Differential tumor infiltration by T-cells characterizes intrinsic molecular subtypes in breast cancer.

BACKGROUND: Molecular subtypes of breast cancer and presence of tumor-infiltrating immune cells have both been implicated as important predictive and prognostic factors for improved risk stratification and treatment individualization of breast cancer patients. Their association, however, has not been studied in detail. The aim of this study was to evaluate the expression of the T cell markers CD8, FoxP3, CD3 and ζ-chain in molecular subtypes of the invasive margin and tumor center of breast cancer and corresponding sentinel nodes and to deduct prognostic information from these findings. METHODS: Tumor and sentinel node sections from 177 patients with primary, invasive, unilateral early-stage breast cancer were stained by immunohistochemistry and T-cell phenotypes quantified manually. Clinical data were collected from medical records. RESULTS: The degree of T-cell infiltration and expression of all markers differed significantly among the molecular subtypes, being highest in non-luminal, more aggressive tumors: more T-cell infiltration and higher expression of all markers were associated with hormone receptor negativity, higher proliferation and higher histological grades, but also with larger tumor size. Basal-like tumors, and most remarkably their tumor centers, hosted the highest number of FoxP3+ T-cells with an unfavorable ratio to cytotoxic CD8+ T-cells. T-cell infiltration was generally higher in the invasive margin than the tumor center. A scoring system based on densities of CD3 and CD8 could significantly separate molecular subtypes (p < 0.001). CONCLUSIONS: Thus, immunological patterns with functional implications within each subtype are associated with prognostic factors. These findings should be further validated in studies using larger patient populations and longer follow-up.

Tumour-induced immune suppression: role of inflammatory mediators released by myelomonocytic cells.

Tumour-induced immune dysfunction is a serious challenge to immunotherapy for cancer, and intact adaptive and innate cellular immunity is key to its success. Myelomonocytic cells have a central role in this immune suppression, and tumour-associated macrophages, eosinophils, neutrophils and myeloid-derived suppressor cells have all been shown to be of major importance. These myelomonocytic cells secrete a broad repertoire of inflammatory mediators providing them with powerful tools to inhibit tumour-reactive T cells and natural killer cells; free oxygen radicals including reactive oxygen species and NO, arginase, indoleamine 2,3-dioxygenase, prostaglandins, the pro-inflammatory heterodimer S100A8/9 and cytokines, such as granulocyte-macrophage colony-stimulating factor and transforming growth factor-ß, have proven particularly potent in suppressing antitumour cellular immunity. Determining which of these factors prevail in individual cancer patients and designing methods aimed at neutralization or inhibition of their effects on target tissues have the potential to greatly enhance the clinical efficacy of immunotherapy.

Tumor-dependent down-regulation of the ζ-chain in T-cells is detectable in early breast cancer and correlates with immune cell function.

Suppressive factors produced by tumors or tumor-associated cells represent a major obstacle to immune-mediated tumor eradication and immunotherapy. We studied tumor-dependent changes in expression of the ζ-chain, a key molecule for the transduction of stimulatory signals through the T-cell receptor and activating receptors on natural killer (NK) cells, in patients with early (stages 1 and 2) breast cancer. Ex-vivo levels of ζ-chain expression, proliferation and cytokine expression in lymphocyte subpopulations were measured in breast tumors, their draining lymph nodes and in pre- and postoperative blood samples of cancer patients and healthy controls by multi-parametric flow cytometry. We found that T-cell ζ-chain expression in peripheral blood of breast cancer patients (n = 29) was down-regulated compared with healthy controls (n = 10) (p ≤ 0.033), which was most pronounced in stage 2 (n = 15, p ≤ 0.004). T- and NK-cell ζ-chain loss was most distinct in the tumor and decreased with increasing distance (p ≤ 0.015). After surgical tumor resection, peripheral blood ζ-chain levels normalized to those observed in healthy controls. ζ-chain expression in peripheral blood T-cells correlated with lymphocytic proliferative activity (p ≤ 0.035), and with the expression of proinflammatory cytokines in CD8+ T-cells (p ≤ 0.035). Breast cancer patients had lower numbers of circulating early memory CD8+ T-cells than healthy donors (p ≤ 0.000), correlating with lower T-cell ζ-chain levels (p ≤ 0.011). Our findings suggest that phenotypic and functional alterations in lymphocytes can be detected even in early stage breast cancer patients. The observed immunosuppression appears to be systemic and tumor dependent, as it is strongest in the tumor, correlates with tumor stage and normalizes after surgery.

Tumor-induced changes in the phenotype of blood-derived and tumor-associated T cells of early stage breast cancer patients.

Tumor-induced immune suppression has mainly been studied in patients with advanced cancer. Despite the fact that they are most likely to benefit from immunotherapy, patients with early stage cancers were under-represented in these studies. We analyzed blood and tumor-derived T cells from patients with stage 1 (n = 20), stage 2 (n = 23) or stage 3 (n = 1) breast cancer and found that, even early stage tumors induced T cell differentiation. Breast cancer patients had significantly more circulating CD8+ memory and fewer CD8+ naïve T cells than healthy controls (n = 10). Up-regulation of CD69 and PD1 on cancer patient T cells suggests previous activation, and increased expression of the chemokine receptors CCR5 and CXCR3 on CD8+ T cells indicates that their homing capacity differs from that of healthy individuals. Comparison of blood-derived and tumor-associated T cells from patients with different metastatic status and tumor grades revealed that tumor progression and aggressiveness seem to favor the expansion of memory T cells over naive T cells. We have previously shown that immunosuppression in this patient population is stronger in the tumor than in the blood. Here, we report signs of exhaustion, such as loss of CD28, on tumor-associated as compared to blood-derived CD8+ T cells, despite the fact that tumor-associated T cells are predominantly effector memory cells and express high levels of CD69. The finding that the presence of a tumor potentially induces immunosenescence early during tumorigenesis indicates that efficient immunotherapy might be difficult even in patients with early stage cancer due to T cell exhaustion and tolerance.

Myeloid-derived suppressor cells impair the quality of dendritic cell vaccines.

Myeloid-derived suppressor cells (MDSC) are important regulators of the immune system and key players in tumor-induced suppression of T-cell responses. CD14+HLA-DR-/low MDSC have been detected in a great number of malignancies, including melanoma. MDSC are known to be impaired in their ability to differentiate along the myeloid lineage, e.g., into dendritic cells (DC). This is a concern for utilization of monocyte-derived DC for vaccination of patients with melanoma or other cancers exhibiting accumulation of CD14+ MDSC. When producing DC according to standard operating procedures of two currently ongoing clinical trials, we found that MDSC co-purified with monocytes isolated by elutriation. MDSC frequencies did not affect yield or viability of the produced DC, but induced a dose-dependent decrease in DC maturation, ability to take up antigen, migrate and induce T-cell IFNγ production. Changes in DC characteristics were most notable when 'pathological' frequencies of >50% CD14+HLA-DR- cells were present in the starting culture. The impaired DC quality could not be explained by altered cytokine production or increased oxidative stress in the cultures. Tracking of HLA-DR- cells throughout the culture period revealed that the observed changes were partially due to the impaired maturation and functionality of the originally HLA-DR- population, but also to their negative effects on HLA-DR+ cells. In conclusion, MDSC could be induced to differentiate into DC but, due to the impairment of overall DC vaccine quality when >50% HLA-DR- cells were present in the starting culture, their removal could be advisable.

Generation of natural killer cells: an autonomous function of the bone marrow.

Generation of natural killer (NK) cells in spleens from radiation chimeras produced between pairs of histocompatible 'high' and 'low' NK-reactive mouse strains has been investigated. Spleen cells of high-reactive recipients reconstituted with bone marrow from low-reactive mice were found to be low reactive. Conversely, spleen cells of low mice grafted with bone marrow or fetal liver cells from high donors were high reactive. Similarly, the age-related changes of NK activity were shown to be expressed at the bone marrow precursor cell level. These results indicate that the generation of natural killer cells is an inborn and autonomous function of the bone marrow and does not depend on the genotype or other influences of the host environment.

The recombinant 65-kD heat shock protein of Mycobacterium bovis Bacillus Calmette-Guerin/M. tuberculosis is a target molecule for CD4+ cytotoxic T lymphocytes that lyse human monocytes.

Since little is known about Tc cells in the human immune response to intracellular parasites, we have studied the role of Tc cells in response to M. bovis Bacillus Calmette-Guerin (BCG). Donors whose PBMC responded to BCG, purified protein derivative (PPD), and the recombinant 65-kD heat shock protein (HSP) of BCG generated BCG/PPD-specific CD4+ effector T lymphocytes that lysed PPD as well as recombinant 65-kD-pulsed monocytes. Nonpulsed or irrelevant antigen-pulsed target cells were lysed to a much lower but still significant extent. PPD-stimulated effector lymphocytes of a recombinant 65-kD nonresponder lysed PPD but not recombinant 65-kD-pulsed monocytes. Recombinant 65-kD-educated effector lymphocytes lysed both recombinant 65-kD- and PPD-pulsed monocytes. In addition, these effector cells efficiently lysed nonpulsed target cells. These results demonstrate that in recombinant 65-kD responders, the recombinant 65-kD HSP of BCG is an immunodominant target as well as a triggering molecule for BCG/PPD-specific CD4+ cytotoxic T cells that lyse autologous monocytes. The implications of these findings with respect to the role of the 65-kD HSP in autoimmunity are discussed.

Killer cells: a functional comparison between natural, immune T-cell and antibody-dependent in vitro systems.

Previous reports have shown that spleen cells from nonimmune adult mice of certain strains do regularly kill Moloney leukemia virus-induced lymphomas in short-term 51Cr release assays. This naturally occuring killer (NK) cell had low adherent properties and had the morphological appearance of a lymphocyte. Still it lacked surface characteristics of mature T or B lymphocytes. In the present report a functional study was carried out, comparing in parallel the NK system, the T-cell killing across an H-2 barrier (anti-P815), and the antibody-dependent cell-mediated chicken red blood cell (CRBC) system. In contrast to the effector cells in the CRBC system, the NK cells were insensitive to erythrocyte antibody complement (EAC) rosette depletion and would pass through nylon wool columns. NK activity was not inhibited by the presence of heat-aggregated human or mouse gamma globulin, in contrast to the strong inhibition noted in the CRBC system. Sensitivity to trypsin pretreatment was noted in the NK system as well as in the immune P815 system, whereas the CRBC system was relatively trypsin resistant. Antitheta plus complement eliminated the anti-P815 activity, but did not touch the NK activity. The present results thus further distinguish the NK cell from cytotoxic T lymphocytes or from antibody-dependent killer cells.

Interleukin 10 pretreatment protects target cells from tumor- and allo-specific cytotoxic T cells and downregulates HLA class I expression.

Interleukin 10 (IL-10) is a cytokine with a variety of reported effects including inhibition of monocyte major histocompatibility complex (MHC) class II-dependent antigen presentation, type 1 helper T cell cytokine production, and inhibition of T cell proliferation. Herein we report the effect of IL-10 pretreatment on antigen presentation to tumor- and allo-specific CD8+ cytotoxic T lymphocytes (CTL). Prior incubation of human melanoma cells with recombinant IL-10 (rIL-10) for 48-72 h resulted in a dose-dependent, up to 100% inhibition, of autologous CTL-mediated, HLA-A2.1-restricted, tumor-specific lysis. Allo-specific CTL cytotoxicity against Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) was also inhibited, demonstrating a protective effect also on lymphoid cells. In contrast, IL-10 pretreatment of allogeneic LCL or K562 targets had either no effect or slightly enhanced cytotoxic activity mediated by freshly isolated or IL-2-activated natural killer cells. Flow cytometric analysis with monoclonal antibodies against HLA-A2, or nonpolymorphic determinants of MHC class I proteins, revealed a 20-50% reduction in cell-surface expression, whereas intercellular adhesion molecules 1, and 2, and lymphocyte function-associated antigen 3 levels were not affected. In addition, relative to untreated target cells, IL-10 pretreated tumor cells were unaltered in their capacity to affect CTL-mediated lysis by cold target inhibition, demonstrating that the effect of IL-10 is unrelated to the initial binding of CTL to their targets. These results are compatible with an effect of IL-10 on the MHC class I antigen presentation pathway, and suggest a novel mechanism of immune tolerance, based on escape from CTL-mediated tumor and allo-transplant rejection.

Immunization with a lymphocytic choriomeningitis virus peptide mixed with heat shock protein 70 results in protective antiviral immunity and specific cytotoxic T lymphocytes.

Heat shock proteins (hsp's) isolated from murine cancer cells can elicit protective immunity and specific cytotoxic T lymphocytes (CTLs) by channeling tumor-derived peptides bound to hsp's to the major histocompatibility class I antigen presentation pathway. Here we have investigated if hsp70 can be used in a novel peptide vaccine for the induction of protective antiviral immunity and memory CTLs. A CTL epitope from the well-defined lymphocytic choriomeningitis virus (LCMV) system was mixed with recombinant hsp70 in vitro under conditions that optimize peptide binding to hsp70. Mice were immunized with the hsp70-peptide mixture and challenged with LCMV. Virus titers were reduced 10-100-fold in these mice compared to control mice. Immunization with the hsp70-peptide mixture resulted in the development of CTL memory cells that could be reactivated during LCMV infection, and that in a 51Cr-release assay could lyse cells pulsed with the same peptide, but not cells pulsed with another LCMV peptide. These results show that hsp70 can be used with CTL epitopes to induce efficient protective antiviral immunity and the generation of peptide-specific CTLs. The results also demonstrate the usefulness of hsp70 as an alternative to adjuvants and DNA vectors for the delivery of CTL epitopes to antigen-presenting cells.

The reconstitution of cell-mediated immunity in the cutaneous lesions of lepromatous leprosy by recombinant interleukin 2.

Human rIL-2 (10-30 micrograms) was injected intradermally into the skin of patients with lepromatous leprosy with high bacillary loads. All patients responded to the lymphokine with local areas of induration that peaked at 24 h and persisted for 4-7 d irrespective of whether the site was "normal skin" or a nodular lesion. Within 24 h there was an extensive emigration of T cells and monocytes into the site. The percentage of the dermis infiltrated by mononuclear cells increased by more than sevenfold, peaking at 4 d and persisting for greater than 15 d. Both CD4+ and CD8+ T cells entered the site. T cells of CD4+ phenotype predominated at 2-7 d but by 11 d, CD8+ cells were predominant. Considerable numbers of T6+ Langerhans' cells appeared in the dermis by 72 h and persisted for 3 wk. By 4 d the thickness of the overlying epidermis had increased twofold, and keratinocytes were expressing MHC class II antigen and the IFN-gamma-induced peptide IP-10. Starting at 48 h, there was an extensive destruction of mononuclear phagocytes that contained structurally intact or fragmented M. leprae observed at the electron microscope level. The organisms, either free or contained within endocytic vacuoles, were discharged into the extracellular space and then reingested by blood-borne monocytes. This was followed by marked reductions in the number of acid-fast organisms in the injected site, evident as early as 4-7 d and more marked at 2-3 wk after injection. 13 of 15 patients exhibited a disposal of acid-fast bacilli ranging from 5- to 1,000-fold with a mean value of approximately 100-fold. The administration of IL-2 leads to the generation of an effective cell-mediated immune response, recapitulating an antigen-driven event and leading to striking local reductions in M. leprae. In comparison with the purified protein derivative of tuberculin reaction, bacilli are cleared more promptly, although emigratory cells persist for a shorter time.

Modulation of sensitivity to natural killer cell lysis after in vitro explantation of a mouse lymphoma.

On the basis of studies indicating that natural killer (NK) cells of the mouse can selectively kill certain syngenetic, allogeneic, and xenogeneic tumor cells in short-term Cr release assays and that cell lines established in vitro are more sensitive than the corresponding ascites tumor cells passaged in vivo, the kinetics of the modulation to increased sensitivity was studied after in vitro explanation of the A/Sn mouse-derived YAC ascites lymphoma. Sensitivity to NK lysis appeared after 3 weeks of culturing and reached the level of the continuously cultured line after 2 months. With the more sensitive competition assay, a change could be demonstrated as early as 2--24 hours of culture. The expression of the Moloney murine leukemia virus-determined, cell-surface antigen, measured by quantitative absorption with intact cells, increased in parallel with the NK sensitivity. In contrast, the H-2 alloantigen concentration decreased during in vitro culture.

Demonstration of natural antibodies in normal rabbit serum with similar specificity pattern as mouse natural killer cells.

Normal rabbit sera (NRS) obtained from noninbred animals were shown to contain antibody to YAC-1, a mouse lymphoma, in a complement-dependent 51Cr release assay. A positive correlation was found between the sensitivity to lysis by NRS antibody and the susceptibility to lysis by mouse natural killer (NK) cells when 7 mouse tumors and 1 guinea pig hepatoma were tested in both assay systems. This correlation was further established with a quantitative absorption assay in which NRS was absorbed with various mouse tumors and subsequently tested for cytotoxicity against YAC-1, the most NRS- and NK-sensitive tumor. A striking positive correlation appeared between the capacity to absorb the NRS anti-YAC-1 activity and the NK sensitivity of various mouse tumors to CBA spleen cells. Thymocytes from strain A/Sn mice less than 2 weeks old absorbed a substantial amount of the NRS anti-YAC-1 activity; these cells were also sensitive to mouse NK cytolysis, whereas thymocytes from older mice lacked both properties. These natural rabbit antibodies could not "arm" peripheral blood lymphocytes in the rabbit because 1) the peripheral blood lymphocytes from these animals used as serum donors showed only low cytotoxic activity in an NK assay against NK-sensitive mouse tumors, and 2) no positive correlation appeared between the levels of cytolytic activity and the levels of natural serum antibodies observed in the rabbits. The present data suggest that natural antibodies in NRS showed the same specificity pattern for mouse tumors displayed by mouse NK cells.

Comparison between murine natural antibodies and natural killer cells: recognition of separate target structures as revealed by differential in vitro expression and dependence on glycosylation.

The specificity of complement-fixing, cytotoxic antibodies against the YAC lymphoma in sera of normal young adult (A X C57BL)F1 mice was studied. In vivo-maintained, immunoselected sublines of the YAC lymphoma expressed low amounts of the natural antibody (NAb) target structure. These cell lines were also resistant to natural killer (NK) cell-mediated lysis. After 2-3 weeks of in vitro culture the immunoselected cell lines became NK sensitive, but they remained resistant to NAb. When several independently derived variants selected for low NK sensitivity were tested for their ability to absorb NAb, the degree of absorption varied considerably among the variants. NAb could be inhibited by purified C-type virus particles and also by bacterial sonicates and various glycoprotein preparations. Treatment of target cells with tunicamycin, an inhibitor of asparagine-linked glycosylation, decreased the sensitivity to NAb lysis but had no impact on NK sensitivity. Thus the results indicated that a) NAb and NK cells recognized separate target structures and b) the target structure(s) for NAb but not for NK cells were saccharides of the N-glycosidic type.

Comparison of the allospecific and viral-specific immune responses to irradiated versus formaldehyde-fixed allogeneic Moloney lymphoma cells in CBA mice.

Two groups of adult CBA mice were immunized with 10-7 allogeneic Moloney lymphoma (YAC) cells. These YAC (H-2a) cells, which were either irradiated with 6000 R (Group i) or were formaldehyde fixed (Group II), were injected i.p. at weekly intervals for 3 weeks. Four days following the last injection, sera and lymphocytes were collected and tested in vitro for activity against either allospecific antigens (H-2d target cells) or viral-specific antigens, namely, Moloney leukemia virus (MLV). Both groups of animals developed measurable cellular and humoral immunity to the virally determined antigens. However, only the animals in Group i, immunized with irradiated cells, developed detectable immunity to H-2d. Immune and control lymphocytes were tested in microcytotoxicity tests and by 51Cr release. Antibody was assessed by complement-dependent cytotoxicity, indirect membrane immunofluorescence, virus neutralization, and antibody-dependent lymphocyte cytotoxicity. Group I serum, which had both anti-MLV and anti-H-2 antibodies, was absorbed with either living or formaldehyde-fixed YAC cells. The living cells were able to remove both H-2 and MLV antibodies. On the other hand, the formaldehyde-fixed cells removed no H-2 antibody but were able to remove MLV antibody, although less efficiently than living cells. These data indicate that formaldehyde fixation selectively impaired the H-2 antigens, leaving the viral antigenicity relatively intact. Differences between the immune responses to MLV-determined antigens and to H-2 antigens were demonstrated in many of the parallel in vitro tests.

Primary chemically induced tumors induce profound immunosuppression concomitant with apoptosis and alterations in signal transduction in T cells and NK cells.

Whereas transplantable tumors can be readily cured with immunotherapeutic approaches, similar therapies in cancer patients have been less effective. This difference may be explained by an immunosuppression resulting from the presence of a slowly growing primary tumor in the patient, whereas the immune system in a mouse with a rapidly proliferating transplantable tumor would be less affected. As a more appropriate model to the immune dysfunction in patients, slowly progressing primary tumors were induced by the carcinogen methylcholanthrene (MC) in mice. Their ability to induce immunosuppression in T cells and natural killer (NK) cells was compared to that of rapidly growing transplanted MC-induced tumors. The results demonstrate that mice bearing primary MC tumors had significantly diminished T-cell and NK-cell functions, impaired capacity to produce Th1 cytokines, and markedly reduced levels of the signal-transducing zeta chain in T cells and NK cells, similar to that described in cancer patients. Moreover, a substantial number of CD8+ T cells in mice with large primary MC tumors were undergoing apoptosis, correlating with alterations in CD4/CD8 ratios. In contrast, T cells and NK cells from mice bearing rapidly growing transplanted tumors were only marginally affected. These findings could explain the apparent discrepancy between the consistent findings of a diminished immune response and alterations in signal transduction in cancer patients as compared to the less reproducible observations in murine transplantable tumors. In addition, they could explain the differences in the high efficacy of immunotherapy in mice with transplantable tumors and the low therapeutic results in cancer patients.

Methylcholanthrene-induced mouse sarcomas express individually distinct major histocompatibility complex class I-associated peptides recognized by specific CD8+ T-cell lines.

Mouse sarcomas induced by methylcholanthrene (MC) are immunologically distinct even if they are induced in the same strain of mice. T-cell lines were derived from mice immunized against a series of syngeneic MC sarcomas on B6 background, known to carry unique tumor-specific transplantation antigens. Tumor necrosis factor-alpha (TNF-alpha) release assays concurred with the in vivo rejection tests. The strongest response in the TNF-alpha release was always obtained with the corresponding tumor, with very limited cross-reactivity against five other MC tumors or two virally induced B6 lymphomas. The specific TNF-alpha release from the anti-MC tumor CTL lines was mainly mediated by CD8+ cells. T-cell lines from intact and CD4-/- mice gave a similarly specific pattern. In contrast, T-cell lines derived from CD8-/- mice cross-reacted with several other MC-induced tumors. Peptides eluted from MC sarcomas under mild acid conditions were fractionated by reverse-phase high performance liquid chromatography and tested for their ability to sensitize the processing- and presentation-defective mutant RMA-S line. Only one high performance liquid chromatographic fraction from each of the three different tumor-derived peptide eluates capacitated RMA-S to induce TNF-alpha release and sensitized the cell to the cytotoxic effect of the corresponding tumor-specific T-cell line. A different Kb-restricted peptide fraction was active for each of the three MC sarcomas tested, indicating that they all expressed individually distinct peptide epitopes.

Human heat shock protein 70 peptide complexes specifically activate antimelanoma T cells.

Members of the heat shock protein 70 (HSP70) family display a broad cellular localization and thus bind a repertoire of chaperoned peptides potentially derived from proteins of different cellular compartments. In this report, we show that HSP70 purified from human melanoma can activate T cells recognizing melanoma differentiation antigens in an antigen- and HLA class I-dependent fashion. HLA class I-restricted anti-melanoma T cells were susceptible to MHC-restricted, HSP70-dependent stimulation, indicating that HSP70 complexed peptides were able to gain access to the class I HLA presentation pathway. In addition, MHC matching between the melanoma cells used as a source of HSP and the responding T cells were not required, indicating that HSP70 activation may occur across MHC barriers. Besides the MHC-restricted and peptide-dependent activation pathway, HSP70 with no endogenous complexed peptides or HSP70 purified from antigen-negative cells was also able to induce IFN-gamma release by antimelanoma T cells by a MHC-independent mechanism. In this case, however, higher doses of HSP70 were required. The capacity to activate class I-restricted, antitumor T cells as well as antigen-presenting cells, together with the finding that the HSP70 chaperoned peptide repertoire includes melanoma-shared epitopes, holds promise for a HSP70-based cancer vaccine.

Decreased expression of the signal-transducing zeta chains in tumor-infiltrating T-cells and NK cells of patients with colorectal carcinoma.

An impaired immune response is frequently observed in cancer patients and tumor-bearing mice. T-cells from mice with an experimental colon carcinoma were recently shown to express T-cell receptors that completely lacked the signal-transducing molecule CD3 zeta. Here, we have investigated the expression of the signal-transducing molecule zeta on lymphocytes from 14 patients with colorectal carcinomas using flow cytometric analysis of permeabilized cells with a monoclonal antibody (TIA-2; IgG1) specific for the cytoplasmic domain of the zeta chain as well as with immunoprecipitation and analysis on diagonal gel electrophoresis. We demonstrate that T-cells isolated from the tumors of the patients express significantly less CD3 zeta than T-cells in the peripheral blood of the same patients and that the peripheral blood of the patients express decreased levels of zeta chains, as compared to the levels found in lymphocytes from healthy controls. This decreased expression was also observed on zeta chains associated with the low affinity Fc receptor for IgG found in tumor-infiltrating NK cells (Fc gamma RIIIA alpha; CD16).

Synthetic peptides derived from the melanocyte-stimulating hormone receptor MC1R can stimulate HLA-A2-restricted cytotoxic T lymphocytes that recognize naturally processed peptides on human melanoma cells.

Human melanoma-specific HLA-A2 restricted CTLs have recently been shown to recognize antigens expressed by melanoma lines and normal melanocytes, including Melan-A/Mart-1, gp100, gp75, and tyrosinase. Herein, we define HLA-A2-restricted CTL epitopes from a recently cloned melanocortin 1 receptor (MC1R), which belongs to a new subfamily of the G-protein-coupled receptors expressed on melanomas and melanocytes. Thirty-one MC1R-derived peptides were selected on the basis of HLA-A2-specific motifs and tested for their HLA-A2 binding capacity. Of a group of 12 high or intermediate HLA-A2 binding peptides, three nonamers, MC1R244 (TILLGIFFL), MC1R283 (FLALIICNA), and MC1R291 (AIIDPLIYA), were found to induce peptide-specific CTLs from peripheral blood mononuclear cells of healthy HLA-A2+ donors after repeated in vitro stimulation with peptide-pulsed antigen-presenting cells. The CTLs raised against these three HLA-A2+-restricted peptides could recognize naturally processed peptides from HLA-A2+ melanomas and from Cos7 cells cotransfected with MC1R and HLA-A2. CTLs induced by the MC1R291 peptide (but not induced or induced only to a very low extent by the other two MCR1 peptide epitopes) showed cross-reactions with two other members of the melanocortin receptor family, which are more broadly expressed on other tissues. Taken together, our findings have implications in relation both to autoimmunity and immunotherapy of malignant melanomas.