Immunology Today April 1984

Thymocytes are morphologically homo- geneous upon microscopic examination but functionally diverse, so the develop- ment of monoclonal antibodies that recognize different T-cell subsets and the advent of the fluorescence-activated cell sorter have contributed immeasurably to the current view of thymocyte hetero- geneity. These techniques have now been combined with quantitative assays of T-lymphocyte function that allow the discrimination of large differences in the frequency of functional cells within dif- ferent separated subsets.

There are now monoclonal reagents that recognize on certain mouse T cells the equivalent of the human T-cell anti- gen (T4). Termed L3T4, the antigen is found on M H C class-II-restricted T cells. Together with Lyt 2,3, an antigen found on M H C class-I-restricted T cells (the murine equivalent of T8), it segre- gates thymocytes into at least five sub- populations (K. Shortman, Melbourne; R. MacDonald, Lausanne; B. Mathie- son, Bethesda). Cells presumed to have newly arrived from the bone marrow appear in the subcapsular thymic cortex, account for 3% of thymocytes, and do not express Lyt 2, L3T4 or M H C anti- gens, although they have a high density of Thy 1 and a low density of Lyt 1 (R. Ceredig, Lausanne; R. Scollay, Melbourne; Shortman; Mathieson). These cells are blastoid and are in the proliferative phases of the cell cycle. Several lines of evidence suggest that they are thymic stem cells. A proportion of them differentiate in vitro to express both Lyt 2 and L3T4 (Ceredig). In addi- tion, when purified and injected back into irradiated congenic mice, these cells can ,give rise to all of the other subpopula- tions normally found in the thymus (B. J. Fowlkes, Bethesda). Of the remaining cells in the thymus, most (80-85%) express both L3T4 and Lyt 2. These cells, which comprise mo.~t of the cortical thymocytes, when examin~.d by limiting dilution analysis do not produce inter- leukin 2 (IL2) or become cytoutic T cells in response to alloantigen (Ceredig) or lectin (Shortman and Scollay). They can be separated into two subpopulations on

Inside the thymus from Kendall Smith

The thymus has fascinated immunologists since the discovery that it influenced the maturation ofT lymphocytes. However, only within the past five years have investigations of the thymus progressed beyond descriptive histology or functional studies performed at the population level. Our closeness to an understand- ing of the raison d'etre of the thymus was discussed at a recent meeting*.

the basis of size (25 % are large Mastoid cells and 75 % are small). Based on iso- topic precursor studies of DNA synthesis in vivo and cytofluorometric analysis of DNA content it is estimated that only the large L3T4 +, Lyt 2 + cells are actively proliferating (R. Scollay; Ceredig; Shortman). In addition, it is estimated that most of these cells are destined to die in situ so that only a small proportion actually may become functionally mature medullary thymocytes (Scollay).

The remaining thymocytes (15 %) are found in the medulla and are the classic 'mature' thymocytes as previously defin-

* A w o r k s h o p o n t h y m u s f u n c t i o n w as h e l d at the Base l I n s t i t u t e for I m m u n o l o g y , o n 1 7 - 1 8 N o v e m b e r 1983.

ed by functional tests (resistance to lysis by cortisone in vivo and lack of reactivity with peanut agglutinin). Through the use of the L3T4 and Lyt 2 markers, to- gether with limiting dilution analysis, these cells can be separated into two sub- populations: 2/3 are L3T4 +, Lyt 2 - and contain all of the ceils capable of pro- dueing IL2; 1/3 are L3T4 -, Lyt 2 + and comprise all of the ceils Capable of be- coming eytolytic cells (Ceredig). The cells comprising both of these subpopula- tions are somewhat larger than peri- pheral T cells and thus are morphologic- ally distinct from those cells that presum- ably have left the thymus. Moreover, these cells are in the G0/G ~ phase of the

cells in the absence of antigenic stimula- tion.

M. Davis (Stanford) provided data which a//ow one to propose a scheme for thymocyte maturation based upon the T-cell antigen receptor. By classic hybrid subtraction techniques, several T-cell- specific eDNA clones have been isolated that appear to represent the genes coding for one of the chains of the T-cell antigen receptor (see box on p. 84). An observation of considerable interest to theories of thymocyte development is that some of the nucleotide sequences isolated from thymocyte-thymoma T-cell hybrids contain stop codons within coding regions. Thus, it can be postulated that within the thymus the genetic rearrangements necessary for the formation of competent T-cell antigen receptors occur. In a fashion analogous to immunoglobulin gene expression, if a productive genetic change does not take place, transcription and translation of the membrane receptor cannot occur, thus leading to the cell's demise. This concept would fit the functional data showing that only the phenotypically mature medullary thymocytes can respond to signals presumed to trigger the T-cell antigen receptor (i.e. lectin and alloantigen).

The definitive demonstration of the T-cell antigen receptor on the different thymocyte subpopulations must await future studies. However, the functional

© 1984, Elsevier Science Publishers B.V., Amsterdam 0167 4919/84/502.00

For technical reasons we are unable to reproduce these figures in colour in this edition--see the April issue of Immunolagy Today for full colour illustration.

84

tests suggest that only the mature medul- lary thymocytes express the receptor that recognizes ant igen and self M H C . Moreover , studies by Reinherz and co- workers indicate that T3, the 23 000 mol. wt ant igen that is non-covalently linked to the h u m a n T-cell ant igen receptor, is only found on ' m a t u r e ' medul lary thymocytes. Consequent ly , we m a y have to search for other struc- tures, cells or factors which p rog ram the discr iminat ion between self and non-self that has long been thought to occur within the thymus . T he clue, perhaps, m a y be found in the support ing cells of the t hymus . Monoclonal antibodies directed toward M H C - d e r i v e d ant igens and other tissue-specific ant igens have permit ted a view of the un ique character- istics of thymic support ing cells. E. J . J enk inson (Bi rmingham, U K ) presen- ted revealing immunohis tochemica l studies showing that the suppor t ing cells of the thymic cortex express a h igh den- sity of dass- I I M H C antigens. In con- trast, the medul lary support ing ceils ex- press a h igh density o fdass - I M H C gene products bu t not class-II. Addit ional evi- dence for the distinct na ture of cortical and medul lary epi thel ium was provided by W. V o n Ewijk (Rot terdam) who des- cribed a new monoclonal ant ibody which dear ly dist inguishes between the two. Even more provocatively, a new mono- clonal ant ibody that appears specific for neuroendocr ine cells stains a large n u m b e r of support ing cells that are pre- sent at the corticomeduilary junct ion. The functional implications, if any, of the strict anatomical compar tmental iza- tion of these thymic suppor t ing cells re- mains to be determined. However , any

model that a t tempts to explain the na tu re of the thymocytes populat ing these areas of the t h y m u s m u s t include a functional role for these suppor t ing ceUs.

O n e would hope that soluble factors might be identified that direct the pro- liferation and ma tu ra t ion of thymocytes . It is clear that IL2 is not involved in thymocyte proliferation, since thymo- cytes do not express IL2 receptors. It is somewhat disappoint ing that none of the previously identified thymic ho rmones can be shown to have any effects on the proliferation or matura t ion of thymo- cytes to become functionally reactive ceils when tested by limiting dilution analysis (Shor tman) . As antibodies become available that are reactive to c o m m o n epitopes on the T-cell ant igen receptors, it would appear that a major effort should be expended to identify sources of factors that promote the appearance o f these receptors.

A n y model that a t tempts to explain the functional role of the t h y m u s m u s t take into considerat ion the fact that the organ is of max ima l size in childhood and involutes at puberty. Studies of cell migra t ion indicate that 1% of the total thymocytes enter and leave the t h y m u s daily (Scollay). O f those cells that popu- late the peripheral lymphoid tissue, most studies indicate that the life span o f T cells is extremely long, perhaps the entire life of the o rgan i sm (J. Sprent , LaJol la) . Moreover , expansion of the total peripheral T-cell mass appears to be largely ant igen dependent , since germ- free an imals have only 10% of the T-cell mass found in no rma l animals , despite an intact funct ioning thymus . Conse- quent ly , a picture emerges of the t h y m u s

Immunology Today, ont. 5, No. 4, 1984

as a developmental organ that is active dur ing the growth period of the animal and ceases to funct ion when adult stature is achieved. Therefore , if one assumes that T cells leaving the t h y m u s survive for the life span o f the animal , then the cells that are processed by the t h y m u s dur ing embryonic and early develop- menta l life m a y very well make up the total T-cell repertoire of the adult. Expans ion of this repertoire would .then depend upon ant igenic s t imulat ion th roughout life.

It could be postulated on the basis of these da ta that an identifiable organ is necessary for the matura t ion of T cells because these cells are primari ly produced only dur ing the actively grow- ing stages of the organism. In contrast, all other hematopoiet ic cells have a finite life span of hours or days and are produc- ed by the bone mar row th roughout life. Accordingly, the t h y m u s might be a more efficient sys tem for the expansion and selection of functionally ma tu r e T cells. Th i s concept would fit the observa- tions that functionally reactive T cells are present in a thymic mice bu t with a m u c h lower f requency than in no rma l mice (MacDonald ; T . Hfinig, Wfirzburg). T h e knowledge that such T cet!s increase in f requency in a thymic mice throughout life (MacDonald) supports the impres- sion that the t h y m u s serves quanti ta- tively to expand the peripheral T-cell mas s and that T cells can ma tu re to func- tionally reactive cells expressing compe- tent T-cell ant igen receptors in the ab- sence of thymic influence.

Kendall A. Smith is in the Department oJ Medicine, Dartmouth Medical School, Hanover, N H 03756, USA.

A gene

These data, (see p. 83, column 3) discussed by Jay Berzofsky (lramunol. Today, 1983, 4,300) were reported by Stephen Hedrick, Mark Davis and colleagues from Stanford in Nature (London) 1984, 308, 149; 153. Remarkably similar findings emerged from an independent study of human cells report- ed in the same issue (p. 145) by Tak Mak and colleagues in Toronto. The Stanford group prepared cDNA copies of RNA from an antigen-specific mouse T-cell hybridoma and removed the sequences common to B cells by hybridization. This T-specific cDNA generated 10 cloned copies of mRNAs that were bound to polysomes--a characteristic expected of a message for a re- ceptor polypeptide. One clone represented a gene that was rearranged only in a lym- phoma and hybridomas of T-cell origin. A receptor gene, like immunoglobulin (Ig) genes, would need such rearrangement to diversify the repertoire of antigen recogni- tion. The amino acid sequences predicted

f o r t h e T c e l l r e c e p t o r f o r a n t i g e n ?

by the cDNA of this clone and three similar clones from thymncytes indicated that the proteins, like Igs, had 5' variable, inter- mediate joining and 3' constant regions of about the same size as Igs. A search of a sequence data bank r~vealed that the most similar sequences were from an HLA-DCI alpha chain and from Igs. The closest match was found within the V H region of the monoclonal antibody 93G7, one of the sequences listed by Renate Dildrop on the centre pages of this issue.

Mak's group first made cDNA clones from a leukemic T-ceU line and screened a selection of them, eventually finding a message that was expressed not in B cells but in T lymphoblasts, thymocytes and PHA-stimulated lymphocytes. The protein deduced from its sequence had a long region of similarity to human and mouse light chains.

In the absence of published functional data both groups are cautious about what

the deduced proteins actually do in T cells. But their size (e 34 000 mol. wt), resem- blance to Igs and the evidence that the gene is rearranged in different T-cen lines strongly suggest that it is part of the antigen receptor, perhaps one chain of a dimer.

For both groups an urgent goal now will be to determine the relationship between these proteins and the clonotypic polypep- tide dimers identified with monoclnnal anti- bodies as surface structures on cloned T cells from man (E. Reinherz et al. lmmunol. Today, 1983, 4, 5) and mouse (J. Allison et al. J. hnmunol. 1982, 129, 2293), Once this is known there will be fresh fuel for the still un- resolved arguments about how the T-cen re- ceptor sees antigen in an MHG-restricted fashion. The cloning data, if confirmed, represent the most important clue obtained so far but one of the central mysteries of the immune response is still not solved.