Clin. exp. Immunol.

(1988) 74 454-458

MHC Class I and Class I-like gene product expression by malignant T cells: relationships between CD1a, HLA-ABC and f2-microglobulin
R. A. JONES, C. S. SCOTT, F. E. KATZ* & J. A. CHILDt Department of Haematology, Cookridge Hospital, Leeds. *Department of Haematology/Oncology, Great Ormond St. Hospital, London and tDepartment of Haematology, Leeds General Infirmary, Leeds.

(Acceptedfor publication 29 July 1988)

SUMMARY light chain of the MHC-encoded HLA-ABC and the invariant the forms Beta2-microglobulin (f12m) non-MHC-encoded CDI molecules. While HLA-ABC (MHC Class I) molecules are virtually ubiquitious in tissue distribution, CD1 determinants by contrast are more restricted. We have assessed, by indirect immunoenzymeassay, the relative membrane densities of these molecules on malignant thymic and post-thymic T cells. It was found that the T cells of mature post-thymic proliferations expressed significantly more f2m-associated protein, predominantly HLA-ABC in nature, than thymic-ALL blasts. This parallels the situation found in normal peripheral T cells and thymocytes. In contrast to post-thymic T cells, thymic-ALL blasts showed considerable case to case variation with respect to non-HLA-associated f2m and, of particular interest, not all of this excess 132m could be accounted for by CD 1a. We therefore conclude that other f32m-containing molecules may be expressed on thymic-ALL blasts and possibly also on post-thymic leukaemic T cells. In addition, it was found that T cells from CD4+ cases of post-thymic proliferations expressed more &2m-associated determinants than other T cells, whether of either normal or malignant origin, and that certain post-thymic malignancies express significantly increased levels of f2m-associated protein relative to normal peripheral T-cells. This is in direct contrast to the situation seen in many solid malignancies. Keywords T cell leukaemia CDlI32m HLA-ABC

INTRODUCTION
The 12 kD protein beta2-microglobulin (f32m) constitutes the light chain of several structurally related glycoproteins. The most widespread of these are the MHC-encoded 45 kD HLAABC (MHC Class I) molecules (Cresswell et al., 1974; Peterson, Rask & Lindblom, 1974). Three other J32m-containing HLA-like glycoproteins of 43-49 kD form the first cluster of differentiation (CDla, CDlb and CDlc) and are not MHC-encoded (McMichael & Gotch, 1987; Milsten et al., 1987). Functionally, HLA-ABC molecules are involved in MHCrestricted T cell recognition events (Zinkernagel & Doherty, 1979; McMichael, 1980) and are expressed on the surface of most nucleated cells (Daar et al, 1984; Natali et al., 1984). CD1 molecules, by contrast, have restricted tissue distribution and it has been suggested that they may be homologues of murine TI and/or Qa antigens, defining functionally discrete lymphocyte
Correspondence: R. A. Jones, Department of Haematology, Cookridge Hospital, Leeds LS16 6QB, UK.

populations (Flaherty, 1981; Knowles, 1984). The CDla antigen is expressed on thymocytes in reciprocal quantity to HLAABC (Jones, Scott & Child, 1988), and is also expressed on dermal dendrocytes and the blasts from a proportion of cases of thymic-acute lymphoblastic leukaemia (ALL) (Boumsell et al., 1986), and may function as a receptor for extracellular /2m (Kefford et al., 1984).

It is well documented that malignant cells in some solid tumours exhibit defective expression of f2m-associated determinants compared with their normal counterparts (Fleming et al., 1981; Turbitt & Mackie, 1981; Natali et al., 1984), but little is known about the situation in lymphoid malignancies. In a previous report, we detailed the expression of 12m, HLA-ABC and CD1 a antigens by normal thymic and peripheral blood T cell subpopulations (Jones, Scott & Child, 1988) as part of a wider investigation into the expression and secretion of f2m by lymphoid tissue. We have now extended this investigation to include the T cell malignancies, in which 32m, HLA-ABC and CD 1 a antigen expression was examined, as assessed by indirect immunoenzymeassay, by the lymphoid cells from 39 cases of T cell malignancy.

454

Expression of f2m-associated molecules on T cells

Table 1. Antibodies used

455

Antibody
B2.62.2 W6/32 NA1/34 HRPx-ramlgt
*

Specificity

Subclass

Source

Reference

IgG 1 HLA-ABC core IgG 2a CDIa antigen IgG 2a Mouse Ig Polyclonal

fl2m

ARS* Liebeufet al 1981 Serotec Barnstable et al 1978 Serotec McMichael et al 1979 Dako -

kindly donated by Dr A.R. Sanderson. t Horseradish peroxidase conjugated rabbit anti-mouse Ig.
Table 2. Classification of TdT- post-thymic disorders

Disorder CD3 CD4

T-PLL V

CD5
+ + + +

CD7
+ +

CD8
V

CD16 CD25
V V V

T-NHL SS ATLL LDGL

+ + + +

+ +(-) + +
-

-(+) +

washing away unbound second antibody, the cells were assayed for bound peroxidase using 2 1 mm o-phenylenediamine in citrate/phosphate buffer pH 5*0 containing hydrogen peroxide at 2 9 mm. The rate of change in absorbance at 492 nm was determined and peroxidase activities expressed as U/106 cells, where 1 U was defined as a rate of change in A492 of 0 01 /min
under standard assay conditions.
Statistical analysis To test the significance of difference between two populations the Mann-Whitney u-test was used, or for paired data, the Wilcoxon matched pairs signed-ranks test. Correlation analysis was performed using Spearman's rank correlation coefficient (rj). All the above tests are suitable for non-parametric data.

+ V

-(+)-(+)

Cases were classified by combined morphological and immunological characteristics.

MATERIALS AND METHODS T cell malignancies A total of 39 T cell malignancies were examined, classified as either TdT+ leukaemias (thymic-ALL; n=20) or TdT- postthymic disorders (n = 19). Diagnoses were established according to standard immunological criteria. The post-thymic disorders were subdivided, as detailed in Table 2, into T prolymphocytic leukaemia (T-PLL; n = 3), T cell non-Hodgkin's lymphoma (T-NHL; n = 4), adult T cell leukaemia/lymphoma (ATLL; n = 1), Sezary Syndrome (SS; n = 4) and lymphoproliferative disease of granular lymphocytes (LDGL; n = 7). Of this latter group, all cases had large granular lymphocyte (LGL) morphology, and the T cells from four out of seven cases were CD 16 (Leu I Ib) +. Peripheral blood or bone marrow mononuclear cells were isolated using density gradient centrifugation (Lymphoprep; Nyegaard), and resuspended in HBSS/BSA. T cell components exceeded 85% in each case, as determined by multiple criteria, with at least one of the following applicable: indirect rosetting with monoclonal antibodies against T cell determinants (MacKarill et al., 1987), nuclear TdT expression, focal acid phosphatase reaction (I case), or peripheral leucocyte count in excess of 100 x 106/ml coupled with CD14 and CD19 determinants < 5% (2 cases). HLA-Dr (la) was also determined in each case, and considered positive when > 30% HLA-Dr+ cells were present. Quantitation of cell surface f32m-associated membrane determinants by indirect saturation binding assay
This method is described in detail elsewhere (Jones, Scott & Child, 1988) but, briefly, consisted of incubating test cells with monoclonal antibodies at saturation, removal of the unbound component by washing, and resuspending the cells in saturating concentrations of HRPx-labelled rabbit anti-mouse IgG. After

RESULTS

Cell-surface fl2m and HLA-ABC antigen expression was determined by immunoenzymeassay on the lymphoid cells from 39 cases of T cell malignancy, and CDla antigen expression was also determined in 25 of these. In a replicate study (n = 20), estimation of /32m:HLA-ABC ratio gave a value for 1 s.d. of 0 08, and therefore cases with ratios greater than s.d. above unit (i.e. > 1 16: 1) were considered significantly increased. The twostage determination of cell-surface f2m density gave a coefficient of variation (CV) of 12 8%. Mean t2m and HLA-ABC values for the TdT+ thymic-ALL blasts (n = 20) were 17 6 (s.e. 2 6) and 14 9 (s.e. 2-2)) U/106 cells respectively (Table 3). In contrast, TdT- leukaemic T cells from post-thymic disorders (n = 19) (Table 4) expressed significantly more (P<0 001) f32m and HLA-ABC determinants, with mean values of 52 9 (s.e. 6 2) and 50 6 (s.e. 6-4) U/ 106 cells respectively. Comparison by Wilcoxon matched pairs test showed that thymic-ALL blasts expressed significantly more (P < 0-0) fJ2m than HLA-ABC. There was no significant difference between f2m and HLA-ABC expression for the T cells from post-thymic disorders, however. This is further illustrated by the finding of significantly different (P <005) mean th2M: HLA-ABC ratios for thymic-ALL blasts (mean 1-26:1; range 0-84:1 to 1-83:1) compared to post-thymic leukaemic T cells (mean 1 06: 1; range 0-87: 1 to 1 26: 1). These two sets ofdata indicate that other f2mcontaining molecules in addition to HLA-ABC are present on the blasts from some cases of thymic-ALL. In this context, it was apparent that CD 1 a expression, as a function of the total t2M component, was significantly higher (P < 0 001) on thymic-ALL blasts (mean 0-14, s.e. 0-05) compared to post-thymic leukaemic T cells (mean 0-01, s.e. 0-0). Specifically, on the blasts from six out of 14 cases of thymic-ALL, proportionate CDI a expression

456

R. A. Jones et al.
Table 4. Cell-surface I?2m, HLA-ABC and CD la expression TdT- postthymic T-cells
Ia
/2m

was equal to or in excess of 0 1, and of these, four exhibited 12m:HLA-ABC ratios in excess of 1 16: 1. In the post-thymic leukaemic T cells, by contrast, proportionate CD la expression in excess of 0-1 was seen in none of I I cases, and fl2m: HLAABC ratio above [ 16: 1 was seen in one out of 1 1. This apparent correlation between proportionate CDla expression and nonHLA f2m was tested by Spearman's rank correlation analysis and found to be significant at P < 0 05 (r, = 0 44). It can be seen however that in many cases of thymic-ALL and post-thymic proliferations, the apparent excess 32m expression is not wholly accounted for by expression of the CD 1 a antigen, possible reasons for which are discussed below. No significant difference existed between f2m expression by the leukaemic T cells from HLA-Dr+ (mean 54 2, s.e. 10 1 U/106 cells; n= 10) compared to HLA-DR- (mean 51 5, s.e. 7-4 U/106 cells; n =9) post-thymic disorders. However, when comparing these disorders as a function of CD4 or CD8 expression, T cells from the CD4+ group were found to express significantly higher (P < 0 025) levels of f2m (mean 64 3, s.e. 9 7 U/106 cells; n= 10) compared to those from the CD8 + group (mean 41 1, s.e. 5-8 U/ 106 cells; n = 8). Comparing the data from this study with that from the study of normal T cells (Jones, Scott & Child, 1988; data summarised
Table 3. Cell-surface

HLA-ABC CD1a

fi2m:HLA* 103+004 1-03 +0 05

1-08 0-94 1-05 1-18 1-18 1-15 0-87 1 13 1-04 0-92

Normal PB T cellst (mean + s.e.) - 22-2+1-2 21-8+1-3 CD4+830-4+ 1-5 30-2+2-0 CD-8+

ND ND
1-0 (0-01) ND 0-7 (0-0) ND 2 5 (005) 0-2 (0 0) ND ND 1 0 (0 02) 0 0 (0 0)

CD4+ post-thymic T cells

21 22 23 24 25 26 27 28 29 30 T-PLL
T-PLL

SS Ss Ss Ss T-NHL T-NHL T-NHL ATLL

79000 32-0 L300 4 46-0 47-5 4 41 5 78-0 465 465 96-0

730 34-0 123-5 39-0 400 36-0 900 41 0 44.5 104-5

CD8+ post-thymic T cellsl T-NHL - 28-0 31

32 33 34 35 36 37 38 39 LDGL LDGL LDGL LDGL LDGL LDGL LDGL T-PLL
-

27-5
59-0 29-0 44-0 28 5 38-0 25 5 51 0 34-0

+
+ + +
-

f32m, HLA-ABC and CDla expression by TdT+

thymic ALL blasts
HLA-ABC

74-0 290 425 330 400 27-0 55-0 34-0

0-6 (0 02) ND ND 0-7 (0-01) ND 0 0 (0 0) 0-2 (0 1) 1-7 (0-03) ND

1-02 1-25 1-00 0-97 1-16 1-05 1-06 1 08 1-00

f2m
Normal

CDla
15 7 + 1-9 05+0-2 0-7 (0-07) 0 1 (0 02) 0 7 (0-04) 10 (003) ND 0 8 (0-07) ND ND 1-7 (0 11) 1 1 (0-07) 1-0 (0-13) 0 0 (0 0) 0-2 (0-03) 4-7 (0-1) 1 5 (0 23) 20-0 (0 58) ND 9-6 (0-51) ND ND

f2m:HLA*
Results are relative fi2m, HLA-ANC and CDla antigen densities, shown as enzyme (peroxidase) units/106 cells (mean of duplicate assays). Figures in parentheses indicate CD I a expression as a proportion of total fl2m expression. ND not determined. *#2m: HLA-ABC ratio tCell-surface (J2m and HLA-ABC expression by normal CD4+8(n = 10) and CD4- 8 + (n = 10) peripheral blood T-cells as determined previously (Jones, Scott & Child, 1988). 1 LDGL cases numbered 33, 34, 36 and 38 were CD16 (leu I1 b)+ Co-expressed both CD4 and CD8 determinants.

thymocytesf (mean+ s.e.) 1 7+04 16-4+2-0 CDla+

CDla17-6+1-7

16-0+1-9

Malignant thymic-ALL blasts 125 10-5 Case 1 7-0 65 2 21-0 19-5 3 38-0 4 36-5 26-0 5 26-0 11 0 6 11-5 25-0 26-0 7 14-0 15-5 8 14-0 16-0 9 14 5 10 16-5 65 11 7.5
12 13 14 15 16 17 18 19 20
22-0 7.5 47-5 6-5 34 5 5-0 19-0 7-0 110
18 5 60 34 0 4.5 22-0 30

0-84 0-93 0-93 0-96 1-00 1-04 1-04 1I11 1-14 1 14 1-15 1-19 1-25 1-40
1-44

11-0
40 6-0

1-57 1-67 1-73 1-75 1-83

in Tables 3 and 4), we found that ti2m expression by TdT+ CDla+ thymic-ALL blasts (mean 22 8, s.e. 4 7; n=9) was not significantly different to that of TdT+ CDla+ normal thymocytes (mean 16 4, s.e. 2 0; n = 14). Similarly, there was o significant difference between the level of expression on T cells from CD8+ post-thymic disorders (mean 41 1, s.e. 5 8; n=8) and the normal peripheral CD4-8+ subpopulation (mean 30 4, s.e. 1 5; n = 10). However, when the level of,2m expression by T cells from CD4+ post-thymic disorders (mean 64 3, s.e. 9 7; n = 10) was compared to that of the normal peripheral CD4+8subpopulation (mean 22 2, s.e. 1-2; n= 10), significantly higher (P < 0-001) expression was found.
DISCUSSION This study is an extension of a previous investigation (Jones, Scott & Child, 1988) which examined the expression of /2m, HLA-ABC and CDla antigens on monoclonal antibodydefined normal thymic and peripheral blood T cell subpopulations. In the present study of 39 cases of T cell malignancy, we found that the expression of fl2m-associated protein was

Results are relative f32m, HLA-ABC and CDla antigen densities, shown as enzyme (peroxidase) units/106 cells (mean of duplicate assays). Figures in parentheses indicate CD I a expression as a proportion of total /32m expression. ND not determined. *fl2m:HLA-ABC ratio. tCell-surface fl2m, HLA-ABC and CDla expression by normal CDla + (n = 14) and CDIa- (n = l1) thymocytes as determined previously (Jones, Scott & Child, 1988).

Expression of f32m-associated molecules on T cells

significantly higher on the T cells from post-thymic disorders compared with thymic-ALL blasts. This parallels the situation in normal lymphoid tissue, where /2m expression by peripheral T cells is significantly higher than that of thymocytes (Jones, Scott & Child, 1988). It has been proposed that T-CLL (designated LDGL in this study; Pandolfi, 1986) and Sezary cells correspond, in terms of immunological differentiation, to subsets of normal peripheral T cells (Boumsell et al., 1981). Mean fi2m expression by the T cells from these subgroups (n = 11) was 51-4 (s.e. 88) U/106 cells, which was significantly higher (P<0 001) than mean f2m expression by the cells from normal CD4+ and CD8+ subpopulations (mean 26-3, s.e. 1 3). We have also shown that leukaemic TdT- CD4+ T cells express significantly more f2m-associated protein than leukaemic TdT -CD8+ T cells. This is an interesting observation and is the reverse of the situation in normal lymphoid tissue, where i2m expression by the peripheral blood CD4- 8 + subpopulation is significantly higher (P < 0-O01) than the CD4+8- subpopulation (Jones, Scott & Child, 1988). It is apparent that post-thymic CD4+ leukaemic T cells express significantly more f2m-associated molecules than all other types of normal or malignant T cell. We are not aware of the precise significance of this increased level of expression of fl2m-associated molecules, relative to normal peripheral T cells, in certain T lineage lymphoid proliferations, but it clearly contrasts with the situation seen in many solid tumours where progression to malignancy may be associated with decreased cell-surface MHC Class I antigens (Fleming et al., 1981; Turbitt & Mackie, 1981; Natali et al., 1984), a process which may facilitate evasion of T cell cytotoxicity (Festenstein & Schmit, 1981; Gooding, 1982). It has recently been reported, however, that susceptibility to NK activity varies inversely with HLA-ABC expression on the target cell (HaralBellan et al., 1986), and therefore cells which evade T cell cytotoxicity by virtue of reduced MHC Class I antigen expression may become targets for the natural killer cell mediated pathway. The clinical significance of these findings requires further investigation. A previous study (Hokland et al., 1982) reported high density membrane f2m (but with no corresponding increase in HLA-ABC) in 25% of peripheral lymphocytes, and this appeared to correlate with natural killer (NK) activity. The majority of NK cells are associated with the characteristic LGL morphology and label with CD 16 antibodies (Lanier, Phillips & Warner, 1986), and it can be seen from Table 4 that the T cells from the four cases of CD 16 + LDGL do not have an apparent increase in either fl2m expression or fl2m:HLA-ABC ratio, relative to normal peripheral T cells. We are currently examining the normal peripheral CD4-8-14-19- subpopulation, which contains a large CD16+ component, to determine the level of expression of f2m-associated molecules. No significant difference was detected in the level of expression of tiM-associated determinants between T cells from 'activated' (HLA-Dr+) and 'non-activated' (HLA-Dr-) stages of post-thymic disease, suggesting that the level of expression of these molecules is not activation-related. This is in contrast to the situation in vitro, whereby mitogenic stimulation of peripheral T cells induces a marked increase in the expression of 32massociated protein (Dorval et al., 1977) in parallel with HLA-Dr induction (Accolla, Moretta & Carrel, 1984).

457

We could detect no significant difference in the level of expression of t2M-associated molecules between TdT+CDla+ thymic-ALL blasts and TdT+CDla+ thymocytes. However, t2m expression in these latter cells is almost exclusively CD 1 aassociated, with only a very minor HLA-ABC component, whereas TdT+CDla+ thymic-ALL blasts co-express both CDla and HLA-ABC, and may therefore represent a different stage of differentiation to the TdT+CDla+HLA-ABC- thymocyte (Bradstock et al., 1980). We could not compare the expression of th2M-associated protein by the TdT+ CDlathymic-ALL blasts with their corresponding normal thymocyte phenotypes, since CDla- thymocytes comprised less than 10% of the whole thymocyte fraction, and of these, about 90% were also TdT-, representing a phenotype more closely corresponding to that of the mature peripheral T cell. By examining the ratio of fl2m to HLA-ABC, it is possible to assess the potential expression of non HLA-ABC molecules. We have shown that excess (non-HLA-associated) fl2m is present in significant quantity on the surface of blasts from some cases of thymicALL, and that there is a correlation between this excess, as determined by fi2m:HLA-ABC ratio, and CDla expression (as a proportion of total fl2m). In some of these cases, however, the apparent non-HLA-ABC associated f32m is not wholly accounted for by CD 1 a expression, and it is therefore possible that other th2M-containing molecules are present. In this respect, four distinct f2m-containing gp43-49,12 surface antigens other than HLA-ABC have been identified (Knowles & Bodmer, 1982; Olive, Dubreuil & Mawas, 1984; Amiot et al., 1986) three of which have since been ascribed to the CD1 cluster (McMichael & Gotch, 1987). It is likely that some of these molecules in addition to CD I a may be present on the T cells from thymic and possibly also post-thymic disorders and may therefore account for the apparent non-HLA and non-CDla-associated 32m. We are currently examining the expression of other t2M-associated molecules in malignant B and T cells. In conclusion therefore, we have shown that TdT- leukaemic T cells express significantly more t2M-associated determinants than TdT+ thymic-ALL blasts, which parallels the situation in normal lymphoid tissue. No significantly different level of expression of these molecules was detected between thymic-ALL blasts and thymocytes, or between TdT-CD8+ leukaemic T cells and CD4-8+ normal peripheral T cells. We did however detect significantly elevated expression in TdT-CD4+ leukaemic T cells compared to peripheral CD4+8-T cells and TdT-CD8+ leukaemic T cells, and also in the T cells from the Sezary syndrome and LDGL group compared to the normal (phenotypically similar) peripheral T cell. Finally, we have demonstrated a correlation between apparent non-HLA-ABC associated fl2m and CDla expression, and also found evidence pointing to an additional f2mcontaining component.

ACKNOWLEDGMENTS
We wish to express our gratitude to Dr Dario Campana, Dept. of Immunology, Royal Free Hospital, London, and the Clinical Haematologists of the Yorkshire Region for supplying much of the material, and Dr A. R. Sanderson for supplying the anti-fl2m antibody. This work was supported by the Friends of the Leukaemia Unit, and the Special Trustees of the General Infirmary, Leeds.

458
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