T CELL RECOGNITION OF THE POSTTRANSLATIONALLY CLEAVED INTERSUBUNIT REGION OF INFLUENZA VIRUS HEMAGGLUTININ
G- PBS, glucose containing phosphate buffered saline
in this stage of viral infection presenting viral peptides for virus-specific helper T cells (Doherty et al., 1992;
Long, 1992). If infective viruses enter the appropriate host cell, virus replication occurs generating newly syn-thesized viral proteins which can predominantly be handled as e n d o g e n o u s proteins in terms o f antigen processing. In this process, primarily M H C class I but, depending o n the type o f the host cell, also M H C class II molecules are involved (Brooks et al., 1991; Nuchtern et al., 1990; Jaraguemada et al., 1990; Long, 1992).
Influenced by the intracellular destination o f viral pro-teins, M H C class I and class II molecules have the chance to select from different sets o f viral peptides, but some fragments o f viral proteins might be able to bind to both types of M H C molecules (Sweetser et al., 1989;
Perkins et al., 1991).
The hemagglutinin ( H A ) spike protein o f influenza viruses has multiple biological functions and it is one o f the main targets o f immune recognition (Wilson and Cox. 1990; T h o m a s et al.. 1989; Yewdell and Hackett.
1989). T cell epitopes recognized either by helper or cytotoxic T cells in the context o f different H - 2
1404 É. RAJNA VÖLGYI el al.
(Eisenlohr and Hackett, 1989; Jackson and Brown, 1991) or H L A ( L a m b et al., 1982) haplotypes have been localized in distinct regions of the molecule including the C-terminal membrane spanning segment o f the H A 2 subunit (Braciale et al., 1989) or the head region ( H A 1 ) accumulating most o f the type- and subtype-specific variations and the corresponding B cell epitopes (Mills et al., 1986; Mills, 1986; Wilson and Cox, 1990).
T h e most conserved sequence of the H A molecule from different influenza viruses is the functionally essen-tial fusion peptide representing the N-terminal 1 - 1 2 amino acids o f the H A 2 subunit (White, 1990). In newly synthesized, immature hemagglutinin (HAO) molecules, this peptide is covalently linked to the C-terminal o f the H A 1 subunit via an Arg residue in position. 329- This Arg will be deleted in the course of HAO maturation by subsequent posttranslational enzymatic cleavages (Garten et al., 1991) giving rise to mature H A built up from disulphide-bridge-ljnked H A 1 amd H A 2 subunits (Webster and Rott, 1987). This event is the prerequisite of viral infectivity ensuring a pH-dependent confor-mational change of the H A molecule in e n d o s o m e s where membrane fusion will be catalysed by the exposed fusion peptide (White, 1990). The amino acid sequence o f the C-terminal stretch of the H A 1 subunit also shows restricted amino acid variations and comprises an en-zyme cleavage motif determining enen-zyme susceptibility and thus influencing tropism o f the virus (Nagai et al.,
1991; G o t o h et al., 1990; Barr, 1991; Walker and K a w a o k a , 1993). The enzyme involved in the posttrans-lational modification was identified as a trypsin-like serine protease acting in the post-Golgi compartment o f defined cell types such as the epithelial cells o f the respiratory tract (Webster and Rott, 1987). Professional antigen-presenting cells such as B cells, macrophages or dendritic cells lack the appropriate enzyme for HAO cleavage and thus upon infection generate immature, uncleaved molecules giving rise to non-infective viruses (Doherty et al., 1992; Webster and Rott, 1987).
A s we demonstrated (Rajnavolgyi. 1992; N a g y et al., 1994), the 25 amino acid-long synthetic peptide IP corresponding to the uncleaved form o f the intersubunit region o f the A / P R / 8 / 3 4 ( H 1 N I ) human influenza A virus hemagglutinin (see Table 1) is able to elicit an IgG response in B A L B / c mice without the introduction o f any carrier. This result implies that T cell epitope(s) may
also be present in this region. This paper reports a study on the possible recognition o f the H A 1 - H A 2 intersub-unit region by T cells in relation to the influence o f the posttranslational modification. This question was approached by the use of synthetic peptides comprising the C-terminal sequence of the HA1 and the N-terminal sequence of the H A 2 subunits. These peptides corre-spond to the covalently linked intact or cleaved forms of the immature HAO and posttranslationally modified H A molecules, respectively. The conformational properties o f these overlapping peptides have been characterized by circular dichroism ( C D ) and Fourier-transform infrared (FT-IR) spectroscopic methods (Hollósi et al., 1992;
Holly et al., 1993; N a g y et al., 1994). They have been used for specificity analysis of peptide or virus-primed T cells. Our results revealed that the intersubunit region encompasses an I-Ed-restricted T cell epitope located in the C-terminal of the H A 1 subunit. This epitope can be recognized in peptides corresponding both to the cleaved and uncleaved forms o f the hemagglutinin molecule (Table 1). Peptides o f different length covering the core region of this epitope are able to prime and activate T cells which also recognize influenza virus-infected APC.
These results reveal that the natural hemagglutinin processing pathway can generate peptides comprising this T cell epitope.
MATERIALS AND METHODS Animals
Eight-ten-week-old female B10 (H-2b) and CBA (H-2k) mice were purchased from LATI (Gödöllő, Hungary). Female B A L B / c (H-2d) mice were delivered by the Institute of Oncology (Budapest, Hungary).
Animals were housed under conventional conditions.
Virus preparation
The A / P R / 8 / 3 4 influenza virus ( H 1 N 1 ) stock bearing cleaved hemagglutinin was grown in chorio-allantoic fluid of hen eggs and was prepared as described pre-viously ( N a g y et al., 1994). Heat inactivation o f the virus was performed at 5 6 ' C for 30 min.
Synthetic peptides
The description o f peptide synthesis, purification and characterization has been reported elsewhere (Hollósi
Table I. Description, amino acid sequence and purity of the synthetic peptides
Localization Code Sequence
Purity"
(%)
HA 1 + HA2|.|, IP VTGLRNIPSIQSRGLFGAIAGFIEG 96
HAlj|;.j-9+ H A 2M : [o-Argl3]IP VTGLRNIPSIQSRGLFGAIAGFIEG 97
HA1J|7 o : 9 HAlC(Arg) VTGLRNIPSIQSR 98
HAljn.j;, HA1C VTGLRN1PSIQS 99
HAlj|().j;j HACJ k j GLRNIPSIQS 97
HAjm-J:» HAC„mj LRNIPSIQS 98
H A 2m 2 FP GLFGAIAGFIEG 97
"Determined by HPLC.
T cell recognition of influenza virus hemagglutinin 1405 et al., 1992; H o l l y et al., 1993; T ó t h et al., 1993).
Characteristics o f the peptides used in this study are summarized in Table 1.
In vivo induction of A/ PR /8/34 influenza virus-primed T cells
B A L B / c mice were aerosol-infected with 0.03 LDJO
d o s e o f the m o u s e pathogenic A / P R / 8 / 3 4 influenza virus and 3 m o n t h s later red blood cell lysed spleen cells were enriched with T cells by passing the single cell suspension through a N y l o n w o o l (Fenwal Laboratories) column.
In another series of experiments, B A L B / c mice were infected as described above but 3 weeks later they were re-challenged with 1 LD». T cells were isolated 3 m o n t h s after secondary infection as described above. In a third series of experiments, the A / P R / 8 / 3 4 virus was injected subcutaneously as 1 0 0 / t g purified virus emulsified in c o m p l e t e Freund's adjuvant and introduced to the base o f the tail.
In vivo induction of peptide-primed T cells
BIO, C B A or B A L B / c mice were immunized subcu-taneously at the base o f the tail and in the hind foot-pads with 100 pg peptide dissolved in sterile PBS and emulsified in C F A . Fourteen days later the T cell enriched lymph node cells were used for the prolifer-ation assay. In some experiments, peptide adminis-tration was repeated on day 21 in combination with incomplete Freund's adjuvant and the T cell-enriched l y m p h n o d e cells were isolated 7 - 1 0 days after the secondary peptide challenge. T cell enrichment of the popliteal, inguinal and paraaortic lymph node cells of subcutaneously immunized mice was performed either by N y l o n w o o l (Fenwal Laboratories) elimination of adherent cells or by panning o n anti-mouse immunoglobulin (Ig) coated Petri dishes. The T cell-enriched, eluted cell population (subsequently termed as T cells) was contaminated with 1 - 3 % B cells detected by cytofluorimetry using FITC-labelled anti-mouse Ig (Sigma, Deisenhofen, Germany).
T cell proliferation assay
T h e T cell proliferation assay was performed by the m e t h o d o f Corradin et al. (1977). Briefly, 5 x 10s enriched lymph node T cells were cultured in the pres-ence of 2 % 10s irradiated syngeneic spleen cells as A P C in the absence or the presence of different concentrations of synthetic peptides. Triplicate microcultures in a final v o l u m e of 2 5 0 p \ were established in flat-bottomed 96-well plates (Greiner) in complete R P M I m e d i u m supplemented with 10% fetal calf serum (Gibco), 1 m M s o d i u m pyruvate, 2 m M glutamine, 10 m M H E P E S buffer, 5 x 1 0 ~5M 2-mercaptoethanol, antibiotics, non-essential a m i n o acids, and BME-vitamins (all purchased from Sigma).
Influenza virus infection of normal B A L B / c spleen cells was performed by a 4 hr preincubation of the cells
with different doses o f the infective A / P R / 8 / 3 4 influenza virus. After the preincubation period, cells were washed twice with glucose-containing phosphate saline (G-PBS), irradiated or m y t o m y c i n - C (Sigma) treated ( 2 5 / t g / m l , 37°C, 30 min) and used as A P C at 2 x 10s cell/well.
Cultures were pulsed with 1 pCi (3.7 kBq) JH-thymidine for the final 16 hr of the 4-day cultures.
Experiments performed in the presence o f the enzyme inhibitor leupeptin (acetyl-Leu-Leu-Arg-SOjH,
Boehringer, M a n n h e i m , G e r m a n y ) o r the tripeptide B o c - D - P h e - P r o - A r g - H 1/2 H:S 04 (GKI-14451; D - F P R , the generous gift o f S. Bajusz, Institute of D r u g Re-search, Budapest, Hungary) (Bajusz et al., 1978) were performed as described for the T cell proliferation assay except that the inhibitor w a s present during the whole period of cell culture at 0.5 or 1 m M final concentration.
The inhibitor effect o f M H C specific monoclonal antibodies ( m A b ) 1-47 rat IgG2a, anti-H-2Dd (generous gift of G . László, Department o f Immunology, "L-.-E ö t v ö s University); 14.4.4 m o u s e IgG2a, anti-I-"L-.-Ek,d (Ozato et al., 1980); M 5 / 1 1 4 rat IgG2b, anti-I-A/Dd ( A T C C T I B 120) (Bhattacharya et al., 1981); MK.D6 m o u s e IgG2a, anti-I-A ( A T C C H B 3 ) (Kappler et al.,
1981) was detected in the presence o f 5 % hybridoma cell culture supernatants.
Isolation of T cell lines and the IP -12-29 T cell hybridoma
Spleen cells (2 x 106) isolated form virus-infected mice were restimulated weekly with irradiated syngeneic spleen cells and 1 0 / t g HI A virus or 20 p g peptide.Twenty-four hr after the third or fourth restimulation, cell culture supernatants were tested for IL2 pro-duction. Three days after restimulation, 200 /zl of cell suspensions were tested in triplicate microcultures for proliferation. The IP-12-9 T cell hybridoma was selected from the spleen o f a H A l C [ A r g ] - i m m u n i z e d and virus-challanged B A L B / c mouse. H A l Q A r g ] immunization was performed as described above and 4 weeks before cell fusion the preimmunized mouse w a s aerosol-infected with 5 LD30 of A / P R / 8 / 3 4 influenza virus. Spleen cells o f the peptide-immunized and subsequently virus-challenged m o u s e were reactivated in vitro with the H A l C J A r g ] peptide and 5 days later Ficoll separated T cells were fused with the BW-1100.129.237 alpha-/beta-(White et al., 1989) t h y m o m a cells according to the method o f Rock (1985).
Detection of IL2 production
Peptide- or virus-induced IL2 production of activated polyclonal T cells was measured in cell culture super-natants of the proliferation assay harvested after 24 hr incubation in the presence of the peptides or virus-in-fected A P C . The detection o f IL2 in the supernatant of the IP-12-29 T cell hybrid was measured in samples taken at 24 o r 48 hr o f cell cultures containing 1 x 104 hybridoma cells and 2 x 105 irradiated syngeneic normal spleen cells. The relative level of IL2 of cell culture supernatants was quantitated by the proliferation of
1406 É. R A J N A V Ö L G Y I et al.
T a b l e 2. T h e
in vitro IP-specific proliferative response a n d IL2 p r o d u c t i o n of l y m p h n o d eT cells isolated f r o m I P - p r i m e d B A L B / c . BIO o r C B A mice
T cell IL2
M o u s e H - 2 IP
proliferationp r o d u c t i o n
strain
KI-A I-E
D (/tg/ml) (cpm x 10"3) (cpm x 10"3)BIO
b b b b b- b b 10 2.8 ± 0.4 0.2 ± 0 . 14 3 . 7 + 1 . 2
0.1 + 0 . 1
0.8 5 . 0 + 1.4 0.8 + 0.2
—
3.8 + 2.6 0.2 + 0.1
C B A kk
k k kk kk 202.0 + 0.7 7 . 8 + 1 . 2
4 1.5 + 0.3
4.9 + 0.7
0.8 0.7 + 0.1 1.2 + 0.1
— 0.5 + 0.1 0.3 + 0.1
B A L B / c
dd d d dd d d 20 86.3 + 10.7 131.3 + 23.24
45.6 + 8.8
128.2 + 5.10.8 13.5 + 9.4
7 1 . 5 + 14.1
—
1.9 + 0.6 38.8 + 5.4
20" 4.9 + 0.5 n.t.
"Tested in t h e p r e s e n t e o f C B A n o r m a l spleen cells a s A P C . n.t., n o t tested.
T cell proliferation w a s m e a s u r e d by 3H - t h y m i d i n e i n c o r p o r a t i o n in the 4-day cultures.
IL2 p r o d u c t i o n was m e a s u r e d by the p r o l i f e r a t i o n o f H T 2 cells in a secondary culture containing 50 p\ of culture s u p e r n a t a n t harvested 24 h r after p r i m a r y culture in t h e presence o f A P C a n d peptide. M e a n values o f triplicates + S.D. of a typical experiment are d o c u m e n t e d .
H T 2 cells d e t e c t e d b y
3H - t h y m i d i n e i n c o r p o r a t i o n ( g i v e n a s c p m ) o r b y ( 3 [ 4 , 5 d i m e t h y l t h i a z o l 2 y l ] 2 . 5 d i p h e n y l -t e -t r a z o l i u m b r o m i d e ( M T T ) u p -t a k e ( g i v e n a s OD
550) i n s e c o n d a r y c u l t u r e s u s i n g a c a l i b r a t i o n c u r v e i n t h e p r e s e n c e o f d i f f e r e n t a m o u n t s o f r e c o m b i n a n t h u m a n I L 2 ( k i n d l y p r o v i d e d b y H o f f m a n L a R o c h e ) .
RESULTS
The proliferative response of CBA, B10 and BALB/c mice to the synthetic IP peptide
T h e p r o l i f e r a t i v e r e s p o n s e o f
in vivop r i m e d l y m p h n o d e T cells o f d i f f e r e n t i n b r e d m o u s e s t r a i n s t o t h e
Anti-MHC Ab 100
90 80 70
© 60
o o
K
50E
U
4030 20 10 0
IP
• H A I C ( R )
l-A/E l-A L-E H-2 Dd
Fig. 1. M H C restriction o f the IP- a n d H A l C [ A r g ] - p r i m e d T cell response. Proliferation of IP-(dark c o l u m n s ) o r H A l C [ A r g ] - ( e m p t y c o l u m n s ) p r i m e d T cells was studied in the presence of 1 /Jg/ml I P o r H A I C [ A r g ] , respectively in t h e absence o r presence o f 5 % cell culture s u p e r n a t a n t s c o n t a i n i n g m A b reacting with I-A. I - E o r H - 2 DJ molecules. Percentage inhibition w a s calculated f r o m c p m values o b t a i n e d f o r positive cultures ( I P , 4696 + 262, H A l C [ A r g ] , 7316 + 642) c o n t a i n i n g t h e c o r r e s p o n d i n g peptide in the absence of the m A b . B a c k g r o u n d m e a s u r e d in negative cultures was
982 + 108.
T cell recognition of influenza virus hemagglutinin 1407 synthetic IP p e p t i d e (see T a b l e 1) representing the
u n c l e a v e d f o r m o f the intersubunit region o f the influ-e n z a virus h influ-e m a g g l u t i n i n m o l influ-e c u l influ-e is s u m m a r i z influ-e d in T a b l e 2. D a t a d e m o n s t r a t e that IP is able to induce a peptide-specific, M H C - r e s t r i c t e d proliferative response in I P - i m m u n i z e d B A L B / c but n o t in BIO or C B A mice.
It is a l s o s h o w n that I P i n d u c e s proliferation a n d IL2 p r o d u c t i o n in a d o s e - d e p e n d e n t m a n n e r in the presence o f syngeneic A P C . I n h i b i t i o n by M H C - s p e c i f i c anti-b o d i e s revealed the I-Ed restriction o f the IP-specific T cell response ( F i g . 1, d a r k c o l u m n s ) .
Specificity of the IP-induced T cell response of BALB/c mice
T h e reactivity o f IP-specific T cells w i t h o v e r l a p p i n g synthetic peptides c o v e r i n g the intersubunit region w a s studied with p o l y c l o n a l T cells as well as with T cell h y b r i d o m a s . A s s h o w n in T a b l e 3, IP-primed T cells c a n be activated by the H A I Q A r g ] s u b f r a g m e n t peptide but n o t with the F P f u s i o n Reptide. T h i s o b s e r v a t i o n p o i n t s t o the l o c a t i o n o f a T cell e p i t o p e to the C-terminal s e g m e n t o f the H A 1 s u b u n i t . T h e presence o f a T cell e p i t o p e in the H A I Q A r g ] peptide w a s a l s o verified by the capability o f this peptide, but n o t o f the FP f u s i o n p e p t i d e to prime T cells ( T a b l e 3). H A l C [ A r g ] - p r i m e d T cells are cross-reactive w i t h the IP peptide (Table 3) a n d are a l s o recognized in the c o n t e x t o f the I-Ed m o l e c u l e (Fig. 1, plain c o l u m n s ) .
T o a n s w e r the q u e s t i o n w h e t h e r the C-terminal arginine o f the H A I Q A r g ] peptide has a n y role in T cell recognition, the c a p a c i t y o f the A r g - t r u n c a t e d H A 1 C d o d e c a p e p t i d e to i n d u c e the a c t i v a t i o n o f H A 1 C , H A I Q A r g ] or I P - p r i m e d T cells w a s studied. A l t h o u g h n o striking difference in the T cell p r i m i n g or activating
capacity o f H A I Q A r g ] or H A 1 C c o u l d be observed (Table 3), it s h o u l d be n o t e d that the A r g e x t e n d e d peptide g a v e a higher response in m o s t cases.
T h e o b s e r v a t i o n s o b t a i n e d with polyclonal T cells raised the possibility that T cells o f differing specificity might be responsible for the recognition o f IP a n d H A I Q A r g ] a n d their a n a l o g s . T h e fine specificity pat-tern o f a typical T cell h y b r i d o m a (IP-12-29) depicted in Fig. 2 s h o w s full cross-reactivity o f IP and H A I Q A r g ] s u p p o r t i n g the interpretation that H A I Q A r g ] c a n be recognized b o t h in its 13-mer a n d fusion peptide e l o n g a t e d 2 5 - m e r f o r m s . A s in the c a s e o f p o l y c l o n a l T cells ( T a b l e 3), A r g truncation at the C-terminal o f the H A I Q A r g ] peptide d o e s n o t a b o l i s h recognition by the IP-12-29 T cell h y b r i d o m a (Fig. 2B) d e m o n s t r a t i n g the identical specificity pattern o f p o l y c l o n a l T cells and that o f this m o n o c l o n a l T cell.
O n the basis o f these results, a T cell e p i t o p e localized in the 3 1 7 - 3 2 8 region o f the H A 1 subunit representing the H I serotype s e q u e n c e o f influenza virus h e m a g -glutinin c o u l d be identified. N - t e r m i n a l l y truncated a n a l o g s o f H A 1 C up to a deca- ( H A l Cd e c a) or n o n a p e p -tide ( H A 1 Cn 0 i u) are n o t able to activate the IP-12-29 T cell h y b r i d o m a (Fig. 2B).
Processing requirements of IP recognition
T h e cross-reactivity o f IP- and H A l C [ A r g ] - p r i m e d T cells in b o t h directions d o e s not clarify w h e t h e r IP is recognized in its full length or will be further processed giving rise to a f r a g m e n t c o m p r i s i n g the H A I Q A r g ] peptide. T h e processing requirements o f IP were investigated by s t u d y i n g the activity o f the D-Arg sub-stituted a n a l o g o f IP ([D-Arg]1 3IP) a n d using different e n z y m e inhibitors k n o w n to interfere with the activity
Table 3. Cross-reactivity of peptide-primed lymph node T cells with the intersubunit IP and subunit peptides H A I Q A r g ] , H A 1 C and FP
In vitro stimulation (cpm x 10~3 + S.D.)
Immunization /tg/ml IP H A I Q A r g ] H A I C FP
IP 20 48.9 + 9.9 36.3 + 6.6 33.4 + 4.3 12.2 + 2.3
4 6 3 . 1 + 2 . 9 33.5 + 3.0 2 8 . 3 + 0 . 5 14.2 + 6.2 0.8 50.2 + 4.0 19.9 + 5.3 2 3 . 0 + 1 . 7 1 2 . 5 + 1.4
— 7.1 + 0 . 7
H A I Q A r g ] 20 43.3 + 3.49 66.1 + 17.4 41.9 + 2.7 n.t.
4 50.1 + 16.0 4 I . S + 10.5 2 3 . 8 + 12.4 n.t.
0.8 65.5 + 4.8 33.4 + 6.3 26.7 + 5.5 n.t.
— 14.6 + 5.2
H A I C 20 22.3 + 6.9 34.8 + 2.3 32.9 + 3.9 n.t.
4 28.0 + 3.9 2 2 . 8 + 12.7 24.0 + 9.5 n.t.
0.8 2 1 . 1 + 0 . 3 6 18.8 + 5.3 2 1 . S + 10.4 n.t.
— 2.8 + 0.3
FP 20 10.7 + 2.9 n.t. n.t. 5.9 + 0.5
4 8.8 + 3.4 n.t. n.t. 5.4 + 0.7
0.8 7.2 + 0.S n.t. n.t. 5.7 + 2.4
— 5 . 5 + 1.1
T cell proliferation was measured as described under Table 2.
1408 É. R A J N A V Ö L G Y I et al.
(A) IP-12-29
3 0 0 0 0 0
2 5 0 0 0 0
-•
200000 |
150000
-100000
5 0 0 0 0 -I I
(B)
ug/ml H A 1 peptides
— o
n o
60000
5 0 0 0 0
-4 0 0 0 0
5. 3 0 0 0 0 -1
-20000
10000
1
control HA1CIRI HA1C H A I d e c a H A I n o n a
Fig. 2. Reactivity of the IP-12-29 h y b r i d o m a with synthetic peptides representing the i n t e r s u b u n i t region o f influenza virus h e m a g g l u t i n i n . ( A ) T h y b r i d o m a cells were c u l t u r e d in t h e presence of i r r a d i a t e d B A L B / c spleen cells
a n d different c o n c e n t r a t i o n s o f the synthetic peptide I P ( • — • >[ D - A r g " ] I P ( • — • ) o r H A l C [ A r g ] ( • — • ) . Relative IL2 c o n t e n t of cell culture s u p e r n a t a n t s was m e a s u r e d by H T 2 cell p r o l i f e r a t i o n a n d given a s c p m . M e a n values o f triplicates of a typical e x p e r i m e n t a r e d o c u m e n t e d . ( B ) T h y b r i d o m a cells were cultured in t h e presence of 2 . 5 / r g / m l
, peptides. Negative cultures (control) did n o t c o n t a i n peptide.
H A 1 C[Arg], ( H A 1 C(R), H A 1C, H A
1 Cdecao r H A 1
Cn o n a,o f t r y p s i n - l i k e e n z y m e s , w h i c h h a v e a n i n h i b i t o r y e f f e c t f o r e x o g e n o u s a n t i g e n p r o c e s s i n g a n d a l s o f o r c e r t a i n v i r a l i n f e c t i o n ( A n d e r s e n , 1983). A s s h o w n in F i g . 3 t h e p r e s e n c e o f l e u p e p t i n ( F i g . 3 A , B ) o r t h e D - F R P ( F i g . 3 B ) t r y p s i n i n h i b i t o r p r e v i o u s l y s h o w n
t oi n h i b i t t r y p s i n - m e d i a t e d f u s i o n o f i n f l u e n z a v i r u s ( u n p u b l i s h e d o b s e r v a t i o n ) d i d n o t a b o l i s h I P r e c o g n i t i o n b y p o l y -c l o n a l ( F i g . 3 A ) o r m o n o -c l o n a l ( F i g . 3 B ) T
cells,w h i l e t h e s a m e c o n c e n t r a t i o n o f i n h i b i t o r s c a u s e d c o m p l e t e i n h i b i t i o n o f t h e p r o l i f e r a t i o n o f o v a l b u m i n - p r i m e d T cells i n d u c e d b y 2 0 o r 4 / j g / m l o v a l b u m i n ( d a t a n o t s h o w n ) . T h i s r e s u l t , i n g o o d a c c o r d a n c e w i t h t h e s i m i
-l a r d o s e - r e s p o n s e c u r v e s o f H A -l C [ A r g ] , I P o r i t s [D-Arg
n]IP a n a l o g t o a c t i v a t e t h e I P - 1 2 - 2 9 T h y b r i d o m a ( F i g . 2 A ) o r p o l y c l o n a l T cells ( d a t a n o t s h o w n ) r e v e a l e d t h a t t h e I P p e p t i d e c a n b e r e c o g n i z e d w i t h o u t f u r t h e r p r o c e s s i n g a t t h e A r g / G l y c l e a v a g e site.
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T cell recognition o f influenza virus hemagglutinin 1409 (A) 60 T