LYMPHOCYTE DEVELOPMENT AND DIFFERENTIATION
INTO MEMORY

KYOKO HAYAKAWA, M.D., Ph.D., Member

Generation of immunological memory to foreign antigens is a complex process. T cells expressing the CD4 glycoprotein (CD4⁺ T cells) play a pivotal role through the generation of newly differentiated memory lymphocytes and maintenance of their clones throughout life, as an "acquired" immune system. The lifelong maintenance of lymphocyte clones can also occur for cells specific to certain self-antigens, as an "innate" immunity. Our research focuses on investigating the developmental mechanisms for these two types of "memory" systems and seeks to understand the reason why two such "memory" systems should co-exist.

We have been seeking the key elements that specifically define "long-term memory helper T cells" that recognize foreign antigens. Of several characteristics associated with memory helper T cells that we identified, high 6C10 antigen expression serves as a good marker for memory helper T cells in contrast to 6C10^- anergic cells after antigen stimulation. High 6C10 expression is also characteristic of superantigen responsive-cells (1). 6C10 is highly expressed by immature thymocytes and a fraction of CD4⁺ T cells in the periphery. It is recognized by the IgM natural autoantibody SM6C10. Although we have demonstrated previously that its surface expression is dependent on Thy1 (CD90) expression, whether it is actually a part of the Thy-1 glycoprotein has remained unclear. Answering this question is critical, however, since Thy-1 is known to transduce signals, resulting in cell activation.

Immunoprecipitation with the IgM autoantibody has been difficult. However, based on several different experimental approaches, we are now able to conclude that 6C10 is an epitope on Thy1. SM6C10 immunoprecipitates only a 26-29 kilodalton molecule from thymocyte and T cell line lysates. In addition, the ability to detect this band is correlated with Thy-1 (i.e., it is absent in immunoprecipitates from Thy-1^- cells). Furthermore, the SM6C10 species can be pre-cleared by anti-Thy-1, and the 6C10 antibody binds to affinity purified Thy-1 in an ELISA assay. Two-dimensional electrophoresis (NEPHGE/SDS-PAGE) showed a similar heterogeneity of isoelectric points between 6C10 and thymic Thy-1. Finally, Nglycosidase F treatment eliminated SM6C10 reactivity. Thus, 6C10 is a carbohydrate epitope of the Thy-1 (CD90) glycoprotein, expressed on most thymic Thy-1 glycoforms. Importantly, we found that upregulation of 6C10⁺ Thy-1 glycoform occurs on CD4⁺ T cells during peripheral maturation. This led us to inquire whether 6C10⁺ Thy-1 expression itself is important for memory helper function, or instead, whether 6C10⁺ is simply a phenotype associated with mature T cells that memory T cells maintain.

We first reported generation of autoreactive CD4⁺ ab T cells in the thymus of normal healthy mice in 1992 (Hayakawa et al., J. Exp. Med. 176:269, 1992; Kariv et al., J. Exp. Med. 177:1429, 1993). This CD4⁺ thymocyte subset reacts with autologous cells and secretes a diverse array of cytokines, similar to memory helper T cells; therefore, we proposed the name "Thy0" (for thymic TH0 type cells). The mechanism for development and maintenance of Thy0, and its immunologic significance are not fully understood at present. Within the Thy0 population, we found that there is considerable surface phenotypic heterogeneity with a significant fraction expressing a molecule considered characteristic of natural killer cells, NK1.1 (Kariv et al., J. Exp. Med. 180:2419, 1994). This NK1.1⁺ (NK1⁺) cell fraction, commonly referred to as NKab cells, has recently been extensively studied by several investigators. Such cells are considered to be a unique T cell subset, based on their non-classical class I (CD1)-dependent development, their restricted TCR usage (canonical Va14 rearranged to Ja281; Va14-J281), their reactivity to a galactosylceramide (aGalCer), and their involvement in tumor rejection.

In contrast to the NKab cell subset, the NK1.1^- (NK1^-) Thy0 fraction is comprised of a relatively diverse set of ab TCR combinations; however, our data strongly suggests a common underlying mechanism for generating these two autoreactive T cell populations. In particular, we found specific enrichment of Va14-J281⁺ cells in the neonatal NK1^-Thy0 population before NKab cell appearance, suggesting that the NK1^- Thy0 cells are a precursor for the NK1⁺ subset. To test this possibility, we took advantage of the unique reactivity of NK1⁺ Va14-J281⁺ T cells to a-GalCer. We have established hybridomas from NK1^- and NK1⁺ Thy0 or NK1⁺CD4^- 8^- ab cells in the thymus. Their reactivity was tested by IL-2 secretion upon stimulation with either aGalCer pulsed CD1 transfectants or direct a-GalCer stimulation (3). Our data demonstrated that all Va14-J281⁺ T cells, predominantly derived from NK1⁺ ab cells, are specifically reactive to a-GalCer/CD1, revealing a clear relationship between Va14-J281⁺ TCR usage and aGalCer reactivity. However, we also found three examples of Va14-J281⁺ a-GalCer-reactive hybridomas from the NK1^- Thy0 cells in which two uniquely express Vb14 (rare in NKab), supporting a developmental relationship between the NK1^- Thy0 and NK1⁺ab cells. Furthermore, we found an NK1⁺CD4^-8^- cell derived hybridoma expressing Va5/Vb14, which is also reactive to a-GalCer-pulsed transfectants. Interestingly, this Va5/Vb14⁺ hybridoma and several Va14⁺ hybridomas exhibited CD1-dependent autologous cell reactivity without a-GalCer pulsing, but this was not generally the case with Va14⁺ hybridomas. This suggests an endogenous mechanism(s) for controlling CD1-mediated self reactivity, even when identical TCRs reactive to a-GalCer are used. Our working hypothesis is that self-antigen is important for the survival of autoreactive T cells and also for their further differentiation into NK1ab cells, which is controlled by other non-antigen receptor mediated signals. We are currently carrying out several experimental approaches to test this hypothesis.

The presence of autoantibody in the serum of healthy animals, known as "natural autoantibody," has been long recognized. Considering the exquisite sensitivity of B cells to be "tolerized", the normal presence of such autoantibody has been a puzzle. In collaboration with Dr. R. Hardy, we have been investigating this issue from the view of an ontogeny-based difference in B cell development at the early B cell repertoire establishment stage (2) and at the late B cell selection stage. Our previous data suggested that, after the generation of immature B cells, self-antigen positively influences the development and maintenance of natural autoreactive B cells, establishing an autoreactive B cell "memory" pool (Hardy et al., Adv. Immunol. 55:297, 1994). However, demonstrating the importance of self-antigen has been difficult.

To test whether self-antigen influences natural autoreactive B cell development, we have studied one such natural autoantibody in mice, anti-thymocyte autoantibody (ATA), which is a specificity encoded by V_H3609/V_k21C germline genes. This ATA binds specifically to T cells by recognition of a carbohydrate epitope on the Thy-1/CD90 glycoprotein and ATA B cells are exclusively present among the CD5⁺ B cell population, an enriched source for natural autoantibody. By establishing the V_H3609 Ig m heavy chain transgenic mouse lines and generating self-antigen deficient transgenic mice due to a lack of the Thy-1 gene (provided Dr. J. Silver, Division of Molecular Medicine, North Shore University Hospital, Cornell University Medical College, Manhasset, NY), we have been able to demonstrate that self-antigen is indeed critically involved in the normal accumulation of ATA B cells, induction of CD5, and antibody secretion into serum (Figure 1). Thus, our ATA B cell system provides the first evidence for a positive role of self-antigen in the generation and maintenance of B cells. Our data will open several opportunities to assess the influence of natural antigens in the development of autoimmune disease and in disregulated B cell growth in aged animals due to repeated exposure to self-antigen.

PUBLICATIONS

1.   MAIER, C.C., BHANDOOLA, A., BORDEN, W., YUI, K., HAYAKAWA, K., GREENE, M.I. Unique molecular surface features of in vivo tolerized T cells. Proc. Natl. Acad. Sci USA. 95:4499-4503, 1998.

2.   WASSERMAN, R., LI, Y.-S., SHINTON, S.A., CARMACK, C.E., MANSER, T., WIEST, D.L., HAYAKAWA, K., HARDY, R.R. A novel mechanism for B cell repertoire maturation based on response by B cell precursors to pre-B receptor assembly. J. Exp. Med. 187:259-264, 1998.

Paper in press at time of previous report:

3.   BURDIN, N., BROSSAY, L., KOEZUKA, Y., SMILEY, S.T., GRUSBY, M.J., GUI, M., TANIGUCHI, M., HAYAKAWA, K., KRONENBERG, M. Selective ability of mouse CD1 to present glycolipids: a-galactosylceramide specifically stimulate Va14⁺ NK T lymphocytes. J. Immunol. 161:3271-3281, 1998.

^§   Fox Chase researcher

^a   C.C. Maier, M.I. Greene: Department of Pathology, University of Pennsylvania, Philadelphia, PA 19104

^b   M. Kronenberg: The La Jolla Institute for Allergy & Immunology, San Diego, CA 92121

^c   Y. Koezuka: Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Takasaki-shi, Gunma 370-12, Japan

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998