9 1991 S, Karger AG, Basel 0257-277X/9 I/0104-022652,75/0
lmmunol Res 1991:i0:226-229
Anti-CD3- or PHA-Induced Cytotoxicity in Human Intraepithelial and Lamina Propria Lymphocytes Joachim Riithlein, Gabriele Heinze Medizinische Universit/i,tsklinik, Immunologisches Labor, WCirzburg, BRD
The mucosal i m m u n e system can be separated into an afferent and an efferent limb. The afferent part is found in specialized areas, called lymphoid aggregates. The efferent limb consists of mononuclear cells spread in between the lamina propria and the epithelial cells without any visible organisation [1]. By immunofluorescence it could be shown that lamina propria mononuclear cells (LPL) contain B cells, CD4 + and CD8 + T cells, whereas intraepithelial mononuclear cells (IEL) do not contain B cells and few CD4 + cells [2]. Both compartments have very low numbers of cells with surface markers that are associated with killer function in peripheral blood (CD56, CD57, CD16). Freshly isolated EPL do not exhibit spontaneous cell-mediated cytotoxicity [3-6]. However, previous studies have suggested that LPL exhibit mitogen-induced cytotoxicity and antibody-dependent cellmediated cytotoxicity [7]. In peripheral blood certain effector T cells can be triggered by phytohemagglutinin (PHA) or anti-CD3 to exert their cytotoxic potential and become non-human lymphocyte antigen (HLA)-restricted cytotoxic cells. It was proposed that those 'unnatural' stimuli would trigger anti-
gen-specific, HLA-restricted cytolytic T cells. Thus, it would be possible to demonstrate the function of these T cells without knowing the specific antigen. We have shown previously that CD3 § CD8 + and CD57 + cells are the main population in peripheral blood that has this ability [8]. In LPL and IEL we do not find cells with this phenotype. However, it was shown by Shanahan et al, [9, 10] that one effector function of LPL is anti-CD3-mediated cytotoxicity in short-term assays, proposing that L P L contain cytolytic T cells in vivo. As it was shown by electron microscopy that about 20% of h u m a n IEL are large granular lymphocytes [1 I] - an appearance associated with cytotoxic function in h u m a n peripheral blood and in mouse IEL [ 12] - we tested if h u m a n IEL would also mediate cytotoxic function in short-term assays.
Cytotoxic Function of Human IELs and LPLs Samples were obtained from patients aged 25-72 years, who underwent colon resection for cancer (n = 6) or inflammatory bowel disease (Crohn's disease n = 5, ulcerative colitis n = 2). Mucosal cells were ob-
227
Function of Human Mucosal Lymphocytes
tained by a modified enzymatic method [13]. After 90 min incubation in Ca- and Mg-free Hank's solution with 0.7 m M EDTA for the isolation of IEL, we further purified IEL by several meshes, by Ficoll and Percoll gradients. LPL were released from the remaining tissue after 90 min incubation in collagenase CLSPA (Worthington, 8,000 U in 100 ml) and hyaluronidase (Sigma, 40-75,000 U in 100 ml). LPL were also purified by density gradients. IEL and LPL populations were used fresh for cytotoxicity studies and for flow cytometry. Cytotoxicity assays were performed in 96-well round-bottom microtiter plates using 51Cr-labeled K562 cells as targets. We used 5,000 target cells and added effector cells to achieve effector/target (E/T) ratios of 12/1, 25/I, 50/l and 100/1. We always tested lbr spontaneous cytotoxicity (= natural-killer activity), for PHA (Gibco) and anti-CD3-mediated cytotoxicity. PHA was used at 1/50 dilution. For anti-CD3mediated lysis we used anti-OKT-3 ascites (ATTC cell line CRL 8001) in optimal concentration for peripheral blood cells. Cytotoxicity assays were incubated for 6 h at 37 ~ in 5% CO2 and 95% air atmosphere. After this time cells were centrifuged at 500 g for 5 min. Then supernatants were harvested with a Skatron harvester and counted with a gamma counter. Spontaneous 5~Cr release was always less than 10 % of the total release induced by triton X-100. All effector/target ratios were done in triplicate and mean counts per minute were used. Specific release was calculated by the following formula: % cytotoxicity = test release - spontaneous release • 100. total release - spontaneous release
Spontaneous cell-mediated cytotoxicity (= natural-killer activity) was always subtracted for calculation of PHA or anti-CD3-induced cytotoxicity.
Results Isolated human
L P L (n = 10) w e r e a n a -
l y z e d b y f l o w c y t o m e t r y . W e f o u n d 59 _+ 2 4 % C D 3 § cells, 14 _+ 6 % C D 2 0 + cells a n d b e t w e e n 8 a n d 3 3 % H M L - 1 + cells. In 3 o f 11 i n d i v i d u a l s we o b s e r v e d s p o n t a n e o u s
cell-
m e d i a t e d c y t o t o x i c i t y a b o v e 5 % s p e c i f i c lysis t h a t is E / T r a t i o d e p e n d e n t ( t a b l e 1). L P L from the colon do have PHA or anti-CD3m e d i a t e d c y t o t o x i c f u n c t i o n in m o s t cases. As c y t o t o x i c i t y was a l w a y s d e p e n d e n t o n t h e E/T ratio, we only show the results with the h i g h e s t n u m b e r s o f e f f e c t o r cells (table 1). I n some
patients
we c o u l d
not
PHA or anti-CD3-mediated
demonstrate
cytolytic func-
t i o n o f L P L . T h e i n a b i l i t y to d e m o n s t r a t e t h i s c y t o t o x i c i t y was n o t d u e to less p u r e T cell p r e p a r a t i o n s , as d e t e r m i n a t i o n s o f C D 3 + cells w e r e n o t d i f f e r e n t in L P L p r e p a r a t i o n s with and without cytotoxic function. There was n o d i f f e r e n c e b e t w e e n p a t i e n t s w i t h col o n c a n c e r o r i n f l a m m a t o r y b o w e l disease. I s o l a t e d h u m a n I E L w e r e 38 + 1 7 % C D 3 + (n = 11, r a n g e 1 2 - 8 8 % ) . B e t w e e n 80
Table 1. Cytotoxic function of IEL and LPL IEL
Natural killer activity PHA Anti-CD3
LPL
specific cytotoxicity, %
individuals with > 5% lysis
specific cytotoxicity, %
individuals with > 5% lysis
0.8 2.8 I. l
0/10 3/10 1/7
2.6 9.5 10.4
3/l l 8/1 ! 8/10
228
a n d 100% o f CD3* I E L were also H M L - I +. C D 2 0 § cells were always less t h a n 5%. I E L f r o m the c o l o n h a v e v e r y low P H A o r d o n o t have any PHA- or anti-CD3-mediated cytotoxic f u n c t i o n in s h o r t - t e r m assays (table 1). In s o m e s a m p l e s o f I E L we f o u n d a low yet d e f i n i t e c y t o t o x i c i t y . T h i s c y t o t o x i c i t y is m o s t likely d u e to c o n t a m i n a t i o n w i t h L P L , as we f i n d specific c y t o t o x i c i t y e x c e e d i n g 5 % in I E L o n l y w h e n we f i n d c o n s i d e r a b l y h i g h e r c y t o t o x i c i t y in the c o r r e s p o n d i n g L P L population.
Discussion T h e flow c y t o m e t r y d a t a o f I E L a n d L P L p r e p a r a t i o n s a r e in a g r e e m e n t with histologic e x a m i n a t i o n s . T h e d i s t r i b u t i o n o f B cells a n d t h e p e r c e n t a g e o f H M L - 1 + T cells allow the c o n c l u s i o n t h a t we are able to sepa r a t e the m a j o r i t y o f I E L a n d L P L into dist i n c t p o p u l a t i o n s [ 14]. As there s h o u l d be no B cells in IEL, we must, h o w e v e r , a s s u m e t h a t a s m a l l p o p u l a t i o n o f cells f r o m the l a m i n a p r o p r i a is l e a k i n g i n t o the I E L p o p u l a tion. We find that human LPL have a definite c y t o t o x i c f u n c t i o n using P H A a n d a n t i - C D 3 m e d i a t e d assays. O u r d a t a w i t h a n t i - C D 3 suggest t h a t a n t i g e n - s p e c i f i c cytolytic T cells are p r e s e n t in L P L . In p r e l i m i n a r y d a t a we f o u n d no d i f f e r e n c e s b e t w e e n p a t i e n t s w i t h c o l o n c a n c e r a n d p a t i e n t s with i n f l a m m a t o r y b o w e l disease. T h e f u n c t i o n o f I E L in h u m a n s is still u n k n o w n . It is v e r y likely that they do h a v e a n effector f u n c t i o n . In spite o f t h e i r m o r p h o l o g y as large g r a n u l a r l y m p h o c y t e s it c o u l d n o t b e s h o w n t h a t these cells in hum a n s h a v e c y t o t o x i c f u n c t i o n in s h o r t - t e r m assays. O u r d a t a p r o p o s e t h a t h u m a n I E L do
R/.ithlein/Heinze
e i t h e r n o t c o n t a i n c y t o t o x i c T cells o r that r e c o g n i t i o n o r triggering o f I E L is d i f f e r e n t from LPL.
Summary Morphological data in humans and rodents and functional data of intraepithelial lymphocytes of mice support the idea that cytotoxic cells are a significant population of the human mucosa. Previously it was shown that human IEL have no spontaneous cellmediated cytotoxicity and that human LPL cells have anti-CD3-mediated cytotoxicity. We confirm that most individuals have anti-CD3- or PHA-mediated cytotoxicity of LPL. In IEL we do not find cytotoxic function in short-term assays. There is no difference between patients with colon cancer and inflammatory bowel disease.
References 1 Targan SR: Immunologic mechanisms in intestinal disease. Ann Intern Med 1987;106:853-870. 2 Jarry A, Cerf-Bensussan N, Brousse N, Selz F, Guy-Grand D: Subsets of CD3 * (T cell receptor a/f3 or y/8) and CD3- lymphocytes isolated from normal human gut epithelium display phenotypical features different from their counterparts in peripheral blood. Eur J Immunol 1990;20:10971103. 3 Bland PW, Britton DC, Richens ER, Pledger JV: Peripheral, mucosal, and tumor infiltrating components of cellular immunity in cancer of the large bowel. Gut 1981;22:744-751. 4 Falchuk ZM, Barnhard E, Machado I: Human colonic mononuclear cells: Studies of cytotoxic function. Gut 1981;22:290-294. 5 Gibson PR; Dow EL, Selby WS, Strickland RG, Jewell DP: Natural killer cells and spontaneous cell mediated cytotoxicity in the human intestine. Clin Exp Immunol 1984;56:438-444. 6 Auer IO, Rfithlein J, R6der A, Reineke C, Grudzinsky K: Untersuchungen der intestinalen spontanen zellvermittelten Zytotoxizit~it und ihrer Immunoregulation bei Patienten mit chronisch-entziindlichen Darmerkrankungen und Kontrollen. Immun Infekt 1986;14:22-23.
Function of Human Mucosal Lymphocytes
7 MacDermott RP, Franklin GO, Jenkins KM, Kodner I J, Nash GS, Weintrieb IJ: Human intestinal mononuclear cells. I. Investigation of antibody-dependent, lectin-induced and spontaneous cell-mediated cytotoxic capabilities. Gastroenterology 1980;78:47-56. 8 Riithlein J, James SP, Strober W: Role of CD2 in activation and cytotoxic function in CD8/Leu-7 positive T cells. J Immunol 1988;141:37913793. 9 Shanahan F, Brogan M, Targan S: Human mucosal cytotoxic effector cells. Gastroenterology 1987;92:1951-1957. 10 Shanahan F, Deem R, Nayersina R, Leman B, Targan S: Human mucosal T-ceU cytotoxicity. Gastroenterology 1988;94:960-967. 11 Cerf-Bensussan N, Guy-Grand D, Griscelli C: Intraepithelial lymphocytes of the human gut: Isolation, characterization and study of natural killer activity. Gut 1985;26:81-88. 12 Tagliabue A, Befus AD, Clark DA, Bienenstock J: Characteristics of natural killer cells in the routine intestinal epithelium and lamina propria. J Exp Med 1982;155:1785-I796.
229
13 Zeitz M, Quinn TC, Graeff AS, James SP: Mucosal T cells provide helper function but do not proliferate when stimulated by specific antigen in lymphogranuloma venereum proctitis in nonhuman primates. Gastroenterology 1988;94:353366. 14 Cerf-Bensussan N, Jarry A, Brousse N, LisowskaGrospierre B, Guy-Grand D, Griselli C: A monoclonal antibody (HML-1) defining a novel membrane molecule present on human intestinal lymphocytes. Eur J Immunol 1987;17:1279-1285.
Dr. med. Joachim Riithlein Medizinische Universit~itsklinik Immunologisches Labor Josef-Schneider-Strasse 2 D-W-8700 Wfirzburg (FRG)
lmmunol Res 1991:i0:226-229
Anti-CD3- or PHA-Induced Cytotoxicity in Human Intraepithelial and Lamina Propria Lymphocytes Joachim Riithlein, Gabriele Heinze Medizinische Universit/i,tsklinik, Immunologisches Labor, WCirzburg, BRD
The mucosal i m m u n e system can be separated into an afferent and an efferent limb. The afferent part is found in specialized areas, called lymphoid aggregates. The efferent limb consists of mononuclear cells spread in between the lamina propria and the epithelial cells without any visible organisation [1]. By immunofluorescence it could be shown that lamina propria mononuclear cells (LPL) contain B cells, CD4 + and CD8 + T cells, whereas intraepithelial mononuclear cells (IEL) do not contain B cells and few CD4 + cells [2]. Both compartments have very low numbers of cells with surface markers that are associated with killer function in peripheral blood (CD56, CD57, CD16). Freshly isolated EPL do not exhibit spontaneous cell-mediated cytotoxicity [3-6]. However, previous studies have suggested that LPL exhibit mitogen-induced cytotoxicity and antibody-dependent cellmediated cytotoxicity [7]. In peripheral blood certain effector T cells can be triggered by phytohemagglutinin (PHA) or anti-CD3 to exert their cytotoxic potential and become non-human lymphocyte antigen (HLA)-restricted cytotoxic cells. It was proposed that those 'unnatural' stimuli would trigger anti-
gen-specific, HLA-restricted cytolytic T cells. Thus, it would be possible to demonstrate the function of these T cells without knowing the specific antigen. We have shown previously that CD3 § CD8 + and CD57 + cells are the main population in peripheral blood that has this ability [8]. In LPL and IEL we do not find cells with this phenotype. However, it was shown by Shanahan et al, [9, 10] that one effector function of LPL is anti-CD3-mediated cytotoxicity in short-term assays, proposing that L P L contain cytolytic T cells in vivo. As it was shown by electron microscopy that about 20% of h u m a n IEL are large granular lymphocytes [1 I] - an appearance associated with cytotoxic function in h u m a n peripheral blood and in mouse IEL [ 12] - we tested if h u m a n IEL would also mediate cytotoxic function in short-term assays.
Cytotoxic Function of Human IELs and LPLs Samples were obtained from patients aged 25-72 years, who underwent colon resection for cancer (n = 6) or inflammatory bowel disease (Crohn's disease n = 5, ulcerative colitis n = 2). Mucosal cells were ob-
227
Function of Human Mucosal Lymphocytes
tained by a modified enzymatic method [13]. After 90 min incubation in Ca- and Mg-free Hank's solution with 0.7 m M EDTA for the isolation of IEL, we further purified IEL by several meshes, by Ficoll and Percoll gradients. LPL were released from the remaining tissue after 90 min incubation in collagenase CLSPA (Worthington, 8,000 U in 100 ml) and hyaluronidase (Sigma, 40-75,000 U in 100 ml). LPL were also purified by density gradients. IEL and LPL populations were used fresh for cytotoxicity studies and for flow cytometry. Cytotoxicity assays were performed in 96-well round-bottom microtiter plates using 51Cr-labeled K562 cells as targets. We used 5,000 target cells and added effector cells to achieve effector/target (E/T) ratios of 12/1, 25/I, 50/l and 100/1. We always tested lbr spontaneous cytotoxicity (= natural-killer activity), for PHA (Gibco) and anti-CD3-mediated cytotoxicity. PHA was used at 1/50 dilution. For anti-CD3mediated lysis we used anti-OKT-3 ascites (ATTC cell line CRL 8001) in optimal concentration for peripheral blood cells. Cytotoxicity assays were incubated for 6 h at 37 ~ in 5% CO2 and 95% air atmosphere. After this time cells were centrifuged at 500 g for 5 min. Then supernatants were harvested with a Skatron harvester and counted with a gamma counter. Spontaneous 5~Cr release was always less than 10 % of the total release induced by triton X-100. All effector/target ratios were done in triplicate and mean counts per minute were used. Specific release was calculated by the following formula: % cytotoxicity = test release - spontaneous release • 100. total release - spontaneous release
Spontaneous cell-mediated cytotoxicity (= natural-killer activity) was always subtracted for calculation of PHA or anti-CD3-induced cytotoxicity.
Results Isolated human
L P L (n = 10) w e r e a n a -
l y z e d b y f l o w c y t o m e t r y . W e f o u n d 59 _+ 2 4 % C D 3 § cells, 14 _+ 6 % C D 2 0 + cells a n d b e t w e e n 8 a n d 3 3 % H M L - 1 + cells. In 3 o f 11 i n d i v i d u a l s we o b s e r v e d s p o n t a n e o u s
cell-
m e d i a t e d c y t o t o x i c i t y a b o v e 5 % s p e c i f i c lysis t h a t is E / T r a t i o d e p e n d e n t ( t a b l e 1). L P L from the colon do have PHA or anti-CD3m e d i a t e d c y t o t o x i c f u n c t i o n in m o s t cases. As c y t o t o x i c i t y was a l w a y s d e p e n d e n t o n t h e E/T ratio, we only show the results with the h i g h e s t n u m b e r s o f e f f e c t o r cells (table 1). I n some
patients
we c o u l d
not
PHA or anti-CD3-mediated
demonstrate
cytolytic func-
t i o n o f L P L . T h e i n a b i l i t y to d e m o n s t r a t e t h i s c y t o t o x i c i t y was n o t d u e to less p u r e T cell p r e p a r a t i o n s , as d e t e r m i n a t i o n s o f C D 3 + cells w e r e n o t d i f f e r e n t in L P L p r e p a r a t i o n s with and without cytotoxic function. There was n o d i f f e r e n c e b e t w e e n p a t i e n t s w i t h col o n c a n c e r o r i n f l a m m a t o r y b o w e l disease. I s o l a t e d h u m a n I E L w e r e 38 + 1 7 % C D 3 + (n = 11, r a n g e 1 2 - 8 8 % ) . B e t w e e n 80
Table 1. Cytotoxic function of IEL and LPL IEL
Natural killer activity PHA Anti-CD3
LPL
specific cytotoxicity, %
individuals with > 5% lysis
specific cytotoxicity, %
individuals with > 5% lysis
0.8 2.8 I. l
0/10 3/10 1/7
2.6 9.5 10.4
3/l l 8/1 ! 8/10
228
a n d 100% o f CD3* I E L were also H M L - I +. C D 2 0 § cells were always less t h a n 5%. I E L f r o m the c o l o n h a v e v e r y low P H A o r d o n o t have any PHA- or anti-CD3-mediated cytotoxic f u n c t i o n in s h o r t - t e r m assays (table 1). In s o m e s a m p l e s o f I E L we f o u n d a low yet d e f i n i t e c y t o t o x i c i t y . T h i s c y t o t o x i c i t y is m o s t likely d u e to c o n t a m i n a t i o n w i t h L P L , as we f i n d specific c y t o t o x i c i t y e x c e e d i n g 5 % in I E L o n l y w h e n we f i n d c o n s i d e r a b l y h i g h e r c y t o t o x i c i t y in the c o r r e s p o n d i n g L P L population.
Discussion T h e flow c y t o m e t r y d a t a o f I E L a n d L P L p r e p a r a t i o n s a r e in a g r e e m e n t with histologic e x a m i n a t i o n s . T h e d i s t r i b u t i o n o f B cells a n d t h e p e r c e n t a g e o f H M L - 1 + T cells allow the c o n c l u s i o n t h a t we are able to sepa r a t e the m a j o r i t y o f I E L a n d L P L into dist i n c t p o p u l a t i o n s [ 14]. As there s h o u l d be no B cells in IEL, we must, h o w e v e r , a s s u m e t h a t a s m a l l p o p u l a t i o n o f cells f r o m the l a m i n a p r o p r i a is l e a k i n g i n t o the I E L p o p u l a tion. We find that human LPL have a definite c y t o t o x i c f u n c t i o n using P H A a n d a n t i - C D 3 m e d i a t e d assays. O u r d a t a w i t h a n t i - C D 3 suggest t h a t a n t i g e n - s p e c i f i c cytolytic T cells are p r e s e n t in L P L . In p r e l i m i n a r y d a t a we f o u n d no d i f f e r e n c e s b e t w e e n p a t i e n t s w i t h c o l o n c a n c e r a n d p a t i e n t s with i n f l a m m a t o r y b o w e l disease. T h e f u n c t i o n o f I E L in h u m a n s is still u n k n o w n . It is v e r y likely that they do h a v e a n effector f u n c t i o n . In spite o f t h e i r m o r p h o l o g y as large g r a n u l a r l y m p h o c y t e s it c o u l d n o t b e s h o w n t h a t these cells in hum a n s h a v e c y t o t o x i c f u n c t i o n in s h o r t - t e r m assays. O u r d a t a p r o p o s e t h a t h u m a n I E L do
R/.ithlein/Heinze
e i t h e r n o t c o n t a i n c y t o t o x i c T cells o r that r e c o g n i t i o n o r triggering o f I E L is d i f f e r e n t from LPL.
Summary Morphological data in humans and rodents and functional data of intraepithelial lymphocytes of mice support the idea that cytotoxic cells are a significant population of the human mucosa. Previously it was shown that human IEL have no spontaneous cellmediated cytotoxicity and that human LPL cells have anti-CD3-mediated cytotoxicity. We confirm that most individuals have anti-CD3- or PHA-mediated cytotoxicity of LPL. In IEL we do not find cytotoxic function in short-term assays. There is no difference between patients with colon cancer and inflammatory bowel disease.
References 1 Targan SR: Immunologic mechanisms in intestinal disease. Ann Intern Med 1987;106:853-870. 2 Jarry A, Cerf-Bensussan N, Brousse N, Selz F, Guy-Grand D: Subsets of CD3 * (T cell receptor a/f3 or y/8) and CD3- lymphocytes isolated from normal human gut epithelium display phenotypical features different from their counterparts in peripheral blood. Eur J Immunol 1990;20:10971103. 3 Bland PW, Britton DC, Richens ER, Pledger JV: Peripheral, mucosal, and tumor infiltrating components of cellular immunity in cancer of the large bowel. Gut 1981;22:744-751. 4 Falchuk ZM, Barnhard E, Machado I: Human colonic mononuclear cells: Studies of cytotoxic function. Gut 1981;22:290-294. 5 Gibson PR; Dow EL, Selby WS, Strickland RG, Jewell DP: Natural killer cells and spontaneous cell mediated cytotoxicity in the human intestine. Clin Exp Immunol 1984;56:438-444. 6 Auer IO, Rfithlein J, R6der A, Reineke C, Grudzinsky K: Untersuchungen der intestinalen spontanen zellvermittelten Zytotoxizit~it und ihrer Immunoregulation bei Patienten mit chronisch-entziindlichen Darmerkrankungen und Kontrollen. Immun Infekt 1986;14:22-23.
Function of Human Mucosal Lymphocytes
7 MacDermott RP, Franklin GO, Jenkins KM, Kodner I J, Nash GS, Weintrieb IJ: Human intestinal mononuclear cells. I. Investigation of antibody-dependent, lectin-induced and spontaneous cell-mediated cytotoxic capabilities. Gastroenterology 1980;78:47-56. 8 Riithlein J, James SP, Strober W: Role of CD2 in activation and cytotoxic function in CD8/Leu-7 positive T cells. J Immunol 1988;141:37913793. 9 Shanahan F, Brogan M, Targan S: Human mucosal cytotoxic effector cells. Gastroenterology 1987;92:1951-1957. 10 Shanahan F, Deem R, Nayersina R, Leman B, Targan S: Human mucosal T-ceU cytotoxicity. Gastroenterology 1988;94:960-967. 11 Cerf-Bensussan N, Guy-Grand D, Griscelli C: Intraepithelial lymphocytes of the human gut: Isolation, characterization and study of natural killer activity. Gut 1985;26:81-88. 12 Tagliabue A, Befus AD, Clark DA, Bienenstock J: Characteristics of natural killer cells in the routine intestinal epithelium and lamina propria. J Exp Med 1982;155:1785-I796.
229
13 Zeitz M, Quinn TC, Graeff AS, James SP: Mucosal T cells provide helper function but do not proliferate when stimulated by specific antigen in lymphogranuloma venereum proctitis in nonhuman primates. Gastroenterology 1988;94:353366. 14 Cerf-Bensussan N, Jarry A, Brousse N, LisowskaGrospierre B, Guy-Grand D, Griselli C: A monoclonal antibody (HML-1) defining a novel membrane molecule present on human intestinal lymphocytes. Eur J Immunol 1987;17:1279-1285.
Dr. med. Joachim Riithlein Medizinische Universit~itsklinik Immunologisches Labor Josef-Schneider-Strasse 2 D-W-8700 Wfirzburg (FRG)
