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Molecular Oncology, Markers, Clinical Correlates |
Departments of Medicine [A. T. S., T. P. M., E. M. H.], Pathology [A. G., Y. F., T. M. G.], and Biometry [C. S. J., H. C.], Arizona Cancer Center, University of Arizona, Tucson, Arizona 85724
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CAM, HLA, and CSMs are important determinants of B-cell stimulation and activation of T cells. Whereas the MHC class I and II antigens are necessary for presentation of specific antigenic epitopes, T-cell recognition is insufficient for effective CTL responses. ICAM-1/LFA-1 interactions permit the leukocyte adhesion required for CTL generation (8 , 9) . B7.1 and B7.2 are CSMs that provide the second signal required for stimulated T cells to become activated. Without costimulation, T cells will undergo apoptosis, and an anergic immune response will prevail, even in the presence of optimal recognition and presentation of antigens via the MHC-T-cell receptor interaction (10, 11, 12) . Thus, multiple interactions between the T cell and antigen-presenting cell are necessary for optimal and effective T-cell responses.
Because HLA, CAM, and CSMs play an important role in generating cytotoxic T-cell responses, we hypothesized that their expression on malignant B cells may determine the host T-TIL response. In this study, we examine the role of B7.1, B7.2, ICAM-1, and LFA-1 expression in host T-TIL responses. Our results significantly expand our previous data, suggest a hypothesis by which malignant B cells may evade host immune surveillance, and provide a rationale for immunotherapy strategies for treating B-cell lymphoma.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tissue Immunohistochemistry.
B- and T-cell antigens, percentage of T-TILs, and immunoregulatory
molecules [recognition molecules, MHC I (MAS and MCA) and MHC II
(HLA-DR, DP, and DQ); cellular adhesion molecules, LFA-1
(CD11a),
LFA-1ß (CD18), and ICAM-1 (CD54); and costimulatory molecules, B7.1
(CD80) and B7.2 (CD86)] were analyzed using a three-stage
avidin-biotin horseradish peroxidase 3,3'-diaminobenzidine
immunoperoxidase technique on snap-frozen tissue sections as described
previously (5
, 7)
. Negative controls excluded the primary
antibody and substituted an irrelevant isotype-matched monoclonal
antibody. Positive control specimens were reactive tonsil tissue
sections placed at the bottom of each glass slide assay ("same slide
control").
Positive expression of a specific HLA, CAM, or CSM was determined as surface staining in any of the tumor cells. Loss of expression (negative expression) constituted an absolute absence of staining in the malignant B cells of the tumor specimen, whereas the "same slide control" was positive. The immunoregulatory molecules were also grouped into families. Loss of expression of any of the tested antigens included in the family classified the family as negative (lost expression). All cases were reviewed independently and then reviewed jointly by two of the authors (A. G. and T. M. G.), who were blinded to the clinical staging and percentage of T-TILs of the specimens. The tabulated results are the consensus findings of the two authors.
The number of T-TILs was determined as described previously (4) . Briefly, host T-TILs were quantitated by counting the number of CD8+ cells among the total number of lymphoid cells counted as indicated by methylene blue counterstaining. A cut point of 6% T-TILs was established previously to distinguish tumor specimens with low (<6%) and adequate (>6%) T-TIL expression as determined by predictive power for relapse-free survival in the lymphoma patients (4) .
Antibodies used in detecting B cells, cytotoxic (CD8+) T cells, HLA-DP,
HLA-DR, HLA-DQ, and class I HLA-A, -B, and -C have been described
previously (4, 5, 6)
. LFA-1, LFA-1ß, and ICAM-1 (CD54)
antibodies were purchased from Immunotech (Marseilles, France). B7.1
(CD80) antibody was purchased from Becton Dickinson (Mountain View,
CA), and B7.2 (CD86) antibody was generously provided by Repligen Corp.
(Cambridge, MA).
Statistical Methods.
Two-sample t tests were used to test the association between
individual molecules or molecular families and T-TILs as a continuous
measurement. P < 0.05 was considered statistically
significant. Several regression models were fit with all HLAs, CAMs,
and CSMs entered as independent variables to examine the correlation
between T-TILs, specific molecules, and molecular families. Logistic
regression models were also fit with the outcome variable being T-TIL
response (<6% versus >6%). Statistical
significance was determined as P < 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Table 1
shows the expression of the
10 HLA, CAM, and CSMs analyzed in the lymphoma specimens. B cells
function as effective antigen-presenting cells in vivo and
thus normally express all examined molecules. This finding was
confirmed in the "same slide control" tonsil sections. Overall, MHC
class II expression was most conserved because 94% of the malignant B
cells expressed HLA-DR and HLA-DP. The CSM B7.2 was also highly
conserved, being present in 90% of the cases. In nine patients, the
malignant B cells expressed only one of the CSMs, B7.1 or B7.2, and in
five patients, expression of both B7.1 and B7.2 was lost.
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. Loss of B7.2 (P = 0.04), MAS
(P = 0.02), HLA-DR (P = 0.0004), and
ICAM-1 (P = 0.0004) expression was significantly
associated with low T-TIL (Figs. 1
and 2)
. The number of absent HLA, CAM, and
CSM on the surface of the malignant B cells was highly associated with
host T-TIL response (P = 0.0001). In the 18 patients
with a low host T-TIL response, a mean number of 2.67 (range, 1- 7)
HLA, CAM, or CSM were lost as compared with a mean number of 0.89
(range, 0–4) molecules lost in the lymphomas characterized by a high
host T-TIL response. In addition, every lymphoma specimen with a low
host T-TIL response had loss of expression of at least one HLA, CAM, or
CSM on the malignant B cells. In contrast, 26 (49%) of the B-cell
lymphomas with high host T-TIL response had not lost expression of any
of the 10 HLA, CAM, and CSMs analyzed. Expression of each HLA, CAM, and
CSM was highly correlated with expression of the other molecules, thus
only B7.2, HLA-DR, and ICAM-1 expression independently correlated with
host T-TIL response at a statistically significant level. The total
number of tumor specimens examined for each molecule (MAS, MCA, and
HLA-DR, n = 70; B7.1, B7.2, HLA-DP, HLA-DQ, and LFA-1a,
n = 69; LFA-1b, n = 65; and ICAM-1,
n = 59) is less than 71 because not all patients were
evaluable secondary to inadequate tissue or indeterminate results.
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, the data are consolidated
into HLA, CAM, and CSM families. Tumors were considered negative or to
have lost family expression if any of the antigens tested in that
family were absent on the malignant B cells (see "Materials and
Methods"). Expression in the LFA family was lost most frequently
(32%) but had no effect on host T-TIL responses. Host T-TIL responses
were statistically correlated to expression of the B7, ICAM-1, and MHC
(class I) families.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor cells use multiple strategies in obtaining a growth advantage over normal cells, including overexpression of growth factors or their receptors, loss of apoptotic controls, and increased cell cycling. Despite these genetic mutations that increase the cell growth of tumor cells, survival also requires tumor cells to evade immune recognition. Because tumor cells are potentially susceptible to T-cell-mediated cytotoxicity, tumor cells have also developed mechanisms to escape immune recognition. Examples of these strategies include the release of immunosuppressive cytokines such as interleukin-10 and transforming growth factor ß as well as the loss of surface molecules crucial to T-cell recognition and stimulation (14, 15, 16) . B cells normally express or can be stimulated to express the molecules necessary for optimal T-cell recognition and stimulation (17) . Thus, malignant B cells, by retaining expression of HLA, CAM, and CSMs, would be able to initiate and stimulate T-cell cytotoxic and helper responses. Prominent infiltrates of normal and proliferating T cells can often be observed in lymphoma specimens (7) . In addition, the ability of malignant B cells to stimulate a strong host T-cell response has been associated with longer disease-free survival and improved prognosis in patients with B-cell malignancies (4 , 18) . This suggests that cytotoxic (CD8+) T cells can inhibit the growth and progression of B-cell malignancies in patients.
A variety of surface molecules are necessary for optimal T-cell-B-cell interactions (10 , 11) . T-cell activation is thought to require two distinct signals. The first signal (recognition) requires interaction of the T cell receptor with a MHC-peptide complex. The second signal is provided by the CSMs, B7.1 and B7.2, on the antigen-presenting cell and CD28 and CTLA-4 on the reacting T cell. If a costimulatory signal is not received, the T cell undergoes apoptosis instead of activation (12) . Adhesion molecules, including LFA-1 and ICAM-1, further promote immune recognition and T-cell stimulation by docking B and T cells such that effective antigen presentation and T-cell stimulation occur (9 , 19 , 20) . Antibodies directed against LFA-1 or ICAM-1 block lysis of target cells by CTLs and abrogate lymphokine-activated killer cell and natural killer-mediated cell killing in vitro (8 , 9 , 21) . Thus, each of these surface molecules, MHC class I, MHC class II, B7.1, B7.2, LFA-1, and ICAM-1, plays a major role in T-cell activation and the generation of cell-mediated immunity.
Our results suggest that loss of immune recognition, adhesion, or costimulatory molecules on tumor cells is a mechanism by which B-cell lymphomas inhibit host cytotoxic T-cell responses. Every tumor with a low host T-TIL response also had loss of expression of at least one HLA, CAM, or CSM important for B-cell-T-cell interaction and cytotoxic T-cell generation. In fact, in the majority of patients with low T-TIL responses, not just one but several HLA, CAM, or CSM were undetectable (a mean of 2.67 molecules lost in each low T-TIL tumor). This suggests that evading host T-TIL responses is a multifactorial process involving the progressive loss of several surface molecules necessary for T-cell recognition and activation.
Others have also found that loss of immunoregulatory molecules effects lymphoma biology and prognosis (22 , 23) . Terol et al.(22) found that absent or weak ICAM-1 expression predicted disseminated (stage IV) disease and poor survival in several subtypes of NHL, including 61 patients with DLCL. They noted that ICAM-1 maintained prognostic importance within the subset of patients with stage IV disease and thus suggested that ICAM may play other important biological roles. Our results are consistent with this hypothesis as we found ICAM expression to play an important role in generating host T-TIL responses.
Terol et al. (23) also published results on LFA-1 expression in 64 DLCL specimens. Their results of 41% LFA loss are in excellent agreement with our own results, in which 32% of DLCL specimens lost LFA expression (23) . Interestingly, they did not find that LFA expression predicted overall or relapse-free survival. In our cases, LFA expression was the most common defect observed, yet it was not associated with low T-TIL response and thus may explain the failure of LFA to predict prognosis.
Dorfman et al. (24) analyzed CSM expression on NHL specimens and found low levels of expression on all germinal center-derived lymphomas examined using a highly sensitive immunohistochemical technique. The low level of B7 expressed on the surface of malignant B cells was nonetheless functional and able to prevent T-cell anergy but was insufficient to initiate T-cell proliferation in vitro (24) . Only 7 specimens with DLCL were analyzed in their study, as compared with 70 patients in our series. Thus, the increased number of specimens with undetectable B7.1 and B7.2 expression found in our series may be related to the increased number of patients analyzed, the selection of all stages of NHL for our study, and antibody clone selection. For each specimen examined, we similarly analyzed B cells from tonsillar tissue and confirmed positive CSM expression in germinal center B cells, thus verifying the sensitivity of our assay method. The association we found between undetectable B7.2 expression and T-TIL response may reflect ineffective B7 function including loss of T-cell proliferation as suggested by Dorfman et al. (24) or an inability to recruit T cells.
In the literature, there are also reports in which lymphoma ICAM or LFA-1 expression does not predict prognosis (25 , 26) . These results may be due to imbalances in important clinical prognostic features such that clinical outcome relative to the biological variable could not be accurately tested. In our series, we have chosen a biological end point (T-TIL response) to avoid the limitations that confounding prognostic variables might have on a relatively small group of patients (n = 71) analyzed retrospectively. Differences in immunohistochemical technique including paraffin or fresh tissue and choice of antibody clone might also account for the variability in results, as suggested by Medeiros et al. (26) . The importance of our findings to the clinical outcome of patients with B-cell NHL awaits the results from prospective clinical trials involving uniformly staged and treated patients.
The mechanism for progressive loss of immune molecules in DLCL is not known but is important for determining optimal immunorestorative therapies. Because the genes for these molecules are located on different chromosomes, allelic loss is an unlikely mechanism to explain our results. Others have found that loss of HLA-DR expression in lymphomas is not associated with gene deletions but rather with transcriptional inhibition (27) . Transcriptional or translational inhibition is an attractive hypothesis for explaining aberrant expression because altered expression or binding of a single regulatory factor that targets a common enhancer or promoter sequence can inhibit the transcription and expression of several proteins. Whereas allelic loss may be best corrected by gene therapy strategies, transcriptional and posttranscriptional events may be corrected with cytokine therapies (28) . We are currently pursuing additional studies to determine the mechanism by which malignant B cells alter surface molecular expression.
Our results predict that therapies designed to increase
expression of a single immunoregulatory molecule will be successful in
only a minority of cases. Interferon- (IFN-) 32 has been linked
to restoration of LFA-1 (CD11a) and ICAM-1 (CD54) expression on
malignant B cells (29
, 30)
and enhanced susceptibility to
CTL killing in hairy cell leukemia (29)
. From our results,
17 of 71 (24%) patients with DLCL have altered expression of MHC class
I, ICAM-1, and/or LFA-1, and, of these, 59% have additional HLA,
CAM, or CSM losses as well. Thus, the multitude and heterogeneity of
altered HLA, CAM, and CSM expression in DLCL may be one reason for the
disappointing and conflicting results of IFN- treatment in B-cell
NHL (31, 32, 33)
. More effective antitumor responses may be
obtainable by designing therapies that target more immunoregulatory
molecules.
To summarize, the results presented in this study suggest that in B-cell DLCL, a deficient host T-TIL response is a multifactorial event characterized by combined loss of cellular adhesion, recognition, and costimulation molecules. Our results suggest that a combination of immunorestorative cytokine or gene therapies may be required to generate effective antitumor responses in B-cell lymphoma.
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FOOTNOTES |
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1 Supported in part by gifts to the Arizona Cancer
Center from Anne Mangelsdorf and Mary Dabney. 
2 To whom requests for reprints should be
addressed, at Arizona Cancer Center, University of Arizona, P. O. Box
245024, 1515 North Campbell Avenue, Tucson, AZ 85724. Phone:
(520) 626-2816; Fax: (520) 626-2225; E-mail: astopeck{at}azcc.arizona.edu
3 The abbreviations used are: DLCL, diffuse
large-cell lymphoma; T-TIL, tumor-infiltrating T-lymphocyte; CAM,
cellular adhesion molecule; CSM, costimulatory molecule; NHL,
non-Hodgkin’s lymphoma; ICAM-1, intracellular adhesion molecule 1;
LFA, leukocyte function antigen; IFN-, interferon .
Received 4/25/00; revised 8/ 1/00; accepted 8/ 2/00.
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REFERENCES |
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