Thymocyte Development in Major Histocompatibility Complex- CD4 and CD8 Lineages

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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10739-10743, November 1993 Immunology Thymocyte development in major histocompatibility complex- deficient mice: Evidence for stochastic commitment to the CD4 and CD8 lineages (T-cel selection/T-cell subsets/thymocyte differentiation) ANNE L. CRUMP*, MICHAEL J. GRUSBYt, LAURIE H. GLIMCHERt, AND HARVEY CANTOR*t *Department of Pathology, Harvard Medical School, Laboratory of Immunopathology, Dana-Farber Cancer Institute, Boston, MA 02115; and tDepartment of Cancer Biology, Harvard School of Public Health, Department of Rheumatology and Immunology, Harvard Medical School, Boston, MA 02115 Communicated by Lewis Thomas, August 6, 1993

ABSTRACT The mechanism resulting in commitment of or premature expression of these transgenic products on precursor cells in the thymus to either the CD4 or CD8 lineage thymocytes. remains poorly understood. In principle, this may reflect a Two general models have been proposed to explain the stochastic process or may reflect instructional signals from host mechanism by which a single DP precursor cell becomes major histocompatiblity complex (MHC) molecules. We have committed to either the CD4 or the CD8 lineage. One model examined the role of MHC products in subset commitment by suggests that TCR/coreceptor ligation by MHC class I or using mice deficient in class I or class II MHC products. class II molecules instructs the cell to differentiate into the Normal numbers of committed CD4 intermediates (CD4+ CD8 and CD4 pathways, respectively (14). A second model CD8I0) develop in the thymus in the absence of class II of subset commitment assumes that coreceptor downregula- molecules. Similarly, CD8 transitional cells (CD4IOCD8+) are tion occurs stochastically on- DP thymocytes. Lineage- present in the thymus of mice lacking class I products. These committed intermediaries that display a coreceptor and a findings suggest that commitment ofCD4+8+ precursor cells to TCR that recognize the same MHC product are further either lineage is a stochastic process that does not depend on selected for development into mature SP progeny. Experi- instructive signals from MHC molecules (i.e., expression of ments using TCR transgenic mice have been interpreted in alternative differentiative options by uncommitted precursor favor of the instructional model (14, 15). However, evidence cells is independent ofthis environmental signal). These studies for transient independent expression of coreceptors and a also suggest that an interaction between the T-cell antigen TCR transgene in neonatal but not adult mice may be more receptor (TCR) and MHC molecules that is independent of consistent with a stochastic commitment process (16). CD4/CD8 coreceptor engagement enhances stochastic core- We have chosen to study thymocyte development by using ceptor downregulation substantially and leads to upregulation mutant mice that do not express. class 11 (6) or class I MHC of TCR expression as a prelude to selective events that require molecules (8, 17) and that therefore lack potential instruc- joint coreceptor/TCR engagement. We suggest that this initial tional signals for CD4 and CD8 development, respectively. interaction molds the TCR repertoire of stochastically gener- This approach has the advantage that subset development ated T-cell subsets toward recognition of self-MHC products. will not be affected by transgenic manipulation of TCR or coreceptor expression. We summarize experiments that an- alyze the effects of MHC deficiency on intrathymic progres- The thymus gives rise to two lineages of T lymphocytes that SP regulate immune responses. T cells that express the CD4+8- sion from DP precursors through mature progeny. surface phenotype (CD4 cells) recognize antigen associated with class II major histocompatibility complex (MHC) prod- MATERIALS AND METHODS ucts and mediate helper activity. T cells that express the Mice. /2-Microglobulin (j32m)-deficient (class I-deficient) CD4-8+ phenotype (CD8 cells) recognize antigen associated (8) and class II-deficient (1) mice and an intercross deficient with class I MHC products and mediate killer activity (re- in both class II and 832m (MHC double-deficient) (17) were viewed in ref. 1). used for these studies along with C57BL/6 (normal control) The developmental steps resulting in the formation ofthese mice (The Jackson Laboratory) at 6-12 weeks of age. Class independent T-cell lineages remain poorly understood. Pre- II-deficient mice had a C57BL/6 x 129 background. All other cursor cells in the thymus that expresses the CD4+8+ surface mice were on a B6 background. Mice were age matched phenotype [termed double-positive (DP) thymocytes (2, 3)] within 1 week for experimental use. downregulate either CD4 or CD8 to generate lineage- Flow Cytometric Analysis. Thymocytes and lymph node committed intermediaries expressing the CD4+CD8l0 or cells were isolated in phosphate-buffered saline/2% fetal CD41OCD8+ phenotype (4, 5). Development of mature single- bovine serum at 4°C and used immediately. Washes and positive (SP) CD4 or CD8 progeny depends upon positive incubations (30 min each) were carried out at 4°C. One million selection by intrathymic MHC molecules (6-9). The timing thymocytes or lymph node cells were incubated in a total and regulation ofthis process have been studied primarily by volume of 0.1 ml with the following antibodies: anti-CD4 using mice that express transgenic T-cell antigen receptors (GK1.5; Becton Dickinson) conjugated to phycoerythrin, (TCRs) and/or coreceptors (CD4 and CD8) (10-13). Al- anti-CD8 (53-6.7; Becton Dickinson) conjugated to fluores- though this experimental model has contributed substantially cein, and anti-TCR ap (H57-597; PharMingen) or anti-CD69 to our understanding of thymocyte development, interpreta- (H1.2F3; PharMingen) conjugated to biotin. After washing, tion of these studies may be complicated by abnormally high Abbreviations: DP, double positive; SP, single positive; TCR, T-cell The publication costs ofthis article were defrayed in part by page charge antigen receptor; MHC, major histocompatibility complex; P2m, payment. This article must therefore be hereby marked "advertisement" T2-microglobulin . in accordance with 18 U.S.C. §1734 solely to indicate this fact. TTo whom reprint requests should be addressed. 10739 Downloaded by guest on October 1, 2021 10740 Immunology: Crump et al. Proc. Natl. Acad. Sci. USA 90 (1993) streptavidin red 670 (Life Technologies, Grand Island, NY) lymph node T cells were used to define mature SP thymo- was used to detect cell-bound TCR or CD69 antibody. cytes. Mature CD4+ CD8- cells were not detected in thy- Thymocytes (50,000) or lymph node cells (10,000) were muses ofclass II-deficient mice, as reported (6, 7). However, analyzed in each sample. Fluorescence analysis was carried a subpopulation of thymocytes expressing the surface phe- out on a Becton-Dickinson FACScan and the data were notype of CD4 transitional cells (Fig. 1, region B) was readily analyzed with LYSYS II software, or the analysis was per- detected. In a series of six experiments, the proportion of formed on a Coulter Epics and the data were analyzed with putative CD4 transitional thymocytes (CD4+810) was indis- Elite software. tinguishable in thymocytes from class II-deficient and control Regions defining mature CD4 and CD8 thymocytes were mice, although the percentages varied from experiment to delineated by gating of mature SP lymph node populations. experiment (Table 1). Expression of the peanut agglutinin The DP thymocyte population was defined by the intersec- antigen and heat-stable antigen, which distinguishes thymo- tion of the lines delineating the lower limits of the CD4 and cyte maturational stages (3, 18, 19), did not differ significantly CD8 regions in lymph node cells, which determine the lower in transitional thymocytes from class II-deficient and normal left corner of the DP region (see Fig. 1). The transitional mice (data not shown). populations were placed between the mature SP thymocytes We evaluated the level of TCR expression on CD4 transi- and DP thymocytes. These transitional (CD4+CD8l0 and tional thymocytes in class II-deficient and normal mice using CD41OCD8+) regions contained 0.1-0.6% of total cells from three-color immunofluorescence (Fig. 2 and Table 2). TCR lymph nodes. The distribution ofthymocytes in these regions expression was deemed high (TCRhi) if it fell within gates set is discussed in the text. using similarly stained lymph node cells. Transitional thymocytes from class II-deficient mice and normal mice contained similar proportions of TCRhi cells, which ranged from RESULTS 35% to 70% (Table 2). Analysis of Maturation within the CD4 Lineage in MHC- The finding that transitional cells from class II-deficient Deficient Mice. The mechanisms of commitment to the CD4 mice expressed elevated TCR levels despite the absence of lineage can be directly addressed by analysis of thymocyte the correct MHC ligand opened the possibility that they had development in mice deficient in class II MHC products. If received a signal from "incorrect" MHC class I products in coligation ofthe TCR and CD4 by class II molecules normally the thymus. This possibility was consistent with the finding instructs DP cells to downregulate CD8 and commit to the that the proportion ofCD4 transitional thymocytes from class CD4 pathway, thymocytes from class II-deficient mice II-deficient mice that expressed CD69 [a surface antigen should not contain committed CD4 intermediaries expressing expressed by recently activated thymocytes (20)] was similar the CD4+810 surface phenotype, which are the immediate to the proportion of CD69+CD4 transitional cells from class precursors of SP cells (4, 5). Thymocytes and lymph node II+ controls (Table 2). cells frosi age-matched class II-deficient and normal mice We therefore determined whether thymocytes from mice were incubated with antibodies to CD4 and CD8 and ana- lacking both class I and class II (MHC double-deficient) might lyzed by fluorescence-activated cell sorting (Fig. 1). The contain CD4 transitional cells. We found that thymocytes from levels of CD4 and CD8 immunofluorescence on mature these mice contained approximately one-third to one-half the Normal (I+II+) Class II-deficient (I+II-) Class I-deficient (I-II+)

Thymus

CD4

Lymph node

CD8 FIG. 1. Definition of CD4 and CD8 lineage cells in class II-deficient and class I-deficient mice. Thymocytes and lymph node cells from age-matched normal, class II-deficient, and class I-deficient mice were stained with antibodies to CD4 and CD8. Regions defining mature CD4 and CD8 thymocytes were delineated by enclosing mature SP populations according to lymph node immunofluorescence (bottom row). The DP thymocyte population was defined by using the lower limit of CD4 and CD8 expression of the lymph node cells (the intersection of the lines delineating the lower limit ofCD4 and CD8 regions determines the lower left corner ofregion C, the DP thymocytes). The transitional populations (regions B and D) were placed between the mature SP thymocytes (regions A and E) and DP thymocytes (region C). The percentages of CD4 and CD8 transitional thymocytes in normal, class II-deficient, and class I-deficient mice are shown. Downloaded by guest on October 1, 2021 Immunology: Crump et al. Proc. Natl. Acad. Sci. USA 90 (1993) 10741 Table 1. Percentage of CD4 transitional cells in the thymus of Table 2. TCR and CD69 expression on CD4+CD8l0 transitional normal and class II-deficient mice cells in normal and class II-deficient mice % CD4+CD81' Expression of Exp. Class II+ Class II-deficient Exp. MHC I/II TCRhi, % CD69+, % 1 8.4 13.3 1 +/+ 62.9 60.5 2 5.6 11.8 +/- 58.2 53.2 3 4.0 3.6 2 +/+ 70.7 58.0 4 5.2 5.4 +/- 34.3 35.4 5 3.3 3.5 3 +/+ 31.7 59.5 6 2.2 1.6 +/- 48.3 60.7 These data indicate the percentage oftotal thymocytes expressing The percent TCRhi thymocytes was determined from a histogram the CD4+CD8l0 transitional phenotype from six separate expeni- of TCR immunofluorescence of CD4 cells from region B in Fig. 1. ments using one mouse from each strain. The CD4+ CD8l0 transi- The gates delineating TCRhi cells were set using lymph nodes staining tional population is shown in Fig. 1, region B. as described in the legend to Fig. 2. Analysis of Maturation Within the CD8 Lineage and MHC- level of CD4 transitional cells compared to normal mice in Deficient Mice. The above findings suggested that commit- three separate experiments (Fig. 3). However, unlike CD4+810 ment to the CD4 pathway can occur in the absence of MHC cells in the class II-deficient mice, CD4 transitional cells from products but that the efficiency of this developmental step MHC double-deficient mice did not express either high surface and induction of high levels of surface TCR depended on levels of TCR or surface CD69 (Table 3). Therefore, an expression of MHC molecules. We asked whether commit- interaction with class I MHC products expressed in the class ment to the CD8 lineage followed similar rules. We examined 11-deficient thymus may enhance CD8 coreceptor downregu- this lineage in f32m mutant mice that are deficient in class I lation and upregulate TCR expression. expression (8, 9, 17). We found that CD8 transitional cells [CD8+410 (4, 5)] were present in the thymus of class I-defi- CD4+81o C D41o8 + cient mice, albeit at levels slightly lower than class I+ controls (Fig. 1). The proportion of CD8 transitional cells in I+ll+ the thymus of class I-deficient and normal mice that expressed high TCR levels was also similar, although the absolute percentage varied from experiment to experiment (Fig. 1 and Table 3). Finally, although thymocytes from MHC 48%i double-deficient mice also contained significant numbers of CD8 transitional cells (Fig. 3), virtually none of these 0I IA. CD4108+ thymocytes expressed high TCR levels or CD69, similar to the surface phenotype of CD4 transitional thymocytes in MHC double-deficient mice (Table 3). Thus, an 0 l0o 0 1lo lo: 103 1°4 interaction with host class II products in class I-deficient l +i _ Il-l mice may enhance CD4 downregulation and increase TCR expression on CD8 transitional cells. DISCUSSION 3_ 437% The presence ofCD4 and CD8 transitional cells in the thymus SL of mice lacking class II and class I MHC, respectively, indicates that lineage commitment is stochastic and does not 5s 4e f I .j~

CD4

-- CD8 Thihmocvte pQOpUlali 1-M 4%1 l%) 4+8- 10.5 1.6 12.3 0.4 4+810 3.7 4.2 2.1 2.2 4+8+ 74.7 82.2 78.4 89.7 4108+ 2.2 2.2 1.6 1.3 4-8+ 3.9 5.2 0.9 0.9 FIG. 3. Analysis of CD4 and CD8 transitional thymocyte populations in mice deficient in MHC expression. Thymocytes from normal, class II-deficient, class I-deficient, and MHC double-deficient mice were stained as described in Fig. 1. Regions were drawn as described in Materials and Methods and Fig. 1. The percentage of cells within the indicated thymocyte subpopulations is shown for each mouse and represents one of three experiments with similar results, except that the levels of SP CD4 cells in class II-deficient and MHC double-deficient mice are not reproducibly different. leads to upregulation of the TCR on CD4+810 cells and CD4 or CD8 coreceptor molecules. The proportion of large similarly for class II and CD4108+ cells). Moreover, this cells, as judged by forward light scatter, in these SP inter- coreceptor-independent interaction is necessary but not suf- mediaries is 4- to 5-fold less than in intermediaries that ficient for maturation into SP CD4 or CD8 thymocytes. One differentiate in an environment that allows development of effect of this interaction may be molding of the TCR reper- SP thymocytes (data not shown), suggesting that increased toire toward recognition of self-MHC products, since only size may distinguish transitional cells that have undergone those thymocytes that express TCRs that can interact with complete positive selection from stochastically generated host MHC products undergo subset commitment and TCR intermediaries that have not. upregulation as a prelude to further selectional events. This In sum, these studies suggest that subset development in interaction may also stabilize expression of useful TCRs in the thymus may reflect three distinct events. First, DP thymocytes, since TCR ligation ofDP thymocytes can inhibit thymocytes may undergo stochastic coreceptor downregu- RAG-2 expression (23, 24) and thus prevent the generation of lation in the absence ofMHC products. Second, TCR ligation new TCRs by these cells. By contrast, DP cells that express by host MHC molecules, without coreceptor ligation, sub- TCRs that cannot interact with host MHC molecules will not stantially enhances the efficiency of this developmental step undergo this maturational step and, as a consequence, are and upregulates TCR expression on these cells. Finally, winnowed out of the developmental process. Stochastically coengagement of the TCR and coreceptor by the same MHC generated transitional cells displaying inappropriate TCR/ molecule is necessary for positive selection of mature SP coreceptor combinations (class I-specific CD4+810 cells or progeny from intermediaries within the two lineages. Critical class II-specific CD4108+ cells) are also eliminated because questions that require further study include the nature of the they cannot receive the second signal for positive selection. initial TCR interaction with host MHC molecules and its Previous studies with TCR transgenic mice have suggested functional relationship to TCR/coreceptor engagement nec- that upregulation of surface TCR is a specific consequence of essary for maturation and survival of T-cell subsets. positive selection (25). Our studies suggest that TCR upregulation can result from TCR ligation by MHC molecules that Note added in proof. While this manuscript was under review, two do not allow coengagement of the TCR and coreceptor papers containing evidence consistent with a stochastic mechanism necessary for the development of mature SP thymocytes. of intrathymic subset commitment were published (26, 27). This initial developmental step is associated with an increase in surface CD69 expression and downregulation of either We thank Sarah Boyerfor expert technical assistance; John Daley, Peter Lopez, and Robin Pratt for FACS analysis; and Alison Angel Table 3. TCR and CD69 expression on CD4 and CD8 for assistance in the preparation of this manuscript. This work was transitional cells supported in part by National Institutes of Health research grants A112184 and A113600 and March of Dimes and Arthritis Foundation Expression of CD4+CD8l0 CD4lOCD8+ grants to H.C. and National Institutes of Health research grants Exp. MHC I/II TCRhi CD69+ TCRhi CD69+ A131541 and A121569 and a gift from the Mather Foundation to L.H.G. A.L.C. is a National Institutes of Health Postdoctoral AIDS 1 +/+ 74.1 54.0 49.4 29.8 Training Fellow. M.J.G. is supported by an Arthritis Foundation +/- 48.9 58.5 - Investigator Award and a Leukemia Society Special Fellowship. -/+ - - 7.6 10.7 -/- 7.1 8.1 0.9 1.9 1. Patarca, R., Singh, R. P., Wei, F.-Y., Iregui, M. V., Singh, P., 2 +/+ 25.4 11.8 26.4 14.7 Schwartz, J. & Cantor, H. (1990) Immunological Reviews 116, +/- 41.3 14.6 1-16. -/+ - - 18.1 12.2 2. Petrie, H. T., Hugo, P., Scollay, R. & Shortman, K. (1990) J. -/- 3.9 0.9 4.9 1.5 Exp. Med. 172, 1583-1588. 3 +/+ 86.0 70.3 48.1 25.6 3. Hugo, P., Boyd, R. L., Waanders, G. A. & Scollay, R. (1991) +/- 93.0 62.5 Eur. J. Immunol. 21, 2655-2659. -- 4. Guidos, C. J., Weissman, I. L. & Adkins, B. (1989) Proc. Natl. -/+ 43.5 14.6 Acad. Sci. USA 86, 7542-7546. The percentage of cells expressing TCRhi or CD69+ within each 5. Guidos, C. J., Danska, J. S., Fathman, C. G. & Weissman, population was determined as described in the legend to Fig. 2. I. L. (1990) J. Exp. Med. 172, 835-842. Downloaded by guest on October 1, 2021 Immunology: Crump et al. Proc. Natl. Acad. Sci. USA 90 (1993) 10743

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