In vitro generation of tumour-specific lymphocyte reactivity to colonic carcinoma cells

Summary Purified tumour cells and normal mucosa cells from fresh human colorectal cancer resection specimens, and T-cell-enriched autologous peripheral blood lymphocytes, were mixed in short-term (6 day) mixed lymphocytetumour cell (MLTC) microcultures. Lymphocyte stimulation was measured by ³H-thymidine uptake, and a stimulation index (SI=[lymphocytes vs tumour cells (cpm)–tumour cells (cpm)]/[lymphocytes (cpm)])>3 was regarded as significant. Significant lymphocyte reactivity was found in 10/15 patients with colon carcinoma. However, 1 patient with autologous tumour reactivity, also showed significant stimulation against autologous normal mucosa cells, suggesting tumour-associated reactivity. Maximum stimulation occurred most frequently at a lymphocyte:tumour cell ratio of 2:1 and with nylon wool-passaged lymphocytes.Project was supported by a grant from the Cancer Research CampaignSupported by New South Wales Cancer Council Fellowship     

Immunological Reactivity in Patients with Carcinoma of Colon

Sixty cases of colonic carcinoma have been investigated for antitumour immunoreactivity. The tests employed were blood lymphocyte reactivity and complement-dependent serum cytotoxicity against cultured tumour cells, and immunofluorescence for membrane staining of viable tumour cells and cytoplasmic staining of dried tumour cells in films. Nineteen cases were positive by one or more tests and the most frequent positive response, lymphocytotoxicity, was detected in 8 of the 24 cases tested in this way. The lymphocytotoxicity persisted in a case tested three times over a year. Immunoreactivity against tumour cell surface, as by lymphocyte or serum cytotoxicity or membrane immunofluorescence, was restricted to colonic carcinomas but there was an additional element of individual specificity; cross-reactivity with other tissues and tumours was not observed. Lymphocytes from regional lymph nodes were non-reactive even in a case with positive blood lymphocyte cytotoxicity against the carcinoma cells.     

Immunogenicity and immunosensitivity of ex vivo human carcinomas: interferon γ and tumour necrosis factor α treatment of tumour cells potentiates their interaction with autologous blood lymphocytes

Human carcinoma cells vary appreciably in the expression of MHC class I, class II, ICAM-1 (CD54) and B7 (CD80) molecules. Short-term in vitro exposure of ex vivo carcinoma cells to interferon and tumour necrosis factor elevated/induced the surface expression of MHC class I, class II and ICAM-1, but only rarely of B7. We found that cytokine treatment elevated the cytotoxic susceptibility and the stimulatory potential of ex vivo tumour cells. This was demonstrated (a) by the increased frequency and elevated level of auto-tumour lysis and (b) by induction of DNA synthesis and generation of cytotoxic lymphocytes in autologous mixed lymphocyte/tumour cell culture (MLTC). The MHC class I and ICAM-1 molecules on the tumour cells were required for interaction with the lymphocytes as indicated by the inhibitory effect of specific mAb both in the stimulation and in the cytotoxic tests. While the cytokine-induced increases in MHC and ICAM-1 on the low-expression tumours were probably important for the modification of functional interaction with the autologous lymphocytes, it is likely that alterations in other properties of tumour cells were also induced which contributed to the phenomenon. This was indicated by the results obtained with several tumours, which expressed indigenously high levels of these molecules but activated the autologous lymphocytes only after cytokine treatment. In several experiments the untreated targets that did not activate the lymphocytes were sensitive to the cytotoxicity of the effectors activated in MLTC. The results show that the afferent and efferent arms of the immune response have different requirements for functional interactions between lymphocytes and tumour cells.     

In search of specific cytotoxic T lymphocytes infiltrating or accompanying human ovarian carcinoma

Summary Lymphocytes infiltrating human ovarian carcinoma obtained directly from the tumour mass (tumour-infiltrating lymphocytes, TIL) or from the carcinomatous ascites (tumour-associated lymphocytes, TAL) were expanded in vitro in long-term cultures with interleukin-2 and tested for their specific cytolytic activity. Killing of the autologous tumour was detected only in a proportion of the patients, less frequently in TIL compared to TAL. In fact two out of ten TIL and four out of nine TAL cultures tested showed significant levels of lysis against the autologous tumour. This cytotoxic activity was not restricted to the autologous tumour, as other tumour cell lines, including non-ovarian ones, were lysed as well. The cultures that were not cytotoxic against the autologous tumour were in most cases able to lyse other tumour cell lines of ovarian or other histology. Cloning of TIL from one patient was performed: of 22 clones tested, 4 displayed higher cytotoxicity against the autologous tumour compared to the uncloned population and 3 out of these 4 did not kill an irrelevant carcinoma cell line. In order to stimulate the expansion of putative specific effectors we performed mixed lymphocyte/tumour cultures (MLTC) with autologous or allogeneic tumour cells. No stimulation of cytotoxicity against the autologous tumour was detected after MLTC in nine different TAL populations, using autologous or allogeneic tumours as stimulators. On the contrary, peripheral blood lymphocytes from two patients after MLTC with the autologous tumour showed increased killing of the autologous and decreased killing of an allogeneic target. In conclusion TIL and TAL from ovarian carcinoma expanded in vitro with interleukin-2 usually have non-MHC-restricted cytotoxicity and variable degrees of reactivity against the autologous tumour. A preferential killing for the autologous tumour was not observed even after MLTC. These results do not exclude the existence of tumour-specific cytotoxic T lymphocytes in ovarian carcinoma; nevertheless they suggest that putative specific effectors have very low frequency and that culture techniques for expanding their growth more selectively are still to be optimized.     

Supernatant derived from a human hepatocellular carcinoma cell line (PLC/PRF/5) activates a population of T-suppressor cells

Harvest fluid derived from a primary hepatocellular carcinoma cell line (PLC/PRF/5) inhibited the incorporation of 3H-thymidine into PHA-activated human lymphocytes. A similar effect was observed when lymphocytes were pre-incubated with the tumour supernatant and washed prior to mitogen activation. Not only did the tumour supernatant inhibit 3H-thymidine incorporation by mitogen-activated lymphocytes, but it also inhibited production of the lymphokine leucocyte inhibitory factor (LIF). In experiments designed to establish whether a component of the tumour harvest fluid was activating a population of suppressor cells, normal mononuclear (MN) cells were treated with the PLC/PRF/5 or embryonic fibroblast supernatant for 48 h, after which they were washed and added to normal mitogen-activated lymphocyte cultures. Only cells pretreated with the PLC/PRF/5 supernatant suppressed mitogenesis. The cell responsible for the suppressor effect was a T cell, which after a further 24 h in culture liberated a suppressor factor responsible for inhibiting lymphocyte function. Although the nature of the factor/s in the PLC/PRF/5 supernatant responsible for activation of the T-suppressor cell population is unknown, it is suggested that this mechanism may be important in protecting the tumour from the immune response.     

Human ex vivo carcinoma cells produce transforming growth factor β and thereby can inhibit lymphocyte functions in vitro

 We tested 20 human carcinoma samples for the production of transforming growth factor β (TGFβ) in vitro. Tumour cell suspensions without obvious contamination with non-malignant cells were kept in culture conditions for 16 h and their supernatants were added to CCL-64 cells. The proliferation of these cells is inhibited by TGFβ. According to this assay, the supernatants contained both active and latent TGFβ. In addition, the supernatants were found to suppress the spontaneous cytotoxic function and activation of T-cell-enriched lymphocyte populations. A specific monoclonal antibody (mAb) counteracted these effects and therefore we concluded that they were mediated to a large extent by TGFβ. In line with the results obtained with the supernatants, activation of lymphocytes could also be inhibited by tumour cells and their inhibitory effect was weaker in the presence of the TGFβ-specific mAb. It is important to note that, when TGFβ-specific mAb was added to autologous mixed lymphocyte/tumour cell cultures, lymphocyte activation occurred more often. These results thus substantiate the assumption that production of TGFβ may help the survival of potentially immunogenic tumour cells in immunocompetent patients. Received: 21 August 1996 / Accepted: 12 November 1996     

Tumour-associated proliferative responses in vitro of regional lymph nodes draining solid cancers in man

Summary The proliferative responses in vitro of tumourdraining lymph node lymphocytes were evaluated against autologous colon and lung carcinoma cells. The reactivity of lymphocytes appeared to be directed against tumour-associated rather than tumour-specific antigens. The lymphocyte reactivity detected was not due to an autologous mixed lymphocyte reaction. Recombinant interleukin-2 augmented the responses detected but not their tumour specificity. Phenotypic characterisation indicated the presence of T suppressor/cytotoxic (TS/C) cells as well as natural killer (NK) cells. Only the latter, however, were active in functional cytotoxicity assays. The inability to generate both tumour-specific proliferation of tumour-draining lymph node lymphocytes and tumour-specific cytotoxic killer cells may be due to the presence of suppressor cells in the regional lymph nodes; preliminary data suggest the presence of such cells.Part of this project was supported by a grant from Cancer Research Campaign     

The polysaccharide K (PSK) potentiates in vitro activation of the cytotoxic function in human blood lymphocytes by autologous tumour cells

Summary We have studied the effect of polysaccharide K (PSK) in the in vitro recognition of ex vivo carcinoma, sarcoma and lymphoma cells by the autologous blood lymphocytes. In 4/25 experiments PSK treatment activated the lymphocytes for auto-tumour lysis. Tumour cells alone generated lytic activity both in short- (16 h) and in longterm (6 days) mixed lymphocyte/tumour cell cultures (MLTC), in 2/12 and 3/13 cases respectively. The tumours that activated the lymphocytes expressed high levels of major histocompatibility complex (MHC) class I molecules. In vitro cytokine (interferon and tumour necrosis factor ) treatment of the tumour cells elevated the amounts of class I antigens and the treated cells acquired stimulatory potential. When PSK was added to the MLTC, in which untreated tumour cells were used, lytic potential was induced in 9/13 short-term and in 11/12 long-term cultures. It is noteworthy that in the presence of PSK the untreated, negative or low-class-I-expressor tumours also activated the cytotoxic function of the lymphocytes in 4/5 long-term and in 6/7 short-term cultures. Even in the case of those lymphocytes that could be activated by PSK or tumour cells alone, the simultaneous exposure was more efficient. The effect of PSK was dose-dependent, being optimal at 1 µg/ml and 10 µg/ml. The presence of EDTA and/or cytochalasin B in the cytotoxic test performed with the activated effectors abrogated the lysis, indicating the requirement of contacts with the effector cells.     

Correlation between lymphocyte-mediated auto-tumor reactivities and the clinical course

Summary T-cell-enriched lymphocyte populations of 69 lung carcinoma (44 squamous cell, 23 adeno-, and two large cell carcinoma) patients were investigated at the time of surgery for proliferative response to, and/or cytotoxic potential against, freshly separated autologous tumor cells. Tumor-free period and survival time of the patients were correlated with the reactivity obtained in the in vitro tests. The observation time varied between 20 and 78 months (mean 52). Tumor-free period and survival time were longer and survival rate higher in the group with lymphocyte reactivity toward their tumors. In the non-reactive group, all patients but one died within 3 years. Almost all patients had cytotoxic lymphocytes against K562, the three who did not belonging to the category with short survival time.     

Cholesterol inhibits glutamine metabolism in LLC WRC256 tumour cells but does not affect it in lymphocytes: possible implications for tumour cell proliferation

The effect of cholesterol on proliferation and glutamine metabolism of lymphocytes and tumour cells was investigated. The addition of cholesterol to the culture medium did not cause a significant effect on [2-(14)C]-thymidine incorporation in lymphocytes. In the presence of concanavalin A, lymphocyte proliferation was increased by cholesterol (from 0.013 up to 1.3 microm). At high concentrations (234 and 468 microm), however, a marked inhibition of lymphocyte proliferation occurred. The same inhibitory effect was observed in the presence of lipopolysaccharides. Cholesterol also caused a marked decrease of LLC WRC256 tumour cell growth at 117 and 234 microm. The same findings were obtained by the measurement of [2-(14)C]-thymidine incorporation in these cells. The effect of cholesterol on phosphate-dependent glutaminase activity was then tested in cultured lymphocytes and LLC WRC256 tumour cells. Cholesterol at concentrations of 117 and 234 microm did not alter this enzyme activity in lymphocytes. However, this sterol, already at 26 microm, caused a 44 per cent reduction in glutaminase activity. Similar to the changes observed for glutaminase, cholesterol reduced glutamine oxidation in LLC WRC256 tumour cells, whereas no effect was observed on lymphocytes. Therefore, cholesterol might control lymphocyte and tumour cells proliferation by different mechanisms. The significance of these findings for the immune function in tumour-bearing patients remains to be investigated.     

Inhibition of protein tyrosine kinases or protein kinase C prevents nonspecific killer T lymphocyte-mediated tumoricidal activity

The signal transduction events which govern major histocompatibility complex-unrestricted tumour cell destruction by nonspecific killer T lymphocytes induced with anti-CD3 antibody have not yet been determined. In this study we used pharmacologic inhibitors to investigate the role of protein tyrosine kinases (PTK) and protein kinase C (PKC) in this process. The PTK-inhibitors herbimycin A, genistein, and methyl 2,5-dihydroxycinnamate blocked anti-CD3-activated killer T (AK-T) lymphocyte-mediated killing of tumour target cells. The PKC-inhibitors staurosporine, calphostin C, and myristoylated PKC pseudosubstrate peptide, as well as PKC desensitization by phorbol 12-myristate 13-acetate pretreatment, also suppressed the cytolytic effector function of AK-T lymphocytes. Lack of tumoricidal activity was not due to reduced AK-T lymphocyte binding to tumour target cells but was associated with the abrogation of granule exocytosis, indicating that PTK and PKC are involved in the postbinding process which results in delivery of the `lethal hit' by AK-T lymphocytes. © 1997 Elsevier Science B.V. All rights reserved.     

Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture

Summary Lymphocytes from cancer patients were stimulated in mixed culture with autologous tumour (MLTC) or pooled allogeneic lymphocytes (MLC). Both protocols induced increased uptake of ³H-thymidine at 5 days and the appearance of lymphoblasts. Blasts were isolated on discontinuous Percoll gradients and either expanded as bulk cultures or cloned directly under limiting dilution conditions in the presence of conditioned medium containing IL-2. Results with MLTC-blast-CTC have been reported elsewhere. MLC-activated cultures lysed autologous tumour but not autologous lymphoblasts. Lysis of some allogeneic tumours, lymphoblasts from members of the inducing pool, and K562 was also apparent. MLC activated cultures did not undergo restimulation in response to autologous tumour or lymphocytes but were restimulated by leukocytes from pool members.MLTC clones showed autologous tumour-specific cytotoxic activity or cross-reactive proliferative responses with tumours of the same site and histology. The majority of MLC clones cytotoxic for autologous tumour were also specific and did not lyse allogeneic tumour, K562, or lymphoblasts from the inducing pool. Two clones lysed autologous tumour and pool members. None of the clones tested proliferated in response to autologous tumour following MLC activation but some were responsive to pool members and one clone was restimulated by autologous monocytes. No association was found between clone phenotype and function. The implication of these data is that the effector cells with activity against autologous tumour induced in MLC arose largely by transstimulation of in vivo-activated tumour reactive lymphocytes by IL-2 release rather than expansion of NK-like effectors or sharing of antigenic specificities between tumour and allogeneic lymphocytes. Since MLC activation of cancer patients lymphocytes does not induce proliferative responses to autologous tumour it is unlikely to be a useful procedure in preparing cells for immunotherapy protocols. Abbreviations used in this paper: PBL, peripheral blood lymphocytes; TIL, tumour infiltrating lymphocytes; MLTC, mixed lymphocyte tumour culture; IL-2, interleukin-2; MLC, mixed lymphocyte culture; LSM, lymphocyte separation medium; BSS, balanced salt solution; HuSe, human serum; PBS, phosphate-buffered saline; CTC, cultured T cells; PHA, phytohaemagglutinin; CM, cultured medium; NK, natural killer; FcR, receptor for the Fc portion of IgG     

Immunosuppressive factors from human breast carcinoma cell lines that affect initiation of lymphocyte proliferation

Summary Supernatants from two human breast carcinoma cell lines, 734B and 231, have been shown to inhibit lymphocyte activation by mitogens and antigens. This inhibition appears to be specific for lymphocytes or recently stimulated cells, while having no effect on the growth of established cell lines. Studies of the mechanism of inhibition revealed that the factors inhibit lymphocyte activation and that the factors must be present at the initiation of lymphocyte stimulation for inhibition to occur. The supernatants do not inhibit lymphocyte activation by blocking binding of PHA to lymphocytes. Preliminary purification steps have shown that the inhibitory factors present in the tissue culture supernatants are precipitated at 50% ammonium sulfate saturation and their molecular weights are greater than 100 000. The inhibitory capacity of the 734B supernatants was destroyed by heating at 70° C, while the factors present in the 231 supernatants were only partially destroyed by heating to 90° C. The possible mechanism of action of the inhibitory substances released by tumors and their relevance to tumor growth are important to understanding of immune responses to neoplasia.     

In vivo infiltration of mononuclear cells in squamous cell carcinoma of the head and neck correlated with the ability to expand tumour-infiltrating T cells in vitro and with the expression of MHC class I antigens on tumour cells

A series of 18 head and neck squamous cell carcinoma biopsies, 6 primary and 12 recurrent, were investigated for tumour-infiltrating mononuclear cells with monoclonal or polyclonal antibodies. Our results suggest that the number of T cells at the tumour edge in vivo correlates well with their ability to expand in vitro in the presence of high-dose interleukin-2 (2000 U/ml). High MHC class I antigen expression on tumour cells was found to be positively correlated with p53 overexpression, suggesting that p53-derived peptides. wild-type or mutated ones, presented by MHC class I antigens, are potential targets for MHC-restricted cytotoxic T cells in head and neck squamous cell carcinomas. However, lack of correlation between peritumoural T cell infiltration in vivo and T cell expansion in vitro on the one hand, and p53 over-expression on tumour cells, on the other hand, suggests absence of p53-peptide-specific T cells in the patients. Eight out of ten expanded tumour-infiltrating lymphocyte (TIL) cultures showed T-cell-mediated cytotoxicity. ?Promiscuous” cytotoxic T cell activity against the natural-killer-cell-sensitive K562 target cell line was observed in three out of ten TIL expansion cultures.     

Conversion of human lymphocytes to antitumor immune reactivity by xenogeneic and allogeneic imune RNA detected by leukocyte-adherence inhibition tests

Summary Using leukocyte adherence inhibition (LAI) tests, we studied the activity of xenogeneic immune RNA (I-RNA) extracted from the spleen and lymph nodes of sheep after immunization with human breast carcinoma tissue or keyhole limpet hemocyanin (KLH) in inducing lymphocytes from normal healthy donors to mediate immune responses in vitro. Mononuclear cells isolated from venous blood of normal donors, depleted of monocytes and, in some experiments, separated into T cells and non-T cells, were incubated with and without anti-breast carcinoma I-RNA or anti-KLH I-RNA for 20 min at 37° C. Then, lymphocyte adherence was determined by a Coulter counter method in the presence of 3 M KCl extracts of breast carcinoma tissues, control tissue, or KLH. Following incubation with anti-breast carcinoma I-RNA, the adherence of lymphocytes from normal donors was found to be inhibited only in the presence of breast carcinoma extracts. Following incubation with anti-KLH I-RNA, lymphocyte adherence was inhibited only in the presence of KLH. The principal effector cells involved appeared to be T lymphocytes. I-RNA treatment with RNase, but not with DNase or pronase, completely abrogated the LAI responses. In a blind study utilizing coded samples of xenogeneic and allogeneic I-RNA of unknown origin, samples containing activity against breast cancer extracts were identified correctly by LAI. Abbreviations used: I-RNA, immune RNA; LAI, leukocyte adherence inhibition; KLH, Keyhole limpet hemocyanin; PBS, phosphate buffered saline; RNase, ribonuclease; DNase, deoxyribonuclease     

Cellular characteristics of peripheral blood lymphocytes and tumour-infiltrating lymphocytes in patients with gynaecological tumours

 Immunotherapy of gynaecological cancer with tumour-infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL) has become a valid treatment modality with varying degrees of success in obtaining an antitumour response. TIL consist of lymphocytes, mainly T cells and minor populations of natural killer cells or B cells. Conventional cytogenetic studies of tumour cells from patients with breast and ovarian cancer have shown multiple chromosomal abnormalities including chromosomes 7 and 12. This study was designed to analyse the surface further, as well as investigate the intracellular, characteristics of TIL by multicolour flow cytometry and the cytogenetic features by fluorescence in situ hybridization. Tumour cell, peripheral blood and TIL samples from 25 patients (15 ovarian tumours, 8 breast cancers, 1 uterine sarcoma, 1 cervical carcinoma) were analysed for their phenotype, the expression of major cytokines [interleukin-2 (IL-2), IL-4 and interferon γ (IFNγ)], their proliferation rate, their cytotoxic ability and for the presence of numerical aberrations of chromosomes 7 and 12. All the tumour cells showed a high frequency of numerical aberration in chromosomes 7 and 12, especially trisomies or tetrasomies and combined aberrations. Trisomies of both chromosomes also occured at a low percentage in TIL and PBL. Received: 20 June 1996 / Accepted: 4 January 1997     

In vitro sensitization of human lymphocytes to a myeloma cell-related antigen

Peripheral blood lymphocytes from normal human donors were cocultivated with cells from two established human multiple myeloma cell lines, RPMI 8226 and K-737, and with lymphoblastoid cells from a third B cell line, RAMM. After a comparison of three methods of lymphocyte sensitization, a 6-day incubation protocol with equal numbers of normal lymphocytes and mitomycin C-treated tumor cells was selected. Cells from the RPMI 8226 myeloma line stimulated the differentiation of lymphocytes into cytotoxic effector cells as measured by ⁵¹Cr release from labeled target cells. The RPMI 8226-sensitized lymphocytes were cytotoxic for myeloma cells (RPMI 8226 and K-737) and for lymphoblastoid cells (RAMM) but not for cells from human lung tumor lines (A549, A427, MB9812), a breast carcinoma line (ALAB), a normal diploid fibroblast line (HSBP), or normal lymphocytes.     

Lymphocyte subsets as prognostic markers for cancer patients receiving immunomodulative therapy

Immunogenic features of some malignancies have aroused interest in immunotherapy of cancer. Immunotherapy seems most effective in patients with a small tumour burden, and the focus of immunotherapy trials has, thus, lately been on adjuvant treatment. To enable further development of immunotherapy we need to know more about the mechanisms involved in host defence, especially when the system is influenced by extrinsic factors, that is, immunomodulative agents. T lymphocytes play an important role in the host defence against tumour cells trying to escape from immune surveillance. The mechanisms that regulate the host defence systems are complex, and the influence of extrinsic factors such as immunotherapeutic agents is poorly understood. Most data on lymphocyte subsets in malignant disease originate from melanoma or renal cell carcinoma (RCC) studies, although there are scattered data on lymphocyte subsets also in other malignancies. There are several studies implying that the relative amount of CD4+, CD8+, and natural killer (NK) cells may be important and that, by reducing the tumour burden or by using different therapeutic agents, we can stimulate the host defence. However, only some of these studies imply that these changes can have an impact on clinical outcome and prognosis. The findings of the studies reviewed in this paper are mostly encouraging, but whether the lymphocyte subsets have any value as prognostic markers in patients with malignancies receiving immunotherapy is still unclear. Large randomized immunotherapy trials including an observation arm give an ideal opportunity to recognize those immunological changes that are due to therapy, related to the natural host defence, or whether they have any prognostic value.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Selective Cytotoxicity of Anti-Kappa Serum for B Lymphocytes

MOUSE lymphocytes can be divided into two distinct populations according to the density of immunoglobulin determinants on their surface. Lymphocytes with a high density of immunoglobulin are marrow-derived, nonthymus-processed, B cells, whereas lymphocytes with little or no immunoglobulin are thymus-derived, T cells^1–3. Since more than 95% of mouse immunoglobulin light chains are of the kappa type⁴, treatment of lymphocyte suspensions with an appropriate dilution of rabbit anti-mouse kappa serum and complement should be cytotoxic for only B lymphocytes. This prediction was tested by using lymphocyte populations enriched for either T or B cells or containing the two cell types in a known proportion.