraminidase”

HI H2 H3 H3 H3 H3 H3 H3 H3 H4 H4 H4 H4 H4 H4 H6 H8 Hll H12

Nl N2 N2 N2 N2 N2 N2 N? N8 ? N6 ? ? ? N2 N4 ? N2 N5

Human

Duck

Horse

A/PR8 A/Leningrad A/Texas A/Hong Kong A/Philippine A/X3 1d A/X41’ A/Duck/France/MJ90 A/equi/Miami A/Duck/France/MY73 A/Duck/Czech A/Duck/Ger/E409 A/Duck/Ger/E384 A/Duck/Ger/E4 12 A/Duck/Ger/E855 A/Duck/France/MT75 A/Duck/France/MY247 A/Duck/France/MT82 A/Duck/USA/14739

1933c 1980 197-I 1968 1982 1977 1963 1982 1956

1980 1982 1980

’ Current subtypes designation. b Viruses were isolated from human, horse, and duck species. ‘Year of appearance of each virus strain. d A/X31 is a recombinant strain between external components of A/Hong Kong and internal components of A/PRI. ‘A/X47 is a recombinant strain between external components of A/Victoria and internal components of A/PRI.

396

STERKERS ET AL.

MATERIALS

AND METHODS

Viruses The virus strains used in this study are described in Table 1. The human strains were prototypes from the collection of the National Influenza Reference Center as well as A/equi/Miami. The strains A/duck/France were isolated from wild ducks during a longitudinal surveillance study in the North of France by one of us from 1980 to 1982. The strains A/duck/Ger were obtained from Dr. P. Bachman (WHO Collaborating Center, Munich). A/duck/Czech/56 was received from Dr. Douglas (World Influenza Center, London). A/duck/USA 14739 was received from Dr. V. Hinshaw (Memphis, Tennessee). The viruses were grown in embryonated eggs.The titers of virus containing allantoic fluids are given in hemagglutinin units (HAU). Partially uv-inactivated A/Texas virus was used for initial and weekly restimulations of the cloned cell lines. Partially uv-inactivated or infectious viruses were used in proliferation assaysas indicated in the legends while infectious viruses were used in cell-mediated cytotoxicity assays.All viruses were stored at -70°C. In each experiment, B/Singapore virus was used as a control.

T-Cell Lines The T-cell lines were issued from three different donors whose peripheral blood mononuclear cells (PBM) were stimulated with A/Texas virus (H3N2/77). They were obtained and maintained in long-term cultures as previously described (24). One of them, L2, is an uncloned proliferating long-term cultured cell line that specifically helps B cells to produce anti-influenza antibodies (25). The five others are cloned cell lines; they proliferate specifically when stimulated with the A/Texas/ l/77 virus strain and are specifically cytolytic for influenza A/Texas-infected target cells. All activities are restricted by HLA class II molecules (25, 26, manuscript in preparation). All cell lines described here are OKT4+, OKT8-, OKT3+.

Culture Medium All cultures were performed in RPM1 1640 culture medium supplemented with L-glutamine ( l%), 2 X 10m5M mercaptoethanol, nonessential amino acids (l%), sodium pyruvate ( 1%) and heat-inactivated pooled human AB sera (5%) as previously described (24).

Proliferation Assays The antigenic specificities of these lines were tested in a proliferation assay as described elsewhere (24). Briefly, cloned cell lines were tested 6 days or more after the last stimulation, in IGZfree medium. Five to ten thousand cells were seededin round-bottom microtiter plates and cocultured for 3 days in a total volume of 0.2 ml with lo5 irradiated (4000 rad) autologous PBM and different influenza virus strains (5 HAU). [3H]Thymidine [O.S &i ([3H]Tdr)] was added during the last 18 hr of culture. The results are expressedas mean counts per minute (cpm) f standard deviation (SD) of triplicate cultures or as relative percent response: %RR:

cpm experimental - cpm with B/Singapore x 100. cpm with A/Texas - cpm with B/Singapore

SPECIFICITY OF ANTI-INFLUENZA

HUMAN

T CELLS

397

Assay of Cell-Mediated Cytotoxicity Cytotoxicity was assayed by release of 51Cr as described elsewhere (27). Briefly, autologous EBV-transformed cell lines were infected by various influenza virus strains, labeled with 51Cr,and used as target cells. Cloned cells were used as effecters 5 to 8 days after their last antigenic stimulation and washed twice before testing. Six-hour cytotoxicity assayswere carried out by mixing various numbers of effector cells with a constant number (5 X lo3 per well) of target cells in a ratio of effector/ target cells (E/T) as indicated in the legends. After 6 hr at 37°C 0.1 ml of supernatant was harvested and assayed for gamma radiation on a gamma counter. Results are expressed as Specific lysis % =

experimental counts - spontaneous release x 100. total counts - spontaneous release RESULTS

1. L2 and TAIO Recognize a Determinant on HA Molecule Shared by H3 and H4 Strains L2 is a long-term cultured proliferating cell line issued from an HLA DR 1/DR7 donor and strictly restricted by HLA DRl molecule (24). While uncloned, its very precise restriction and antigenic specificity suggestsa spontaneous cloning during the culture as already observed in the mouse (23). This cell line specifically helps B cells to produce anti-influenza antibodies (25). TAlO is a proliferating CTL cloned cell line issued from an HLA DR4/DRw8 individual. It is restricted by class II molecule segregating with the HLA-DRw8 haplotype in a familial study and closely related with the HLA DRw8 phenotype in a population study (not shown). L2 and TA 10 have the same pattern of reactivity when tested in proliferation assays (as shown in Figs. 1 and 2). They proliferate with several influenza strains including A/Texas, A/X3 1, and A/Hong Kong but not A/PR8 (Table 2). Since A/X31 is a recombinant strain between the external components of A/Hong Kong and the internal components of A/PR8, these results indicate that both cell lines recognize the external components of A/Texas. Moreover, these cell lines proliferate with viruses sharing with A/Texas the same hemagglutinin (H3) but not with viruses sharing the same neuraminidase (N2) (Figs. 1 and 2), suggesting that they do recognize the HA of H3 subtype. Note that the reacting determinant is highly conserved among H3 influenza strains. More surprisingly, these two cell lines also recognize all avian strains of the H4 subtype. As illustrated in Fig. 7 TAlO has the same specificity when tested for CTL activity or in proliferation assay. The same pattern of restriction is also observed for both activities (not shown). 2. TA4 Recognizes a Component of the N2 Molecule TA4 is a CTL cloned cell line issued from the same donor as TA 10 and restricted by an HLA class II molecule segregating with the HLA DRw8 phenotype (not shown). As shown in Table 2, when tested in proliferation assaysTA4 recognizes A/X3 1 but does not proliferate with A/PR8, indicating that, like TAlO and L2, this cell line recognizes a determinant on HA or NA. However this cell line proliferates with all strains sharing the A/Texas N2 specificity irrespective of their HA specificities,

398

STERKERS ET AL. X Response (c) Virus

strains

20

(b)

40

60

80

100

I A/Philippines(H3Nt)

1

A/X47

I

(H3N2)

A/eq/Miami A/OK/M.l/W A/Leningrad

(H3NB) (H3N?) (H2N2)

A/OK/MT/62

(HllN2)

A/OK/MT/75

(H6N4)

A/OK/MY/247

I

(HEN?)

w B

I

A/OK/USA/14739(H12N5) A/OK/MY/73

(H4N?)

I

A/OK/Czech

(H4N6 j

L

A/OK/E409

(H4N?)

A/DK/E384

(H4N?)

A/DK/E412

(H4N?)

A/D~iE855

(H4N2)

L

A 4 &

L2 (a) RG. 1. Antigenic specificity of L2 cells. lo4 L2 cells (a) are cultured with lo5 autologous irridiated PBM and partially w-inactivated viruses of different strains (b). [‘H]Tdr incorporation was determined on the third day of the culture. Results are expressed at %RR (c) (see Materials and Methods).

while it does not proliferate with the strains that share with A/Texas the H3 specificity only (Fig. 3). These results indicate that this cell line recognizes an epitope borne by the neuraminidase N2 of influenza viruses. Here again this determinant is highly conserved on all N2 strains. As illustrated in Fig. 7, TA4 has the same antigenic specificity when tested for cytolytic activity. The same restriction pattern is also observed for both activities (not shown). 3. CotC2, CotC6, and CotC8 Recognize More Broadly Reactive Determinants CotC2, CotC6, and CotC8 are CTL cloned cell lines issued from an HLA DRw 131 wl3 donor. CotC2 is restricted by the HLA DRwl3 molecule (26). CotC6 and CotC8 are restricted by a very private class II specificity inherited from the paternal haplotype (26). These three cell lines proliferate with A/X31, A/PR8, and A/Hong Kong (Table 2). So it is not possible to determine if they recognize an external or internal antigen of the vii-ion. CotC2 (Fig. 4) recognizes all influenza strains tested while CotC6 and CotC8 (Figs. 5 and 6) have the same pattern of specificity by recognizing a cross-reacting determinant shared by all human strains but not

SPECIFICITY OF ANTI-INFLUENZA

HUMAN

T CELLS

399

% Resp~nse(~'

20 Virus

40

60

80

100

strains(b)

A/Philippine&l3NZ) A/Eq/Miami(H3N8)

I

A/DK/MJ/%l

(H3N?)

A/Leningrad(HLNL) A/DK/MT/D2

(H11N2)

A/DK/MT/75(HbN4) A/DK/USA/14739

(H12N5)

A/DK/MV/73(H4N?) A/DK/Czech(H4Nb) A/DK/E4Dg(H4N?) A/DK/E384&4N?) A/DK/E412(H4N?

)

A/DK/E855(H4N2)

I FIG.

TAIO(a)

2. Antigenic specificity of TAlO cells (see Fig. 1 legend).

recognizing the A/duck/Czech strain. Here again, the same specificities are observed when these clones are tested for proliferative or cytolytic activities (Fig. 7). In particular, CotC6 and CotC8 do not recognize A/duck/Czech influenza-infected target cells while CotC2 does. DISCUSSION Three patterns of influenza virus specificities have been shown for mouse or human T cells: type specific (7, 15), subtype specific (7-9, 15), and strain specific ( 15). Subtype- and strain-specific T cells recognize surface glycoproteins of the virus (7-9). The predominant target antigens of cross-reactive CTL are highly conserved internal components of the virion (10, 11, 29) but may also be the surface glycoproteins HA (7, 8). In this paper, we report the fine specificity analysis of one long-term cultured and five cloned cell lines. All these lines of helper phenotype (OKT4+, OKT8-, OKT3+) proliferate specifically when stimulated with A/Texas influenza virus and these reactions are restricted by class II molecules. Moreover all of them evidence specific functional properties. One of them, L2, helps specifically B cells to produce antiinfluenza antibodies. The five remaining lines (TA4, TAlO, CotC2, CotC6, and CotC8) have CTL activity. Each cell line has the same pattern of restriction when tested in proliferation assay and for helper (24, 25) or CTL activity (26, manuscript in preparation). All of them were raised against A/Texas (H3N2) influenza virus.

400

STERKERS ET AL. TABLE 2 L2, TA4, and TAIO Recognize a Surface Viral Antigen Virus strainb

Cell line”

A/Texas

B/Singapore

A/Hong Kong

A/PRI

A/X3 1

L2

44,266 + 6,369’ lOO%d

275 +

178

24,687 + 1412 55%

1,936 + 4%

35

38,997 + 7578 88%

TAlO

15,676 + 3,782 100%

3239 f

472

12,094 + 1275 71%

2,669 + 229 0%

20,358 + 1895 131%

TA4

59,759 + 12,985 100%

4218 k 1870

49,060 k 3828 81%

1,853 + 873 0%

63,360 f 2603 106%

cotcz

42,196 + 1,572 100%

2624 f 1756

39,021 f 1382 91%

41,506 f 9156 98%

22,708 f 1788 51%

CotC6

39,135 k 6,090 100%

2348 + 335

34,462 + 3651 88%

35,956 + 2568 91%

25,955 + 7329 64%

CotC8

48,627 + 3,409 100%

3345 + 1021

45,153 + 9062 93%

47,152 + 708 97%

47,398 f 6140 97%

a,bCloned cell lines (104) are cultured with 10’ autologous irradiated PBM and different virus strains. HTdr incorporation was determined on the third day of culture. c*dResults are expressed as mean cpm f SD’ of triplicate cultures or %RRd (see Materials and Methods).

Three of them, CotC2, CotC6, and CotC8, recognize broadly cross-reacting determinants as usually do most CTL (3-5). CotC2 is specific for an epitope shared by all type A viruses tested. The two other clones CotC6 and CotCS share between them the same pattern of reactivity, suggestingthat they recognize the same epitope: they react with all type A mammalian strains and almost all avian strains but in contrast to CotC2 they do not recognize the A/DK/Czech avian strain. This is true when proliferative response as well as cytolytic activity are tested. Interestingly, most partially uv-inactivated virus strains are less effective than infectious viruses in inducing proliferative responses of CotC2, CotC6, and Cot03 cells (not shown). On the contrary, both uv-treated and infectious viruses induced strong proliferation when TA4, TAlO, and L2 cell lines that recognize the surface glycoproteins were used as effecters. Since virus replication is required for matrix-protein (M) appearance on plasma membranes (28), these results might suggestthat the target molecules of CotC2, CotC6, and CotCS could be the matrix protein. However, the precise target of these three cloned cell lines cannot be given here since T cells recognizing ail or almost all type A influenza virus strains have been shown to be specific either for the matrix protein (29) or for other molecules like the hemagglutinin (18), the nucleoprotein (1 l), or the polymerase (10). Finally, other conservative internal components of the virion might be involved. The remaining cell lines exhibited a very clear specificity for the surface glycoprotein antigens, since all of them can be stimulated in proliferation assay by A/X3 1 virus which shares HA and NA with A/Texas virus, but not by A/PRS virus which shares only internal determinants with A/X3 1. When tested with a large panel of mammalian and avian strains which shares with the immunizing virus either H3 or N2 subtype,

SPECIFICITY OF ANTI-INFLUENZA

HUMAN

401

T CELLS % Response(c)

20 Virus

40

60

80

100

strains(b)

A/Philippine(H3N2) A/X47

(H3N2)

A/Eq/Miami A/DWMJI90 A/Leningrad

(H3N8) (H3N?) (H2N2)

A/DK/MT/82

(HllN2)

A/DK/MT/75

(H6N4)

A/DK/USA/14739(H12N5) A/DK/MV/73

(H4N?)

A/DK/Czech

(H4NG)

A/DK/E409

(H4N?)

I

A/DK/E384

(H4N?)

I

A/DK/E412

(H4N?)

I

A/DK/E855

(H4N2) TA4(a)

FIG. 3. Antigenic specificity of TA4 cells (see Fig. 1 legend).

it is clear that TA4 recognizes a determinant on the neuraminidase N2 molecule. To our knowledge, this specificity has never been described for CTL, suggestingthat it might be rare. On the other hand, L2 and TAlO recognize a determinant on the hemagglutinin H3 molecule. Surprisingly L2 as well as TAlO also recognize all the strains of the H4 subtype, suggesting that T cells might recognize a cross-reactive determinant shared by H4 and H3 molecules. Another possibility is that L2 and TA4 contain two different populations, one of them reacting with H3 and the other with H4 molecule. However this hypothesis appears unlikely for the following reasons. First, L2 cells are very probably oligoclonal if not monoclonal, according to their very precise H3-H4 specificity and DRl restriction as previously discussed (24). TAlO is a cloned cell line evidencing the same very precise HLA restriction (manuscript in preparation) for both proliferative and CTL activities and it is shown here that the same antigenic cross-reactivity is also observed for both activities. Secondly, it is well known that “in viva” priming is necessary to observe an “in vitro” proliferative response of T cells in such a system (30). Since HA seropositive donors are-apparently devoid of anti-H4 antibodies as found in a series of 75 samples (results not shown), it is probable that human beings have never met H4

402

STERKERS

ET AL. % Response

20 Virus

40

60

I

strains(b)

100

80

I

A/Philippins(H3NZ) A/X47

(HJNE)

A/Eq/Miami

(H3N8)

A/Leningrad

(H2N2)

A/DK/MT/75

(H6N4)

A/DK/USA14739(H12N5) A/DK/MY/73

(H4N?)

A/DK/Czech

(H4N6)

A/DK/E409

1

(H4N?)

A/DK/E384/

(H4N?)

A/DK/E412/

(H4N?)

A/DK/E855

'

I, , I'

(H4N2)

CDTC2(") FIG. 4. Antigenic specificity of CotC2 cells. Cloned CotC2 cells ( lo4 (a)) are cultured with 10’ autologous irradiated PBM and infectious viruses of different strains (b). [‘H]Tdr incorporation was determined on the third day of the culture. Results are expressed as %RR (see Materials and Methods).

% Response(c) 20 Virus

40

60

80

100

strains(b)

A/Philippines

(H3N2)

A/X47(H3N2) A/eq/niami(H3N8) A/Leningrad(H2N2) A/DK/HT/75(H6N4) A/DK&ech(lMN6)

I

A/DK/E409bMN?) A/DK/E412(H4N?)

/P COTC6(a)

FIG.

5. Antigenic specificity of CotC6 cells (see Fig. 4 legend).

% Response (') Virus

60

40

20

st.rains(b)

80

100

I AjPhilippines(H3N2) A/X47 (H3N2) A/Eq/Miami

(H3N8)

A/Leningrad

(H2N2)

A/OK/MT/75

(H614)

A/DK/Czech

(H4N6)

A/DK/E40g

(H4N?)

A/OK/E412

(H4N?)

c

I

FIG. 6. Antigenic specificity of CotC8 (see Fig. 4 legend). %specific

Lo

Cr release

\

COTCP (a)

% specifi?+ Cr release

40

40

30

20

\

\

4 0 10

. 4/l

(c) % specific Cr release

1.30

0.4/l

l d.15/1

db)

%specific

7;1

2.311

0.8/l

Oh/l

E/Ttb)

(cl lAIOca)

Cr release

30

30

E/db)

FIG. 7. Specificities of CTL activity. Cloned cell lines (a) were seededwith 5’Cr-1abe1edautologous EBV transformed target cells infected with A/Texas (H3N2) (A); A/Leningrad (H2N2) (Cl); A/PRI(HlNI) (0); A/X31(H3N2) (v); A/Hong Kong (H3N2) (m); A/DK/Czech (H4N6) (+), or B/Singapore (0) viruses. Target cells (5 X 104)were seeded with various numbers of effector cells in an effecter/target cell ratio (E/T) (b) as indicated. Results are expressed as percentage specific Cr releaseas indicated under Materials and Methods (c). 403

404

STERKERS ET AL.

Hl HZ H5 Hll

H7 HlO H4 H3

FIG. 8. Dissimilarity between HA1 molecules. Relationship between the 12 HA subtypes from direct amino acid sequence comparisons. Data from Air et al. (34).

viruses and cannot be primed for this antigen. One may therefore conclude that the most likely hypothesis is that these lines recognize a unique epitope common to H3 and H4 molecules which has not been observed previously. Interestingly, when the sequencesof HA1 molecules were compared by Air et al. (34) a greater analogy of H3 with H4 was found than with the other subtypes (Fig. 8), reinforcing the idea that the T cells reported here recognized a shared determinant between H3 and H4 molecules. Note that the same cross-reacting pattern is observed with two different cell lines, issued from two different donors, suggesting that this T-cell specificity might not be so rare. Moreover, these two cell lines having either CTL activity or helper function, it can be concluded that this cross-reactivity can be observed irrespective of the functional properties of the cell lines. To our knowledge such cross-reactivity between H3 and H4 molecules has never been described for T cells and this epitope is apparently not seen by B cells. Recently Lamb and Green (3 1) have described a cross-reactive population of influenza-specific T-cell clones specific for HA peptides distinct from the antibody-binding sites of hemagglutinin. These two peptides were located at the carboxyl terminus of HA1 molecule including, respectively, the residues 105-140 and 306-329. Known HA1 sequencesof human (32) and bird (33) viruses of H3 subtype recognized by L2 and TAlO show great homology in these two regions. However, to our knowledge the hemagglutinins of H4 subtype strains have not been sequenced in these two regions. So it is not possible to be precise as to whether or not the specificities recognized by TAlO and L2 could be mapped to these peptides. The results reported here confirm that cloned cell lines are useful tools for determining the structures involved in the recognition process of T cells, which is still poorly defined. Moreover, the determination of their functional properties for each of them seems to offer unique possibilities for detailed investigations of the role of these cells in the defense against viral infections. ACKNOWLEDGMENTS We thank Dr. Van Reganmortel for helpful discussionsand D. Hirtzlin for typing the manuscript.

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SPECIFICITY OF ANTI-INFLUENZA

HUMAN

T CELLS

405

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