Activated tumor-infiltrating T lymphocytes (TIL-T) were quantitated prospectively in excisional biopsy specimens of 49 B-cell non-Hodgkin's lymphomas.
American Journal of Pathology, Vol. 139, No. 3, September 1991
Copyright ©) American Association of Pathologists
Activated T-cell Subsets in Benign Lymphoid Hyperplasias and B-cell Non-Hodgkin's Lymphomas Jose 1. Diaz, Mark G. Edinger, Mark H. Stoler and Raymond R. Tubbs
lations from neoplastic tissue. (Am J Pathol 1991, 139:503-509)
From the Department of Pathology, Cleveland Clinic Foundation, Cleveland, Ohio
Activated tumor-infiltrating T lymphocytes (TIL-T) were quantitated prospectively in excisional biopsy specimens of 49 B-cell non-Hodgkin's lymphomas (NHL) of various grades and compared with eight benign lymphoid hyperplasias (BLH) to identify any potential difference in host T-cell response. Immunotyping of tissue-cell suspensions was done by threecolor flow cytometry, which was complemented by immunocytology by using cytocentrifuged preparations. Two activated T-cell subsets were studied: acutely activated TIL-T (CD3 + CD25 + HLADr-) and chronically activated TIL-T (CD3 + CD25 HLADr+). Results showed an association of the more aggressive intermediate/high-grade B-cell NHL with a higher percentage of late-phase activated TIL-T and a progressive increase with the grade of malignancy: 10.29%, 23.25%, 33.87%, and 47.78% (means) for BLH and for low-, intermediate- and high-grade B-cell NHL, respectively. Differences for this subset were significant (P < 0.050) for the following comparisons: hyperplasia versus intermediate-grade NHL (P < 0.0012), hyperplasia versus high-grade NHL (P < 0.0002), and low versus high-grade NHL (P < 0.0080). The percentage of acutely activated TIL-T cells did not show a statistically significant difference between the groups. The results suggest a host T-cell response to proliferating neoplastic cells in B-cell NHL. Paradoxically, the response does not appear to be protective for the host since the intensity is directly proportional to the grade of malignancy. However, the recognition of this response may have clinical applications since its amplification with biological response modifiers may result in effective adoptive immunotherapy of B-cell NHL. Further clarification of the specificity and biologic significance of host T-cell activation in B-cell NHL will require functional studies of isolated lymphocytic subpopu-
Activated tumor-infiltrating T lymphocytes (TIL-T) have become the subject of intensive investigation to better understand the immunobiologic responses to cancer cells. TIL-T cells are always present in B-cell NHL1 as well as numerous nonlymphoid neoplasias.2 Although their role in the pathogenesis of cancer remains unknown, TIL-T are often so numerous that their presence as a simple residual element is difficult to accept;3 also, TIL-T usually accompany extranodal extensions of B-cell NHL. Therefore, it has been postulated that TIL-T may represent a host-immune response to neoplastic B cells and that they may proliferate in situ3 or accumulate in the tumor by extravasation or specific entrapment from the
blood.' During the course of an immune response, naive T cells encounter antigen and become activated. New surface molecules (activation markers) such as CD25, CD71, HLADr, and CD45RO are then sequentially expressed. Activation is initiated by the triggering of the T CD3 molecule T-cell receptor complex with antigen.4 Within the first few hours of stimulation, acutely activated T cells begin to express a variety of early markers such as the interleukin-2 receptor (IL2-r, CD25) and transferrin receptor (T9, CD71). approximately 24 to 48 hours after stimulation, early activation molecules decline and almost entirely disappear by day 15. A second subset of chronically activated T cells then evolves and is characterized by the expression of late activation molecules many of which belong to the Class II major histocompatibility complex family. HLADr, one of the most representative molecules of this family, is normally present in B lymphocytes and macrophages and is absent in naive T cells. HLADr appears on T cells to augment communication between lymphocytes during late activation.5'6 A new subset of memory T lymphocytes, which is characterized by irreAccepted for publication April 29, 1991. Address reprint requests to Dr. Jose I. Diaz, Department of Pathology L25, Cleveland Clinic Foundation, One Clinic Center, 9500 Euclid Avenue, Cleveland, Ohio 44195-5138.
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versible loss of CD45RA and expression of CD45RO and CDw29, evolves.7 This study compared host T-cell responses in patients with BLH and various grades of B-cell NHL. We studied the potential host T-cell response by identification of two T-cell subsets that sequentially appear after T-cell activation: CD3 + CD25 + HLADr - (acutely activated T cells) and CD3+ CD25- HLADr+ (chronically activated T cells). The current study reports the percentage of these two subsets of activated T lymphocytes, which were obtained by three-color flow cytometry of cell suspensions prepared from excisional biopsy specimens that harbored BLH and low-, intermediate-, and high-grade B-cell NHL.
Materials and Methods Fresh tissue from excisional biopsy specimens from 57 patients was examined: 49 untreated B-cell NHL (40 nodal and 9 extranodal surgical specimens) and 8 BLH (nodal).
Histopathology Representative tissue was fixed overnight in B5, methacarn, formalin, and Hollandes solutions. Sections of paraffin-embedded tissue were cut 3,um thick, mounted on
glass slides and stained with eosin-hematoxylin. All the slides were evaluated and the lymphoma specimens were classified according to the International Working Formulation.8
Three-Color Immunocytometry Immunocytometric analysis of tissue-cell suspensions was performed using the three-color immunofluorescence method.911 Lymphocytes were isolated from
fresh tissue via enzymatic digestion with inhibition of trypsin activity. The tissue was minced and digested with 70 mg of collagenase Type 11 (Sigma, St. Louis, MO, #C 6885, 3.5 mg/ml) and 3 ml of egg white trypsin inhibitor (Sigma #T-9253, 3 mg/ml) in 20 ml of Minimal Essential Media (M.A. Bioproducts, Walkersville, MD), (#12-126B) for 1 hour at 37°C. The tissue was then filtered through 150 ,um Nitex mesh (Tetko 3-150/151). A cell count and percent viability by trypan blue exclusion was obtained using a hemocytometer. The cell suspension was adjusted to 5 x 106 cells/ml using Hanks balanced salt solution (HBSS) (Whittaker, Walkersville, MD, #10-5434) as a diluent. The suspension was maintained on ice until needed. A 1 00-pI aliquot of the cell suspension and 20 each of an optimal dilution of one fluorescein isothiocyanate
(FITC), one phycoerythrin (PE), and one biotinconjugated monoclonal antibody were added to each individual tube. Each antibody was optimally tittered using peripheral blood mononuclear cells (PBMC). This mixture was incubated 15 minutes in the dark at room temperature, with vortexing every 5 minutes. The cells were then washed twice using HBSS that contained 1% bovine serum albumin (BSA) and 0.1% sodium azide (Sigma A-6793, Sigma S-2002). Any contaminating red blood cells were lysed by incubating 10 minutes in the dark at room temperature with FACS-lyse (BectonDickinson, San Jose, CA, #9026). The cells were washed and incubated in 80 ,ul HBSS/1.0% BSA/0.1% sodium azide and 20 pI streptavidin-Duochrome (PE & Texas Red; Becton-Dickinson) for 30 minutes in the dark at 40C, vortexing every 10 minutes. After two final washings with HBSS/1.0% BSA/0. 1% sodium azide, the cells were fixed in phosphate-buffered saline that contained 0.5% paraformaldehyde and 1.0% BSA solution for anal-
ysis. Commercial antibodies to leukocyte-cluster designations antigens (CD Ags) were used to phenotypically identify and quantitate lymphocytic subsets as follow: biotinylated-anti-HLADr, PE-CD25, PE-CD19, PE-CD14, FITC-CD45 from Becton-Dickinson Monoclonal Center (San Jose, CA); and FITC-non-immune rabbit Ig, FITCanti-human kappa light chains, and FITC-anti-human lambda light chains, from Dako Corporation (Carpinteria, CA). FITC-CD3, PE-CD19, and biotinylated anti-HLADr were used to quantitate B cells (CD3- CD19+ HLADr+). FITC-CD3, PE-CD25, and biotinylated antiHLADr were used to quantitate T cells and activated T-cell subsets (Total CD3+ cells, CD3+ CD25+ HLADr- and CD3+ CD25- HLADr+ cell subsets). Kappa/lambda ratios were derived from quantification of kappa+ CD19+ HLADr+ and lambda+ CD19+ HLADr + populations. Data were acquired using a FACScan flow cytometer (Becton-Dickinson) equipped with a 15 mw argon laser at 488 nm excitation. 10,000 list mode gated events were acquired per tube. Live gating of the forward and orthogonal light scatter as well as backgating of the CD45+/ CD14- population was used to isolate the lymphocyte populations. Separate 10,000 event list mode files were acquired for large and small lymphocyte populations. When two separate populations of small and large lymphocytes were gated, the percentage of small and large T cells of each subset were integrated via the formula:
(%SMLx %SMsL) + (%LLx %LsL) 100
where SML represents the proportion of total small
lymphocytes gated, SMSL represents the proportion of
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the T cell subset in the small cell population, LL represents the proportion of total large lymphocytes gated, and L,L represents the proportion of the T-cell subset in the large cell population. Instrument optimization was achieved using similarly prepared peripheral blood mononuclear cells. FACScan Research Software, LYSYS, and Paint-a-Gate (BectonDickinson, San Jose, CA) software were used in the acquisition and analysis of the cell suspension specimens, and data were collected and statistically analyzed using RS1 (BBN) software running on a Microvax 11 GPX (Digital, Nashua, NH) workstation.
Immunocytology Three-color flow cytometry was complemented by immunocytologic analysis. Mononuclear cells isolated from the cell suspension using Ficoll-Paque density sedimentation were used in the cytocentrifuge preparations. The cell suspension, adjusted to 1.5 x 1 05 cells/1 00 ,ul was added to the cytocentrifuge cuvette containing an equal volume of calf bovine serum (Hyclone #A-2151-D). Slides precoated with poly-l-lysine (P 8920) were used. The cytocentrifuge was operated at low acceleration, 1000 rpm for 5 minutes. The cytospins were allowed to air dry for 15 minutes then dip fixed in absolute acetone. Immunotyping of the cytocentrifuge prepared slides was performed by the Avidin-Biotin Complex method.12'13 Slides were stained with CD3, CD22, and anti-HLADr commercial antibodies (Becton-Dickinson). Thereafter, sections were washed and incubated with biotinylated horse-anti-mouse IgG (Vector Laboratories, Burlingame, CA) followed by preformed avidin-DH-biotinylated peroxidase complex (Vector Laboratories). The color reaction product was developed with the chromogen 3-amino, 9-ethylcarbazole (AEC) in the presence of 0.005% H202 and then permanently mounted with Crystal Mount (Biomeda, Foster City, CA). The direct immunoperoxidase technique that employed peroxidase-conjugated F(ab)2' goat anti-kappa and anti-lambda antibodies was used to identify surface and cytoplasmic immunoglobulins (Slg and Clg),.12
Quality Control for Immunocytology and Immunocytometry To maintain quality control, a human peripheral blood sample was stained daily for three-color immunocytometry and analyzed in a similar manner to clinical samples. Results were reported only if the control values were within mean + 2 SD of the laboratory established values for that population. Alignment and compensation were checked daily. Alignment was checked with yellow-
green microspheres (Polysciences 18604). Compensation was checked using a 50% mixture of previously stained FITC-lgG1/FITC-CD45 (B-D), PE-lgG1/PE-CD3 (Gentrak), and Biotin-lgG-Duochrome/Biotin-CD3Duochrome (Vector and Olympus) for each corresponding fluorescence channel. Tissue sections from normal tonsillar frozen archival tissue were used as in-run controls with each immunocytologic test. These controls for immunocytology included equivalently titered nonimmune IgG fraction from the species from which the antibody was derived applied to both the test and the archival normal tissue specimen. In addition, the archival normal tissue was immunostained with the marker to be tested. Also, these archival specimens were used to establish antibody titers and check the performance of new lots of reagents.
Statistical Analysis The percentage of each activated T-cell subset was originally derived from total cells in suspension (B plus T cells) and then divided by the percentage of total T cells (total CD3 + cells) and multiplied by 100 to obtain the percentage of this subset per total T cells. This is a more accurate manner to express the results since selective loss of neoplastic B cells is not uncommon in intermediate- and high-grade NHL,14 which ultimately will produce falsely elevated percentage of total T cells and T-cell subsets. The results obtained for intermediate- and high-grade B-cell NHL were considered separate and as a combined group since the biology and prognosis of most intermediate NHL is similar to high-grade NHL. The percentage of both activated T-cell subsets were compared by paired t-tests (RS1 Scientific software BBN Inc.) in the five diagnostic groups: BLH, low, intermediate, high, and combination intermediate/high-grade B-cell NHL.
Results Using the International Working Formulation system, the 49 B-cell NHL were classified as follows: 21 low-grade lymphomas (9 small lymphocytic cell type, 4 follicular small-cleaved cell type and 8 follicular mixed smallcleaved and large cell types); 18 intermediate-grade Iymphomas (4 follicular large cell types, 1 diffuse smallcleaved cell type, 1 diffuse mixed small and large cell type and 12 diffuse large cell types); and 10 high-grade lymphomas (8 large cell immunoblastic types and 2 small noncleaved cell types). Three-color flow cytometric analysis and immunocy-
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Table 1. Activated T-cell Values
Hyperplasia Low-grade NHL Intermediate-grade NHL High-grade NHL Combination
Acutely activated (AA) T cells
Cronically activated (CA) T cells
AA/CA T-cell ratio
CD3+ CD25+ HLADr- cells
CD3+ CD25HLADr+ cells 10.3 -- 1 9 23.2 -- 4.6 33.9 + 5.6 47.8 + 8.6 38.8 -- 9.5
CD3-+ CD25+ HLADr-/CD3+ CD25- HLADr+ cells 1.31 0.42 0.24 0.44 0.32
13.5 + 2.3 9.8+ 1.7 8.2 -- 1.6 21.2 + 10.1 12.8 + 3.8
Mean percentage + se per total T Iymphocytes. Acutely (CD3 + CD25 + HLADr -) and chronicaliy (CD3 + CD25 HLADr + ) activated T-cell percentage per total T cells are presented in column 1 and 2. The ratio of acutely activated T cells per chronically activated T-cells is presented in column 3. Combination represents the integrated results from intermediate- and high-grade NHL as a unique group.
tologic results demonstrated a B-cell origin for all NHL. The immunocytologic phenotype of the neoplastic cells was consistently CD3- CD22+ and or HLADr+. Kappa or lambda light-chain monoclonality (kappa/ lambda ratio 3) was present in all NHL. Monoclonality was absent in all BLH. When two separated populations of small and large lymphocytes were gated, activated T cells appeared to distribute randomly. A final percentage of activated T cells was obtained by using the formula explained in the Materials and Methods section. The three-color flow immunocytometric results of both activated TIL-T are summarized in Table 1 and the histogram is shown in Figure 1. Two distinct populations of (CD3+ HLADr+) chronically activated T cells and (CD3- HLADr+) B cells in two typical samples of lowand high-grade B-cell NHL are shown in the three dimensional contour plot of Figure 2. Interestingly, there was a progressive increase of the percentage of chronically activated TIL-T from BLH to B-cell NHL, as well as with the grade of malignancy: 10.3%, 23.2%, 33.9%, and 47.8% (means) for BLH-, low-, intermediate-, and high-grade B-cell NHL, respectively. Statistically significant P values (P < 0.05) were obtained for the following comparisons: hyperplasia versus intermediate grade NHL (P < 0.0012), hyperplasia versus high-grade NHL (P < 0.0002), hyperplasia versus combination intermediate high-grade NHL (P < 0.0001), lowversus high-grade NHL (P < 0.0080) and low versus combination intermediate high-grade NHL (P < 0.0260). BLH were characterized by a greater percentage of acutely activated T cells with an early/late activated T-cell ratio of 1 .3. In contrast, B-cell NHL were characterized by at least a two-fold increase in the percentage of chronically activated T cells with early/late activated T-cell ratios of 0.4, 0.2, and 0.4 for low-, intermediate-, and high-grade B-cell NHL, respectively. The percentage of both activated TIL-T were not significantly different when nodal or extranodal NHL of the same grade were compared.
Discussion TIL-T are always present in NHL-involved lymph nodes. Regardless of their origin (in situ proliferation vs. bloodborne sequestration) their relationship to and interactions with neoplastic B cells remain an unresolved and challenging question. Since immune homeostasis results from a delicate balance between inducer and suppressor T cells,15 and imbalance of these immunoregulatory subsets has been demonstrated in some human diseases,16 17 it is conceivable that a T-cell immunoregulatory failure may be implicated in the pathogenesis of B-cell NHL. Alternatively, neoplastic B cells may have a THREE COLOR IMMUNOCYTOMETRY ACTIVATED T CELL SUBSET
[(CD3+CD25-HLADr+ (% of total CD3+ cells)] 1 nn-
svv
900
80-
F
70-
T L y M p H 0
60-
4 0-
C Y T
3 0-
IE
20-
I
s
I
50-
T T
1 0A.
vO-.HYPERPLASIA
LOW
INTERMEDIATE HIGH
I7hefilled bars r-epre%sent the mtean pCentcq q/e of cronicilit' ati'vated 1 cels ((1) 3 C)2 - IILAI)r + ) pe- total T livmv Iphoc'tes (total (I) 3 + cell/). A fowbiln-d iimc-ecs%e of this lmtf)hocTte' s1bs)e is Ix pesent atin ig-g,iade B cell wlin comtIpareied owith Bl-l. Figure 1.
Activated T-cell Subsets in BLH and B-cell NHL 507 AJP Septemnber 1991, Vol. 139, No. 3
Figure 2. Three dimensional contour plot of small lymphocytes from a low-grade (left panel) and a high-grade (right panel) B-cell non-Hodgkin's lymphoma. CD3 + cells are displayed on the x axis and HLADr + cells are displayed on the y axis. The percentage or numeric proportion of the cell subsets is represented in axis Z CD3 - HLADr + cells on the y axis are B cells. Very few CD3 + HLADr+ (chronically activated TIL-T) cells are present in the low-grade NHL (left), but numerous dual-labeled activated TIL-T cells (arrow) are present in the high-grade NHL (right). The CD3-HLADr cells present in the high-grade NHL represent neoplastic B cells with HLADr deletion.
proliferation rate beyond the functional limits of the immune system to contain neoplastic growth or may lack some surface molecules, which are important for immune-mediated cytotoxicity. Therefore, a host-immune response may appear during the natural course of some neoplasms, although this may have little or no protection for the host. The self recognition of B cells by T cells and the proliferation of activated T lymphocytes when naive T cells are under autologous B-cell stimulation is well established.18 This phenomenon, known as the autologous mixed lymphocyte reaction (AMLR), occurs as a T-cell response to recognition of other normal T and non-T cells (B cells and macrophages). A similar autologous T-cell response has been observed when T cells are challenged with tumor cells in the so-called mixed lymphocyte tumor culture (MLTC).19 Although positive MLTC between peripheral blood T cells and malignant solid tumor cells have been demonstrated,19'20 TIL-T responsiveness to neoplastic B cells in NHL has only recently been explored.3 Some studies have reported a low percentage of activated T cells in NHL-involved lymph nodes and no significant difference with healthy control lymph nodes.3'21 In contrast, other studies have found a greater percentage of activated T cells in NHL than in benign lymphoid hyperplasia.2223 We have found a similar proportion of acutely and chronically activated T cells in BLH with slight predominance of the former (ratio of 1.3), whereas a greater proportion of chronically activated TIL-T is present in B-cell NHL (ratio
