Signaling defects in T lymphocytes of patients with malignancy

In patients with cancer, alterations in the expression of T-cell receptor-associated molecules in tumor-infiltrating lymphocytes (TIL) as well as in circulating lymphocytes have been reported. By quantitative flow cytometry analysis, decreased or absent expression of the ζ chain in CD4⁺ or CD8⁺ T cells as well as in natural killer (NK) cells was demonstrated in patients with malignancies. Changes in the expression of ζ are biologically significant, because the absence or low expression of this signaling molecule in TIL of patients with stage III or IV head and neck cancer predicts a significantly shorter 5-year survival than that of patients with normal ζ expression in TIL. Preliminary evidence indicates that expression of ζ in TIL may not only influence survival but also predicts a favorable response to biologic therapies. Patients with cancer also show significantly greater spontaneous ex vivo apoptosis in peripheral blood mononuclear cells (PBMC) compared to normal controls, as measured by a terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay. While no correlation could be established between the proportions of cells with low ζ chain expression and those that spontaneously apoptose ex vivo, the ζ chain has been shown to be cleaved by caspases in T cells coincubated with tumor cells or with T cells exposed to CH-11 antibody, which induces apoptosis upon crosslinking Fas on the cell surface. The results suggest that low/absent ζ chain expression and lymphocyte apoptosis may be manifestations of negative effects of the tumor on the host immune system. Received: 20 March 1999 / Accepted: 3 May 1999     

Ex vivo activation of tumor-draining lymph node T cells reverses defects in signal transduction molecules

 The adoptive transfer of tumor-draining lymph node (LN) T cells activated ex vivo with anti-CD3 and interleukin 2 (IL-2) mediates the regression of the poorly immunogenic murine melanoma D5. The efficacy of the activated LN cells is augmented when the sensitizing tumor is a genetically modified variant (designated D5G6) that secretes granulocyte/macrophage-colony-stimulating factor. In contrast to anti-CD3/IL-2-activated LN cells, adoptive transfer of freshly isolated tumor-draining LN T cells has no therapeutic activity. To determine whether the acquisition of antitumor function during ex vivo activation is associated with modifications in signal transduction capacity, the protein tyrosine kinases p56 ^lck and p59 ^fyn and proteins of the NF-κB family were analyzed in tumor-draining LN T cells. The levels of p56 ^lck and p59 ^fyn were lower in tumor-draining than in normal LN T cells and production of tyrosine-phosphorylated substrates was markedly depressed following anti-CD3 stimulation. After 5-day anti-CD3/IL-2 activation, levels of p56 ^lck and p59 ^fyn and protein tyrosine kinase activity increased. Interestingly, the levels of p56 ^lck , p59 ^fyn , and tyrosine kinase activity were higher in activated T cells derived from LN that drained D5G6 than they were in those from D5 tumors. In contrast, the cytoplasmic levels of c-Rel and Rel A were normal in freshly isolated tumor-draining LN, as was nuclear κB DNA-binding activity induced by anti-CD3 mAb or phorbol myristate acetate. Stimulation of activated LN cells with D5 tumor cells induced the nuclear translocation of NF-κB. These findings indicate that the recovery of proteins mediating signal transduction through the T cell receptor/CD3 complex in LN T cells activated ex vivo was associated with the acquisition of antitumor function. Received: 28 August 1997 / Accepted: 23 February 1998     

Cytotoxic T cells infiltrating a glioma express an aberrant phenotype that is associated with decreased function and apoptosis

 In this study, we report on novel alterations found in rat intracranial (i.c.) tumor-infiltrating T lymphocytes (TIL) that are indicative of T cell defects and death. FACS analysis showed that the cytotoxic T cells (CTL) infiltrating rat T9.F gliomas were CD3ɛ⁺, αβTCR⁺, CD8α ⁺, but CD8β ⁻. These lymphocytes also stained positive for the B cell-specific marker, CD45RA, as well as Annexin-V, signifying apoptotic changes. Functional and biochemical analyses were performed to assess whether the aberrant phenotype was linked to other defects. When CD8α ⁺ TIL were purified and stimulated in vitro, their proliferative capacity was markedly diminished in comparison with CD3⁺CD8α ⁺CD8β ⁺ T cells isolated from the spleens of naive, non tumor-bearing rats. Furthermore, the mean fluorescence intensity of surface CD3ɛ was dramatically reduced in the CD3⁺CD8α ⁺CD8β ⁻ TIL population as compared with CD3⁺CD8α ⁺CD8β ⁺ TIL from the same tumor-bearing animal. Biochemical studies revealed that the expression of TCRζ and LAT were reduced in lysates generated from CD8α-purified TIL with respect to CD8α-purified T cells from naive spleen. We believe that these degenerative changes are reflective of chronic T cell receptor ligation, because in vitro culture of rat splenocytes or purified T cells with ConA or anti-CD3 mAb induced the same alterations. In vitro, the downregulation of CD8β could be inhibited by the caspase inhibitor, z-VAD. These results suggest that the aberrant CTL phenotype found in the TIL of glioma-bearing rats may be novel signals for their impending death and degenerating anti-tumor immune function. Received: 27 February 2001 / Accepted: 26 April 2001     

Crosslinking of Fas/CD95 suppresses the CD3-mediated signaling events in Jurkat T cells by inhibiting the association of the T-cell receptor ζ chain with src-protein tyrosine kinases and ZAP70

Crosslinking of Fas (APO-1/CD95) on the surface of T cells initiates a biochemical cascade leading to programmed cell death. We have previously shown that crosslinking of Fas with an apoptosis-inducing IgM anti-Fas mAb results in suppression of the CD3-initiated cell signaling including Ca²⁺ mobilization and protein tyrosine phosphorylation. We conducted experiments to decipher the mechanisms whereby the cross talk between the Fas- and CD3 signaling pathways occur. We used lysates from Jurkat T and examined the composition of the TCR chain-precipitated immune complexes using immunoblots. While crosslinking of Fas affected the association of p59^fyn and p56^lck tyrosine kinases with the TCR chain to a limited degree, it dramatically inhibited the association of the protein tyrosine kinase ZAP70 with the chain. In cells that were preincubated with an apoptosis-inducing anti-Fas mAb, the binding of the protein tyrosine phosphatases SHP-1 to the TCR chain was increased. These experiments indicate that crosslinking of Fas interferes with early T cell signaling events by promoting the recruitment of SHP-1 and decreasing the association of protein tyrosine kinases with TCR chain. Therefore, crosslinking of Fas antigen may regulate the antigen-induced T cell response and play an active role in the T cell anergy.     

Systemic immune effects of adjuvant chemotherapy with 5-fluorouracil,epirubicin and cyclophosphamide and/or radiotherapy in breast cancer: a longitudinal study

Immunotherapy is being increasingly utilized for adjuvant treatment for breast cancer (BC). We have previously described immune functions during primary therapy for BC. The present study describes immune recovery patterns during long-term, unmaintained follow-up after completion of adjuvant therapy.A group of patients with primary BC had been treated with adjuvant radio-chemotherapy (RT + CT) 5-fluorouracil, epirubicin and cyclophosphamide (FEC) (n = 21) and another group with radiotherapy (RT) (n = 20) alone. Immunological testing of NK and T-cell functions was performed initially at the end of adjuvant treatment and repeated after 2, 6 and 12 months. NK cell cytotoxicity was significantly higher (P < 0.05) at all time-points in patients than in age-matched controls and did not differ between the two treatments groups during one year observation. In contrast, lower numbers of CD4 T-cells and lower expression of CD28 on T-cells was observed particularly in RT + CT patients and did not normalize during the observation period. The numbers of T_reg cells (CD4⁺CD25^high) were low in the RT + CT group during follow-up, as well as expression of TCRξ, Zap70, p56^lck, P59^fyn and PI3 k in CD4⁺ cells. In contrast, expression of intracellular cytokines (IFN-γ, IL-2, IL-4) in CD4 and CD8 T cells were significantly higher in RT + CT patients than in the RT group and the difference increased during follow-up. In conclusion, NK-cell cytotoxicity increased during unmaintained long-term follow-up whereas CD4 and regulatory T cells as well as signal transduction molecules remained low following adjuvant radio-chemotherapy.     

Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+renal-cell-carcinoma-infiltrating lymphocytes

The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3⁺ tumor-infiltrating lymphocytes (CD3⁺ TIL) and in autologous CD3⁺ peripheral blood lymphocytes (CD3⁺ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4⁺ TIL and CD8⁺ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3⁺ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3⁺ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4⁺ TIL and simultaneously obtained CD8⁺ TIL revealed a significantly higher expression of IFN in the CD8⁺ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8⁺ TIL. Received: 14 January 1999 / Accepted: 30 April 1999     

Association between GM3 and CD4-Ick complex in human peripheral blood lymphocytes

The aim of this study was to further elucidate our previous observation on molecular interaction of GM3, CD4 and p56^lck in microdomains of human peripheral blood lymphocytes (PBL). We analyzed GM3 distribution by immunoelectron microscopy and the association between GM3 and CD4-p56^lck complex by scanning confocal microscopy and co-immunoprecipitation experiments. Scanning confocal microscopy analysis showed an uneven signal distribution of GM3 molecules over the surface of human lymphocytes. Nearly complete colocalization areas indicated that CD4 molecules were distributed in GM3-enriched plasma membrane domains. Co-immunoprecipitation experiments revealed that CD4 and p56^lck were immunoprecipitated by IgG anti-GM3, demonstrating that GM3 tightly binds to the CD4-p56^lck complex in human PBL. In order to verify whether GM3 association with CD4 molecules may depend on the presence of p56^lck, we analyzed this association in U937, a CD4+and p56^lck negative cell line. The immunoprecipitation with anti-GM3 revealed the presence of a 58[emsp4 ]kDa band immunostained with anti-CD4 Ab, suggesting that the GM3-CD4 interaction does not require its association with p56^lck. These findings support the view that GM3 enriched-domains may represent a functional multimolecular complex involved in signal transduction and cell activation.     

Assembly of the CD8α/p56 protein complex in stably expressing rat epithelial cells

We have previously characterized the biogenesis of the human CD8α protein expressed in rat epithelial cells. We now describe the biosynthesis, post-translational maturation and hetero-oligomeric assembly of the human CD8α/p56^lck protein complex in stable transfectants obtained from the same cell line. There were no differences in the myristilation of p56^lck, or in the dimerization, O-glycosylation and transport to the plasma membrane of CD8α, between cells expressing either one or both proteins. In the doubly expressing cells, dimeric forms of CD8α established hetero-oligomeric complexes with p56^lck, as revealed by co-immunoprecipitation assays performed with anti-CD8α antibody. Moreover, p56^lck bound in these hetero-oligomeric complexes was endowed with auto- and hetero-phosphorylating activity. The present study shows that: (1) the newly synthesized p56^lck binds rapidly to CD8α and most of the p56^lck is bound to CD8α at steady state; (2) CD8α/p56^lck protein complexes are formed at internal membranes as well as at the plasma membrane; and (3) about 50% of complexed p56^lck reaches the cell surface.     

Adjuvant adoptive immunotherapy with tumour-infiltrating lymphocytes and modulated doses of interleukin-2 in 22 patients with melanoma, colorectal and renal cancer, after radical metastasectomy, and in 12 advanced patients

 Adoptive tumour infiltrating lymphocytes (TIL) in combination with a modulated dosage of interleukin-2 (IL-2) can be used with acceptable toxicity in the treatment of immunogenic tumours. Following an experience of reinfusion in advanced melanoma, colorectal and renal cancer patients, treatment was given to disease-free patients after metastasectomy. The high risk of relapse and favourable ratio between reinfused TIL and possible microscopic residual disease determined this choice of adjuvant treatment. A group of 12 patients with advanced disease (7 melanoma, 4 colorectal carcinoma, 1 kidney carcinoma) were treated with TIL (median 5.8×10¹⁰ cells) and IL-2 (West’s schedule) modulated towards a lower dosage (from 12 to 6 MIU/day) in order to maintain an acceptable level of toxicity. As treatment was well tolerated, it was offered to another 22 patients in an adjuvant setting after metastasectomy (11 melanoma, 10 colorectal carcinoma, 1 renal cancer), the median dose of TIL reinfused being 4.95×10¹⁰ cells. No objective response was observed in advanced patients: all patients progressed after a median of 1.5 months (0–8 months) and median survival was 8 months (3–22+ months). Thirteen patients from the second group are still disease-free after a median of 23+ months (9+–47+ months). The remaining 9 patients relapsed after a median of 5 months (3–18 months). Toxicity was moderate as clinical and hepatic/renal function parameters were used to assess the need for dose reductions. Consequently, there was great diversity in IL-2 dosages administered. In particular, there seemed to be a difference in IL-2 doses administered between disease-free cases and those who progressed (17.5 MIU/day versus 7 MIU/day in melanoma patients; 11.2 MIU/day versus 7.1 MIU/day in colorectal cancer patients). By contrast, no differences were observed between number of TIL reinfused and clinical response. Phenotypical characteristics of reinfused TIL were similar to those reported in the literature: 97% were CD3 and 92% were CD8. Aspecific cytolytic activity was evaluated on 12 cases whereas, in 2 melanoma cases, autologous tumour tissue was available for the specific cytotoxicity test. Perforin levels in TIL measured at the end of culture were generally high or very high. Cytokine levels were measured on the supernatant at the end of culture, with an estreme variability in results. Finally, ζ chain and p56^lck were histologically assessed on the resected tissue from which TIL were cultivated. There were virtually none of the former and a complete absence of the latter, which concurs with data reported in the literature. The same immunocytochemical analysis was carried out on TIL at the end of culture. This time an almost complete restoration of both functions was seen, especially in melanoma patients, who are still free from disease. The study is on-going and it has been decided to focus on disease-free patients after metastasectomy in order to increase the number and possibility of clinical and histological correlations.     

Induction of Interleukin 2-Responsiveness in Thymocytes of the Transgenic Mice Carrying lck-Transgene

The role of lck gene in T cell proliferation and differentiation was investigated with transgenic mice carrying human lck cDNA whose expression was regulated by the promoter of mouse H-2K^b and the enhancer element of mouse IgH. RNase protection assay revealed that the lck transgene was expressed in the thymus and spleen, whereas immunoblot analysis demonstrated that amounts of p56^lck in freshly isolated lymphoid organs were almost equal between transgenic mice and negative littermates. Cell-surface marker analyses of the thymocytes and peripheral lymphocytes revealed no remarkable difference between both groups. Notable finding is that the thymocytes from transgenic mice showed a significant proliferative response to the stimulation with IL-2, but not the thymocytes from negative littermates. Further analysis revealed that CD4⁺8^– single positive thymocytes proliferated in response to IL-2. While surface expression levels of IL-2Rα and IL-2Rβ of these CD4⁺8^– thymocytes from transgenic and control mice were almost equal before stimulation with IL-2, the expression of IL-2Rβ was induced only in transgenic thymocytes after stimulation with IL-2. Immunoblot analysis demonstrated that the expression of p56^lck of transgenic thymocytes was not down-reguated at 4 hr after stimulaion with IL-2, whereas p56^lck of control ones were not detectable any more at 4 hr after stimulation with IL-2. Moreover, in vitro kinase assay substantiated such unchanged expression of p56^lck in the thymocytes from transgenic mice: the kinase activities of p56^lck did not decrease in thymocytes from transgenic mice after stimulation with IL-2, while kinase activities of control ones were significantly down-regulated by stimulation of IL-2. These results suggested that a significant proliferative response found in the thymocytes from lck-transgenic mice after the stimulation with IL-2 was caused by a constitutive expression of p56^lck in these thymocytes even after the stimulation. Our findings, therefore, support a possibility that p56^lck may play a role in the IL-2R-mediated signaling system in CD4⁺8^– thymocytes.     

Retrovirus-mediated gene transfer into CD4+ and CD8+ human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells

Summary Studies were undertaken to test the susceptibility of individual T cell subpopulations to retroviral-mediated gene transduction. Gene transfer into human tumor-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) was carried out by transduction with an amphotropic murine retroviral vector (LNL6 or N2) containing the bacterialneo ^R gene. The presence of theneo ^R gene in the TIL population was demonstrated by Southern blot analysis, detection of the enzymatic activity of the gene product and by the ability of transduced TIL to proliferate in high concentrations of G418, a neomycin analog that is toxic to eukaryotic cells. The presence of theneo ^R gene in TIL did not alter their proliferation or interleukin-2 dependence compared to nontransduced TIL. The differential susceptibility of CD4⁺ and CD8⁺ lymphoid cells to the retro-virus-mediated gene transfer was then tested. Transduction of heterogeneous TIL cultures containing both CD4⁺ and CD8⁺ cells resulted in gene insertion into both T cell subsets with no preferential transduction frequency into either CD4⁺ or CD8⁺ cells. In other experiments highly purified CD4⁺ and CD8⁺ T cell subpopulations from either TIL or PBMC could be successfully transduced with theneo ^R gene as demonstrated by Southern blot analysis and detection of the gene product neophosphotransferase activity. No such activity or vector DNA could be detected in controls of nontransduced cells. In these highly purified cell subsets the distinctive T cell phenotypic markers were continually expressed after transduction, G418 selection and long-term growth. Clinical trials have begun in patients with advanced cancer using heterogeneous populations of CD4⁺ and CD8⁺ gene-modified TIL. Current address: Bone Marrow Transplantation, Hadassah University Hospital, 91120 Jerusalem, Israel     

Pre-operative intracellular glutathione levels of peripheral monocytes as a biomarker to predict survival of colorectal cancer patients

The ability to predict anti-tumor immune responses at local tumor growing sites using only peripheral blood specimens would be helpful in determining therapeutic options for patients with solid tumors. Here, we show that the glutathione intracellular content (icGSH) of peripheral monocytes (Mo) correlates positively with T cell infiltration within tumor islets and overall survival in patients with colorectal carcinoma. IcGSH redox status was determined in CD14⁺ Mo prior to surgery by staining with monochlorobimane. The tumor-infiltrating T cells (TIL) were quantified as CD45RO⁺ T cells in resected tumors using paraffin sections. A positive association was found between the GSH index and TIL in tumor islets (P < 0.001). The 50% cut-off value for the GSH index, that is the determinant between TIL presence or absence in tumor islets, was calculated to be almost 0.7 through logistic regression analysis. Mo with a GSH index of ≥0.7 were termed reductive (R)-Mo, and those with <0.7 were designated as oxidative (O)-Mo. Cox’s proportional hazards regression analysis of patients with R-Mo or O-Mo prior to surgery, and the presence or absence of TIL, was found to correlate significantly with the overall survival rate of stage II and III patients. Kaplan–Meier analysis also showed a significant correlation. These results indicate that the Mo icGSH index is a useful biomarker parameter for better understanding the host/tumor relationship prior to surgery, thereby enabling the development of an individual patient-oriented therapeutic strategy.     

Inhibition of T Cell Receptor-mediated Signal Transduction by Erbstatin

Erbstatin, a tyrosine kinase inhibitor, inhibited p59^fyn- and p56^lck-kinases in vitro with IC₅₀s of 0.21 and 0.18 μg/ml, respectively. Inositol phosphates formation, enhanced by anti-CD3, was also inhibited by erbstatin. Moreover, erbstatin treatment prevented anti-CD3-induced interleukin 2 (IL-2) production, but phorbol ester-induced IL-2 production was not affected by erbstatin.     

Cytokine biosynthesis by tumor-infiltrating T lymphocytes from human non-small-cell lung carcinoma

The purpose of this work was to assess the capacity of tumor-infiltrating leukocytes (TIL) from human non-small-cell lung carcinoma (NSCLC) specimens to synthesize type-1 and type-2 cytokines. Methods: TIL were isolated from tumors following digestion with collagenase/DNase and further enriched by ficoll-hypaque gradient centrifugation. Membrane phenotypes and intracellular cytokine protein expression of TIL were assessed by flow cytometry. Results: The majority of TIL expressed the CD3 antigen with a CD4:CD8 ratio of approximately 2:1. Other leukocytes such as macrophages (CD14), B lymphocytes (CD20), and natural killer (NK) cells (CD56) were also found to infiltrate the tumors, but in significantly lower numbers. Owing to the limited recovery of non-CD3⁺ leukocytes, our analysis of cytokine biosynthesis has focused on T lymphocytes. In the absence of activation, a small percentage of CD3⁺ TIL synthesized cytokines ( <4%). Following activation with anti-CD3+interleukin-2 (IL-2), CD3⁺ TIL synthesized predominantly a type-1 cytokine profile; however, the type-2 cytokines, IL-6 and IL-10, were also detected in a small percentage of infiltrating cells. Following activation with phorbol 12-myristate 13-acetate + ionomycin, CD3⁺ TIL also expressed more type-1 than type-2 cytokines and in significantly greater numbers of cells. The CD3⁺CD8⁺ component of the TIL synthesized only type-1 cytokines, whereas the CD3⁺CD4⁺ component synthesized both type-1 and type-2 cytokines. Conclusion: These results show that the majority of the TIL isolated from NSCLC specimens are T lymphocytes with the capacity to synthesize type-1 cytokines. Received: 24 March 1999 / Accepted: 9 September 1999     

Emergence of a dominant cytotoxic T lymphocyte antitumor effector from tumor-infiltrating cells in the anterior chamber of the eye

Summary Previous studies in mice revealed that resolving intraocular tumors (UV5C25 fibrosarcoma) were infiltrated with mononuclear cells and invoked potent systemic delayed-type hypersensitivity responses without nonspecific tissue destruction. The present study characterized the tumor-infiltrating lymphocyte (TIL) population and established its role as the mediator of specific intraocular tumor rejection. This was accomplished by (a) isolating TIL from resolving intraocular tumors; (b) identifying characteristic surface markers on TIL; and (c) demonstrating in vitro and in vivo antitumor functions. Fluorescence-activated cell sorter analysis of TIL showed 33.4% Thy1⁺, 19.8% CD8⁺, 11.1% CD4⁺, 17.2% MAC–1⁺, 10.4% F4/80⁺, and 7.7% B220⁺. Functional studies indicated that TIL were directly cytolytic for UV5C25 tumor cells. Additionally a tumor-necrosis-factor(TNF)-sensitive cell line (WEHI 164.1) was lysed on cocultivation with TIL, whereas UV5C25 tumor cells were insensitive to lysis by TNF. Precursor CTL analysis demonstrated a high frequency (1/251) of tumor-specific precursors and a low frequency of alloresponsive cells in the TIL population. In vivo analysis by a Winn-type assay demonstrated that only TIL could effect tumor resolution in immunosuppressed hosts. These results demonstrate that although CD4⁺ T cells and macrophages were present and TNF activity was detected in the TIL population, there was no evidence for nonspecific tissue destruction within the eye. Therefore, this pattern of intraocular tumor rejection is mediated by a lymphocyte population expressesing cell-surface phenotypes and functional characteristics of conventional cytotoxic T lymphocytes. Moreover, the results suggest that a regulatory mechanism within the eye allows for the emergence of one dominant antitumor effector (CTL) while controlling a more destructive mechanism (delayed-type hypersensitivity).Supported in part by USPHS grants EYO5631 and CA30276, an unrestricted grant from Research to Prevent Blindness Inc., and a grant from the Texas Department of the Ladies Auxiliary, Veterans of Foreign Wars. Dr. Niederkorn is a Research to Prevent Blindness Olga Keith Wiess Scholar     

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     

T cell augments the antitumor activity of tumor-targeting <Emphasis Type="Italic">Salmonella</Emphasis>

Systemic administration of Salmonella to tumor-bearing mice leads to preferential accumulation within tumor sites and retardation of tumor growth. However, the detailed mechanism of Salmonella-induced antitumor immune response via host T cell remains uncertain. Herein, we used wild-type, CD4⁺ T-cell-deficient, and CD8⁺ T-cell-deficient mice to study the role of T cell in the antitumor immune responses induced by Salmonella enterica serovar Choleraesuis (Salmonella Choleraesuis). When systemically administered into mice bearing tumors, Salmonella Choleraesuis significantly inhibited tumor growth by 50%. In contrast, in T-cell-deficient mice, there was only 34–42% inhibition of tumor growth. We found that treatment with Salmonella Choleraesuis significantly upregulates interferon-γ in wild-type and CD8⁺ T-cell-deficient mice, but not in CD4⁺ T-cell-deficient mice. Furthermore, immunohistochemical staining of the tumors revealed more infiltration of macrophages and neutrophils in wild-type mice after Salmonella Choleraesuis treatment compared with those in T-cell-deficient mice. The antitumor therapeutic effect mediated by Salmonella Choleraesuis is associated with an inflammatory immune response at the tumor site and a tumor T helper 1-type immune response. In conclusion, these results suggest that tumor-targeted therapy using Salmonella Choleraesuis, which exerts tumoricidal effects and stimulates T cell activities, represents a potential strategy for the treatment of tumor.     

GD3-reactive antibodies can inhibit the lysis of autologous tumor cells by tumor-infiltrating lymphocytes

Summary GD3 is expressed in high concentrations on melanoma cells and may serve as a useful target antigen for mAb-mediated immunotherapy. Monoclonal antibodies (mAbs) against GD3 stimulate cell-mediated immune responses against tumor cells in vitro and this activity may contribute to antitumor effects in patients with melanoma treated with GD3-reactive mAbs. In the present study the effects of GD3-reactive mAbs on autologous tumor cell lysis by a human melanoma-derived tumor-infiltrating lymphocyte (TIL) population were examined. Unlike results reported for other GD3⁺ T cells isolated from melanoma patients, the tumor-specific lytic activity of the TIL line was inhibited by incubation with mAbs against GD3. Other melanoma-reactive mAbs, including those against GD2 and the high-molecular-weight melanoma-associated Ag, had no effect on the TIL lytic activity. Overall, these results indicate that mAbs against GD3 may have different effects on T cell/tumor cell interactions.     

Reduced naïve CD8+ T‐cell priming efficacy in elderly adults

Aging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8⁺ T‐cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling naïve CD8⁺ T‐cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8⁺ T‐cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8⁺ T‐cell responses specific for a model antigen. Reduced CD8⁺ T‐cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the naïve CD8⁺ T‐cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging.