Expression of CD45 isoforms in lymph node reactive hyperplasia

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

Expression

of CD45 Isoforms in Lymph Node Reactive Hyperplasia CHARLES

Department

of Pathology,

57, 411-419 (1990)

W. CALDWELL

University of Missouri School of Medicine, Columbia, Missouri 65212

1 Hospital

Drive,

The CD45 antigen family consists of multiple molecular isoforms ranging from 180 to 220 kDa. The highest M, isoforms are recognized by monoclonal antibodies (MoAbs) designated CD45RA, while those recognizing the low M, isoforms are designated CD45RO. T cells expressing CD45RA are “naive” or unpruned, while those expressing CD45RO have “memory.” Further, stimulation of CD45RA+ T cells induces an isoform switch to the CMSRA-/CD45RO+ phenotype. The present study examined this in vitro process by determining the in vivo CD45 isoform expression of T cells from human hyperplastic lymph nodes. Hyperplastic, as opposed to nonhyperplastic, lymph nodes exhibited the expected CD45 isoform switch from CD45RA+ to CD45ROf T cells that has been described in vitro. The percentage of CD45ROf T cells did not correlate with other parameters of lymphoid activation. Thus, CD45RO expression probably represents a marker of differentiation and acquisition of “memory” or late cellular activation.

0 1990 Academic

Press, Inc.

INTRODUCTION As reviewed by Thomas (l), CD45, the leukocyte common antigen or T200, consists of a family of glycoproteins with cell type-specific expression. This antigen family is found on all cells of lymphohemopoietic lineage with the exception of mature erythrocytes and platelets. At least four isoforms of M, 18C~220 kDa are produced by differential splicing of three exons within the mRNA (l-4). All four isoforms differ in M,, but share a large cytoplasmic region, transmembrane region, and variable portions of the external domain. The higher M, isoforms have inserts of 47-161 amino acids in the distal external domain and are heavily glycosylated. It is likely that CD45 plays an essential role in transmembrane signaling, perhaps as a result of the cytoplasmic protein tyrosine phosphatase (PTPase) activity (5-9), but the significance of selective isoform expression or isoform switching is not understood. Ostergaard et al. (10) demonstrated that murine CD45 isoforms have equivalent basal PTPase activity associated with the cytoplasmic domain, but this may not necessarily be true of activated cells. Considerable evidence supports the concept that expression of cell-surface CD45RA and CD45RO represent sequential stages of mature T-cell activation and differentiation (11-16). The CD45RA+ subset of T cells appears to be “naive” with respect to antigen exposure, while CD45RO+ T cells have properties of antigen-experienced “memory” cells (11, 14-16). Upon activation in vitro, CD45RAt T cells lose their CD45RA and acquire CD45RO (11, 13-15, 17). This process is associated with a rapid change in mRNA and a much slower loss of surface membrane CD45RA (13). Acquisition of CD45RO occurs only after at 411 0090-1229/90 $1.50 Copyright B 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

412

CHARLES

least one round of mitotic division,

W.

CALDWELL

but once present, remains on the cell surface

(12).

In certain clinical situations associated with immune activation, alterations in the percentage of CD45RA+ T-cell subsets in peripheral blood have been reported (la21). These alterations were initially thought to represent changes in percentages of cells with a stable phenotype, but more recent information has made it clear that such alterations in CD45 isoform expression are a dynamic event-(ll16). In peripheral lymphoid tissue, approximately 60% of T cells normally express CD45RA and ~40% express CD45RO in a predominantly nonoverlapping distribution (16). Double-labeling studies have shown that these subsets are also reciprocal in normal peripheral blood (11, 22, 23). In lymphoid tissues, there is a particular histological distribution of CD45RA+/CD45ROV and CD45RA-/ CD45RO+ subsets (23), thus suggesting a physiological role in cellular activation and/or cellular trafficking. If this experimentally documented sequence of events occurs during T-cell activation and differentiation in vitro has physiologic relevance, then it should be demonstrable in vivo in situations characterized by lymphoid hyperplasia and reactivity. The current study demonstrates this reciprocal relationship between the low and high M, CD45 isoforms on T cells and other immunophenotypic markers of cellular activation in lymphoid hyperplasia. Overall, the situation that was demonstrated in vivo with hyperplastic lymph nodes is that predicted based on previous in vitro laboratory studies. Lymph nodes from those with reactive hyperplasia exhibited increased percentages of CD45ROt T cells (“memory”) with a simultaneous decrease in CD45RA+ T cells (“naive”), but the percentage of CD45ROt T cells did not correlate with other accepted parameters of cellular activation. MATERIALS

AND METHODS

Lymph Node Preparations Cells were obtained from lymph nodes submitted to the flow cytometry laboratory for evaluation of lymphadenopathy (n = 20). Portions of all specimens were also submitted for routine histopathologic examination. In all cases, the specimens included in this study were nonmalignant and demonstrated a polyclonal T- and B-cell reactive hyperplasia. Lymph nodes used as controls (n = 10) were obtained from patients undergoing abdominal or thoracic surgery for correction of noninfectious, nonimmune anatomic pathologies such as traumatic injuries and from patients undergoing cardiac bypass surgery. Histopathologic examination was also performed on these specimens. All specimens were obtained in compliance with regulations of the Institutional Review Board of the University of Missouri School of Medicine. Portions of lymph nodes were mechanically dissociated in RPMI 1640 tissue culture media, large clumps (~40 Frn) were removed by filtrations through nylon mesh, and the remaining cells were resuspended in medium at a concentration of

CD45 ISOFORMS IN REACTIVE

1 x 10’ cells/ml. 2 hr of receipt.

LYMPH

NODES

In all cases, the cells were immunostained

and examined

413 within

Immunostaining and Flow Cytometric Analysis The monoclonal antibodies (MoAbs) employed in this study and their major reactivities relevant to this project are listed in Table 1. All MoAbs were used as direct fluorochrome (fluorescein isothiocyanate (FITC) or phycoerythrin (PE)) conjugates at concentrations previously determined in this laboratory to be saturating for the number of cells examined (24). In the case of single MoAb staining, aliquots of SO ~1 containing 5 x IO5 cells were incubated with 50 ~1 of optimally diluted MoAbs at 4°C for 30 min. For dual staining, 50 pl of PE-conjugated MoAb was added after the first 5 min and incubated an additional 30 min. After incubation, cell suspensions were washed once in cold phosphate-buffered saline (PBS), resuspended in 0.5 ml of cold PBS containing 0.1% sodium azide, and submitted to flow cytometric analysis. These cells were not postfixed in order to obviate the potential loss of CD45RA (2H4) reactivity (25). A Coulter Profile flow cytometer (Coulter Electronics, Hialeah, FL) was used for all analyses. This instrument was operated under standardized conditions for measurement of light scatter and fluorescence as previously described (24). Fluorescence measurements were taken from 10,000 cells from within the “lymphoid” light scatter gates and single- or dual-parameter histograms were generated from the immunostained cells. In all cases, isotypic negative control MoAbs were used to set the positive and negative cutoffs. Percentages of singleand double-positive cells were determined from these cutoffs. Data Analysis The percentages of cells positive for each of the MoAb of interest were tested for correlation by use of the Spearman rank sum test and the one-way analysis of variance. MONOCLONAL

ANTIBODIES

USED IN

TABLE 1 THIS STUDY AND THEIR MAIN REACTIVITIES

CD”

MoAbb

Main Reactivity’

Sourced

CD2 CD4 CD8 CD20 CD45 CD45RA CD45RO N.C.

CCTll CCT4 CCT8 Leu 16 Hle-1 2H4 UCHL 1 HLA-DR

T cells, E-rosette receptor Helper/inducer T cells Suppressor/cytotoxic T cells B cells All known isoforms of CD45 antigen 200- to 220-kDa isoform(s) of CD45 antigen 180-kDa isoform of CD45 antigen Class II MHC antigen

C C C BD BD C D BD

LIClusters of differentiation, as defined by the Fourth International Workshop on Leukocyte Differentiation Antigens (39). NC., not clustered. b The specific antibody used in this study. c Main reactivity as pertinent to this study. d Sources of MoAb: C, Coulter Immunology, Hialeah, FL; BD, Becton-Dickinson, Mountain View, CA; D, Dako, Carpinteria, CA.

414

CHARLES W. CALDWELL

RESULTS Histopathology

The reactive histology included specimens with follicular, diffuse, and mixed patterns, with variable degrees of sinus hyperplasia (26). Although the specific etiology of lymphoid hyperplasia was not known in most of the cases, the lymph nodes were characterized by follicular and/or interfollicular hyperplasia. No evidence of lymphoid or nonlymphoid malignancy was found in any of the lymph nodes. Control lymph nodes contained predominantly primary follicles and occasionally small mildly reactive secondary follicles or germinal centers. In general, these lymph nodes were small and not considered “reactive” by standard eriteria. Single MoAb Positivity

Single-cell suspensionsof the above lymph nodes were prepared and submitted for flow cytometric examination. The results of single-parameter MoAb immunostaining of reactive and control lymph nodes are listed in Table 2. There was generally an increase in the ratio of T to B cells (CD2/CD20), an increased Tcell-associated class II MHC antigen (HLA-DR), and an increased CD4/CD8 ratio in the reactive nodes compared to controls. These are commonly associated with reactive hyperplasia of lymph nodes (27-30). In all cases, the pattern of K and A immunoglobulin light chain staining was superimposable by flow cytometry and there was no suggestion of either T- or B-cell clonal proliferation. Overall, the percentages of lymphoid cells positive for each MoAb were within published ranges (27, 31). The patterns of CD45RA and CD45RO on T cells demonstrated an increased percentage of T cells expressing the low M, isoform, CD45R0, which is associTABLE 2 MoAb POSITIVITY FROMREACTIVEANDCONTROL

CD2 CD20 CD4KD8 ratio CD45ROKD2 CD45RAKD2 HLA-DR/CDZ CDUCD20 ratio

LYMPH NODES

Reactive

Control

60.59 2 3.12” (30-84)b 35.27 t 2.98 (1 l-66) 6.35 -+ 1.51 (1.2-35.0) 62.18 2 4.58 (23-94) 18.41 2 2.94 (l-49) 9.14 ‘- 2.50 (O-51) 2.27 +- 0.36 (0.45-7.56)

54.2 k 2.98 (45-69) 44.7 -c 2.55 (2%59) 3.1 * 0.44 (1.8-4.3) 19.6 ” 1.8 (12-29) 73.4 -t 5.1 (55-83 ) 3.7 f 0.8 (O-6) 1.21 2 0.14 (0.86-1.56)

LIResults are expressed as the mean percentage, 2 the standard deviation, of 10,000 total lymphoid cells from within the light scatter gates. b Range of observations for each percentage positivity.

CD45

ISOFORMS

IN

REACTIVE

LYMPH

415

NODES

ated with memory T cells and a reciprocal decrease in CD45RA, or naive T cells. Dual-color immunostaining of these cells with CD2 (Tll) and CD45RA or CD45RO confirmed the T-cell association of CD45 isoforms. A few cells (~3%) expressed both isoforms, consistent with the existing literature (12, 23). Correlations

between CD45 Isoforms

and Other Indicators

of Activation

In that increased HLA-DR+ T cells, the CD4/CD8 ratio, and the T- to B-cell ratio have been associated with lymphoid hyperplasia (27-31), and the transition of CD45RA to CD45RO on T cells is associated with a change from naive to memory cells (12, 14, 22), it was of interest to determine if these indicators of lymphoid reactivity or cellular activation correlated with CD45RO expression. The percentage of CD45RO+ T cells did not correlate well with the percentage of HLA-DR+ T cells, CD2, the CD4/CD8 ratio, or the ratio of T to B cells (CD2/ CD20). There was a strong reciprocal correlation between the percentage of CD45RO+ and CD45RA’ T cells (-0.804; P < 0.0001). There were also correlations among the percentages of HLA-DR+ T cells, the CD4/CD8 ratio, and the ratio of CD21CD20, which supports the findings of others (27-31). Therefore, it appears that CD45RO expression on T cells is independent of these parameters (Table 3). DISCUSSION

In vitro, the transition of T cells from naive, or unprimed status, to that of memory, or primed cells, involves a change in the isoform of surface CD45 antigens from CD45RA, the high M, isoform, to CD45R0, the low M, isoform (11, 12, 14-16). It is likely that CD45RO+ T cells in lymph nodes represent dividing or recently divided cells since Akbar et al. (12) have shown that the isoform switch occurs only after at least one round of mitotic division of stimulated CD45RA+ T cells. Once these cells have undergone mitosis and express CD45R0, they retain their positivity for this low M, isoform throughout further cell divisions (11). The change from high to low M, CD45 is preceded by a rapid activation in alternative mRNA splicing and a slow membrane turnover of preexistent surface CD45RA (13). At this time, the physiologic relevance of this activation-associated isoform switch is not known. TABLE 3 CORRELATIONMATRIXOFCELLULARPHENOTYPES

-__ CD45RO” CD4SRA HLA-DR CD2 CD4/CD8

CD45RO

CD45RA

HLA-DR

l.ooo

-0.804 l.ooo

0.224 0.039 l.ooo

CD2

CD4/CD8

CD2KD20

- 0.015 0.246 0.371 l.OOQ

0.071 0.099 0.621 0.371 1.000

0.069 0.252 0.586 0.837 0.675

0 The percentages of positivity for CMSRO, CD45RA, and HLA-DR were all obtained from dualparameter staining with CD2 to obtain positivity of each of these markers on T cells. The CD4/CD8 ratio and the CD2KD20 ratio were obtained from single marking for each of these MoAbs.

416

CHARLES

W.

CALDWELL

The CD45 family of glycoproteins is known to possess protein tyrosine phosphatase (PTPase) activity from two repeating subunits within the cytoplasmic tail of the protein, a finding common to all known CD45 isoforms. It is thought that CD45 functions, at least in part, by its ability to dephosphorylate other regulatory proteins (6, 8). Curiously, CD45 itself is autophosphorylated under stimulatory conditions (32). It is not known what, if any, effect phosphorylation at various sites along the cytoplasmic tail region may have on CD45 function as a PTPase. A close relationship exists between CD45 and lck, a member of the src gene family that has tyrosine protein kinase (TPK) activity (8, 10). CD45 must be present on the cell surface for T cells to undergo activation of the lck gene product by dephosphorylation. Under nonstimulatory conditions in viva, members of the src family, such as lck, are heavily phosphorylated on specific tyrosine residues and the TPK activity is low (33,34). However, upon dephosphorylation, as occurs in lymphoid activation, the TPK activity increases and the activated TPK becomes capable of transforming normal cells to unregulated growth and ditferentiation (IO, 35, 36). CD45 is also capable of regulating T-cell activation by its dephosphorylation of lck (10). Why, how, or if a switch in CD45 isoform might alter this process is unclear since it appears that similar basal PTPase activity is present in the various isoforms of murine CD45 under nonstimulatory conditions (10). Similar studies of PTPase activity from CD45 isoforms obtained from stimulated cells might help answer some of these questions. The present study was undertaken to determine if the conversion from CD45RA to CD45RO that occurs in vitro upon stimulation of T cells, and is associated with acquisition of memory (1 I-15, 17), was demonstrable in vivo under presumably stimulatory conditions. If the in vitro observations have physiological relevance, then a similar process should occur during periods of T-cell activation/stimulation and/or T-cell division in vivo. T cells expressing the high and low M, isoforms of CD45 are known to differ in their anatomic distributions (15, 16,22,23). In a study of peripheral lymphoid tissues (23), it was found that a minority (- 1l-25%) of T cells expressed CD45RO (UCHL 1). This study clearly shows that the majority of T cells in hyperplastic lymph nodes have acquired this antigen. Hyperplastic lymph nodes had increased percentages of CD45ROC T cells and decreased percentages of CD45RA+ T cells compared to control specimens. These percentages were reciprocal, and few (~3%) dual CD45RAf/CD45RO+ T cells were found. Such dual-positive cells are thought to result from the fact that CD45RA is not lost until after the first mitosis, while CD45RO first appears in the Golgi zone of cells just prior to cell division, thus producing dual-positive cells (12). It appears that in specimens from patients with lymphadenopathy characterized by a reactive hyperplasia, many of the T cells undergo a conversion from the presumed naive to the memory stage of activation or differentiation, similar to that demonstrated in vitro (1 I-15, 17). However, this process may be dissociated from the presence of other indicators of “activation.” Immunophenotypic findings associated with lymphoid hyperplasia include class II MHC antigen (HLADR) expression by T cells, increased ratios of CD4/CDS, and increased ratios of CD2/CD20 (27-31). These same correlations were found in the present study. Of interest, however, there was no correlation between the percentage of CD45RO+ T

CD45

ISOFORMS

IN

REACTIVE

LYMPH

NODES

417

cells and HLA-DR expression or any of the other findings traditionally associated with activation. It is possible that cellular memory does not correlate with cellular activation states. Clearly, most resting peripheral blood CD45RO+ T cells do not express markers of activation, although a minor population of these do express low levels of HLA-DR and CD25, the IL-2 receptor (12, 37). In vitro, when CD45RO+ T cells are stimulated with mitogen, markers of activation appear before CD45RO and for the most part are in a state of down-regulation before the induction of CD45RO (37). CD45RO+ T cells do express increased levels of some of the molecules known to be associated with cellular adhesion/signal transduction, such as CD58 (ICAM-I), CD29 (4B4), and CD2 (38). It is therefore likely that CD45RO expression by T cells is a marker of differentiation or late activation, although in the process of transition from CD45RA to CD45R0, some stimulation or activation occurs. This antigen only appears on the cell surface after the first round of mitosis (12) and once present, remains on the cell surface even as other markers of activation are lost from the cell (37). One of the common uses of flow cytometry in clinical hematopathology laboratories is in the evaluation of lymphadenopathy, either malignant or nonmalignant. In the context of differential diagnosis, it is helpful to note that the composite findings of increased HLA-DR+ T cells, T- to B-cell ratio, CD4/CD8 ratio, and an inversion of the usual CD45RO to CD45RA ratio are common findings in lymphoid hyperplasia. Occasional specimens will demonstrate a stronger B-cell component, with a decreased CD21CD20 ratio. Clearly, most non-Hodgkin’s lymphomas are of B-cell type and thus express predominantly CD45RA on the malignant cells. On the other hand, tumors of thymic derivation such as lymphoblastic lymphomas commonly express HLA-DR and CD45RO and will have an increased ratio of T- to B-cells. Some of the T-cell lymphomas may even present an increased ratio of CD4/CD8, dependent of the level of heterogeneity. Thus, histological examination of tissue sections is necessary for complete evaluation. It should also be pointed out that the MoAb UCHL 1 (CD45RO) is commonly used in tissue section immunohistochemical staining methods to detect T cells. Clearly, this should be done with caution since the proportion of T cells reactive with this MoAb depends both on the activation state of the cells and on their stage of differentiation. ACKNOWLEDGMENTS Thanks to Dr. Yohannes W. Yesus, Department of Pathology, for his critical review of this manuscript and helpful discussions and to Susan Hartley, MT(ASCP), Nancy Case, MT(ASCP), Jim Ford, MT(ASCP), Ann Wilson, MT(ASCP), and Pam Puig, MT(ASCP), all of the Flow Cytometry Laboratory, Department of Pathology, for their assistance. This work was supported in part by grants from the Fraternal Order of Eagles of Missouri and the Cancer Research Center, Columbia, Missouri.

REFERENCES 1. Thomas, M. L., The leukocyte common family. Annu. Rev. Zmmunol. 7, 339-369, 1989. 2. Barclay, A. N., Jackson, D. I., Willis, A. C., and Williams, A. F., Lymphocyte specific heterogeneity in the rat common leukocyte antigen “T2OO” is due to differences in polypeptide sequences near the NHZ-terminus. EMBO J. 6, 1259-1264, 1987. 3. Lefrancois, L., Thomas, M. L., Bevan, M. J., and Trowbridge, I. S., Different classes of T

418

4. 5. 6. 7.

CHARLES

W.

CALDWELL

lymphocytes have different mRNAs for the leukocyte-common antigen, T200. J. Exp. Med. 163, 1337-1342, 1986. Ralph, S. J., Thomas, M. L., Morton, C. C., and Trowbridge, I. S., Structural variants of human T200 glycoprotein (leukocyte common antigen). EMBO J. 6, 1251-1257, 1987. Charbonneau, H., Tonks, N. K., Walsh, K. A., and Fischer, E. H., The leukocyte common antigen (CD45): A putative receptor-linked protein tyrosine phosphatase. Proc. Natl. Acad. Sci. USA 85, 7182-7186, 1988. Deans, J. P., Shaw, J., Pearse, M. J., and Pilarski, L. M., CD45R as a primary signal transducer stimulating IL-2 and IL-2R mRNA synthesis by CD3-4-8-thymocytes. J. Zmmunol. 143, 24252430, 1989. Ledbetter, J. A., Tonks, N. K., Fischer, E. H., and Clark, E. A., CD45 regulates signal transduction and lymphocyte activation by specific association with receptor molecules on T or B cells. Proc.

Natl.

Acad.

Sci. USA

85, 8628-8632,

1988.

8. Mustelin, T., Coggeshall, K. M., and Altman, A., Rapid activation of the T-cell tyrosine protein kinase pp56lck by the CD45 phosphotyrosine phosphatase. Proc. Natl. Acad. Sci. USA 86, 6302-6306, 1989. 9. Tonks, N. K., Charbonneau, H., Diltz, C. D., Fischer, E. H., and Walsh, K. A., Demonstration that the leukocyte common antigen CD45 is a protein tyrosine phosphatase. Biochemistry 27, 43534361, 1988. 10. Ostergaard, H. L., Shackelford, D. A., Hurley, T. R., et al., Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines. Proc. Natl. Acad. Sci. USA 86, 8959-8963, 1989. 11. Akbar, A. N., Terry, L., Timms, A., Beverley, P. C., and Janossy, G., Loss of CD45R and gain of UCHL 1 reactivity is a feature of primed T cells. J. Zmmunol. 140, 2171-2178, 1988. 12. Akbar, A. N., Timms, A., and Janossy, G., Cellular events during memory T-cell activation in vitro: The UCHLl (180,000 MW) determinant is newly synthesized after mitosis. Immunology 66, 213-218, 1989. 13. Deans, J. P., Boyd, A. W., and Pilarski, L. M., Transitions from high to low molecular weight isoforms of CD45 involve rapid activation of alternate mRNA splicing and slow turnover of surface CD45R. J. Zmmunol. 143, 1233-1238, 1989. 14. Sanders, M. E., Makoba, M. W., and Show, S., Human naive and memory T cells: Reinterpretation and further characterization of helper-inducer and suppressor-inducer subsets. Zmmunol. Today 9, 195-199, 1988. 15. Serra, H. M., Ledbetter, J. A., Krowka, J. F., and Pilarski, L. M., Loss of CD45R (Lp220) represents a post-thymic T cell differentiation event. J. Zmmunol. 140, 1435-1441, 1988. 16. Smith, S. H., Brown, M. H., Rowe, D., Callard, R. E., and Beverley, P. C., Functional subsets of human helper-inducer cells defined by a new monoclonal antibody, UCHL 1. Immunology 58, 63-70, 1986. 17. Ledbetter, J. A., Rose, L., Spooner, C. E., Beatty, P. G., Martin, P. J., and Clark, E. A., Antibodies to common leukocyte antigen ~220 influence human T cell proliferation by modifying IL 2 receptor expression. J. Zmmunol. 135, 1819-1825, 1985. 18. Morimoto, C., Hafler, D. A., Weiner, H. L., et al., Selective loss of the suppressor-inducer T-cell subset in progressive multiple sclerosis: Analysis with anti-2H4 monoclonal antibody. N. Engl. J. Med.

316, 67-72,

1987.

19. Mylvagnam, R., Ahn, Y. S., Sprinz, P. G., Garcia, R. O., and Harrington, W. J., Sex difference in the CD4+CD45R+ T lymphocytes in normal individuals and its selective decrease in women with idiopathic thrombocytopenia. Clin. Zmmunol. Zmmunopathol. 52, 473485, 1989. 20. Serra, H. M., Mant, M. J., Ruether, B. A., Ledbetter, J. A., and Pilarski, L. M., Selective loss of CD4+CD45R+ T cells in peripheral blood of multiple myeloma patients. J. Clin. Zmmunol. 8, 259-265, 1988. 21. Tilmant, L., Dessaint, J. P., Tsicopoulos, A., Tonnel, A. B., and Capron, A., Concomitant augmentation of CD4 + CD45R + suppressor/inducer subset and diminution of CD4 + CDw29 + helper/inducer subset during rush hyposensitization in hymenoptera venom allergy. Clin. Exp. Zmmunol. 76, 13-18, 1989. 22. Beverley, P. C. L., Human T-cell subsets. Zmmunol. Lett. 14, 263-267, 1987.

CD45 ISOFORMS IN REACTIVE

LYMPH

NODES

419

23. Janossy, G., Bofill, M., Rowe, D., Muir, H., and Beverley, P. C. L., The tissue distribution of T lymphocytes expressing different CD45 polypeptides. Immunology 66, 517-525, 1989. 24. Caldwell, C. W., Maggi, J., Henry, L. B., and Taylor, H. M., Fluorescence intensity as a quality control parameter in clinical flow cytometry. Amer. J. Cfin. Pathol. 88, 447456, 1987. 25. Serra, H. M., Ledbetter, J. A., Neil, D., and Pilarski, L. M., Apparent loss of 2H4+ T cells in peripheral blood of normal donors: A 2H4-specific artifact unique to T cells. Hum. Immunol. 23, 281-288, 1988. 26. Schnitzer, B., Reactive lymphoid hyperplasia. In “Surgical Pathology of the Lymph Nodes and Related Organs” (E. S. Jaffe, Ed.), pp. 22-56, Saunders, Philadelphia, 1985. 27. Hutchison, R. E., Kurec, A. S., and Davey, F. R., Lymphocytic surface markers in lymphoid leukemoid reactions. Clin. Lab. Med. 8, 237-245, 1988. 28. Janossy, G., Tidman, N., Selby, W. S., et al., Human T lymphocyte of inducer and suppressor type occupy different microenvironments. Nature (London) 288, 81-84, 1980. 29. Poppema, S., Bhan, A. K., Reinherz, E. L., McCluskey, R. T., and Schlossman, S. F., Distribution of T cells in human lymph nodes. J. Exp. Med. 153, 3u1, 1981. 30. Reinherz, E. L., Kung, P. C., Pesando, H. M., Ritz, H., Goldstein, G., and Schlossman, S. F., Ia determinants on human T-cell subsets defined by monoclonal antibody. J. Exp. Med. 150, 1472-1482, 1979. 3 1. Cozzolino, F., Torcia, M., Carossino, A. M., et al., Characterization of cells from invaded lymph nodes in patients with solid tumors: Lymphokine requirement for tumor-specific lymphoproliierative response. J. Exp. Med. 166, 303-318, 1987. 32. Autero, M., and Gahmberg, C. G., Phorbol diesters increase the phosphorylation of the leukocyte common antigen CD45 in human T cells. Eur. J. Zmmunol. 17, 1503-1506, 1987. 33. Cooper, J. A., Gould, K. L., Cartwright, C. A., and Hunter, T., Tyr-527 is phosphorylated in pp60f-src: Implications for regulation. Science 231, 1431-1434, 1986. 34. Veillette, A., Horak, I. D., Ho&, E. M., Bookman, M. A., and Bolen, H. B., Alterations of the lymphocyte-specific protein tyrosine kinase (~56”‘) during T-cell activation. Mol. Cell. Biol. 8, 4353-4361, 1988. 35. Amrein, K. E., and Sefton, B. M., Mutation of a site of tyrosine phosphorylation in the lymphocyte-specific tyrosine kinase, p561ck, reveals its oncogenic potential in tibroblasts. Proc. Natl. Acad. Sci USA 85, 4247-4254, 1988. 36. Marth, J. D., Cooper, J. A., King, C. S., et al., Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (~~56’“~). Mol. Cell. Biol. 8, 540-550, 1988. 37. Wallace, D. L., and Beverley, P. C. L., Phenotypic changes associated with activation of CMSRA + and CMSRO + T cells. Immunology 69, -7, 1990. 38. Sanders, M. E., Makoba, M. W., Sharrow, S. O., et al., Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA3, CD2, and LFA-1) and three other molecules (UCHLl, CDw29, and Pgp-1) and have enhanced IFN-gamma production. J. Zmmunol. 140, 1401-1407, 1988. 39. Knapp, W., Dorken, B., Rieber, P., Schmidt, R. E., Stein, H., and von dem Borne A. E. G. Kr., CD antigens 1989. Blood 74, 1448-1449, 1989. Received May 21, 1990; accepted with revision July 20, 1990