Decreased Expression of the Signal-Transducing Chains in Tumor-Infiltrating T-Cells and NK Cells of Patients with Colorectal Carcinoma

[CANCER RESEARCH 53, 5610-5612, December 1, 1993] Advances in Brief

Decreased Expression of the Signal-transducing Chains in Tumor-infiltrating T-Cells and NK Cells of Patients with Colorectal Carcinoma

Hiroshi Nakagomi, Max Petersson, Inger Magnusson, Claes Juhlin, Masanori Matsuda, H~tkan Meilstedt, Jean-Luc Taupin, Eric Vivier, Paul Anderson, and Rolf Kiessling 2 Microbiology and Tumor Biology Center, Laboratory of Immunology, Karolinska Institute [H. N., M. P., M. M., R. K.]; Department of Surgery, SOdersjukhuset [L M., H. M.]; Radiumhemmet, Karotinska sjukhuset [L M., 1-1. M.], Stockholm, Sweden; Department of Surgery, Akademiska sjukhuset, Uppsata, Sweden [C. J.]; and Dana Farber Cancer Institute, Boston, Massachusetts 02115 [J-L. T., E. V., P. A.]

Abstract one of D). Single cell suspensions of the tumors were prepared by enzymatic digestion (4). This "bulk" preparation of tumor-derived cells contained 33 +-- An impaired immune response is frequently observed in cancer patients 27% (Mean -+ SD; n --- 14) of CD3 + cells, and the remaining cells were mainly and tumor-bearing mice. T-cells from mice with an experimental colon composed of tumor cells. Peripheral venous blood was taken from the patients carcinoma were recently shown to express T-cell receptors that completely during or shortly after the surgery, and PBMC were isolated by Ficoll density lacked the signal-transducing molecule CD3 ~. Here, we have investigated centrifugation. The expression of CD3 ~ and CD16 ~ was investigated by flow the expression of the signal-transducing molecule ~ on lymphocytes from cytometric analysis of permeabilized cells using a monoclonal antibody 14 patients with colorectal carcinomas using flow cytometric analysis of (TIA-2; IgG1) specific for the cytoplasmic domain of the ~ chain (5) and permeabilized cells with a monoclonal antibody (TIA-2; IgG1) specific for followed by staining with a rabbit-anti-mouse-fluorescein isothiocyanate anti- the cytoplasmic domain of the ~ chain as well as with immunoprecipitation body as described (6). Cells were double stained with phycoerythrin (FL2)- and analysis on diagonal gel electrophoresis. We demonstrate that T-cells conjugated mAbs to CD3, CD16, CD4, or CD8 (Dako, Glostrup, Denmark) isolated from the tumors of the patients express significantly less CD3 and then subjected to flow cytometric analysis using a FACScan flow cytom- than T-cells in the peripheral blood of the same patients and that the eter (Becton Dickinson). The mean FL1 of PBMC and TIL samples from peripheral blood of the patients express decreased levels of ~ chains, as cancer patients was expressed as the percentage of the average mean FL1 value compared to the levels found in lymphocytes from healthy controls. This of PBMC from healthy donors (n = 9). Statistics were calculated as probability decreased expression was also observed on ~ chains associated with the low for Student's t test (different variance cases). For immunoprecipitations, lym- affinity Fc receptor for IgG found in tumor-infiltrating NK cells phocytes were permeabilized with digitonin and subjected to radio-iodination (FcvRIIIAe~; CD16). with 1251 by the lactoperoxidase method and immunoprecipitated as described (6). The immunoprecipitated samples were analysed with diagonal gel elec- Introduction trophoresis (6). A multitude of different mechanisms have been suggested to ac- count for the poor immune response of lymphocytes from cancer Results and Discussion patients, including an active inhibition by suppressor cells, decreased The expression of CD3 ~ was investigated with flow cytometric production of cytokines, or the presence in the tumor of cytokines analysis comparing T-cells from TIL and PBMC of patients with with known suppressive effects on T-cell functions (1). TIL 3 from T-cells from PBMC of healthy controls (Fig. 1A; n -- 9). A markedly human or experimental tumors often have impaired effector functions reduced CD3 ~ expression was detected in TIL cells of all investigated which could be normalised upon culture in recombinant IL-2 (2). patients (Fig. 1C; n = 14; 23 _ 11% (SD) of control PBMC; P < Mizoguchi et al. (3) recently reported alterations in the signal trans- 0.001). The T-cells in the tumors expressed, on the average, one-half duction molecule CD3 ~ associated with TCR in mice bearing an as many CD3 ~ chains as those in PBMC from the same group of experimental colon carcinoma. Since alterations in the CD3 g chain patients (P < 0.01). Also, the peripheral T-cells from the patients may disrupt the internal signaling of the TCR, leading to a deficient showed decreased expression of ~ compared to T-cells from healthy T-cell activation, this suggests a fundamentally new way of explaining controls (Fig. 1B; n = 12; 47 • 22%; P < 0.001). The decrease was immune defects in tumor-bearing hosts. We have investigated the selective for the ~ chain, as expression of the CD3 9determinant was expression of CD3 ~ in T-cells and of CD16 ~ in NK cells isolated unaffected in the TIL population as well as in the PBMC from this from the tumors of patients with human colorectal carcinoma as well group of patients (data not shown). There was considerable variation as from the peripheral blood of the same group of patients. Our results among different patients, ranging from only a moderate decrease demonstrate for the first time reduced expression of ~ chains in lymphocytes from human cancer patients. (patients 3 and 9) to an almost absent expression (patients 5, 10, and 13) of CD3 ~. Also, PBMC from healthy donors had varying expres- Materials and Methods sion of CD3 ~ (Fig. 1A). Arguing against the possibility that the low CD3 g expression in the Tumor samples were obtained from 14 patients during surgery for their TIL cells may be an artifact due to the procedure involved in isolating primary colorectal carcinoma (three of Dukes stage A; six of B; four of C; and TIL was the observation that the same enzymatic treatment did not significantly affect CD3 ~ expression of control PBMC (n = 5, 92 _ Received 8/19/93; accepted 10/20/93. 13% of nontreated PBMC). The CD4 + as compared to the CD8 + TIL The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with cell subset demonstrated the same degree of decreased CD3 ~ expres- 18 U.S.C. Section 1734 solely to indicatethis fact. sion (data not shown). 1 This work was supported by grants from the Swedish Cancer Society and from the Since the low affinity Fc receptor for IgG on human NK cells Cancer Society in Stockholm. M. M. is the recipient of a fellowshipfrom the Wenner- Grenska samfundet (WennergrenFoundation), Stockholm,Sweden. (Fc3,RIIIAa; CD16) also is expressed in association with a ~:~ ho- 2 To whom requests for reprints should be addressed, at Dept. of Immunology,Karo- modimer and a ~:7 heterodimer (5), the expression of CD16 ~ was also linska institute, S-171 77 Stockholm,Sweden. 3 The abbreviationsused are: TIL, tumor-infiltratinglymphocytes; TCR, T-cell recep- investigated in NK cells from TIL and PBMC in this patient group. tors; PBMC, peripheral blood mononuclearcells; FL1, fluorescenceintensity 1. Compared to the expression of CD16 ~ in NK cells from PBMC of 5610

A! Be CJ 9 CD3+ NORMAL PBMC 9 CD3+ CANCER PBMC 9 CD3+ TIL 140 140 14o 1 gt~ 12o 120 ! o" 12o o |.o F,,., 3= 100 E IO0 _~ 100 tl tl '~ 8o ~ 8o

0 (.~ N ~0 ,.,, 60 ,.- 60

~ 4o E 4o ~ 40 (b (.)

~ 2o =~ 20 ,,~ 20

0 0

9- <: UJ =~ A PBMC NR =~ CANCERPAT NR .~ CANCERPAT NR

D. E. F. 9 CD16+ NORMAL PBMC 9 CD16+ CANCER PBMC 9 CD16+ TIL 14o 14o -~ 14o

12o ~ 12o t ~ 1~o

_~ 100 ~ 100 _~ 100 u. ,';" .';-

'~o 80 r'~ 80 '~ SO

,,,, 60 =~ 60 ,~ SO O. I1,, O.

40 ~ 40 ~ 40 r 0 0 "6 ~ '~ 20 ~ 20 =~ 20

0 0 0

NOaMLA PBMC NR .~ CANCERPAT NR U.I= CANCERPAT NR Fig. 1. The expression of CD3 ~ or CD16 ~ as analyzedby FACScan. The mean FL1 values obtained with PBMC and TIL from cancer patients (Panels B, C, E, and F) were expressed as the percentage of the average mean FL1 value obtained with PBMC from healthy donors (Panels A and D). The expression of CD3 ~ (Panels A, B, and C) as well as CD16 (Panels D, E, and F) is shown.

healthy controls (Fig. 1D), a significantly decreased expression of the gel shown in Fig. 2, one-dimensional electrophoretic analysis CD16 ~ was found in the TIL as well as in the PBMC derived from the indicated that both CD16 and CD3.TCR were associated with 3':3' patients (Fig.IF, n = 8, 24 ___ 13%, P < 0.001 for TIL; Fig. 1E, n = homodimers from FceR1 in TIL isolated from patients with colorectal 12, 39 - 17%, P < 0.001 for PBMC; and TIL:PBMC, P < 0.05) in the carcinoma (data not shown). same range as that seen for CD3 ~. Thus, decreased expression of the It was not clear whether the reduced expression of ~ chains was molecule was apparent in the T-cell as well as the NK cell com- causally related to the malignant disease or was dependent upon some partment of these patients. Biochemical confirmation of the low CD3 factor(s) unrelated to the tumor in this group of patients. Colorectal expression in the TIL was done in a representative patient with carcinoma is a malignancy of advanced age. No correlation was, colorectal carcinoma by separating anti-CD3e immunoprecipitates on however, observed between the CD3 ~ expression and the age of the nonreducing/reducing diagonal gels as shown in Fig. 2. The TCR patients (linear regression; r 2 = 0.0927; n = 12). The strong reduction complex from PBMC of a control individual included the ~:~ ho- of CD3 ~ observed in TIL is particularly interesting in relation to the modimer (Fig. 2A), while the TCR complex from TIL cells (Fig. 2B) comparatively moderate decrease of this molecule on peripheral lym- as well as PBMC (data not shown) of a patient with colorectal car- phocytes from the same group of patients. This may be interpreted as cinoma did not. Mizoguchi et aL (3) found the two ~ chains of the an effect of a locally produced factor affecting, although to a lesser CD3 complex in tumor-bearing mice to be replaced by two structur- degree, peripheral cells as well. Alternatively, lymphocytes located in ally related proteins from the Fc receptor for IgE expressed on mast the gut may generally express lesser amounts of this molecule, and the cells and basophils (FceR1). Although the Mr 8,000-10,000 off-di- low expression in TIL may be unrelated to a tumor-host interaction. In agonal spot corresponding to the 3':3' homodimer is poorly resolved in support of this interpretation, Malissen et al. (7), studying the T-cell 5611

corresponds to the murine thymus-independent gut intraepithelial NORMAL PBL lymphocytes in their report should therfore be considered. If so, how- NR ever, the lower expression of ~ in PBMC reported here and by Mizoguchi et aL (3) must still be a phenomenon associated with cancer and distinct from the suppression observed locally in the tumor.

~.~ ,~- 69 KID The functional consequences of reduced ~ expression on TIL and what role this has in the deficient proliferation and cytotoxicity of freshly 46KD isolated TIL (2) is not dear. Preliminary results indicate that reduced CD3 ~ expression is not unique to malignant diseases and that this A phenomenon occurs in peripheral blood as well as locally in some .... -30KD autoimmune diseases, 4 indicating that this may reflect a dysregulation

C~,~ of immune function common to various conditions characterized by immune deficiency. - 21KD

- 14KD References 1. Oliver, R. T., and Nouri A. M. T-cell immune response to cancer in humans and its relevance for immunodiagnosis and therapy. Cancer Surv., 13: 173-204, 1992. 2. Mischer, S., Whiteside, T. L., Carrel, S., and von Fliedner, V. Functional properties of tumor-infiltrating and blood lymphocytes in patients with solid tumors: effects of TIL COLON tumor cells and their supernatants on proliferative responses of lymphocytes. J. Immunol., 136: 1899-1907, 1986. 3. Mizoguchi, H., O'Shea, J. J., Longo, D. L., Loeffler, C. M., McVicar, D. W., and Nil Ochoa, A. C. Alterations in signal transduction molecules in T-lymphocytes from tumor-beating mice. Science (Washington DC), 258: 1795-1798, 1992. 4. Pisa, P., Halapi, E., Pisa, E. K., Gerdin, E., Hising, C., Bucht, A., Gerdin, B., and Kiessling, R. Selective expression of interleukin 10, interferon 7, and granulocyte- macrophage colony-stimulating factor in ovarian cancer biopsies. Proc. Natl. Acad. Sci. USA, 89: 7708-7712, 1992. 5. Anderson, P., Caligiuri, M., O'Brien, C., Manley, T., Ritz, J., and Schlossman, S. F. Fc3' receptor type IIl (CD16) is included in the/3NK receptor complex expressed by 87: 46KD human natural killer cells. Proc. Natl. Acad. Sci. USA, 2274, 1990. 6. Vivier, E., Rochet, N., Kochan, J. P., Presky, D. H., Schlossman, S. E, and Anderson, P. Structural similarity between Fc receptors and T-cell receptors. J. Immunol., 147: 4263-4270, 1991. 7. Malissen, M., Gillet, A., Rocha, B., Trucy, J., Vivier, E., Boyer, C., Krntgen, E, Brun,

CD3 N., Mazza, G., Spanopoulou, E., Guy-Grand, D. and Malissen, B. T-cell development 9 -~. ~ :: in mice lacking the CD3-~/'q gene. EMBO J., in press, 1993. - 211r, D

- 14KD

Fig. 2. Diagonal gel electrophoresis of anti-CD3 9lmmunoprecipitates of PBMC from a healthy control (A) and TIL from a patient with colorectal carcinoma (B). The molecular components of the TCR complex are indicated. development in mice lacking the CD3 ~ gene, found that gut intraepithelial lymphocytes present in these mice expressed normal levels of TCR complexes which were associated with 7:3' homodimers from FeaR1. The possibility that the human TIL population studied by us 4 p. Anderson and R. Kiessling, unpublished results.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1993 American Association for Cancer Research. Decreased Expression of the Signal-transducing ζ Chains in Tumor-infiltrating T-Cells and NK Cells of Patients with Colorectal Carcinoma

Hiroshi Nakagomi, Max Petersson, Inger Magnusson, et al.

Cancer Res 1993;53:5610-5612.

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