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© 2001 American Society for Clinical Oncology CD4+ T-Cell Immune Response to Large B-Cell Non-Hodgkin’s Lymphoma Predicts Patient OutcomeFrom the Division of Hematology and Internal Medicine, Department of Immunology, Mayo Clinic, Rochester, MN. Address reprint to Thomas E. Witzig, MD, Division of Hematology and Internal Medicine, Mayo Clinic, 200 First St SW, Rochester, Minnesota 55905; email: witzig{at}mayo.edu
PURPOSE: Previous studies in patients with non-Hodgkin’s lymphoma (NHL) and other malignancies have suggested that the presence of host infiltrates in the tumors of these patients may predict a better outcome. This study was undertaken to determine the prognostic importance of the presence of T cells in the biopsy specimens of patients with B-cell NHL. PATIENTS AND METHODS: Seventy-two patients with diffuse large B-cell NHL were prospectively evaluated at a single institution between 1987 and 1994. The percentage of CD3+, CD3+/HLA-DR+, CD4+, CD8+, and natural killer cells was determined by flow cytometry in the pretreatment diagnostic biopsy specimen and correlated with patient outcome. RESULTS: An increase in the percentage CD4+ T cells in the pretreatment tumor biopsies significantly correlated with patient outcome. The percent of CD4+ T cells was also highly correlated with CD3+/HLA-DR+, CD45RO+, and low L-selectin (CD62L) expression, indicating that the CD4+ T cells are activated memory T-helper cells. Those patients with increased numbers of CD4+ T cells, compared with other patients, had a significantly longer 5-year failure-free survival (72% v 43%, respectively; P = .04), as well as a significantly longer 5-year overall survival (65% v 38%, respectively; P = .05). When evaluated in a multivariate model, the International Prognostic Index and more than 20% infiltrating CD4+ T cells in the pretreatment biopsy were significant independent predictors of relapse-free and overall survival. CONCLUSION: The presence of increased numbers of activated CD4+ cells in the area of B-cell diffuse large-cell NHL predicts a better prognosis. This finding provides a strong rationale for the investigation of cellular immunotherapy in B-cell NHL.
THE NON-HODGKIN’S lymphomas (NHL) constitute a heterogeneous group of malignancies of B or T cells. In B-cell NHL, malignant monoclonal B cells arrested at different stages of differentiation usually involve lymph nodes and occasionally extranodal tissues. Cells other than tumor cells are also present in the tumor microenvironment. These cells include T lymphocytes, which are present in proportions that differ from one patient to another. They seem to be more than simple residual elements from the normal lymph node structure. The role and significance of these T cells in the pathogenesis of the disease are not well understood. Previous studies in patients with NHL as well as in patients with other malignancies have suggested that the presence of a host immune response in the tumors of these patients may predict a better outcome.1-7 Lymphoma relapse and patient survival may be related to the type and number of specific T-cell subsets that infiltrate B-cell lymphomas. In one study of large B-cell lymphoma,6 the finding of less than 6% cytotoxic T cells was associated with patient relapse. In a separate study of diffuse small-cell lymphomas,2 the number of infiltrating host T cells predicted survival. The magnitude, subset type, activation state, and memory status of the T-cell response in biopsy specimens from patients with B-cell lymphoma may be independent predictors of clinical outcome. In particular, the poor outcome associated with a low T-cell level might be the object of therapeutic manipulation. Previous studies have focused on the role of cytolytic CD8+ cells as tumor-infiltrating lymphocytes.4,6 Recent studies have suggested that CD4+ T cells may play a central role in the antitumor immune response.8,9 The aim of this study was to measure the presence and extent of CD4+ T cells infiltrating the pretreatment biopsy specimens from patients with B-cell diffuse large-cell lymphoma to assess the prognostic importance of the level of CD4+-cell infiltration.
Patient Selection Patients were eligible for this study if they had a tissue biopsy that on pathologic review showed diffuse large B-cell lymphoma and adequate tissue to perform flow cytometry. The patients were accrued to the study between January 1987 and June 1994, and all patients were newly diagnosed and untreated at the time the biopsy was taken. The biopsy specimens were reviewed and classified using the Revised European-American Lymphoid classification.
Staging was commonly performed with a complete blood count, serum chemistries, chest radiograph, computed tomography scan of the abdomen, and bilateral bone marrow and aspiration and biopsy. The patients were not treated on a specific protocol, but most received an anthracycline-based chemotherapy regimen as initial therapy. The pretreatment prognostic factors, the response to therapy, and overall survival (OS) data were obtained by chart review or correspondence. Patients were followed up to September 1999, and all patients have
Flow Cytometry Methods A panel of antibodies against T-, B-, and natural killer (NK)-cell antigens, including CD3, CD4, CD8, CD16, CD19, CD45RO, CD45RA, CD56, HLA-DR, and CD62L, as well as an isotype control was used for each sample. The fluorescein isothiocyanate– and phycoerythrin-conjugated monoclonal antibodies were used according to manufacturer’s recommendations (Becton Dickinson, Franklin Lakes, NJ). One million cells were incubated with antibody for 30 minutes at 4°C, washed with phosphate-buffered saline, and resuspended in 1 mL of 1% paraformaldehyde in phosphate-buffered saline for flow cytometry. A minimum of 10,000 cells were analyzed on each sample tube. An isotype control was used to gate on the lymphocytes by size and to set the negative fluorescence (FL) markers on either single FL histograms or FL1 versus FL2 dot plots of the dual antibody tubes. The results are reported as the percentage of lymphocytes positive for each antibody. To ensure that the specimen used for flow cytometry was representative of the tumor biopsy, the percents of CD4+ cells, CD8+ cells, NK cells, and CD19+ cells were added together and the sum had to equal 100% ± 15% for inclusion in the study.
International Prognostic Index (IPI) and Other Prognostic Factors
Statistical Analysis
Patient Characteristics Biopsy specimens from 98 patients were initially analyzed. Care was taken to analyze lymphocytes from a representative area of the lymphoma specimen. Most specimens had complete effacement of the normal tissue by large-cell lymphoma, and histologic sections from tissue adjacent to the tissue used for flow cytometry were studied to ensure that the area was involved by lymphoma. Cytospins of each cell suspension, however, were not performed. Twenty-six patients were excluded from the analysis because of the fact that the sum of the percentage CD4, CD8, NK, and CD19+ cells was less than 85% or more than 115%. They were excluded because of quality control concerns that there may have been either inadequate antibody binding caused by debris or nonspecific binding resulting in a selection bias for a subset of cells from the biopsy. The characteristics of the 72 patients with large B-cell NHL who were eligible for this study are listed in Table 1. Thirty-nine patients were men, and 33 were women, with a median age of 65 years (range, 18 to 92 years). Clinical factors that have been shown to influence prognosis were evaluated. Extranodal sites of involvement consisted of bone marrow involvement in 11 patients, gastrointestinal involvement in nine patients, lung in six patients, and liver involvement in two patients. Thirty-eight patients (53%) had a good performance status (Eastern Cooperative Oncology Group performance status 0 to 1), and 34 had a poor performance status (2,3, or 4). The median pretreatment LDH value was 188 U/dL (range, 64 to 1,580 U/dL). When patients were grouped using the IPI, 59 patients (82%) were in the low-risk or low-intermediate–risk group, whereas 13 patients (18%) were in the high-intermediate–risk or high-risk group.
Sixty-five patients (90%) were treated with an anthracycline-containing chemotherapy regimen ( Table 2). Of the remaining seven patients, five were treated with other chemotherapy combinations that excluded anthracyclines because of cardiovascular compromise, one patient received radiotherapy as primary therapy, and one patient died before initiation of treatment. Fifty-six patients (78%) had a CR to their initial therapy, with a median FFS of 38 months (range, 3.5 to 97 months). Twenty-seven (48%) of the 56 patients who had a CR have relapsed, and 30 (42%) of the total group of 72 patients have died. The median survival of all patients is 34 months (range, 1 to 97 months).
Analysis of Prognostic Factors The percentage of T lymphocytes in the B-cell NHL biopsy specimen could be determined in all patients. The median percentage of CD3+, CD4+, CD8+, and CD16+/CD56+ cells was 40% (range, 0.5% to 67%), 19.4% (range, 0.4% to 64%), 8% (range, 0.5% to 48%), and 3% (range, 0.5% to 38%), respectively. An increased percentage of CD4+ T cells in the biopsy significantly correlated with patient outcome, whereas the percentage of CD3+ cells (total T cells), CD8+ cells, and NK cells had no impact on patient outcome in the univariate analysis. The increased percentage of CD4+ T cells in the biopsy significantly correlated with patient outcome as both a continuous and discrete variable. Using the Wilcoxon rank sum test, the percent of CD4+ cells correlated with OS as a continuous variable (P = .01).
Using a scatterplot of residuals, a level of 20% CD4+ cells in the biopsy specimen was shown to be the point that separated the patients into two groups with the most significantly different clinical outcome in terms of FFS and OS. Using
Those patients with
The other factor that was predictive for FFS for the whole patient group was the IPI (P = .01). Other factors predictive for OS were the IPI and lung involvement ( Table 3). Despite the increased number of patients with an elevated LDH in the group with < 20% CD4+ cells, there was no difference in FFS (P = .24) or OS (P = .12) when patients with a normal pretreatment LDH were compared with those with an elevated LDH. Therefore, this variable was not included in the multivariate analysis.
When the significant factors were evaluated in a multivariate model (Cox) for FFS, the percentage of CD4+ cells (P = .05) and the IPI (P = .01) were independent prognostic factors. When a multivariate analysis for OS was performed, the IPI (P < .01) and the percentage of CD4+-cell infiltration (P = .04) were again independent prognostic variables.
Immunophenotype of CD4+ T Cells In this same subset of 41 patients, we also assessed whether the populations of T cells were naïve or memory cells by evaluating whether they expressed CD45RO or CD45RA. The CD4+ cells were found to express CD45RO but not CD45RA+ cells, indicating that the cells are memory CD4+ T cells. To further understand whether the CD4+ cells may be playing a regulatory role in the B-cell lymphoma, we compared the proliferative index of the tumor determined by the bromodeoxyuridine labeling index (LI) to the degree of CD4+-cell infiltration. The LI is a measure of the number of cells in the S phase of the cell cycles. The percentage CD4+ cells showed a significant (P = .001) inverse correlation with the LI, suggesting that the greater the percentage of CD4+ T cells in the B-cell lymphoma, the lower the proliferative rate.
Interactions between host immune cells and malignant cells are complex. T-lymphocytes seem to play a critical role in containing the malignant clone and in immunosurveillance. When growth patterns and phenotypic surface markers have been analyzed, T-lymphocyte subpopulations isolated directly from the tumor tissue differ markedly from those found in the peripheral blood of the same tumor-bearing host.20 The tumor-infiltrating lymphocytes have been considered to be a manifestation of host immune reactions to malignant cells; however, the pathophysiologic significance of infiltrating T-cells in human cancer tissue has remained controversial. One of the major reasons for this was a premise that human cancers arise by evading the host immune surveillance, either as a result of the weak immunogenicity of tumor cells or by certain immunosuppressive effects from tumor cells. Rosenberg et al21 and Muul et al22 have demonstrated that the tumor-infiltrating T lymphocytes are part of a highly potent and specific antitumor immune response in experimental murine tumors and in certain human tumors. Lymphoma relapse and patient survival have been related in a number of studies to the type and number of specific T-cell subsets that infiltrate B-cell NHL. The findings of these studies have suggested that the magnitude, subset type, activation state, and memory status of the T-cell response in biopsy specimens from patients with B-cell lymphoma are independent predictors of clinical outcome. It has been suggested that the poor outcome associated with a low level of infiltrating T-cells might be the object of therapeutic manipulation. Although many studies have focused on the infiltrating CD8+ T cells (cytolytic T cells),23 it seems that the CD4+ T cell component of the infiltrating immune cells may be even more important than CD8+ cells in determining patient outcome. In a study of large B-cell lymphoma by Lippman et al,6 the finding of less than 6% cytotoxic T cells (Leu-2+) was invariably associated with patient relapse (P = .008). The difference in cytotoxic T-cell infiltration was not a significant predictor of OS. In the same study, however, patients with less than 6% T-helper cells had a significantly shorter relapse-free survival (P = .05). However, again there was no detectable difference in OS. In a separate study of diffuse small-cell lymphomas by Medeiros et al,2 the numbers of infiltrating host helper T cells determined by immunohistochemistry predicted OS. Lymphomas with less than 15% Leu-3+ cells (helper/inducer T subset) were associated with a significantly shorter OS (P = .0005). These findings were of particular importance in the patient’s initial biopsy (P = .0007).
In the present study of large B-cell NHL using flow cytometry, we have demonstrated that patients with < 20% infiltrating CD4+ cells have an inferior FFS and OS when compared with patients with The nature and phenotype of the infiltrating T cells has been investigated in a number of studies, and these studies have suggested that these cells should be activated memory T cells. Tumor-infiltrating T cells in activated form (interleukin-2–stimulated) have been found to be related to NHL prognosis by Jacob et al.7 In their study, the influence of irradiated autologous malignant cells on the interleukin-2 responsiveness of infiltrating T cells was investigated. In 38% of the cases, the T-cell proliferation was not modified; in 41%, it was higher; and in 21%, it was lower. The actuarial survival curves showed a significantly better prognosis for patients with higher proliferation. Furthermore, an increase of memory T cells (CD4+/CD45RA-) relative to naïve T cells (CD4+/CD45RA+) has been noted in low-grade compared with high-grade NHL. It has been suggested that a memory T-cell response is active in maintaining a less aggressive NHL status.24 Naïve T-cell subsets were found more frequently and immune response memory T cells less frequently when the lymphoma cells had a higher proliferative rate measured by Ki67 (P = .0007). This suggests that memory T cells play a role in downregulating tumor proliferation rate.24 The results from the present study are compatible with the results from the studies summarized above. In our study, we demonstrated that the infiltrating CD4+ cells are activated with increased expression of HLA-DR and loss of L-selectin expression. We have also shown that these cells are memory T cells and have a CD4+/CD45RO+ phenotype. In addition, we have shown that an increased number of activated memory CD4+ T cells infiltrating areas of B-cell lymphoma correlates with a lower proliferative rate (as measured by the LI) of cells in the malignant lymphoma biopsy specimen. The LI was performed on all cells in the biopsy specimen, and we did not determine whether the proliferating cells were the malignant B cells or the infiltrating T cells. This finding is interesting but should be interpreted with caution. There were also a significantly higher number of patients with an elevated LDH in the group of patients with < 20% CD4+ cells in the lymphoma. Again, this may suggest that low numbers of activated memory CD4+ cells in areas of lymphoma may favor biologically aggressive disease. Not all studies have shown that infiltrating T cells are fully functional or that infiltrating helper T cells are even beneficial. Koulis et al25 have suggested that low-grade B-cell lymphomas of mucosa-associated lymphoid tissue (MALT) contain a significant population of activated helper T cells but that these cells may in fact be supporting tumor growth. This was based on the finding that the T cells expressed CD40L, whereas the B cells expressed CD40, suggesting that the CD40/CD40L interaction plays an important role in the growth of low-grade MALT lymphomas. In the same study, however, high-grade MALT lymphomas did not express CD40L, suggesting that factors other than T-cell help are likely to be important in the growth of high-grade tumors. It has also been shown that the tumor-infiltrating lymphocytes from patients with B-cell NHL may have impaired tyrosine kinase signaling after stimulation with an anti-CD3 antibody.26 The authors suggested that these defects contributed to the poor proliferation and gamma interferon production seen in these cells. Our data would suggest that in large B-cell lymphoma, infiltration by activated memory CD4+ rather than cytotoxic T lymphocytes predicts patient outcome. The mechanism for this finding may be different from the effect of CD8+ T cells. CD4+ cells may be responsible for costimulation of CD8+ cells, and low numbers of CD4+ cells may limit the costimulation process. Furthermore, CD4+ cells may be responsible for stimulating antigen-presenting cells, thereby upregulating the immune response. Additionally, recent studies have suggested that T-helper cells may themselves have cytolytic activity,27 and decreased numbers of these cells may limit this process. Although we postulate that CD4+ T-cell infiltration into areas of large B-cell NHL is an important feature of the body’s immune response and important in predicting patient outcome, it is also equally possible that the infiltration by CD4+ cells may simply be a surrogate for a more efficient immune system. In conclusion, this study has shown in a large cohort of patients with a uniform histology that the presence of increased numbers of CD4+ T cells in the tumor biopsy predicts a better prognosis. This study demonstrates that the prognostic importance of the CD4+ cell infiltration is independent of other established prognostic variables. These cells are activated memory T-helper cells with CD4+/CD45R0+, HLA-DR+, and L-selectin-low phenotype, and tumor biopsies with high numbers of these cells have a low proliferative rate. The immunologic mechanisms underlying these findings remain unclear, and they provide a strong rationale for further investigation of the mechanisms responsible for T-cell recruitment and the development of methods to upregulate this process to provide clinical benefit to patients with these lymphomas. It is important to note that we do not regard the cut point at 20% CD4+ cells to be significant, but rather, we believe that the degree of CD4+-cell infiltration is a continuum with increased numbers of infiltrating CD4+ cells correlating with improved clinical outcome. Additionally, although the degree of infiltration correlates with FFS and OS, we do not wish to describe another prognostic factor. Rather, we regard these results as provocative data that merit further evaluation and that may lead to a better understanding of the immune response and to future innovative immunologic therapies.
We thank Kay Ristow for her assistance with data management, Paul J. Kurtin, MD, for the pathology review, and Curtis A. Hanson, MD, for help with the analysis of the flow cytometric data.
Presented in part at the Thirty-Fifth Annual Meeting of the American Society of Clinical Oncology, Atlanta GA, May 15-18, 1999.
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Copyright © 2001 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
5 years of follow-up. This study was approved by the Institutional Review Board of the Mayo Clinic/Mayo Foundation. 
2 tests for categorical data and the Wilcoxon rank sum test for continuous data.
