ImmunoZ Res 199 l ; 10:199-206

Phenotype and Function of Lamina Propria T Lymphocytes 1 ~i. Z e i t z a, 2, H . L . S c h i e f e r d e c k e r a, R. Ullrich a, H.-U. J a h n a, S.P. J a m e s b, E.-O. R i e c k e n a

aMedical Clinic, Department of Gastroenterology, Klinikum Steglitz, Free University of Berlin, FRG; bMucosal Immunity Section, National Institute of Allergy and Infectious Diseases, Bethesda, Md., USA

Introduction

Lymphocytes at intestinal mucosal sites are in permanent contact with an enormous number of antigens and substances with mitogenic properties in the intestinal lumen. In order to protect the body against invasion or an inappropriate immune response to potentially pathogenic intestinal antigens and microorganisms, the T and B cell system at mucosal surfaces has unique properties with regard to its structure and function [1, 2]. Mucosal lymphocytes include cells in organized sites, such as Peyer's patches, and cells disseminated diffusely in the intestinal lamina propria (lamina propria lymphocytes) and above the basement membrane between epithelial cells (intraepithelial lymphocytes). Due to the localization of the latter two populations, it is likely that these T cells carry out specific effector functions when encountering antigens [3]. Certain T cell surface antigens allow a distinction between different maturational Dedicated to Professor Dr. WolfgangGerok, Freiburg, FRG, on the occasion of his 65th birthday. 2 Supported by a grant from the 'Deutsche Forschungsgemeinschaft' (Ze 188/4-2).

stages of T cells: In vitro activation of CD45RA +, CD29- peripheral blood T lymphocytes by antigen leads to a transition to CD45RA-, CD29 § T cells [4, 5]. This finding in conjunction with functional studies have led to the conclusion that naive (or virgin) and memory (i.e. previously activated) T cells can be distinguished by their high or low expression of different antigen specificities of the CD45 cell surface glycoprotein complex or CD29, the [31 chain of the integrin family [6]. The transition from naive to memory T cell function is accompanied by a shift from the 205-/220-kDa determinant (recognized by 2H4) to the 180-kDa form (recognized by UCHL1) of the CD45 cell surface glycoprotein complex, generated by cell type-specific alternative splicing from a c o m m o n precursor gene [4, 7]. Additional markers have been described which are coexpressed with CD45R or CD29 after the transition from naive to memory T cells [8]. Naive T cells can be characterized as CD45RAhig h, LFA-3 ~ow, CD2 low, LFA-1 low, CD45RO l~ and CD291~ memory T cells are the reciprocal subset. Lamina propria T cells most likely originate from lymphocytes in the organized lymphoid follicles of the mucosa. Here they first

200

Zeitz/Schie ferdecker/Ullrich/Jahn/James/Riecken

Table 1. Phenotypic characteristics of peripheral blood and intestinal lamina propria lymphocytes

ct/f~TCR (WT 31) 7/6 TCR (TCRgl) CD4 (Leu-3) CD8 (Leu-2) CD25 (anti-Tac) Leu-8 CD45RA (2H4) CD45RO (UCHL-I) CD29 (4B4) HML-1

Peripheral blood

Lamina propria

%

range

%

range

97 2 45 26 3 76 33 41 60 2

88-98 2-10 30-55 18-33 0-24 61-84 19-53 33-54 39-79 0-2

95 3 53 35 18 2 10 93 47 38

91-99 1-5 20-90 t4-38 7-33 0-4 2-16 62-99 37-53 9-54

Lamina propria lymphocytes were isolated from human small and large intestine by an enzymatic procedure. Peripheral blood and lamina propria lymphocytes were analyzed by flow cytometry with the monoclonal antibodies indicated. Values are given as median and range [taken from ref. 11 and 9].

encounter antigen, thereupon enter the circulation, and h o m e back to the mucosa. The question arises whether T cells in the effector c o m p a r t m e n t of the mucosa are similar to m e m o r y T cells in other compartments of the i m m u n e system. This chapter will focus on phenotypic and functional characteristics of intestinal lamina propria T cells which indicate that these cells are a specialized subset of m e m o r y T cells.

Phenotypic Characteristics of Lamina Propria T Cells More than 95 % of intestinal lamina propria T cells bear the a/13 isotype of the antigen-specific T cell receptor [9], CD4 + and

CD8 + T cells are present in the lamina propria in a similar proportion c o m p a r e d to the peripheral blood (table 1) [ 10-12]. Thus, regarding the major T cell subpopulations, peripheral blood T cells and lamina propria T cells do not appear to be different. An initial finding indicating maturational differences between circulating and intestinal T cells was the demonstration o f a low expression of the Leu-8 antigen on h u m a n and n o n h u m a n primate lamina propria T cells [12, 13]. As discussed elsewhere, Leu-8 is the h u m a n equivalent of the MEL-14 protein, the mouse homing receptor for peripheral lymph nodes [14] and this finding might explain the lack of Leu-8 expression in the intestinal lamina propria. In additional studies, using cytofluorometry, it was shown that only 10% of lamina propria lymphocytes express C D 4 5 R A (recognized by the monoclonal antibody 2H4) in comparison to 33% CD45RA + cells in the peripheral blood (table 1) [11]. When isolated h u m a n lamina propria lymphocytes were stained with monoclonal antibody U C H L - I , it was found that approximately 90% of this lymphocyte population is C D 4 5 R O + (table I) [11]. This phenotype resembles that of m e m o r y T cells. However, expression of another marker of m e m o r y T cells, CD29 ([31 integrin) is not increased in the lamina propria compared to peripheral blood (table 1). The phenotype of lamina propria lymphocytes therefore only partially corresponds with that of m e m o r y T cells. These phenotypic studies are a first indication that T cells in the intestinal lamina propria represent a specialized m e m o r y T cell subset. Recently, monoclonal antibodies have been developed for both rats ( R G L I [15]) and humans ( H M L - t [16] and Bet ACT8

Phenotype and Function of Lamina Propria T Ls,mphocytes

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[17]) which recognize lymphocyte surface antigens nearly exclusively expressed in the mucosal immune system. By immunohistology and flow cytometry it was shown that approximately 40% of lamina propria lymphocytes and nearly all intraepithelial lymphocytes express the surface antigen recognized by HML- 1 [9, 11, 16]. Dual-color cytofluorometry showed that HML-1 is preferentially expressed on CD8 + lamina propria T cells; however, a significant proportion of CD4 + lamina propria T cells also expresses HML-1 (fig. 1). As discussed elsewhere in this volume, the expression of HML-1 is induced on HML-1- peripheral blood lymphocytes by in vitro activation using various stimuli [11]. HML-1 therefore seems to be an activation marker which is expressed in vivo only in the environment of the intestine. The function of the antigen recognized

101

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by HML-I is unknown so far: there are indications that this antigen defines a subset of memory T cells in the intestinal immune system [see contribution by Schieferdecker et al. in this volume]. In a recent study the differential expression of homing-associated adhesion molecules on human peripheral blood T cells was investigated [ 18]. It was shown that the small proportion of circulating human T cells expressing the mucosal lymphocyte-associated antigen recognized by the monoclonal antibody Ber ACT8 (identifying the same antigen as H M L - I ) also has the CD29 ]~ phenotype. These results are in agreement with our findings of an intermediate expression of CD29 on lamina propria T cells and support the hypothesis of a tissue-specific modulation of memory or homing-associated T cell surface antigens.

202

Activation of Lamina Propria T Cells T cell activation is followed by an early expression of the interleukin-2 (IL-2) receptor on the cell surface, and the interaction of the IL-2 receptor with IL-2 is a critical event in T cell proliferation, differentiation, and function [19]. To address the question whether lamina propria T cells differ in their state of activation from T cells in other sites of the immune system, cytofluorometric and Northern blot analyses were performed in nonhuman primates to study the expression of the gene for the IL-2 receptor a chain (CD25) [20]. mRNA for the IL-2 receptor a chain was clearly detectable in freshly isolated lymphocytes from the intestinal lamina propria, whereas in the other populations from the spleen, mesenteric lymph nodes, or the peripheral blood IL-2 receptor mRNA was only found after activation in vitro. Correspondingly, using flow cytometry, it was shown that 15% (range: 6-29%) of freshly isolated intestinal lamina propria lymphocytes were CD25+, but less than 3% of the other lymphocyte populations. The increased CD25 expression of lamina propria lymphocytes was correlated with a high proliferative response to low doses of IL-2 indicating that the IL-2 receptors are able to transduce the signal after IL-2 binding. In agreement with an increased state of activation of lamina propria T cells, it was demonstrated that these cells are able to synthesize high amounts of IL-2 [20]. Lamina propria lymphocytes also express major histocompatibility complex (MHC) class II antigens [20] and other T cell activation markers [21 ]. Expression of CD25 in the lamina propria in humans has been confirmed by immunohistology of frozen tissue sections [22] and flow cytometry [ 11 ]. The finding that the mucosal

Zeitz/Schieferdecker/Ullrich/Jahn/James/Riecken

T cell-associated antigen recognized by HML-1 is an activation antigen further supports the concept of an increased activation of lamina propria T cells.

T Cell Receptor-Triggered Function of Lamina Propria Lymphocytes The phenotypic findings discussed here indicate that the effector compartment of the mucosa comprises a subset of activated T cells which shows partially overlapping characteristis with memory T cells. Memory T cells differ from naive T cells in their response to antigens and in the profile of cytokines secreted by these cells: memory T cells are capable of mounting a high proliferative response to recall antigen while naive cells have a low proliferative response to triggering the T cell receptor by antigen [6]. Limited data are available on the T cell receptor-triggered function of lamina propria lymphocytes. In a recent study addressing this question, nonhuman primates were immunized by rectal intramucosal injection of Chlamydia trachomatis and lymphocytes from various origins were isolated and tested for their responsiveness to stimulation with specific antigen in vitro [23]. Whereas peripheral blood lymphocytes, lymphocytes from the spleen, and mesenteric lymph node lymphocytes had a clear proliferative response to C. trachomatis antigens, lymphocytes isolated from the intestinal lamina propria, the site of immunization, did not proliferate to specific antigen in vitro. This lack of proliferation was not due to a defect of antigen presentation to lamina propria T cells, since antigen-pulsed peripheral monocytes did not restore the proliferative response, and it was not due to the presence

Phenotype and Function of Lamina Propria T Lymphocytes

203

Helper Function for Immune B Cells

Proliferation to Specific Antigen

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Fig. 2. Antigen-specific proliferation of intestinal lamina propria lymphocytes (LPL) and antigen-induced production of helper factors by lamina propria T cells. Lymphocytes from the peripheral blood (PBL), the spleen, mesenteric lymph nodes (MLN), or the intestinal lamina propria (LPL) were isolated from nonhuman primates infected with C. trachomatis. Cell populations were stimulated with chlamydial antigens in vitro, and proliferation was measured by 3H-thymidine incorporation (zxcpm, left panel).

Spleen B cells from infected animals were cocultured with T cells from uninfected animals (control) or infected animals (LGV-inf.) and cultures were stimulated either with pokeweed mitogen (PWM) or with chlamydial antigens (LGV), and IgG production was measured in culture supernatants (right panel). Lamina propria T cells from immune animals provide help for Ig synthesis in the presence of chlamydial antigens, although they do not proliferate [from ref. 3 with permission].

of CD8 + suppressor T cells, since removal of CD8 + T cells did not result in a proliferative response. Similarly, rectal immunization with other protein antigens did not result in detectable proliferative responses of isolated lamina propria T cells to these antigens. This surprising result was not, however, due to the absence of antigen-reactive T cells in the intestinal lamina propria. Lamina propria T lymphocytes could respond to recall antigen: these T cells provided helper function for immunoglobulin synthesis by spleen B cells after antigenic stimulation with C. trachomatis antigens (fig. 2). These findings show

that lamina propria T cells do not proliferate after stimulation with recall antigen but manifest a T cell effector function, i.e. helper activity, in response to antigen. In contrast, 'classical' m e m o r y T cells have a high proliferative response to recall antigen [6]. In consideration of these findings it can be hypothesized that lamina propria T cells are differentiated effector cells, which respond to antigen stimulation with the production of certain cytokines. This concept is also supported by studies using immunhistology to demonstrate proliferating cells in the intestinal lamina propria: No Ki67 § cells (i.e. pro-

204

Zeitz/Schieferdecker/Ullrich/Jahn/James/Riecken

Table 2. Comparison of surface antigen expression and proliferative responses in naive, memory,

and lamina propria T cells Naive T cells

Memory T cells

Lamina propria

T cells

Surface antigens CD45RA CD45RO CD29

specific T cell receptor but supports the view of differences in signal transduction in lamina propria T cells.

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liferating cells) were present in frozen sections of biopsies of human intestine in the lamina propria [22]. An indication of a different responsiveness of the antigen-specific T cell receptor is also given in recent studies which showed a decreased responsiveness of lamina propria T cells to stimulation with immobilized antibodies to CD3 and an undisturbed responsiveness to mitogenic anti-CD2 monoclonal antibodies [24]. In related studies an impairment of calcium mobilization and phosphatidylinositol response through the T cell receptor/CD3 complex was shown, and it has been speculated that the T cell receptor activation pathway may be down-regulated in the lamina propria [see contribution by Quiao et al. in this volume]. However, the finding of an antigen-induced helper function of lamina propria T cells clearly demonstrates responsiveness through the antigen-

Summary and Conclusions Lamina propria T cells have a low expression of the CD45RA antigen and a high expression of the CD45RO antigen. This phenotype is characteristic for memory T cells (table 2). In addition, T cells in the effector compartment of the mucosa bear surface antigens which are very rarely found in other sites of the immune system. Intestinal T cells also express functional IL-2 receptors and IL-2 receptor a chain mRNA, and are able to synthesize high amounts of IL-2. However, another marker of memory T cells, CD29, is not expressed in high density in the lamina propria indicating that lamina propria T cells differ from 'classical' memory T ceils. This is supported by functional studies in nonhuman primates infected rectally with C. trachomatis which show that lamina propria T cells do not proliferate after stimulation with antigen but rather provide helper function for immunoglobulin synthesis (table 2). The intestinal lamina propria therefore contains highly specialized T cells which have a distinctive phenotype and are activated. Functionally these T cells can be characterized as differentiated effector lymphocytes which respond to triggering the antigen-specific T cell receptor by secreting helper factors for B cells. This concept is supported by recent studies showing that the pattern of lymphokines produced by lamina propria T cells and the responsiveness to certain lymphokines differ from those of other lymphocyte populations [25]. Lamina pro-

Phenotype and Function of Lamina Propria T Lymphocytes

pria T cells thus represent a subset of memory T ceils with a unique maturational state.

References 1 Strober W, Jacobs D: Cellular differentiation, migration, and function in the mucosal immune system; in Gallin JI, Fauci AS (eds): Advances in Host Defense Mechanisms. New York, Raven Press, 1985, vol 4, pp 1-30. 2 James SP, Zeitz M, Kanof ME, Kwan WC: Intestinal lymphocyte populations and mechanisms of cell-mediated immunity; in: Kagnoff MF (ed): Immunology and Allergy Clinics of North America, vol 8, No 3: Gut and Intestinal Immunology. Philadelphia, Saunders, 1988, pp 369-391. 3 Zeitz M, Schieferdecker HL, James SP, Riecken EO: 'Special functional features of T-lymphocyte subpopulations in the effector compartment of the intestinal mucosa and their relation to mucosal transformation. Digestion 1990;46(suppl 2):280289. 4 Akbar AN, Terr L, Timms A, Beverley PCL, Janossy G: Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells. J Immunol 1988;140:2171-2178. 5 Clement LT, Yamashita N, Martin AM: The functionally distinct subpopulations of human CD4 + helper/inducer T lymphocytes defined by antiCD45R antibodies derive sequentially from a differentiation pathway that is regulated by activation - dependent post-thymic differentiation. J Immunot 1988;141:1464-1470. 6 Sanders ME, Malegapuru W, Makgoba MW, Shaw S: Human naive and memory T ceils: Reinterpretation of helper-inducer and suppressor-inducer subsets. Immunol Today 1988;9:195-199. 7 Streuli M, Hall LR, Saga Y, Schlossman SF, Saito H: Differential usage of three exons generate at least five different mRNAs encoding human leucoyte common antigens. J Exp Med 1987;166: 1548-1566. 8 Sanders ME, Makgoba MW, Sharrow SO, Stephany D, Springer TA: Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA-3, CD2, and LFA-I) and three other molecules (UCHL-1, CDw29, and

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Pgp-1) and have enhanced IFN- 7 production. J Immunol 1988; 140:140 !-1407. 9 Ullrich R, Schieferdecker HL, Ziegler K, Riecken EO, Zeitz M: 75 T cells in the human intestine express surface markers of activation and are preferentially located in the epithelium. Cell lmmunol 1990;128:619-627. 10 Selby WS, Janossy G, Bofill M, Jewell DP: Lymphocyte subpopulations in the human small intestine. The findings in normal mucosa and in the mucosa of patients with adult coeliac disease. Clin Exp Immunol 1983;52:219-228. 11 Schieferdecker HL, Ullrich R, Weiss-Breckwoldt AN, Schwarting R, Stein H, Riecken EO, Zeitz M: The HML-I antigen of intestinal lymphocytes is an activation antigen. J Immunol 1990; 144:25412549. 12 James SP, Fiocchi C, Graeff AS, Strober W: Phenotypic analysis of lamina propria lymphocytes. Predominance of helper-inducer and cytolytic T-cell phenotypes in Crohn's disease and control patients. Gastroenterology 1986;91: 1483-1489. I3 James SP, Graeff AS, Zeitz M: Predominance of helper-inducer T cells in mesenteric lymph nodes and intestinal lamina propria of normal nonhuman primates. Cell Immunol t 987; 107:372-383. 14 Camerini D, James SP, Stamenkovic I, Seed B: Leu-8/TQ1 is the human equivalent of the Mel-14 lymph node homing receptor. Nature 1989;342: 78-82. 15 Cerf-Bensussan N, Guy-Grand D, Lisowska-Grospierre B, Griscelli C, Bhan AK: A monoclonal antibody specific for rat intestinal lymphocytes. J Immunol 1986; 136:76-82. 16 Cerf-Bensussan N, Jarry A, Brousse N, LisowskaGrospierre B, Guy-Grand D. Griscelli C: A monoclonaI antibody (HML-I) defining a novel membrane molecule present on human intestinal lymphocytes. Eur J Immunol 1987; 17:1279-1285. 17 Kruschwitz MG, Fritzsche G, Schwarting R, Micklem K, Mason DY, Falini B, Stein H: Ber ACT8: A new monoclonal antibody defining a trimeric antigen associated with activated CD8 T cells, gut mucosa T cells, and hair leukemia cells. Submitted for publication. 18 Picker LJ, Terstappen LWMM, Rott LS, Streeter PR, Stein H, Butcher EC: Differential expression of homing-associated adhesion molecules by T cells subsets in man. J Immunol 1990;L45:32473255.

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19 Greene WC, Leonard WJ, Depper JM: Growth of human T lymphocytes: An anaysis of interleukin 2 and its cellular receptor. Prog Hematol 1986;14: 283-301. 20 Zeitz M, Greene WC, Pfeffer N J, James SP: Lymphocytes isolated from the intestinal lamina propria of normal nonhuman primates have increased expression of genes associated with T-cell activation. Gastroenterology 1988;94:647-655. 21 Peters M, Secrist H, Anders KR, Nash GS, Schloemann S, MacDermott RP: Increased expression of cell surface activation markers by intestinal mononuclear cells. Clin Res 1986;35:462. 22 Ullrich R, Zeitz M, Heise W, L'Age M, Ziegler K, Bergs C, Riecken EO: Mucosal atrophy is associated with loss of activated T cells in the duodenal mucosa of human immunodeficiency virus (HtV)infected patients. Digestion 1990;46(suppl 2): 302-307. 23 Zeitz M, Quinn TC, GraeffAS, James SP: Mucosal T cells provide helper function but do not proliferate when stimulated by specific antigen in lymphogranuloma venerum proctitis in nonhuman primates. Gastroenterology 1988;94:353366.

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24 Pirzer UC, Schiirmann G, Post S, Betzler M, Meuer SC: Differential responsiveness to CD3-Ti vs. CD2-dependent activation of human intestinal T lymphocytes. Eur J Immunol 1990;20:23392342. 25 James SP, Kwan WC, Sneller MC: T celIs in inductive and effector compartments of the intestinal immune system of nonhuman primates differ in lymphokine mRNA expression, lymphokine utilization, and regulatory function. J Immunol 1990;144:1251-1256.

Dr. Martin Zeitz Medical Clinic Klinikum Steglitz Hindenburgdamm 30 D-W-1000 Berlin 45 (FRG)