Interleukin-12 increases bispecific-antibody-mediated natural killer cell cytotoxicity against human tumors

 The combination of CD16/CD30 bispecific monoclonal antibodies (bi-mAb) and unstimulated human resting natural killer (NK) cells can cure about 50% of mice with severe combined immunodeficiency (SCID) bearing subcutaneously growing established Hodgkin’s lymphoma. As interleukin-2 (IL-2) and IL-12 have been shown to increase NK cell activity, we tested the capacity of these cytokines to increase bi-mAb-mediated NK cell cytotoxicity against two types of human tumors (Hodgkin’s disease and colorectal carcinoma). Unstimulated NK cells needed a three- to five-times higher antibody concentration than cytokine-stimulated NK cells to exert similar levels of bi-mAb-mediated cytotoxicity. The augmented tumor cell lysis was achieved with IL-12 at considerably lower concentrations than with IL-2 and was associated with a significantly increased bi-mAb-mediated intracellular Ca²⁺ mobilization. The efficiency of IL-12 in this setting together with its low toxicity make it the ideal candidate for a combination therapy with NK-cell-activating bi-mAb in human tumors that are resistant to standard treatment. Received: 26 July 1995 / Accepted: 16 November 1995     

Further characterization of cytotoxic T cells generated by short-term culture of human peripheral blood lymphocytes with interleukin-2 and anti-CD3 mAb

 In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3⁺ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fcγ receptor (FcγRI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab′)₂, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18⁺ ovarian tumor cell line), was also compared in the presence of a bispecific antibody (OC/TR, anti-CD3 × MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 ×antitumoral bispecific antibodies. Received: 6 December 1995 / Accepted: 4 June 1996     

Nucleosome-dependent escape of tumor cells from natural-killer-mediated lysis: nucleosomes are taken up by target cells and act at a postconjugation level

 Our previous data suggested that chromatin fragments released from dead cells into the extracellular medium could be involved in the impairment of natural-killer (NK)-mediated cytotoxicity reported in cancer patients. In the present study, an inhibition of the NK-mediated lysis was obtained in vitro by nucleosome addition to different tumor target cells, independently of their sensitivity to NK-mediated lysis. We observed a rapid endocytosis and degradation of nucleosomes by K562 tumor target cells and (although to a much lesser extent) a binding to a subpopulation of lymphocytes. Nucleosomes impaired neither the conjugation step nor the expression of adhesion molecules at the effector (CD11a, CD18, CD2) or target (CD54, CD58) cell surface. On the contrary, flow-cytometry analysis of the conjugation suggested that nucleosomes might stabilize the conjugates. Investigations of the killing process showed that nucleosomes decreased the NK cytotoxic potential without modifying Ca²⁺-dependent lethal-hit-delivery kinetics. The cytotoxic potential was not restored by increasing the available magnesium and calcium concentrations in the extracellular medium. Taken together, the results suggest that the inhibition of NK-mediated lysis by nucleosomes may result from alterations of the NK mechanism at the postconjugation level and after lethal-hit delivery. Hence, the inhibition could involve a delay in the recycling of effector cells, or a resistance of tumor target cells to NK cells. Received: 7 October 1996 / Accepted: 12 November 1996     

Perforin and granzyme B induce apoptosis in FasL-resistant colon carcinoma cells

 Cytotoxic lymphocytes may induce apoptosis in their target cells by the FasL (Fas ligand) pathway or the perforin/granzyme B pathway. It has been shown that Fas-expressing colon carcinoma (CC) cells are resistant to FasL-mediated apoptosis. The aims of this study were to determine whether CC cells are also resistant to perforin/granzyme B and whether the FasL resistance lies upstream of caspase-3 activation. The resistance of the Fas-expressing rat CC531s cells to the FasL pathway was confirmed by treating them with recombinant human soluble FasL, using rat hepatocytes as a positive control. The intracellular delivery of granzyme B by sublytic concentrations of perforin, on the other hand, resulted in many features of apoptosis (chromatin condensation, nucleus fragmentation, loss of microvilli and internucleosomal DNA fragmentation) within 3 h. Since both the FasL and perforin/granzyme B pathways converge at caspase-3, we measured caspase-3 activity to learn whether the FasL resistance was due to failure to activate this crucial executioner. Caspase-3 activation occurred in CC531s cells after perforin/granzyme B treatment, but not after the addition of recombinant FasL. Furthermore, we showed that caspase-3 activity is involved in the execution of perforin/granzyme-B-induced apoptosis in CC531s cells, since the cell-permeable caspase-3 inhibitor Z-DEVD-FMK abrogated DNA fragmentation. Together, these results suggest that CC cells are sensitive to perforin/granzyme-B-induced apoptosis by activating caspase-3 and FasL resistance lies upstream of this executioner caspase. Received: 20 November 2000 / Accepted: 8 March 2001     

Perforin, granzyme B, and FasL expression by peripheral blood T lymphocytes in emphysema

Background

It is generally accepted that emphysematous lungs are characterized by an increase in the numbers of neutrophils, macrophages, and CD8⁺ T lymphocytes, the lasts having increased cytotoxic activity. Because systemic inflammation is also a component of emphysema, we hypothesize that peripheral CD8⁺ T lymphocytes of emphysematous smokers who show evidence of systemic inflammation will have higher expression of cytotoxic molecules.

Methods

We assessed parameters of systemic inflammation in normal individuals (smokers or non-smokers) and in emphysematous subjects with an active smoking history by measuring serum interleukine-6, C-reactive protein, and tumor necrosis factor. Expression of perforin, granzyme B, and FasL protein by CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and natural killer cells were assessed by flow cytometry while perforin, granzyme B, and FasL mRNA expression were measured on purified systemic CD8⁺ T lymphocytes by real-time PCR.

Results

Emphysematous smokers had higher levels of serum interleukine-6 than normal subjects. Even with the presence of systemic inflammation in emphysematous smokers, the percentage of peripheral CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and NK cells expressing perforin and granzyme B protein was not different between the three groups.

Conclusion

Despite evidence of systemic inflammation, peripheral T lymphocytes of emphysematous smokers did not show higher levels of cytotoxic markers, suggesting that increase of activated T lymphocytes in the emphysematous lung may be due to either activation in the lung or specific peripheral recruitment.     

Altered effector function of peripheral cytotoxic cells in COPD

Background

There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8⁺ T lymphocytes, natural killer (NK; CD56⁺CD3^-) cells and NKT-like (CD56⁺CD3⁺) cells.

Methods

Peripheral blood mononuclear cells (PBMCs) were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies.

Results

The proportion of peripheral blood NKT-like (CD56⁺CD3⁺) cells in smokers with COPD (COPD subjects) was significantly lower (0.6%) than in healthy smokers (smokers) (2.8%, p < 0.001) and non-smoking healthy participants (HNS) (3.3%, p < 0.001). NK (CD56⁺CD3^-) cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p < 0.001) as were NKT-like (CD56⁺CD3⁺) cells (16.7% vs 52.4% specific lysis, p < 0.001). Both cell types had lower proportions expressing both perforin and granzyme B. Blocking the action of perforin and granzyme B reduced the cytotoxic activity of NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells from smokers and HNS.

Conclusion

In this study, we show that the relative numbers of peripheral blood NK (CD56⁺CD3^-) and NKT-like (CD56⁺CD3⁺) cells in COPD subjects are reduced and that their cytotoxic effector function is defective.     

Disregulation in TH1 and TH2 subsets of CD4+ T cells in peripheral blood of colorectal cancer patients and involvement in cancer establishment and progression

 Recent theories have established that, during an ongoing immune response, the lymphokines produced by TH1 and TH2 subsets of CD4⁺ T cells are critical to the effectiveness of that response. In vivo and in vitro studies have demonstrated that the type of environmental cytokines plays a determinant role in directing the development of naive T cells into TH1 or TH2 effector cells. Disregulated expansion of one or other subset may contribute to the development of certain diseases. To establish whether a similar situation might exist in the cells of the peripheral blood (PBMC) of colorectal cancer patients, we have performed immunological studies on a group of patients and a group of healthy subjects. We examined the interleukin-2 (IL-2), interferon γ (IFNγ), IL-4, IL-6 and tumour necrosis factor α levels in serum; the production of IL-4 and IL-2, with and without activating agents, by PBMC, tumour-draining lymph node lymphocytes and tumour cells; and the proliferative response of PBMC to IL-2, IL-4 and anti-CD3 monoclonal antibody (anti-CD3), which were variously combined. The data of the present study lead us to hypothesize that, because of suppressive effects probably due to environmental IL-4, in the peripheral blood of patients there seems to be a disregulation in the functionality of TH1 and TH2 subsets of CD4⁺ T cells, with an expansion in TH2 and a malfunction in TH1 cells. Moreover it seems that this disregulation increases with as the disease progresses through the stages, suggesting that it can be directly implicated in the mechanisms that allow the tumour to locate and progress in the host. Received: 27 June 1995 / Accepted: 13 November 1995     

Human CD4+ T cells lyse target cells via granzyme/perforin upon circumvention of MHC class II restriction by an antibody-like immunoreceptor

Immune elimination of tumor cells requires the close cooperation between CD8+ CTL and CD4+ Th cells. We circumvent MHC class II-restriction of CD4+ T cells by expression of a recombinant immunoreceptor with an Ab-derived binding domain redirecting specificity. Human CD4+ T cells grafted with an immunoreceptor specific for carcinoembryonic Ag (CEA) are activated to proliferate and secrete cytokines upon binding to CEA+ target cells. Notably, redirected CD4+ T cells mediate cytolysis of CEA+ tumor cells with high efficiencies. Lysis by redirected CD4+ T cells is independent of death receptor signaling via TNF-alpha or Fas, but mediated by perforin and granzyme because cytolysis is inhibited by blocking the release of cytotoxic granules, but not by blocking of Fas ligand or TNF-alpha. CD4+ T cells redirected by Ab-derived immunoreceptors in a MHC class II-independent fashion substantially extend the power of an adoptive, Ag-triggered immunotherapy not only by CD4+ T cell help, but also by cytolytic effector functions. Because cytolysis is predominantly mediated via granzyme/perforin, target cells that are resistant to death receptor signaling become sensitive to a cytolytic attack by engineered CD4+ T cells.     

Tumor regression after adoptive transfer of effector T cells is independent of perforin or Fas ligand (APO-1L/CD95L).

The adoptive transfer of tumor-specific effector T cells can result in complete regression and cure mice with systemic melanoma, but the mechanisms responsible for regression are not well characterized. Perforin- and Fas ligand (APO-1/CD95 ligand)-mediated cytotoxicity have been proposed as mechanisms for T cell-mediated tumor destruction. To determine the role of perforin and Fas ligand (FasL) in T cell-mediated tumor regression in a murine melanoma model, B16BL6-D5 (D5), we generated D5-specific effector T cells from tumor vaccine-draining lymph nodes of wild type (wt), perforin knock out (PKO), or FasL mutant (gld) mice and treated established D5 metastases in mice with the same genotype. Effector T cells from wt, PKO and gld mice induced complete regression of pulmonary metastases and significantly prolonged survival of the treated animals regardless of their genotype. Complete tumor regression induced by PKO effector T cells was also observed in a sarcoma model (MCA-310). Furthermore, adoptive transfer of PKO and wt effector T cells provided long-term immunity to D5. Therapeutic T cells from wt, PKO, or gld mice exhibit a tumor-specific type 1 cytokine profile; they secrete IFN-gamma, but not IL-4. In these models, T cell-mediated tumor regression and long-term antitumor immunity are perforin and FasL independent.     

Serglycin-deficient cytotoxic T lymphocytes display defective secretory granule maturation and granzyme B storage

Cytotoxic T lymphocytes eliminate infected and tumor cells mainly by perforin/granzyme-induced apoptosis. Earlier studies suggested that serglycin-proteoglycans form macromolecular complexes with granzymes and perforin in the cytotoxic granule. Serglycin-proteoglycans may also be involved in the delivery of the cytolytic machinery into target cells. We have developed a serglycin-deficient mouse strain, and here we studied the importance of serglycin-proteoglycans for various aspects of cytotoxic T lymphocyte function. 35SO4(2-) radiolabeling of serglycin-deficient cells demonstrated a dramatic reduction of incorporated label as compared with wild type cells, indicating that serglycin is by far the dominating proteoglycan species produced by the cytotoxic T lymphocyte. Moreover, lack of serglycin resulted in impaired ability of cytotoxic T lymphocytes to produce secretory granule of high electron density, although granule of lower electron density were produced both in wild type and serglycin-deficient cells. The serglycin deficiency did not affect the mRNA expression for granzyme A, granzyme B, or perforin. However, the storage of granzyme B, but not granzyme A, Fas ligand, or perforin, was severely defective in serglycin-deficient cells. Serglycin-deficient cells did not display defects in late cytotoxicity toward target cell lines. Taken together, these results point to a key role for serglycin in the storage of granzyme B and for secretory granule maturation but argue against a major role for serglycin in the apoptosis mediated by cytotoxic T lymphocytes.     

Apoptosis in antibody-dependent monocyte-mediated cytotoxicity with monoclonal antibody 17-1A against human colorectal carcinoma cells: enhancement with interferon γ

 Antibody-dependent cell-mediated cytotoxicity (ADCC) has been considered to be one of the main effector mechanisms by which unconjugated monoclonal antibody (mAb) 17-1A can exert an antitumor effect in vivo. Since the apoptotic pathway as well as the necrotic pathway have been shown to be utilized in various cytotoxic effector mechanisms, we investigated the role of apoptosis in ADCC mediated by monocytes (ADMC) using mAb 17-1A as an antibody and the human colorectal carcinoma cell line, COLO205, as target cells in vitro. The implications of the apoptosis during ADMC was demonstrated by means of both a DNA fragmentation assay and a TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Furthermore, interferon γ (IFNγ) was also found to enhance the induction of apoptosis significantly. The addition of superoxide dismutase did not reduce the level of the apoptosis, although superoxide anion (O₂ ^–) was observed to be produced. However, the release of tumor necrosis factor α (TNFα) was significantly enhanced during ADMC, while, in addition, apoptosis was significantly inhibited by the addition of anti-TNFα antibody. These findings indicated that apoptosis might be implicated in ADMC with mAb 17-1A, which was augmented by IFNγ, while, in addition, TNFα may also be one of the major mediators of apoptosis. Received: 1 August 1996 / Accepted: 27 August 1996     

Contrasting effects of T cell growth factors on T cell responses to melanoma in vitro

 Determinants of T cell responses to tumor cells remain largely unknown. In the present study we have used long-term cultures of human melanoma cells and autologous peripheral blood lymphocytes to examine the influence of cytokines with T cell growth activity on the phenotype and cytotoxic and proliferative response of T cells to melanoma. It was found that addition of interleukin-4 (IL-4) inhibited the response of CD8⁺ T cells and promoted the response of the CD4 subset. IL-2 or IL-7 was effective in increasing melanoma-specific cytotoxic T lymphocyte (CTL) activity in cultures where CD8 T cells were predominant, whereas IL-4 followed by IL-2 was most effective in cultures where CD4 T cells predominated. IL-10 or IL-12 inhibited proliferation and CTL activity against melanoma in long-term cultures. The effects of IL-12 were reproduced in long-term cultures of T cells stimulated with mAb against CD3 and were shown to depend on prior exposure of T cells to IL-12 before IL-2. As yet unidentified factors, such as co-factor expression on melanoma, appear to be as important as exogenous cytokines in determining the nature of T cell responses to melanoma. These results suggest that analysis of responses in long-term culture may assist in defining the role of key cytokines and other determinants of immune responses to melanoma. Received: 4 June 1996 / Accepted: 12 November 1996     

The human cathelicidin, LL-37, induces granzyme-mediated apoptosis in cytotoxic T lymphocytes

LL-37 is a human cationic host defense peptide (antimicrobial peptide) belonging to the cathelicidin family of peptides. In this study, LL-37 was shown to kill stimulated CD8⁺ T cells (Cytotoxic T lymphocytes; CTLs) via apoptosis, while having no cytotoxic effect on non-stimulated CD8⁺ or CD4⁺ T cells or stimulated CD4⁺ T cells. Of interest, the CD8⁺ cells were much more sensitive to LL-37 than many other cell types. LL-37 exposure resulted in DNA fragmentation, chromatin condensation, and the release of both granzyme A and granzyme B from intracellular granules. The importance of granzyme family members in the apoptosis of CTLs following LL-37 treatment was analyzed by using C57BL/6 lymphocytes obtained from mice that were homozygous for null mutations in the granzyme B gene, the granzyme A gene, or both granzymes A and B. Granzymes A and B were both shown to play an important role in LL-37-induced apoptosis of CTLs. Further analysis revealed that apoptosis occurred primarily through granzyme A-mediated caspase-independent apoptosis. However, caspase-dependent cell death was also observed. This suggests that LL-37 induces apoptosis in CTLs via multiple different mechanisms, initiated by the LL-37-induced leakage of granzymes from cytolytic granules. Our results imply the existence of a novel mechanism of crosstalk between the inflammatory and adaptive immune systems. Cells such as neutrophils, at the site of a tumor for example, could influence the effector, activity of CTL through the secretion of LL-37.     

Differentiation of CD8+ T cells from tumor-invaded and tumor-free lymph nodes of melanoma patients: role of common gamma-chain cytokines

Differentiation of CD8(+) T cells at the tumor site toward effector and memory stages may represent a key step for the efficacy of antitumor response developing naturally or induced through immunotherapy. To address this issue, CD8(+) T lymphocytes from tumor-invaded (n = 142) and tumor-free (n = 42) lymph nodes removed from the same nodal basin of melanoma patients were analyzed for the expression of CCR7, CD45RA, perforin, and granzyme B. By hierarchical cluster analysis, CD8(+) T cells from all tumor-free lymph nodes and from 56% of the tumor-invaded lymph node samples fell in the same cluster, characterized mainly by CCR7(+) CD45RA(+/-) cytotoxic factor(-) cells. The remaining three clusters contained only samples from tumor-invaded lymph nodes and showed a progressive shift of the CD8(+) T cell population toward CCR7(-) CD45RA(-/+) perforin(+) granzyme B(+) differentiation stages. Distinct CD8(+) T cell maturation stages, as defined by CCR7 vs CD45RA and by functional assays, were identified even in melanoma- or viral Ag-specific T cells from invaded lymph nodes by HLA tetramer analysis. Culture for 7 days of CCR7(+) perforin(-) CD8(+) T cells from tumor-invaded lymph nodes with IL-2 or IL-15, but not IL-7, promoted, mainly in CCR7(+)CD45RA(-) cells, proliferation coupled to differentiation to the CCR7(-) perforin(+) stage and acquisition of melanoma Ag-specific effector functions. Taken together, these results indicate that CD8(+) T cells differentiated toward CCR7(-) cytotoxic factor(+) stages are present in tumor-invaded, but not in tumor-free, lymph nodes of a relevant fraction of melanoma patients and suggest that cytokines such as IL-2 and IL-15 may be exploited to promote Ag-independent maturation of anti-tumor CD8(+) T cells.     

CD8+ T cells exhaustion induced by myeloid‐derived suppressor cells in myelodysplastic syndromes patients might be through TIM3/Gal‐9 pathway

CD8⁺ T cells play a central role in antitumour immunity, which often exhibit ‘exhaustion’ in the setting of malignancy and chronic viral infection due to T cell immunoglobulin and mucin domain 3 (TIM3) and myeloid‐derived suppressor cells (MDSCs). Our team previously found that overactive MDSCs and exhausted TIM3⁺CD8⁺ T cells were observed in myelodysplastic syndromes (MDS) patients. However, it is not obvious whether MDSCs suppress CD8⁺ T cells through TIM3/Gal‐9 pathway. Here, Gal‐9, as the ligand of TIM3, was overexpressed in MDSCs. The levels of Gal‐9 in bone marrow supernatants, serum and culture supernatants of MDSCs from MDS patients were elevated. CD8⁺ T cells from MDS or normal controls produced less perforin and granzyme B and exhibited increased early apoptosis after co‐culture with MDSCs from MDS. Meanwhile, the cytokines produced by CD8⁺ T cells could be partially restored by TIM3/Gal‐9 pathway inhibitors. Furthermore, CD8⁺ T cells produced less perforin and granzyme B after co‐culture with excess exogenous Gal‐9, and the function of CD8⁺ T cells was similarly restored by TIM3/Gal‐9 pathway inhibitors. Expression of Notch1, EOMES (associated with perforin and granzyme B secretion), p‐mTOR and p‐AKT (associated with cell proliferation) was decreased in CD8⁺ T cells from MDS after co‐culture with excess exogenous Gal‐9. These suggested that MDSCs might be the donor of Gal‐9, and TIM3/Gal‐9 pathway might be involved in CD8⁺ T cells exhaustion in MDS, and that TIM3/Gal‐9 pathway inhibitor might be the promising candidate for target therapy of MDS in the future.     

Synergy between interleukin-2 and prothymosin α for the increased generation of cytotoxic T lymphocytes against autologous human carcinomas

 Peripheral blood mononuclear cells (PBMC) from cancer patients were cultured in vitro with irradiated autologous tumor cells isolated from malignant effusions (mixed lymphocyte tumor cultures, MLTC) and low-dose (50 IU/ml) recombinant interleukin-2 (IL-2). The combination of IL-2 and prothymosin α (ProTα) resulted in a greater PBMC-induced response to the autologous tumor than that brought about by IL-2 alone. In particular, ProTα specifically enhanced the CD4⁺ T-cell-mediated proliferation against the autologous tumor. CD4⁺ T cells seemed to recognize tumor antigens presented by HLA-DR molecules expressed on the autologous monocytes, since preincubation of the latter with an anti-HLA-DR monoclonal antibody (mAb) abrogated the response. In addition, MLTC set up with IL-2 and ProTα also generated more MHC-class-I-restricted cytotoxic T lymphocytes (CTL) against the autologous tumor than did MLTC set up with IL-2 alone. The MLTC-induced CTL contained high levels of cytoplasmic perforin and their development was strictly dependent on the presence of both autologous CD4⁺ T cells and monocytes. In the absence of either population there was a strong impairment of both proliferative and cytotoxic responses which was not restored by the presence of ProTα. In contrast, when both cell populations were present, ProTα exerted optimal enhancement of CD4⁺ T cell proliferation, which was associated with potentiated CTL responses. Our data emphasize the role of ProTα for the enhancement of IL-2-induced CTL responses against autologous tumor cells. Such responses require collaborative interactions between CD4⁺, CD8⁺ T cells and monocytes as antigen-presenting cells. Our data are relevant for adoptive immunotherapeutic settings utilizing IL-2 and ProTα-induced autologous-tumor-specific CTL. Received: 2 March 2000 / Accepted: 1 June 2000     

Analysis of the immune reactivity of infiltrating and peripheral lymphocytes from patients with renal cell carcinoma by measuring cytokine secretion

 The immunological properties of tumor-infiltrating (TIL) and peripheral blood lymphocytes (PBL) from 29 patients with renal cell carcinomas were characterized with respect to their phenotypic expression and cytokine production. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation. To eliminate all non-lymphoid cells, CD3-positive cells were specifically separated from these cell fractions with anti-CD3 magnetic beads. These pure CD3-positive PBL (CD3⁺PBL) and TIL (CD3⁺TIL) were cultured with pokeweed mitogen and the levels of the cytokines interleukin-1α (IL-1α), IL-1β, IL-2, interferon γ (IFNγ), and tumor necrosis factor α (TNFα) measured in the 4-day post-inductional cell culture supernatants. In all cell cultures a wide range of cytokine values was found, indicating a large variation in the immunological activity of the lymphocytes of each individual. When the cell cultures of the CD3⁺TIL and CD3⁺PBL were compared in each patient similar values for IL-1α, IL-1β, IFNγ and TNFα were found. However CD3⁺TIL produced significantly lower levels of IL-2 than CD3⁺PBL upon mitogenic stimulation. This may be due to a lower CD4/CD8 ratio in the CD3⁺TIL as compared to the CD3⁺PBL. These results suggest that there are no fundamental qualitative and quantitative differences in the lymphokine-producing capacity of CD3⁺TIL and CD3⁺PBL derived from patients with renal cell carcinomas. Received: 8 August 1995 / Accepted: 23 January 1996     

Cytotoxic mechanisms of murine lymphokine-activated killer cells: functional and biochemical characterization of homogeneous populations of spleen LAK cells.

A highly purified population of murine lymphokine-activated killer (LAK) cells was obtained by selecting plastic-adherent splenocytes after incubation in high doses of recombinant IL-2. The population obtained was shown to be more than 95% positive for the cell marker asialo-GM1, and negative for both Lyt-1 (CD5) and Lyt-2 (CD8). The cells presented typical large granular lymphocyte morphology, and killed NK-susceptible target cells in an exclusively calcium-dependent fashion. A target cell DNA fragmentation activity of LAK cells could be detected even before target cell death. The presence of Hanukkah Factor/granzyme A/serine esterase 1, CTLA-1/granzyme B/serine esterase 2, and pore-forming protein (PFP/perforin) in these LAK cells was demonstrated by Northern blot analysis, suggesting that these markers are not exclusively associated with cytotoxic T lymphocytes. On immunoblots, antibodies specific for a lymphocyte PFP/perforin reacted with a 70-kDa protein of LAK cells. PFP/perforin was localized by immunofluorescence to the cell granules. A 50-kDa protein antigenically related to the macrophage cytokine tumor necrosis factor (TNF) was detected by immunoblotting and localized by immunofluorescence to both the cell granules and the cytosol. No RNA for TNF, however, could be detected using TNF-specific probes, suggesting that LAK cells may contain a cytotoxic factor which is related to, but distinct from, TNF. The work presented here demonstrates that cytotoxic mediators identified in cell lines are also present in primary cell cultures.     

IL-12 controls cytotoxicity of a novel subset of self-antigen-specific human CD28+ cytolytic T cells

Activated CD8 T cells develop cytotoxicity against autologous cells bearing foreign Ags and self/tumor Ags. However, self-specific cytolysis needs to be kept under control to avoid overwhelming immunopathology. After peptide vaccination of melanoma patients, we studied molecular and functional properties of T cell subsets specific for the self/tumor Ag Melan-A/MART-1. Ex vivo analysis revealed three Ag-specific effector memory (EM) populations, as follows: CD28-negative EM (EM28(-)) T cells strongly expressing granzyme/perforin, and two EM28(+) subsets, one with high and the other with low level expression of these cytotoxic proteins. For further functional characterization, we generated 117 stable CD8 T cell clones by ex vivo flow cytometry-based sorting of these subsets. All EM28(-)-derived clones lysed target cells with high efficacy. In contrast, EM28(+)-derived clones were heterogenous, and could be classified in two groups, one with high and the other with low killing capacity, correlating with granzyme/perforin expression. High and low killer phenotypes remained surprisingly stable for several months. However, strongly increased granzyme expression and cytotoxicity were observed after exposure to IL-12. Thus, the data reveal a newly identified subset of CD28(+) conditional killer T cells. Because CD28 can mediate strong costimulatory signals, tight cytotoxicity control, as shown in this study through IL-12, may be particularly important for subsets of T cells expressing CD28.