Role of effector cell-derived IL-4, IL-5, and perforin in early and late stages of type 2 CD8 effector cell-mediated tumor rejection.

Type 2 CD8 T cells (Tc2) secrete IL-4 and IL-5 and display perforin-dependent cytolysis in vitro. Using an OVA-transfected B16-melanoma model, we show that tumor-reactive Tc2 effector cells accumulated at the tumor site and induced tumor regression that enhanced survival in mice with pulmonary tumors. Transfer of perforin-deficient Tc2 cells generated from perforin gene knockout mice showed no differences in therapeutic efficiency when compared with wild-type Tc2 cells. In contrast, Tc2 cells derived from select cytokine gene-deficient mice showed that therapeutic effects were dependent on effector cell-derived IL-4 and IL-5 that led to a local elevation in lung-derived chemoattractants and accumulation of activated host-derived CD8/CD44(high), CD4/CD44(high), and OVA-specific tetramer-positive CD8 cells in vivo. Host-derived T and non-T immune cells increased in the lung over time and correlated with an elevated production of type 1-related chemokines. Conversely, donor Tc2 cell numbers markedly diminished at later times, suggesting that prolonged therapeutic responses were due to host-derived mechanisms. Moreover, type 1 host responses were detectable with increased levels of IFN-gamma production by lung-derived CD4 and CD8 T cells from surviving Tc2-treated mice. Transfer of Tc2 cells into IFN-gamma-deficient tumor-bearing mice was markedly less effective then into wild-type mice, suggesting that host-derived IFN-gamma-dependent mechanisms play a role in Tc2-mediated antitumor responses.

Immunopotentiating role of IFN-gamma in early and late stages of type 1 CD8 effector cell-mediated tumor rejection.

Type 1 cytolytic CD8 effector T cells (Tc1) characteristically secrete IFN-gamma. Using an OVA-transfected B16 melanoma lung tumor model, we show that OVA Ag-specific Tc1 cells mediate a reduction in tumor growth that significantly prolongs survival in tumor-bearing mice. Transfer of Tc1 cells from OT-I mice crossed to IFN-gamma-KO mice showed that IFN-gamma-deficient Tc1 effector cells were less therapeutically effective than corresponding cells from wildtype mice. Therapeutic effects were dependent, in part, on effector cell-derived IFN-gamma, which not only induced elevated levels of lung-derived IP-10 and RANTES chemokine message in vivo, but also increased the local accumulation of activated host-derived CD4(+)/CD44(High), CD8(+)/CD44(High), and non-T-immune cell populations at the tumor site. Over time, the numbers of host-derived immune cells increased in the lung, which correlated with an elevated production of IP-10 and RANTES and a continued reduction in tumor burden. Conversely, donor Tc1 cell numbers markedly diminished at corresponding times, suggesting that prolonged therapeutic responses were due to the presence of host-derived antitumor mechanisms. Moreover, adoptive transfer of IFN-gamma-deficient Tc1 cells into tumor-bearing IFN-gamma-KO recipients showed that both recipient and donor-derived IFN-gamma play a significant role in Tc1-mediated responses and that Tc1 effector cell immunotherapy is predominantly mediated by IFN-gamma-dependent mechanisms.

Type 1 and type 2 CD8+ effector T cell subpopulations promote long-term tumor immunity and protection to progressively growing tumor.

Cytolytic CD8+ effector cells fall into two subpopulations based on cytokine secretion. Type 1 CD8+ T cells (Tc1) secrete IFN-gamma, whereas type 2 CD8+ T cells (Tc2) secrete IL-4, IL-5, and IL-10. Using an OVA-transfected B16 lung metastases model, we assessed the therapeutic effects of adoptively transferred OVA-specific Tc1 and Tc2 subpopulations in mice bearing established pulmonary malignancy. Effector cell-treated mice exhibiting high (5 x 105) tumor burdens experienced significant (p < 0.05) delays in mortality compared with those of untreated control mice, whereas high proportions (70-90%) of mice receiving therapy with low (1 x 105) tumor burdens survived indefinitely. Long-term tumor immunity was evident by resistance to lethal tumor rechallenge, heightened levels of systemic OVA Ag-specific CTL responses ex vivo, and detection of long-lived TCR transgene-positive donor cells accompanied by an elevation in the total numbers of CD8+ CD44high activated and/or memory T cells at sites of tumor growth. Long-lasting protection by Tc2 and Tc1 effector cells were dependent, in part, on both the level of tumor burden and effector cell-derived IL-4, IL-5, and IFN-gamma, respectively. We conclude that Tc1 and Tc2 effector cells provide immunity by different mechanisms that subsequently potentiate host-derived antitumor responses.

Therapeutic effects of tumor-reactive type 1 and type 2 CD8+ T cell subpopulations in established pulmonary metastases.

Cytolytic CD8+ T cells fall into two subpopulations based on cytokine-secretion. Type 1 CD8+ cells (Tc1) characteristically secrete IFN-gamma, whereas type 2 CD8+ cells (Tc2) secrete IL-4 and IL-5. We assessed the relative therapeutic effects of adoptively transferred OVA-specific Tc1 and Tc2 CD8+ cells in mice bearing established OVA-transfected B16 melanoma lung metastases. Both Tc1 and Tc2 subpopulations mediated a reduction in lung tumor growth that subsequently prolonged survival times in mice with both early (day 7) and more advanced (day 14) levels of tumor development. CD8+ T cell populations recovered from spleens of tumor-bearing mice receiving Tc1 or Tc2 cells showed markedly enhanced tumor Ag-specific cytolytic and cytokine-releasing activities that correlated with delays in tumor cell growth and progression. Initially, both tumor-reactive Tc1 and Tc2 effector cells accumulated at the tumor site with nearly equal frequency. Tc1 cells persisted, whereas Tc2 cell numbers progressively diminished over time. Titration of Tc1 and Tc2 effector cells showed that protection was dose dependent with the former being 5-fold more effective. Tc2 cells achieved a comparable reduction in lung tumor cell growth at higher concentrations of cell transfer. Tc1 effectors from IFN-gamma-deficient mice were less therapeutically effective than wild-type mice, but there was no significant reduction in activity between corresponding Tc2 populations. We speculate that the effectiveness of Tc1 and Tc2 cells may depend on different mechanisms. These studies suggest a potential role for Tc1 and Tc2 CD8+ subpopulations in tumor regression and immunotherapy.

Immunization with a syngeneic tumor infected with recombinant vaccinia virus expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) induces tumor regression and long-lasting systemic immunity.

A recombinant vaccinia virus encoding the gene for granulocyte-macrophage colony-stimulating factor (rV-GM-CSF) was used to infect the poorly immunogenic murine colon adenocarcinoma cell line, MC-38. Infection of MC-38 tumor cells with rV-GM-CSF completely suppressed the growth of the MC-38 primary tumors, whereas progressively growing tumors were formed in mice injected with MC-38 cells infected with wild type V-Wyeth. Irradiation of the recipient B6 mice before implantation of rV-GM-CSF-infected tumor cells resulted in the development of progressively growing tumors. Moreover, in vivo T-cell depletion studies revealed that growth suppression of the rV-GM-CSF-infected tumor cells was dependent on the presence of both CD4+ and CD8+ T-cell subsets. Subsequent studies established that this immunity was long-lasting and antigen specific, as demonstrated by the protection of rV-GM-CSF-immunized mice from MC-38 tumor challenge but not from challenge with another syngeneic tumor cell type. No such effects were observed when MC-38 tumor cells were infected with recombinant vaccinia viruses expressing interleukin (IL)-2 or IL-6. The results demonstrate that paracrine release of biologically active murine GM-CSF by tumor cells infected with rV-GM-CSF enhances the intrinsic immunogenicity of a poorly immunogenic murine tumor. Presumably the augmentation of tumor immunogenicity induces an antigen-specific T-cell-dependent antitumor response that prevents the formation of primary tumors and protects mice from tumor challenge. Thus in this experimental model, GM-CSF functions as a highly effective vaccine adjuvant.

Peptide-specific activation of cytolytic CD4+ T lymphocytes against tumor cells bearing mutated epitopes of K-ras p21.

Alterations in the ras p21 protein have been associated with both rodent and human neoplasia. Thus, mutated ras p21 proteins may bear unique antigenic epitopes for immune recognition, such as by T cells, which have been implicated in host antitumor activity. Synthetic peptides that mimic segments of mutated ras p21 have been reported to be immunogenic in mice in vivo, although detailed functional analyses remains undefined. Here, in a murine model, we explored and characterized distinct effector properties of host-derived T lymphocytes reactive to mutated ras peptides, which was consistent with the CD4+ T helper type 1 (Th1) subset. BALB/c mice (H-2d) were immunized with a purified peptide, 13 amino acids in length, containing the substitution of Gly (G12) to Val (V12) at position 12, which is commonly found in human carcinomas. An alpha beta T cell receptor-positive, CD3+, CD4+, CD8- T cell line was established, which expressed peptide-specific proliferation. Cytokine assays revealed the production of interleukin-2, interferon-gamma, tumor necrosis factor and granulocyte-macrophage colony-stimulating factor. Moreover, antigen-specific cytotoxicity was demonstrable against: (1) Iad-bearing A20 tumor cells incubated with exogenously bound V12 peptide; and (2) A20 tumor cells transduced with the K-ras p21 oncogene encoding the corresponding point mutation. CD4(+)-mediated cytotoxicity was major histocompatibility complex (MHC) class II-restricted, as revealed by the absence of lysis against MHC class II- P815 targets, inhibition of A20 lysis with anti-Iad monoclonal antibodies, and induction of lysis against L cell targets transfected with E alpha A beta d. Independent isolation of a second CD4+ V12 line revealed a very similar cytolytic and MHC class II-restricted profile. Overall, these data demonstrated that peptide immunization produced a CD4+ Th1 response that specifically recognized tumor cells expressing endogenous activated K-ras epitopes, which may have implications for the development of peptide-based active immunotherapies.

Elevated CD4 antigen expression among activated T cells in lymph nodes draining mammary regions chronically exposed to Staphylococcus aureus antigen.

We examined the cell cycle distribution and subset marker characteristics of mucosal-associated supramammary lymph node (MALN) T cell subpopulations at sites proximal and distal to the mammary region of animals repeatedly injected with Staphylococcus aureus antigen. Multiparameter flow cytometric analysis of draining MALNs showed that CD4+ T cells expressed significantly greater amounts of CD4 surface antigen than corresponding cell populations in non-draining MALNs. In contrast, the intensity of CD8 surface antigen expression among draining MALN CD8+ cells remained unchanged. Draining and non-draining MALNs contained nearly equal proportions of large CD4+ T cell subpopulations (CD4/CD8 ratio) with the former having greater cell numbers undergoing active DNA synthesis (S + G2M phase) in vivo. Similarly, draining MALNs had greater cell numbers of small CD4+ lymphocytes in the S + G2M phase, although with lower CD4/CD8 ratios of corresponding cell populations in non-draining MALNs. This study demonstrates that prolonged exposure with Staph aureus antigen enhances the cell number and expression of CD4 surface antigen among T lymphocyte subpopulations actively synthesizing DNA in draining MALNs. The role of the CD4 antigen in inflammatory responses at sites proximal (draining) and distal (non draining) to chronic infection within the mammary/mucosal immune network is discussed.

Phenotypic and functional analysis of mucosal lymph node T cell subpopulations proximal and distal to chronic staphylococcal antigen challenge.

We investigated the functional and subset surface marker characteristics of supramammary lymph node T cell populations at sites proximal and distal to the mammary region of goats repeatedly injected with heat-treated Staphylococcus aureus antigen (HK-SAC). Flow cytometric studies showed quantitative differences in CD4+ and CD8+ T cell subsets among large and small lymphocyte populations in ipsilateral and contralateral supramammary lymph nodes of these animals. Although ipsilateral (draining) lymph nodes were enriched with CD4+ and CD8+ T cells, CD4/CD8 ratios were comparatively lower than those of contralateral (non-draining) lymph nodes (2.30 vs 2.60, respectively). Cell size analysis by flow cytometry showed that nearly 70% of the lymphocytes in ipsilateral nodes were of large cell phenotype with CD4/CD8 ratios of 2.52. In contrast, there were only 56.1% large lymphocytes in contralateral lymph nodes but with similar CD4/CD8 ratios of 2.55. The number of large lymphocytes in corresponding nodes of uninoculated control animals was significantly lower (50%) with much lower CD4/CD8 ratios (2.08). Alloantigenic responses of both ipsilateral and contralateral lymph node T cells were greater than those of uninoculated controls. Antigen-specific proliferation studies showed that ipsilateral lymph node T cells greatly enhanced both primed and non-primed lymph node B cell responses to HK-SAC, whereas those from contralateral lymph nodes were less stimulatory. In contrast, contralateral lymph node T cells had greater enhancing effects on PWM-induced polyclonal B cell responses. These studies indicate that repeated local infection with bacterial antigen induce changes in the numbers, ratios and antigen-specific and non-specific responses among ipsilateral (draining) and distal contralateral (non-draining) lymph node T cell populations in mucosal-associated immune systems such as the mammary gland.

Systemic profiles of antigen-specific lymphocytes in animals chronically exposed to staphylococcus antigen in the mammary region.

Systemic profiles of lymphocytes were assessed in goats exposed chronically with Staphylococcus antigens in the supramammary region. Animals were inoculated three times subcutaneously in the right supramammary region with heat-killed Staphylococcus aureus antigen (HKS) at 1 month intervals. Prior to immunization and 1 week following each injection, 3 and 6 day cultures of peripheral blood mononuclear cells were made to determine proliferative responses of lymphocytes to HKS and the polyclonal T cell mitogen phytohemagglutinin (PHA). Peripheral blood lymphocytes responded significantly to both HKS and PHA in 3 day cultures after the second injection and showed peak responses after the final immunization, suggesting that repeated local injection of S. aureus antigen at the supramammary region, can induce an anamnestic response to the antigen in the peripheral blood of these animals with a concomitant increase in the responsiveness to the polyclonal mitogen, PHA. In contrast, initial antigen challenge induced little, if any, increase in responses to the specific antigen or mitogen when compared to pre-injection states. These data may also suggest that non-reactivity of peripheral blood lymphocytes to the HKS antigen immediately after the primary injection of antigen may be the result of local retention of antigen-reactive cells at the sites of infection.

Differential enhancement and distribution of antigen-specific cells in various lymph nodes in response to locally inoculated bacterial antigens.

The proliferation responses of antigen-specific lymphocytes from various anatomical sites were studied in dairy goats locally immunized with heat-killed Staphylococcus aureus (HKS). Animals were inoculated three times subcutaneously in the right udder with HKS at 1 month intervals. One week following the last inoculation, prescapular, mesenteric and ipsilateral (draining) and contralateral (non-draining) suprammammary lymph nodes were collected and the cells assayed in 3- and 6-day cultures to determine the immune proliferative responses of antigen-specific lymphocytes to HKS and the polyclonal T cell mitogen phytohemagglutinin (PHA). The cells from draining and non-draining supramammary lymph nodes responded to HKS in 3-day cultures. Peripheral lymph nodes, such as the prescapular, showed similar responses. In contrast, mesenteric lymph nodes responded optimally in 6-day cultures, notably to lower concentrations of the antigen. Cells from all lymph nodes tested showed increased responses to PHA in immunized animals, although non-draining lymph nodes demonstrated a greater response to the T cell mitogen than those of draining lymph nodes. These results suggest that unilateral introduction of Staphylococcus cell antigens to the supramammary region can induce an anamnestic response in ipsilateral as well as contralateral supramammary lymph nodes and other distant peripheral lymphoid organs. Furthermore, these data indicate that cells from intestinal lymph nodes respond differently from those of peripheral lymph nodes, suggesting the presence of a unique gastrointestinal lymphoid cell circulation in goats. Concomitant peripheral responses may be attributed to memory cell migration or to antigen leakage and relocation to distant sites from the inoculated region. Analysis with PHA suggests a difference in general responsiveness and perhaps, immunocompetence, by lymphocyte populations in various lymphoid tissues of immunized animals.

Size, CD4 and CD8 marker profiles and functions of lymphocyte subpopulations in mucosal-associated lymph nodes.

We analyzed phenotypic and functional characteristics of T cell populations in mucosal-associated supramammary and mesenteric lymph nodes in goats. Here we demonstrate, by flow cytometry, quantitative differences in CD4 and CD8 T cell subsets among large and small mucosal-associated lymphocyte populations and their differential regulatory activities on resident lymph node B cells stimulated with Staphylococcus aureus Cowan I or pokeweed mitogen. The CD4/CD8 T cell ratio was lower in mesenteric lymph nodes (1.46) when compared to that of supramammary lymph nodes (2.18). Analysis of large and small lymphocyte subpopulations from lymph nodes showed nearly 62% of the lymphocytes from mesenteric lymph nodes being of large cell phenotype with CD4/CD8 ratios of 1.34. In contrast, large cell subpopulations in supramammary lymph nodes showed a significantly lower number (50%) with a higher CD4/CD8 ratio of 2.05. Functionally, mesenteric lymph node T cells, isolated by nylon wool, showed heightened suppressive activity in mitogen-driven B cell proliferation responses, whereas T cells from supramammary lymph nodes were stimulatory. These findings clearly demonstrate distinctive functional properties between resident T cell populations of supramammary and mesenteric lymph nodes, suggesting that different proportions of T cell subsets in these nodes are activated and thus regulate regional immune responses via different pathways.

[A case of neoplastic diathesis]. / Przypadek skazy nowotworowej.

A female patient is described who during 7 years had three operations for neoplasms situated in various sites and exhibiting different histological structure. Seven years ago malignant melanoma was excised from her left thigh, three years later hysterectomy was done for carcinoma, 18 months later an epithelioid carcinoma was removed from the retroperitoneal space.