Programming for cytotoxic effector function occurs concomitantly with CD4 extinction during CD8+ T cell differentiation in the thymus

Avinash Bhandoola, Balaji Kithiganahalli, Larry Granger and Alfred Singer

Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

Correspondence to: A. Singer, Building 10, Room 4B36, National Cancer Institute, Bethesda, MD 20892, USA


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD4+ T cells are generally specialized to function as helper cells and CD8+ T cells are generally specialized to function as cytotoxic effector cells. To explain how such concordance is achieved between co-receptor expression and immune function, we considered two possibilities. In one case, immature CD4+CD8+ thymocyte precursors might first down-regulate expression of one co-receptor molecule, with the remaining co-receptor molecule subsequently activating the appropriate helper or cytotoxic functional program. Alternatively, we considered that the same intrathymic signals that selectively extinguished expression of one or the other co-receptor molecule might simultaneously initiate the appropriate helper or cytotoxic functional program. In the present study, we attempted to distinguish between these alternatives by examining thymocyte precursors of CD8+ T cells for expression of Cathepsin C and Cathepsin W, molecules important for cytotoxic effector function. We report in developing thymocytes that Cathepsin C and Cathepsin W are expressed coordinately with extinction of CD4 co-receptor expression. We conclude that CD4 extinction and initiation of the cytotoxic functional program occurs simultaneously during differentiation of CD8+ T cells in the thymus.

Keywords: Cathepsin C, Cathepsin W, cytotoxic lymphocytes, precursor cytotoxic T lymphocyte, thymic selection


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During T cell development in the thymus, immature CD4+CD8+ [double-positive (DP)] thymocytes expressing both CD4 and CD8 co-receptors differentiate into immunocompetent CD4+CD8 or CD4CD8+ [single-positive (SP)] T lymphocytes. The specificity of the {alpha}ßTCR directs this maturational process, such that DP thymocytes that express TCR specific for MHC class II mature into CD4+ helper T cell precursors, while DP thymocytes that express TCR specific for MHC class I mature into CD8+ cytotoxic T cell precursors. The process by which this occurs in DP thymocytes is referred to as positive selection (14) and has been separated into three distinct developmental stages: induction, co-receptor extinction (lineage commitment) and rescue (5).

Function appears to be matched to co-receptor expression during thymic maturation: CD4+ T cells are specialized to become helper cells and CD8+ T cells are specialized to become cytolytic effector cells, regardless of the MHC specificity of their TCR. Thus, it is possible to experimentally separate TCR specificity from lineage choice so that the MHC specificities of the TCR and co-receptor molecules expressed by mature T cells are discordant (68). Thymocytes expressing an MHC I-restricted TCR can be induced to mature into CD4+ T cells, and thymocytes expressing an MHC II-restricted TCR can be induced to mature into CD8+ T cells (68). Remarkably, in the few cases in which this has been achieved, the functional specialization of these mismatched T cells appears to remain concordant with their co-receptor choice, so that CD8+ T cells expressing an MHC II-restricted TCR can function as cytotoxic effector cells and CD4+ T cells expressing an MHC I-restricted TCR can function as helper cells (68).

To understand how lineage choice and cellular function might be linked, we considered two possibilities: (i) DP thymocytes are first signaled to extinguish expression of one co-receptor, followed by programming for cellular function directed by the remaining co-receptor molecule, or (ii) DP thymocytes are simultaneously signaled in the thymus to extinguish expression of one co-receptor molecule and to initiate the appropriate functional program (9). Consequently, we wished to determine whether lineage choice and functional specialization occur sequentially or simultaneously in signaled DP thymocytes.

To this end we have examined the developmental stage at which programming for cytotoxic effector T cell function begins during thymic maturation. Our approach was to examine cells at different developmental stages in the T cell differentiation pathway for expression of RNAs encoding effector molecules thought to be important for cytotoxic T cell function. Of the effector molecules that are important for cytotoxic T cell function, the pro-granzyme processing enzyme Cathepsin C (also known as Dipeptidyl peptidase I) is expressed in granules (1012) and functions to activate granzymes A and B that are required for nucleolysis of targets by cytotoxic T lymphocytes (CTL) (13). Indeed, expression of Cathepsin C is up-regulated in CD8+ SP T cells in the thymus and spleen (14), and is further up-regulated in activated CTL (15). In addition to Cathepsin C, we have also examined expression of Cathepsin W which also is thought to be important for cytolytic function, and which has been shown to be expressed primarily in peripheral CD8+ T cell and NK cells (16,17). We report that RNAs for Cathepsin C and Cathepsin W are up-regulated simultaneously with extinction of CD4 co-receptor expression during thymic development. These results indicate that programming for cytotoxic effector function begins concomitantly with CD4 co-receptor shut-off during T cell development in the thymus.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Normal B6 mice were obtained from the Frederick Cancer Research and Development Center (Frederick, MD). ß2-Microglobulin 2m)-deficient mice (18) were crossed to the C57BL/6 background. All mice were housed in a specific pathogen-free facility and used at 4–12 weeks of age.

Cell isolation
We isolated each thymic population by a combination of pronase treatment (0.01% pronase; Calbiochem Novabiochem, San Diego, CA), cell culture and electronic cell sorting as described (5) (Fig. 1Go). Cells were stained prior to sorting with anti-CD4–FITC (RM4-5; PharMingen, San Diego, CA), anti-CD5–phycoerythrin (53-7.3; PharMingen) and anti-CD8–CY5 (CT-CD8a; Caltag, Burlingame, CA). DP thymocytes are heterogeneous for CD5 expression, as well as for co-receptor re-expression after pronase treatment. All CD5lo DP thymocytes re-express both CD4 and CD8 after pronase treatment, and so uncommitted DP CD5lo (UDP CD5lo) cells can be isolated by sorting for cells expressing CD4, CD8 and low levels of CD5. To isolate uncommitted DP CD5hi (UDP CD5hi) cells, B6 thymocytes were treated with pronase and cultured overnight, after which cells re-expressing CD4, CD8 and high levels of CD5 were isolated (Fig. 1Go).




View larger version (45K):
[in this window]
[in a new window]
 
Fig. 1. Description and method of isolation of thymic subpopulations used in this study. (A) Precursor–progeny relationships between subpopulations of DP thymocytes. Black circles represent cells which express both CD4 and CD8 surface proteins, and so are phenotypically DP thymocytes. Nevertheless, DP thymocytes are heterogeneous for CD4 and CD8 co-receptor mRNA expression, and the co-receptor mRNAs that are present within each cell are indicated. (B) Isolation of DP and SP T cells. Single-cell suspensions of thymocytes were fractionated by a combination of pronase treatment and cell sorting after staining with antibodies to CD4, CD8 and CD5. As indicated, we isolated the following thymocyte subpopulations from normal B6 mice: UDP CD5lo thymocytes; UDP CD5hi thymocytes; CD4- and CD8-committed DP CD5hi thymocytes; and CD4+ and CD8+ SP thymocytes.

 
We isolated DP CD5hi, CD4 CD5hi cells and CD8 SP CD5hi cells by cell sorting from fresh thymocytes. To prepare CD4- and CD8-committed DP thymocytes, we first isolated DP CD5hi thymocytes, as these have been shown to be enriched in lineage-committed precursors (19). These cells were pronase stripped and cultured overnight to permit co-receptor re-expression. We then sorted CD5hi cells re-expressing CD4 but not CD8 as CD4-committed CD5hi DP cells; and CD5hi cells re-expressing CD8 but not CD4 as CD8-committed CD5hi DP cells.

RT-PCR
We prepared total RNA from 1–2x104 cells of each thymocyte subpopulation using the GlassMAX RNA Microisolation spin cartridge system (Gibco/BRL, Rockville, MD). We reverse transcribed total RNA to cDNA using a poly(dT) oligonucleotide (Superscript II; Gibco/BRL), so that only polyadenylated RNA transcripts were primed and reverse transcribed to cDNA. The following Cathepsin C, Cathepsin W and ß-actin primers were used for PCR amplification of serially diluted cDNA: Cathepsin C: 5'-cca act gca cct acc ctg at-3'; 5'-ctg aac ggt att gat ggc ct-3'; Cathepsin W: 5'-atc tcg tcg gtc aag aac ca-3'; 5'-ccc cag gag ttc ttc agg at-3'; ß-actin: 5'-cag gca ttg ctg aca gga tgc-3'; 5'-aag ggt gta aaa cgc agc tca g-3'. Each PCR reaction contained 150 cell equivalents of cDNA at the top concentration and 3-fold serial dilutions at subsequent concentrations. We performed 36 cycles of PCR amplification (Taq DNA polymerase kit; Gibco/BRL) with 1 min of denaturation at 94°C, 1 min of annealing at 55°C and 1 min of polymerization at 72°C. Amplified products were analyzed on 1.5% agarose gels, transferred to nylon membranes (Hybond-N+; Amersham) and hybridized with the indicated probes: Cathepsin C: 5'-agt atg ccc aag att ttg ggg tggt-3'; Cathepsin W: 5'-tca ccg tga cca tca aca tga aac-3'; ß-actin: 5'-gcc tca ctg tcc acc ttc cag cag-3'. Southern blots were quantitatively analyzed on a Phosphorimager (Molecular Dynamics, Sunnyvale, CA). Band volumes were calculated and expressed as a ratio of the volume of the ß-actin band at the top cDNA input concentration (150 cell equivalents).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have previously determined that DP thymocytes are heterogeneous with regard to CD5 expression and coreceptor extinction, and contain at least three subpopulations of cells. Formal precursor–progeny analysis has identified a linear developmental sequence such that UDP CD5lo -> UDP CD5hi -> lineage-committed DP CD5hi -> SP T cells (Fig. 1AGo). To examine the developmental stage at which programming for cytotoxic effector function first begins during T cell maturation, we isolated B6 DP thymocytes at each of these developmental stages (Fig. 1BGo). We prepared cDNA from each population, and examined expression of Cathepsin C, Cathepsin W and ß-actin using semi-quantitative PCR (Fig. 2Go). The left-most lane in each group corresponds to cDNA from 150 cell equivalents and subsequent lanes correspond to 3-fold serial dilutions of cDNA. All populations expressed roughly equivalent amounts of ß-actin mRNA as visualized by RT-PCR.



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 2. Expression of Cathepsin C and Cathepsin W in purified thymic subpopulations. RT-PCR was performed on 150 cell equivalents (left-most lane of each group) from each sorted thymocyte subpopulation and on 3-fold serial dilutions as indicated. Arrows indicate the identity of each cell population in the positive selection pathway, as well as the precursor–progeny relationships between them. Numbers are band volumes derived from Phosphorimager analysis of Southern blots and are all normalized to the ß-actin band at 150 cell equivalents of input cDNA which was arbitrarily set at 100. The top and bottom panel are each derived from separate experiments. Lineage relationships among thymocytes are diagrammed between the two panels. In this diagram, black circles represent DP thymocytes which express both CD4 and CD8 surface proteins, but which differ in the CD4 and CD8 co-receptor mRNAs they contain, as indicated.

 
It can be seen in Fig. 2Go (upper panels) that Cathepsin C and Cathepsin W mRNA transcripts were not detected in UDP CD5lo cells, CD4-committed CD5hi DP cells or CD4+ SP T cells. In contrast, Cathepsin C and Cathepsin W mRNA transcripts were detected in CD8+ SP T cells and, surprisingly, in CD8-committed CD5hi DP cells (Fig. 2Go). Thus, expression of Cathepsin C and Cathepsin W was up-regulated in developing thymocytes at some time after the UDP CD5lo stage of development, and either before or simultaneously with CD8 commitment (Fig. 2Go). Consequently, we examined UDP CD5hi thymocytes which are developmentally intermediate between UDP CD5lo and CD8-committed DP thymocytes. Interestingly, Cathepsin C and Cathepsin W were not detected in UDP CD5hi cells (Fig. 2Go, bottom panels), which continue to express both CD4 and CD8 proteins. Hence, the developmental stage at which CD8-lineage cells first up-regulate expression of Cathepsin C and Cathepsin W is precisely the same developmental stage at which CD4 protein synthesis is selectively terminated.

CD4 extinction in TCR-signaled precursors of CD8 T cells requires interaction with MHC class I molecules (20). As a result, mice lacking ß2m have very few CD8+ T cells in primary and secondary lymphoid tissues, and have very few CD8-committed cells in their thymi. We wished to determine whether up-regulation of Cathepsin C and Cathepsin W mRNAs in TCR-signaled thymocytes was similarly MHC class I dependent. We therefore compared Cathepsin W expression in DP CD5hi thymocytes from wild-type and ß2m-deficient mice (Fig. 3Go). Cathepsin W mRNA was greatly reduced in DP CD5hi thymocytes from ß2m-deficient mice as compared to DP CD5hi thymocytes from wild-type mice. Thus, MHC I-dependent signals which are required for extinction of CD4 expression are also required for up-regulation of Cathepsin W.



View larger version (44K):
[in this window]
[in a new window]
 
Fig. 3. Expression of Cathepsin W in purified DP CD5hi thymocytes from ß2m-deficient and wild-type mice. DP CD5hi thymocytes include both uncommitted and committed DP CD5hi cells. RT-PCR was performed on 150 cell equivalents (leftmost lane of each group) from sorted DP CD5hi cells and on 3-fold serial dilutions as indicated. Numbers are band volumes derived from Phosphorimager analysis of Southern blots and are all normalized to the ß-actin band at 150 cell equivalents of input cDNA which was arbitrarily set at 100.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have examined expression of transcripts encoding molecules important for eventual cytotoxic effector function during maturation of T cell precursors in the thymus. We focused on the Cathepsins C and Cathepsin W, as Cathepsin C is known to be important for CTL function via the classical granule exocytosis pathway (13,15) and Cathepsin W is selectively expressed in precursor CTL (CD8 and NK cells) (16,17). Our choice of these molecules was based on our reasoning that they are likely to be important in processing other enzymes involved in the effector phase of CTL killing. We report that increased expression of mRNAs for Cathepsins C and W occurred concomitantly with CD4 extinction in developing thymocytes (Fig. 1AGo). Hence UDP CD5lo and UDP CD5hi cells contained undetectable levels of Cathepsin C and Cathepsin W transcripts; expression was first evident during differentiation of UDP CD5hi cells into CD8-committed DP thymocytes. This increase in Cathepsin C and Cathepsin W mRNA levels required interaction with MHC class I molecules in the thymus, demonstrating that MHC class I derived signals are necessary for both CD4 extinction and initiation of the cytolytic functional program.

The positive selection step during which functional programming is initiated has been previously examined and found to occur prior to the appearance of CD4+ and CD8+ SP T cells. In fact, CD3hi DP thymocytes in humans (21) and C69hi DP thymocytes in mice (22) were previously observed to contain RNA transcripts encoding IL-2 and perforin. Our present results are fully concordant with these earlier studies demonstrating that functional programming was initiated during the DP stage of thymocyte development, and extend these previous observations by demonstrating that the cytolytic functional program is initiated in DP thymocytes concomitantly with extinction of CD4 co-receptor expression and in response to the same MHC class I-dependent signals. Indeed, our present observations explain the strict concordance observed in mature T cells between CD8 co-receptor expression and cytolytic immune function (68) as resulting from the fact that the same MHC class I-dependent interactions that signal CD4 co-receptor extinction in developing thymocytes also signal the initiation of the cytolytic program.

Our failure to detect expression of Cathepsins C and W in CD4-committed DP thymocytes deserves further comment, as CD4+ cytolytic T cells have been described that might be expected to express molecules important for cytotoxic function (23). However, a study of the Fas-mediated death pathway has shown that most CD4+ cytolytic T cells mediate cell lysis by the Fas pathway rather than the classical granule exocytosis pathway (23). Thus, killing by the classical granule-mediated exocytosis pathway appears largely restricted to CD8+ T cells and NK cells.

In conclusion, the present results extend our view of the events accompanying CD4 extinction in precursor CD8+ thymocytes. Termination of CD4 transcription during T cell development depends upon a CD4 silencing element found in the CD4 locus control region (24,25), and it is tempting to consider that the same intrathymic signal may activate the CD4 silencer to extinguish CD4 gene transcription (24,25), and also initiate transcription of genes encoding Cathepsin W and C, hence ensuring coordinate regulation of CD8 expression and cytotoxic function.


    Abbreviations
 
ß2m ß2-microglobulin
CTL cytotoxic T lymphocyte
DP double positive
SP single positive
UDP uncommitted DP

    Notes
 
Transmitting editor: L. Glimcher

Received 10 January 2000, accepted 15 March 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. von Boehmer, H., Swat, W. and Kisielow, P. 1993. Positive selection of immature {alpha}ß T cells. Immunol. Rev 135:67.[ISI][Medline]
  2. Robey, E. and Fowlkes, B. J. 1994. Selective events in T cell development. Annu. Rev. Immunol. 12:675.[ISI][Medline]
  3. Kisielow, P. and von Boehmer, H. 1995. Development and selection of T cells: facts and puzzles. Adv. Immunol. 58:87.[ISI][Medline]
  4. Marrack, P. and Kappler, J. 1997. Positive selection of thymocytes bearing {alpha}ß T cell receptors. Curr. Opin. Immunol. 9:250.[ISI][Medline]
  5. Bhandoola, A., Cibotti, R., Punt, J. A., Granger, L., Adams, A. J., Sharrow, S. O. and Singer, A. 1999. Positive selection as a developmental progression initiated by {alpha}ß TCR signals that fix TCR specificity prior to lineage commitment. Immunity 10:301.[ISI][Medline]
  6. Davis, C. B., Killeen, N., Crooks, M. E., Raulet, D. and Littman, D. R. 1993. Evidence for a stochastic mechanism in the differentiation of mature subsets of T lymphocytes. Cell 73:237.[ISI][Medline]
  7. Corbella, P., Moskophidis, D., Spanopoulou, E., Mamalaki, C., Tolaini, M., Itano, A., Lans, D., Baltimore, D., Robey, E. and Kioussis, D. 1994. Functional commitment to helper T cell lineage precedes positive selection and is independent of T cell receptor MHC specificity. Immunity 1:269.[ISI][Medline]
  8. Matechak, E. O., Killeen, N., Hedrick, S. M. and Fowlkes, B. J. 1996. MHC class II-specific T cells can develop in the CD8 lineage when CD4 is absent. Immunity 4:337.[ISI][Medline]
  9. Rothenberg, E. V., Chen, D., Diamond, R. A., Dohadwala, M., Novak, T. J., White, P. M. and Yang-Snyder, J. A. 1991. Acquisition of mature functional responsiveness in T cells: programming for function via signaling. Adv. Exp. Med. Biol. 292:71.[Medline]
  10. Brown, G. R., McGuire, M. J. and Thiele, D. L. 1993. Dipeptidyl peptidase I is enriched in granules of in vitro- and in vivo-activated cytotoxic T lymphocytes. J. Immunol. 150:4733.[Abstract/Free Full Text]
  11. McGuire, M. J., Lipsky, P. E. and Thiele, D. L. 1997. Cloning and characterization of the cDNA encoding mouse dipeptidyl peptidase I (cathepsin C). Biochim. Biophys. Acta 1351:267.[ISI][Medline]
  12. Pham, C. T. N., Armstrong, R. J., Zimonjic, D. B., Popescu, N. C., Payan, D. G. and Ley, T. J. 1997. Molecular cloning, chromosomal localization, and expression of murine dipeptidyl peptidase I. J. Biol. Chem. 272:10695.[Abstract/Free Full Text]
  13. Pham, C. T. and Ley, T. J. 1999. Dipeptidyl peptidase I is required for the processing and activation of granzymes A and B in vivo. Proc. Natl Acad. Sci. USA 96:8627.[Abstract/Free Full Text]
  14. Mabee, C. L., McGuire, M. J. and Thiele, D. L. 1998. Dipeptidyl peptidase I and granzyme A are coordinately expressed during CD8+ T cell development and differentiation. J. Immunol. 160:5880.[Abstract/Free Full Text]
  15. Thiele, D. L., McGuire, M. J. and Lipsky, P. E. 1997. A selective inhibitor of dipeptidyl peptidase I impairs generation of CD8+ T cell cytotoxic effector function. J. Immunol. 158:5200.[Abstract]
  16. Linnevers, C., Smeekens, S. P. and Bromme, D. 1997. Human cathepsin W, a putative cysteine protease predominantly expressed in CD8+ T-lymphocytes. FEBS Lett. 405:253.[ISI][Medline]
  17. Wex, T., Levy, B., Smeekens, S. P., Ansorge, S., Desnick, R. J. and Bromme, D. 1998. Genomic structure, chromosomal localization, and expression of human cathepsin W. Biochem. Biophys. Res. Commun. 248:255.[ISI][Medline]
  18. Koller, B. H., Marrack, P., Kappler, J. W. and Smithies, O. 1990. Normal development of mice deficient in ß2M, MHC class I proteins, and CD8+ T cells. Science 248:1227.[ISI][Medline]
  19. Punt, J. A., Suzuki, H., Granger, L. G., Sharrow, S. O. and Singer, A. 1996. Lineage commitment in the thymus: only the most differentiated (TCRhibcl-2hi) subset of CD4+CD8+ thymocytes has selectively terminated CD4 or CD8 synthesis. J. Exp. Med. 184:2091.[Abstract/Free Full Text]
  20. Suzuki, H., Punt, J. A., Granger, L. G. and Singer, A. 1995. Asymmetric signaling requirements for thymocyte commitment to the CD4+ versus CD8+ T cell lineages: a new perspective on thymic commitment and selection. Immunity 2:413.[ISI][Medline]
  21. Vandekerckhove, B. A., Barcena, A., Schols, D., Mohan-Peterson, S., Spits, H. and Roncarolo, M. G. 1994. In vivo cytokine expression in the thymus. CD3high human thymocytes are activated and already functionally differentiated in helper and cytotoxic cells. J. Immunol. 152:1738.[Abstract/Free Full Text]
  22. Wang, H., Diamond, R. A., Yang-Snyder, J. A. and Rothenberg, E. V. 1998. Precocious expression of T cell functional response genes in vivo in primitive thymocytes before T lineage commitment. Int. Immunol. 10:1623.[Abstract]
  23. Hahn, S., Gehri, R. and Erb, P. 1995. Mechanism and biological significance of CD4-mediated cytotoxicity. Immunol. Rev. 146:57.[ISI][Medline]
  24. Sawada, S., Scarborough, J. D., Killeen, N. and Littman, D. R. 1994. A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development. Cell 77:917.[ISI][Medline]
  25. Siu, G., Wurster, A. L., Duncan, D. D., Soliman, T. M. and Hedrick, S. M. 1994. A transcriptional silencer controls the developmental expression of the CD4 gene. EMBO J. 13:3570.[Abstract]