Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 3, 2001, pp. 273-276

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 273-276

Phenotypic and Functional Analysis of Porcine T Lymphocytes

LI Hua , CHEN Yinghua

Laboratory of Immunology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China

Received: 2000-11-07

Code Number: ts01081

Abstract:   

Porcine and other higher mammals express clusters of differentiation (CD) antigens on the surface of T lymphocytes, such as CD2, CD3, CD4, CD8, etc. However, in porcine, a high percentage of the CD4⁺CD8⁺ T lymphocyte subpopulation exist in the peripheral blood and the ratio of the CD4⁺ and CD8⁺ T lymphocyte subpopulations is reversed. These differences bring new challenges to better understanding of the phenotype and function of porcine T lymphocytes in antigen recognition and immune response.

Key  words:  swine; T lymphocytes; phenotype; function

Introduction   

Porcine is a useful model for the heterogeneity of mammalian immune systems and has also received attention as a possible source of organs for human transplants[1]. T lymphocytes are an important component of immune cells which directly induce cellular immune response and play a key role in immune regulation. The cognition, activation, and function of T lymphocytes depend on interactions between T cells and antigen presenting cells, as well as B lymphocytes and other cells. The bases for the interaction are membrane molecules on the immune cells which include different cell surface molecules, especially clusters of differentiation (CD) antigens, for example CD2, CD3, CD4, CD8, etc. on porcine T lymphocytes[2]. These molecules play an important role in T cell antigen recognition and exercise important biological functions. Here, we analyze some differentiation antigens expressed on porcine T lymphocytes to study the possible biological functions of T lymphocyte subpopulations.

1 CD2 and CD3 Expression  

CD2 antigen, a key surface marker of porcine T lymphocytes, is a 48x10³polypeptide which has been identified as the sheep red blood cells (SRBC) receptor. The monoclonal antibody to CD2 can inhibit SRBC-receptor-based rosette formation. CD2 also triggers T cells[3]. Unlike other species, only some porcine T lymphocytes express CD2 molecules[4]. TCR gd⁺ T cells are CD2-negative and nearly all thymocytes are CD2⁺ cells.

The CD3-complex is composed of five polypeptide chains g, d, e, x, and h, which provide the coupling between the antigen specific signal delivered by the T-cell receptor and the intracellular activation pathways[5]. The porcine CD3e chain was cloned, sequenced and transiently expressed in COS cells in 1996[6]. The extracellular portion of CD3e was found to be 72% homologous to sheep and 65% to human and mouse CD3e, while the intracellular and transmembrane portions were nearly 100% conserved[5]. The predicted sequence was used to generate immunizing peptides, with several mAbs which recognize CD3e used to study the distribution of subpopulations in different tissues[7, 8].

2 CD4 and CD8 Expression as well as T Lymphocyte Subpopulations

CD4 and CD8 are co-receptors on T lymphocytes involved in mediating signal transduction. These glycoproteins also strengthen the interaction between the T lymphocytes and other cells by binding to non-polymorphic regions of class I and II major histocompatibility complex molecules, respectively[9]. The porcine CD4 antigen, which is similar to the CD4 antigen in humans and rodents, has been well characterized as a 55x10³polypeptide that interacts with class II major histocompatibility complex (MHC) molecules on antigen presenting cells. It is expressed on about 50% of thymocytes and a substantial subset of peripheral blood T lymphocytes[10]. The 30x10³-35x10³ CD8 dimeric protein exists as both an aa homodimer and an ab heterodimer. The ab heterodimer is apparently more efficiently expressed than the aa homodimer. As in other species, the CD8 molecule is present on the surface of porcine T cells at both high (CD8⁺high ) and low (CD8⁺low ) densities[11]. Whether this variability in pig is due to the differential expressions of aa homodimer and ab heterodimer is unknown.

In thymus, CD4 and CD8 show an expression pattern comparable to that in other mammals[11]. Besides  CD4^-CD8^-  thymic progenitors and two fractions with the phenotype of more mature thymocytes having either  CD4^-CD8⁺  or CD4⁺CD8^-, the majority of the porcine thymocytes belong to a population which has been defined in other species as common thymocytes with the phenotype CD4⁺CD8⁺[12]. In the extrathymic T lymphocyte, the expression patterns of CD4 and CD8 are completely different from those known in other species[13]. Besides  CD4⁺CD8^-  T lymphocytes with the phenotype of MHC II-restricted T-helper cells and  CD4^-CD8⁺  T lymphocytes showing the phenotype of MHC I-restricted cytolytic T lymphocytes, a CD4⁺CD8⁺ double positive extrathymic T lymphocytes subpopulation exists, which are unique to the porcine immune system[14].

According to their immunological functions, the porcine  CD4^-CD8⁺  T-lymphocyte subpopulation contains two main subsets of lymphocytes, natural killer (NK) cells with spontaneous cytolytic activity against xenogeneic tumor cells and progenitors of MHC class I-restricted cytolytic T lymphocytes[13]. The CD4^-CD8⁺ T-lymphocyte subpopulation is characterized by a heterogeneous CD8 antigen expression with very low to high CD8 antigen density[13, 14]. With regard to other differentiation antigens, e.g., the T-cell specific CD6 antigen, the CD8⁺ subpopulation can be divided into two subsets[15]. CD4^-CD8⁺low  cells are negative for CD6, whereas CD8⁺high  cells show co-expression of CD6 molecules. These features explain differences in the functional behavior of these subsets: CD3^-CD5^-CD6^-CD8⁺low  cells show spontaneous non-MHC-restricted cytolytic activity against tumor cells; while CD3⁺CD5⁺CD6⁺CD8⁺high  T cells contain the MHC I restricted cytolytic T lymphocyte (CTL) subset. The cytolytic activity of CTL can be blocked by the addition of mAb directed against MHC class-I molecules and/or CD8a or CD8b epitopes[11] . This confirms the functional difference of the CD8⁺high  and CD8⁺low  cell subpopulations.

In contrast to the CD4^- T lymphocytes, all porcine CD4 positive T lymphocytes co-express CD2, CD3, CD5, and CD6 antigens. It seems that all CD4⁺ cells belong to the TCR-ab T lymphocyte subset[16, 17]. Functionally, both CD4⁺ T lymphocyte subpopulations are able to respond to alloantigen in mixed leukocyte cultures and show T-helper cell function for the generation of alloantigen-specific cytolytic T lymphocytes[18]. However, porcine CD4⁺ T lymphocytes can be discriminated by their CD8 expression into two subpopulations, the CD4⁺CD8^- cells with the phenotype of classical T-helper cells and the CD4⁺CD8⁺ extrathymic T lymphocytes which are unique to the porcine immune system.  CD4⁺CD8⁺  extrathymic T lymphocytes indeed show the phenotype of CD4⁺CD8⁺ common thymocytes but morphologically and phenotypically differ from thymocytes. With regard to the expression of other differentiation antigens  CD4⁺CD8⁺  extrathymic T lymphocytes, in contrast to CD4⁺CD8⁺ thymocytes, show no expression of the thymocytes-specific CD1 antigen[19]. Furthermore, the extrathymic CD4⁺CD8⁺ T lymphocytes have the morphological phenotype of mature and resting T lymphocytes as shown in histology and electron microscopy[10]. CD4⁺CD8⁺ T lymphocytes as well as  CD4⁺CD8^-  T lymphocytes respond to alloantigen in a primary in vitro immune response. However, in contrast to the CD4⁺CD8^- cells, only the CD4⁺CD8⁺ cells are capable of reacting with an antigen-specific secondary immune response. This indicates that the CD4⁺CD8⁺ T lymphocyte subpopulation contains the porcine memory helper T cell fraction[19, 20]  . The majority of CD4⁺CD8⁺ cells also express high levels of CD29 (CD29⁺high ), which is characteristic of human and porcine memory cells. The absolute number of memory cells in PBL increases gradually with age as confirmed by following flow cytometric analyses which show that CD29⁺high  proliferated when stimulation with recall viral antigen while CD29⁺low  did not[21, 22]. Pescotitz postulated that a CD8 molecule exists on CD4⁺CD8⁺ T cells which might stimulate to respond to low antigen density in swine which does not occur in other species.

3 CD5 and CD6 Expression  

The porcine CD5 molecule is characterized as a monomeric glycoprotein with relative molecular mass of 63x10³under reducing conditions. Analyses of the expression of porcine CD5 molecule revealed that the majority of CD5⁺ cells belonged to the T-lymphocyte lineage while  10%-30%  of B-lymphocytes expressed CD5⁺ with low antigen density[23]. The porcine CD6 is a monomeric molecule with relative molecular mass of 11 000-12 000 under reducing and non-reducing conditions. It shows high specificity for T lymphocytes and is expressed neither on B-lymphocytes nor on cells of the myeloid lineage[24].

CD5 is displayed on nearly all thymocytes with different antigen densities on thymic subpopulations in accordance with data from other species. Suspensions of thymocytes show a biphasic peak containing two subsets of CD5 positive cells characterized by a heterogeneous CD5 antigen density, 13%-32% of thymocytes showed high CD5 expression (CD5⁺high ) and 64%-86% showed low CD5 expression (CD5⁺low )[23] . Flow cytometric analyses showed that CD5⁺low  thymocytes could be characterized as more immature thymocytes, whereas the CD5⁺high  cells belonged to more mature thymic phenotypes, so the CD5 antigen is up-regulated during thymocyte development[13]. Three-color FCM analyses of CD5 expression on peripheral blood T-lymphocytes (PBTL) revealed that CD5⁺high  T lymphocytes (55%) represented the CD4⁺CD8^- T cells, the CD4⁺CD8⁺ T lymphocytes, and the CD4^-CD8⁺high  T lymphocytes. CD5⁺low  T lymphocytes (10%) mostly belong to the CD4^-CD8^- T lymphocyte subpopulation, which have been characterized as CD2^- null cells. CD5^- T lymphocytes (35%) include some CD4^-CD8^- T lymphocytes and CD4^-CD8⁺low  T lymphocytes. Functional analyses showed that the porcine CD5 antigen is an important marker to discriminate between CD5^-CD8⁺ natural killer (NK) cells and CD5⁺CD8⁺ progenitors of MHC-restricted cytolytic T lymphocytes.

The expression of CD6 on porcine thymocytes could be characterized as homogeneous and weak. The majority of porcine thymocytes are CD6⁺, while only a small percentage of thymocytes is negative for CD6[15]. Studies of CD6 expression suggest that the CD6 expression on porcine thymocytes is up-regulated during thyocyte development. Three-color FCM analyses of the CD6 expression on extrathymic-T lymphocytes in combination with antibodies against porcine CD4 and CD8 antigens revealed a clear separation of the CD6⁺ and CD6^- T lymphocyte fractions in the CD4/CD8 defined T lymphocyte subpopulations. All CD4⁺ T lymphocytes co-expressed the CD6 antigen. CD4^-CD8⁺ cytolytic T lymphocytes were separated by their CD6 expression into two fractions: CD4^-CD8⁺high  T lymphocytes showing co-expression of CD6 antigen and CD4^-CD8⁺low  cells without CD6 expression. Nearly all CD4^-CD8^- T lymphocytes were negative for CD6[14]. Functional studies with separated CD6 fractions reveal that the CD6^- cells can be characterized by spontaneous and non-MHC restricted cytolytic activity, whereas the CD6⁺ T lymphocytes are responsible for MHC-restricted T-cell functions.

4 Conclusions  

Further research on the porcine immune system has clarified the phenotypes and functions of various T lymphocyte subpopulations. Porcine has the unique characteristic that a high percentage of the CD4⁺CD8⁺ T lymphocyte subpopulation is in the peripheral blood, the ratio of CD4⁺ and CD8⁺ T lymphocyte subpopulations is reversed, the two subpopulations of CD8⁺ T cells include NK cells and MHC restricted cytotoxic cells and the tissue distribution of CD6⁺ lymphocytes is different, etc. Thus, future research will facilitate the improvement of immune modulating drugs and vaccines on the molecular level, which will result in better quality products derived from animals and a better standard of living for humans.

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