Genetic modulation of immature T lymphocytes and its application

Genetic modulation of immature T lymphocytes and its application

Yousuke Takahama

Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, Japan

Abstract: T lymphocytes are the cells that play an essential role in regulating immune responses. The thymus is the organ in which T lymphocytes are generated. Our laboratory has investigated molecular signals that determine cell fate during T lymphocyte development in the thymus. To this goal, we have devised a technique in which one may efficiently introduce foreign genes into immature T lymphocytes. The somatic gene-transfer into developing T lymphocytes are likely useful to restore various immunodeficiencies and to establish immune tolerance to any introduced genes. The genetically engineered immune tolerance may be applied to reduce allergies and autoimmune diseases, as well as to sustain gene therapies by allowing prolonged survival of therapeutic vectors. J. Med. Invest. 48:25-30, 2001

Keywords:T lymphocyte, thymocyte, thymus, gene therapy, retrovirus, immune tolerance

INTRODUCTION
Most T lymphocytes develop in the thymus. The thymus is a small, isolated organ that can be manipulated in vivo and can be analyzed in vitro. Developmental biology of T lymphocytes investigates how the hematopoietic precursor cells are induced to develop to mature T lymphocytes in the thymus. Specifically, interesting questions to be asked include issues such as (i) how immature T lymphocytes are selected for life and death according to their antigen-recognition specificity, (ii) how multipotent precursor cells are committed to become individual T lymphocyte lineages, and (iii) how developing T lymphocytes relocate along the differentiation into, within and out of the thymus.
In the thymus, immature lymphoid precursor cells begin to rearrange the V (D) J segments of T-cell antigen-receptor (TCR) genes. The V (D) J rearrangement is an irreversible alteration of genomic DNA in the nucleus, so that individual lymphocytes cannot predict the antigen-recognition specificity before they express TCR chains on the cell surface (1, 2). Consequently, immature T lymphocytes in the thymus, also called thymocytes, may express various kinds of antigen-recognition specificity, including harmful specificity, useful specificity, and useless specificity. The developmental fate of immature thymocytes is determined by the interaction between the TCR that they express and its ligand, peptide-MHC complex, expressed in the thymus (3, 4). High avidity TCR interactions cause apoptosis of thymocytes, deleting harmful self-reactive cells, a process referred to as negative selection. Thymocytes expressing TCR that fail to interact with MHC ligands are also destined to die within the thymus, eliminating useless T cells that are unable to recognize foreign peptide-loaded self-MHC molecules. Low avidity TCR interactions, on the other hand, elicit the signal that allows immature thymocytes to differentiate into mature T lymphocytes, a developmental process referred to as positive selection. Thus, positive selection of thymocytes enriches useful T cell clones that can recognize foreign peptides presented by self-MHC molecules. It has been shown that cell surface events such as avidity and valency of TCR ligation by peptide-MHC complex are involved in determining the opposite destinations (life and death) of immature thymocytes (5-8). However, it is still largely unknown how these cell-surface events are transmitted within thymocytes to determine life-death destinations of the cells.
Majority of positively selected thymocytes are induced to develop into either one of CD4+CD8- T cells (mostly helper T cells) or CD4-CD8+ T cells (mostly killer T cells). Even though TCR signal intensity as well as other signals such as Notch have been shown to be involved in lineage choice between CD4+CD8- T cells and CD4-CD8+ T cells (9-11), it is largely unclear how the choice between the T cell lineages is made. It is also unclear how multipotent precursor cells that have migrated to the thymus are destined to become T lymphocytes rather than other cell lineages such as B lymphocytes and other hematopoietic cells.
Developing T lymphocytes relocate through the thymus during differentiation. Immature lymphoid precursor cells immigrate into the thymus, developing thymocytes relocate within the thymus from the cortex to the medulla, and finally mature T lymphocytes emigrate from the thymus (12-14). How the cellular movement along T lymphocyte development is controlled is largely unknown.
Exploring these issues of T cell development would be relevant for better understanding of complex biological systems specialized in multi-cellular organisms, as well as for offering better treatment of various clinical situations in which immune systems are involved. To examine these issues and to aid clinical applications, our laboratory has devised a somatic cell-gene transfer technique in which one can express a given gene in developing T lymphocytes.

GENE-TRANSFER INTO DEVELOPING T LYMPHOCYTES
To genetically modulate developing T lymphocytes, the manipulation of embryonic cells such as transgenesis and ES-mediated homologous recombination has been most widely used (15-18). These techniques have greatly contributed to the current understanding of molecular mechanism for T lymphocyte development. However, genetic manipulation of embryonic cells alone cannot directly allow in situ analysis of the cellular development within the thymus organ.
On the other hand, the analysis of T cell development in organ culture of mouse fetal thymus lobes was first established in the early seventies (19-21). The fetal thymus organ culture (FTOC) technique serves a unique in vitro cell culture system in that functional T cells are differentiated from immature progenitor cells (22). T cell development in FTOC very well represents T cell development during fetal life, even representing the time course. FTOC allows in vitro T cell development isolated from any further cellular or humoral supplies by other organs ; thus, it is suitable for the addition of any reagents, such as drugs and antibodies to the culture, for examining their effects on T cell development. FTOC is also useful for the analysis of T-lymphopoietic capability by hematopoietic progenitor cells.
Recently, retroviral gene transfer has been successfully used for a wide variety of cells including hematopoietic cells (23-28) and developing B lymphocytes (29, 30). Retrovirus-mediated gene transfer has several advantages over the transgenic techniques, including rapid and close analysis of specific cellular events in vitro and potential application for gene therapy. However, retrovirus-mediated gene transfer often suffers from technical difficulties such as low efficiency, hampering applications in various cell types. Consequently, attempts to introduce exogenous genes using retroviruses have gained limited success on developing T lymphocytes (31-37).
During the last three years, a successful and reproducible gene-transfer technique for developing T lymphocytes has been established in our laboratory (22, 38, 39). The short-term co-culture of immature thymocytes in suspension with high-titer retrovirus-producing cells in the presence of interleukin-7, a cytokine that maintain the survival of immature lymphocytes, seems to be the key for highly efficient gene transduction (Fig. 1). We have constructed recombinant retroviruses expressing green fluorescence protein (GFP) along with a protein of interest, using the internal ribosomal entry site (IRES) sequence (Fig. 1). The co-expression of GFP is useful, as gene-transferred cells could be readily detected and sorted using flow cytometry. Immature thymocytes have been successfully infected with these retroviruses in a short-term suspension culture in the presence of interleukin-7, and were examined for their developmental capability by transferring to the thymus organ culture. The fate of isolated GFP-expressing cells can be traced under the fluorescence microscope or by multi-color flow cytometric analysis, so that the developmental potential of genetically manipulated cells may be directly evaluated in FTOC (Fig. 1).
Using the retroviral gene-transfer method, we have shown that ERK kinase and p38 kinase characterize TCR signals that mediate positive and negative selection of developing thymocytes, respectively (40). Quantitative differences in TCR ligation at cell surface have been shown to determine the fate of immature thymocytes during positive and negative selection (5-8). Our results showing differential involvement of ERK and p38-kinase signaling cascade in positive and negative selection suggested that differential TCR ligation in developing thymocytes may activate differential MAP kinase pathways, leading to opposite destinations of immature thymocytes. We have also shown that Pref-1 expressed on the surface of thymic epithelial cells increase the expression of HES-1 transcription factor in developing thymocytes, thereby maintaining the survival of thymocytes (41). Thus, the retroviral technique that we devised is an efficient and reliable method for gene-transfer into developing T lymphocytes.

POTENTIAL APPLICATION OF GENE-TRANSFER IN IMMUNODEFICIENCIES
Gene-transfer for immature T lymphocytes is likely useful for genetic manipulation of T lymphocyte development. The most straightforward application would attempt to restore of immunodeficiencies by the transfer of genes that promote the development of T lymphocytes (Fig. 2). For example, immunodeficiency in ZAP-70 deficient patients could be best restored by transferring the ZAP-70 gene into immature thymocytes (42, 43). Such a restoration could be applied not only for congenital immunodeficiencies such as X-linked severe combined immune deficiency and selective T cell deficiency, but also for acquired immunodeficiencies such as AIDS and immune suppression by chemotherapy or after transplantation.

POTENTIAL APPLICATION OF GENE-TRANSFER IN GENE THERAPIES
The gene-transfer for immature T lymphocytes is also viewed as a very efficient vehicle to introduce gene products into the thymus and developing thymocytes. It is therefore conceivable that the gene-transfer into immature T lymphocytes could efficiently induce immune tolerance to the products of any foreign genes. The gene-specific induction of immune tolerance offers at least two types of clinical applications. First is the treatment of allergy, in which the immune system reacts to foreign molecules that are not expected to be immunogenic. The introduction of the allergen gene to immature thymocytes may cause tolerance to the allergen, so that allergic reactions would be expected to diminish after the gene-transfer. This line of genetic modulation of immune responses can also be applied to reduce auto-reactivity in various autoimmune diseases. Second is immunological support for gene therapies of any cell types (Fig. 3). It has been well appreciated that one of the most serious problems ingene therapy is the rejection of vector-introduced cells by exerting immune responses to vector-derived gene products (44-47). Such immune responses dramatically preclude the prolonged effectiveness of introduced genes. In addition, once the immune response to the introduced vector is established, immune cells would quite efficiently attack the cells that are newly introduced with the vector. It would thus be nearly impossible to expect efficient therapeutic effects by repeated treatment with the same vector during gene therapy.
On the other hand, immune responses are crucially regulated by T lymphocytes. T lymphocytes are the cells that immunologically distinguish self to be protected from non-self to be attacked. It is therefore conceivable that one can specifically repress immune responses to a foreign molecule by displaying that molecule in the thymus as a 'self' molecule. The vector transfer of the therapeutic gene into the thymus, for example by the intrathymic administration of immature thymocytes that have been gene-transferred with the same vector, would induce immune tolerance to the vector (48). Consequently, subsequent gene therapy is expected to be sustained without being disrupted by immune reactions (Fig. 3).
These applications of gene-transfer are currently being evaluated in our laboratory. We believe that the evaluation and further improvement of genetic modulation of the immune system offers a promie of overcoming immune diseases in the new century.

REFERENCES
1. Alt FW, Blackwell TK, DePinho RA, Reth MG, Yancopoulos GD : Regulation of genome rearrangement events during lymphocyte differentiation. Immunol Rev 89:5-30, 1986.
2. Cedar H, Bergman Y : Developmental regulation of immune system gene rearrangement. Curr Opin Immunol 11:64-69, 1999.
3. von Boehmer H : Positive selection of lymphocytes. Cell 76:219-228, 1994.
4. Marrack P, Kappler J:Positive selection of thymocytes bearing αβ T cell receptors. Curr Op Immunol 9:250-255, 1997.
5. Ashton-Rickardt PG, Bandeira A, Delaney JR, Van Kaer L, Pircher HP, Zinkernagel RM, Tonegawa S : Evidence for a differential avidity model of T cell selection in the thymus. Cell 76:651-663, 1994.
6. Hogquist KA, Jameson SC, Heath WR, Howard JL, Bevan MJ, Carbone FR : T cell receptor antagonist peptides induce positive selection. Cell 76:17-27, 1994.
7. Sebzda E, Wallace VA, Mayer J, Yeung RSM, Mak TW, Ohashi PS:Positive and negative thymocyte selection induced by different concentrations of a single peptide. Science 263:1615-1618, 1994.
8. Takahama Y, Suzuki H, Katz KS, Grusby MJ, Singer A : Positive selection of CD4+ T cells by TCR ligation without aggregation even in the absence of MHC. Nature 371:67-70, 1994.
9. Robey E, Fowlkes BJ : Selective events in T cell development. Annu Rev Immunol 12 : 675-705, 1994.
10. Robey E, Chang D, Itano A, Cado D, Alexander H, Lans D, Weinmaster G, Salmon P:An activated form of Notch influences the choice between CD4 and CD8 T cell lineages. Cell 87 : 483-492, 1996.
11. Yasutomo K, Doyle C, Miele L, Germain RN: The duration of antigen receptor signalling determines CD4+ versus CD8+ T-cell lineage fate. Nature 404:506-510, 2000.
12. Dunon D, Imhof BA:Mechanisms of thymus homing. Blood 81:1-8, 1993.
13. van Ewijk W : T-cell differentiation is influenced by thymic microenvironments. Annu Rev Immunol 9:591-615, 1991.
14. Scollay R, Godfrey DI:Thymic emigration: conveyor belts or lucky dips? Immunol Today 16:268-273, 1995.
15. Yeung RS, Penninger J, Mak TW : T-cell development and function in gene-knockout mice. Curr Opin Immunol 6:298-307, 1994.
16. Clevers HC, Grosschedl R : Transcriptional control of lymphoid development : lessons from gene targeting. Immunol Today 17:336-343, 1996.
17. Killeen N, Irving BA, Pippig S, Zingler K : Signaling checkpoints during the development of T lymphocytes. Curr Opin Immunol 10:360-367, 1998.
18. Basson MA, Zamoyska R:Insights into T-cell development from studies using transgenic and knockout mice. Methods Mol Biol 134:3-22, 2000.
19. Owen JJT, Ritter MA : Tissue interaction in the development of thymus lymphocytes. J Exp Med 129:431-442, 1969.
20. Mandel T, Russel PJ:Differentiation of foetal mouse thymus. ultrastructure of organ cultures and of subcapsular grafts. Immunology 21 : 659-674, 1971.
21. Owen JJT:Ontogeny of the immune system. Prog Immunol 2:163-173, 1974.
22. Takahama Y : Differentiation of mouse thymocytes in fetal thymus organ culture. Methods Mol Biol 134:37-46, 1999.
23. Williams DA, Lemischka IR, Nathan IR, Mulligan RC:Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse. Nature 310:476-480, 1984.
24. Dick JE, Magli MC, Huszar D, Phillips RA, Bernstein A. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice. Cell 42:71-79, 1985.
25. Keller G, Paige C, Gilboa E, Wagner, EF : Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors. Nature 318:149-154, 1985.
26. Daley GQ, Van Etten RA, Baltimore D : Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science 247:824-830, 1990.
27. Hawley RG, Fong AZC, Burns ZC, Hawley TS :Transplantable myeloproliferative disease induced in mice by an interleukin 6 retrovirus. J Exp Med 176:1149-1163, 1992.
28. Szilvassy SJ, Cory S : Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells. Blood 84:74-83, 1994.
29. Williams DE, Namen AE, Mochizuki DY, Overell RW : Clonal growth of murine pre-B colony-forming cells and their targeted infection by a retroviral vector : dependence on interleukin-7. Blood 75 :1132-1138, 1990.
30. Corcoran AE, Smart FM, Cowling RJ, Crompton T, Owen MJ, Venkitaraman, AR : The interleukin-7 receptor α chain transmits distinct signals for proliferation and differentiation during B lymphopoiesis. EMBO J 15:1924-1932, 1996.
31. Blaese RM, Culver KW, Miler AD, Carter CS, Fleisher T, Clerici M, Shearer GM, Chang L, Chiang Y, Tolstoshev P, Greenblatt JJ, Rosenberg SA, Klein H, Berger M, Mullen CA, Ramsey WJ, Muul L, Morgan RA, Anderson WF : T lymphocyte-directed gene therapy for ADA-SCID : initial trial results after 4 years. Science 270:475-480, 1995.
32. DeMatteo RP, Raper SE, Ahn M, Fisher KJ, Burke C, Radu A, Widera G, Claytor BR, Barker CF, Markmann JF : Gene transfer to the thymus. A means of abrogating the immune response to recombinant adenovirus. Ann Surg 222 : 229-239, 1995.
33. Plavec I, Voytovich A, Moss K, Webster D, Hanley MB, Escaich S, Ho KE, Bohnlein E, GiGiusto DL : Sustained retroviral gene marking and expression in lymphoid and myeloid cells derived from transduced hematopoietic progenitor cells. Gene Ther 3:717-724, 1996.
34. Gu J, Kuo ML, Rivera A, Sutkowski N, Ron Y, Dougherty JP : A murine model for genetic manipulation of the T cell compartment. Exp Hematol 24:1432-1440, 1996.
35. Kohn DB : Gene therapy for hematopoietic and immune disorders. Bone Marrow Transplant 18S:55-58, 1996.
36. Sharma S, Cantwell M, Kipps TJ, Friedmann T :Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector. Proc Acad Natl Sci USA 93:11842-11847, 1996.
37. Crompton T, Gilmour KC, Owen MJ : The MAP kinase pathway controls differentiation from double-negative to double positive thymocyte. Cell 86:243-251, 1996.
38. Sugawara T, Di Bartolo V, Miyazaki T, Nakauchi H, Acuto O, Takahama Y : An improved retroviral gene transfer technique demonstrates inhibition of CD4-CD8- thymocyte development by kinase-inactive ZAP-70. J Immunol 161:2888-2894, 1998.
39. Spain LM, Law LL, Takahama Y : Retroviral infection of T cell precursors in thymic organ culture. Methods Mol Biol 136:79-86, 2000.
40. Sugawara T, Moriguchi T, Nishida E, Takahama Y : Differential roles of ERK and p38 MAP kinase pathways in positive and negative selection of T lymphocytes. Immunity 9:565-574, 1998.
41. Kaneta M, Osawa M, Osawa M, Sudo K, Nakauchi H, Farr A, Takahama Y:A role for Pref-1 and HES-1in thymocyte development. J. Immunol. 164:256-264, 2000.
42. Dardalhon V, Jaleco S, Rebouissou C, Ferrand C, Skander N, Swainson L, Tiberghien P, Spits H, Noraz N, Taylor N : Highly efficient gene transfer in naive human T cells with a murine leukemia virus-based vector. Blood 96:885-893, 2000.
43. Steinberg M, Swainson L, Schwarz K, Boyer M, Friedrich W, Yssel H, Taylor N, Noraz N: Retrovirus-mediated transduction of primary ZAP-70-deficient human T cells results in the selective growth advantage of gene-corrected cells : implications for gene therapy. Gene Ther 7:1392-1400, 2000.
44. McCormack JE, Martineau D, DePolo N, Maifert S, Akbarian L, Townsend K, Lee W, Irwin M, Sajjadi N, Jolly DJ, Warner J : Anti-vector immunoglobulin induced by retroviral vectors. Hum Gene Ther 8:1263-1273, 1997.
45. Rainov NG, Kramm CM, Banning U, Riemann D, Holzhausen HJ, Heidecke V, Burger KJ, Burkert W, Korholz D:Immune response induced by retrovirus-mediated HSV-tk/GCV pharmacogene therapy in patients with glioblastoma multiforme. Gene Ther 7:1853-8, 2000.
46. Camargo FD, Huey-Louie DA, Finn AV, Sassani AB, Cozen AE, Moriwaki H, Schneider DB, Agah R, Dichek DA:Germline incorporation of a replication-defective adenoviral vector in mice does not alter immune responses to adenoviral vectors. Mol Ther 2:496-504, 2000.
47. Smith-Arica JR, Bartlett JS:Gene therapy:recombinant adeno-associated virus vectors. Curr Cardiol Rep 3:43-49, 2001.
48. Takahama, Y : A method for acquired immune tolerance. Patent pending, K01001PCT for Japan, USA, Canada & EP (1999).

Received for publication November 30, 2000;accepted January 19, 2001.

Address correspondence and reprint requests to Yousuke Takahama, Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, Kuramoto-cho, Tokushima770-8503, Japan and Fax:+81-88-633-9453.