Induction of syngeneic cytotoxic T lymphocytes against a B cell tumor. II. Characterization of anti-idiotypic CTL lines and clones.

In an earlier communication we showed that idiotypic immunoglobulin (Id+ Ig) of a B cell hybrid, 2C3, can induce cytotoxic T lymphocytes (CTL) in the spleens of mice that are hyperimmunized with the irradiated tumor cells. To understand the extent of heterogeneity in the splenic CTL population, stable anti-idiotypic CTL lines and clones were established from 2C3-primed splenocytes. One representative CTL line A102 which exhibited the phenotype of CD3+, CD4-, and CD8+, has been maintained in long-term culture for more than 18 months. Cytotoxic specificity of A102 was determined by cold target inhibition assay using a panel of syngeneic and allogeneic B cell tumors. The CTL line A102 was highly cytotoxic to 2C3, only weakly to other syngeneic tumors, but not at all to allogeneic B cell tumor CH12. Furthermore, CTL-mediated cytolysis was significantly abrogated by blocking 2C3 cells with anti-idiotypic monoclonal and polyclonal antibodies. These results clearly show that 2C3 Id represents the immunodominant epitope(s) recognized by the CTL line A102. To isolate a highly Id-specific effector population, A102 was repeatedly subcloned by limiting dilution. One such clone 102.F5 exhibited considerable specificity toward Id+ 2C3 while another clone 102.E10 showed no such specificity in a competitive cytotoxicity assay. This was further confirmed by the inhibition studies with anti-Id mAb. Thus, hyperimmunization with irradiated 2C3 cells evokes a spectrum of anti-2C3 cytotoxic effector cells, of which a major population is reactive to the idiotypic determinants associated with 2C3 Ig.     

Cell-mediated immune responses to syngeneic tumors. I. Identification of two distinct CTL effector pathways which differ in antigen specificity, genetic regulation, and cell surface phenotype

It has been demonstrated previously that draining lymph nodes (DLN) from tumor-immunized mice contain a population of lymphoid cells that are capable of differentiating into functional antitumor cytotoxic T lymphocytes (CTL) during in vitro culture. In the present studies, it was observed that DLN cells from either C57BL/10 (B10) or C3H mice that had been footpad-immunized with syngeneic tumor cells differentiated into CTL during a 4-day in vitro culture in the absence of added antigen. The specificity patterns of the CTL thus generated, however, were quite different in the two strains. DLN from B10 mice immunized with ultraviolet light-induced fibrosarcoma cells of B10 origin differentiated into CTL which were only capable of lysing target cells from the tumor used for immunization. Thus, the antitumor CTL which differentiate from B10 DLN appeared to be specific for the tumor-specific antigen (TSA) expressed by these tumor cells. In contrast, DLN from C3H mice immunized with a syngeneic ultraviolet light-induced fibrosarcoma differentiated into CTL which effectively lysed not only target cells from the immunizing tumor, but several other fibrosarcomas of both B10 and C3H origin, and which did not lyse normal nontumor targets. These C3H effectors thus appeared to be specific for a tumor-associated antigen (TAA) which is widely shared by a number of tumors. Cold target-blocking studies demonstrated that the CTL generated by C3H DLN cells contained a subpopulation of TSA-specific cells in addition to cross-reactive TAA-specific effectors. (B6 X C3H)F1 (B6C3F1) mice generated cross-reactive TAA-specific CTL in response to in vivo challenge with either B10 or C3H tumors, indicating that the ability to generate a TAA-specific CTL response behaves as a dominant trait of the responding mouse strain and not as a function of the tumor used for immunization. TSA-specific CTL and cross-reactive TAA-specific CTL were distinguishable on the basis of their cell surface phenotypes, because the TSA-specific CTL generated by B10 DLN cells were Thy-1.2+ Lyt-2.2+, whereas TAA-specific B6C3F1 CTL were Thy-1.2+ Lyt-2.2-; alloantigen-specific CTL generated from the same B6C3F1 lymph nodes were Thy-1.2+ Lyt-2.2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel cytotoxic T-cell clone that exhibits differential modes of recognition in the proliferative and cytolytic phase

A T-cell clone (1G8-H7) cytotoxic to P815Y mastocytoma (H-2d) has been established from spleen cells of a C3H/He mouse (H-2k) primed with P815Y cells by means of in vitro stimulation with irradiated C3H.H-2o(H-2KdDk) spleen cells. The clone 1G8-H7 was an interleukin 2 (IL-2)-dependent and H-2Kd antigen-dependent CTL clone and it killed P815Y cells but not Concanavalin A-induced spleen blast cells bearing H-2Kd antigen. The involvement of H-2Kd antigen in the cytolytic recognition mechanism was shown by the inhibition of lysis by anti-H-2Kd monoclonal antibody and also by the cold inhibition experiment that employed H-2Kd-bearing spleen cells. Comparison of cytotoxic activities between 1G8-H7 and Kd-specific CTL clones showed that the killing of P815Y cells by clone 1G8-H7 was not explained by the susceptibility to cell-mediated cytolysis of P815Y cells. These results suggest that H-2Kd antigen on the stimulating cell is sufficient to deliver a proliferation signal in the proliferative phase of this clone, but in the cytolytic phase an additional interaction with surface structure on the target cell other than that with H-2Kd antigen is required for the induction of cytolysis. Possible elucidations for the differential modes of recognition are discussed.     

Identification of a second major tumor-specific antigen recognized by CTLs on mouse mastocytoma P815

Murine mastocytoma P815 induces CTL responses against at least four distinct Ags (AB, C, D, and E). Recent studies have shown that the main component of the CTL response against the P815 tumor is targeted against Ags P815AB and P815E. The gene P1A has been well characterized. It encodes the P815AB Ag in the form of a nonameric peptide containing two epitopes, P815A and P815B, which are recognized by different CTLs. Here, we report the identification of the P815E Ag. Using a cDNA library derived from tumor P815, we identified the gene coding for P815E. We also characterized the antigenic peptide that anti-P815E CTLs recognize on the MHC class I molecule H-2Kd. The P815E Ag results from a mutation within an ubiquitously expressed gene encoding methionine sulfoxide reductase, an enzyme that is believed to be important in the protection of proteins against the by-products of aerobic metabolism. Surprisingly, immunizing mice i.p. with syngeneic tumor cells (L1210) that were constructed to express B7-1 and P815E did not induce resistance against live P815, even though a strong anti-P815E CTL response was observed with splenocytes from immunized animals.     

Mapping of HLA epitopes recognized by H-2-restricted cytotoxic T lymphocytes specific for HLA using recombinant genes and synthetic peptides

Immunization of DBA/2 (H-2d) mice with syngeneic P815 tumor cell transfectants that express HLA class I genes elicits CTL that recognize HLA in the context of H-2Kd molecules. Anti-HLA-CW3 CTL cross-react to a variable extent on the related alleles A3 and A24. Using a panel of target cells expressing native or recombinant HLA genes, we could map the epitope recognized by a CTL clone specific for CW3 to the second external (alpha 2) domain of CW3. Moreover, the epitope recognized by this clone could be mimicked by incubating P815 (HLA negative) target cells with a synthetic peptide corresponding to the C-terminal 12 amino acids of the CW3 alpha 2 domain (residues 171 to 182). Other independent anti-CW3 CTL clones with different fine specificities recognized the same CW3 peptide. In contrast, CTL clones specific for HLA-A24 or HLA-A3 that did not lyse P815-CW3 transfectants did not recognize this peptide. The CW3 peptide could be recognized on other tumor cell targets that were also of H-2d origin, but not on those of H-2b or H-2k origin. The requirement for the expression of H-2Kd by the target cells was directly demonstrated using L cell Kd transfectants. Our results suggest that the CTL response of DBA/2 mice immunized with P815-CW3 transfectants is predominantly Kd restricted and focused on epitopes contained within the 12 C-terminal amino acids of the alpha 2 domain.     

Induction of syngeneic cytotoxic T lymphocytes against a B cell tumor. III. MHC class I-restricted CTL recognizes the processed form(s) of idiotype.

The purpose of this work was to determine the molecular nature of the idiotypic Ig of a B cell tumor, 2C3, involved in the induction of anti-idiotypic cytotoxic T-lymphocytes (CTL). We previously reported that hyperimmunization of mice with irradiated 2C3 cells provides effective tumor protection by inducing MHC class I-restricted CTL. Due to the enormous heterogeneity of the splenic CTL further study could not be undertaken on the idiotype (Id)-CTL interaction. Subsequently an anti-idiotypic CTL line, A102, and a highly Id-specific CTL clone, 102.F5, have been developed. In the present investigation we report that the processed forms of idiotypic determinants are responsible for induction and activation of these specific effector CTL. Inhibition studies using anti-TcR and anti-MHC class I mAbs showed that the TcR-CD3 complex of the anti-idiotypic CTL recognized 2C3 Id in the context of MHC class I antigens. The cytotoxicity of these CTL could not be inhibited with affinity-purified 2C3 Ig used as such or after pulsing with splenic antigen-presenting cells (APC). Furthermore, using brefeldin A (BFA) and chloroquine (CLQ), which are specific inhibitors of cytosolic and endosomal antigen processing pathways, respectively, it has also been observed that exposure of 2C3 to BFA but not CLQ prevents its cytolysis by both anti-idiotypic CTL line and clone. These results clearly indicate that endogenously produced idiotypic determinants of 2C3 Ig are processed in pre-Golgi vesicle, possibly in the ER, along with MHC class I antigens and then are transported to the membrane. Treatment of 2C3 with BFA, however, did not exert any effect on the expression of membrane-associated Ig of 2C3 cells. Therefore, it is the processed form rather than the bona fide receptor Ig on the cell surface that is recognized by the Id-specific CTL.     

Early development of CD8-negative and CD8-positive MHC-unrestricted cytotoxic cells induced by alloantigen inoculation in mice

Peritoneal cells (PC) in C57B1/6 (B6, H-2b) mice receiving an intraperitoneal (i.p.) injection of allogeneic BALB/c (H-2d) spleen cells demonstrated potent cytotoxic activity against syngeneic, xenogeneic, third-party allogeneic tumors as well as H-2d derived tumors. Maximum cytotoxic activity against various tumors other than H-2d derived tumor, B16 (H-2b) was elicited on day 3 post allosensitization and decreased drastically thereafter, whereas cytotoxic activity against P815 (H-2d) peaked 3 days after the inoculation and maintained the peak activity thereafter. Surface phenotype of PC responsible for the cytotoxic activity against B16 was Thy-1+/-, Lyt-2-, L3T4-, asialo GM1 (AGM1)+, and that of PC against P815 was Thy-1+, Lyt-2+ (or Lyt-2+/-), L3T4-, AGM1+. These phenotypes showed similar phenotypes to the counterparts against B16 and against P815 in spleen cells induced by intravenous inoculation of alloantigen. When mice were pretreated i.p. with anti-AGM1 antibody before the allosensitization, anti-P815 cytotoxic-activity in PC was completely diminished. Similar activity in spleen, however, was enhanced by i.v. treatment with the antibody before the i.v. inoculation of alloantigen. The data clearly demonstrate that in vivo inoculation of B6 mice with normal allogeneic cells induces "NK-like" CD8- cytotoxic cells and "anomalous" CD8+ cytotoxic cells in PC.     

CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy.

The outcome of antigen recognition by naive CD8+ cytotoxic T lymphocytes (CTLs) in the periphery is orchestrated by CD4+ T-helper cells, and can either lead to priming or tolerization. The presence of T-helper cells favors the induction of CTL immunity, whereas the absence of T-helper cells can result in CTL tolerance. The action of T helper cells in CTL priming is mediated by CD40-CD40 ligand interactions. We demonstrate here that triggering of CD40 in vivo can considerably enhance the efficacy of peptide-based anti-tumor vaccines. The combination of a tolerogenic peptide vaccine containing a minimal essential CTL epitope with an activating antibody against CD40 converts tolerization into strong CTL priming. Moreover, CD40 ligation can provide an already protective tumor-specific peptide vaccine with the capacity to induce therapeutic CTL immunity in tumor-bearing mice. These findings indicate that the CD40-CD40 ligand pair can act as a 'switch', determining whether naive peripheral CTLs are primed or tolerized, and support the clinical use of CD40-stimulating agents as components of anti-cancer vaccines.     

Fas-mediated T cell deletion potentiates tumor antigen-specific tolerance in a mouse model of prostate cancer

A pivotal obstacle to cancer immunotherapy is peripheral T cell tolerance to tumor-associated antigens (TAAs). Tolerance induction among mature T cells in the periphery operates through a variety of mechanisms, including anergy and apoptosis. Although Fas-FasL-mediated apoptosis is a well-defined tolerance inducing mechanism, direct evidence of its interference with TAA-specific immunity in vivo is still lacking. In this report, we used the TRAMP mouse, which expresses SV40 large T antigen (Tag) preferentially in the prostate and develops prostate tumors, as a model system to address the role of Fas-mediated apoptosis in regulating peripheral T cell tolerance. Using RT-PCR and tetramer staining to quantify TAA-specific TCR-expressing cytolytic T lymphocytes (CTLs), we have shown the presence of TAA-specific CTLs at higher levels in TRAMP mice than in syngeneic C57Bl/6 mice. Tag-specific immunization led to the expansion of Tag-specific CTLs in C57Bl/6 mice, and to their elimination in TRAMP mice. Interestingly, in TRAMP mice with deficient Fas (Hybrid TRAMP-lpr/lpr), Tag-specific CTL elimination in response to Tag immunization did not take place. The results of cytolytic-function assays were consistent with induction and elimination patterns of TAA-specific CTLs and those of RT-PCR and tetramer staining. In conclusion, our data show that Fas-mediated TAA-specific CTL apoptosis contributes to T cell tolerance and suggest that such tolerance could be potentiated following TAA-specific immunization.     

Transmission of HIV-1 CTL escape variants provides HLA-mismatched recipients with a survival advantage

One of the most important genetic factors known to affect the rate of disease progression in HIV-infected individuals is the genotype at the Class I Human Leukocyte Antigen (HLA) locus, which determines the HIV peptides targeted by cytotoxic T-lymphocytes (CTLs). Individuals with HLA-B*57 or B*5801 alleles, for example, target functionally important parts of the Gag protein. Mutants that escape these CTL responses may have lower fitness than the wild-type and can be associated with slower disease progression. Transmission of the escape variant to individuals without these HLA alleles is associated with rapid reversion to wild-type. However, the question of whether infection with an escape mutant offers an advantage to newly infected hosts has not been addressed. Here we investigate the relationship between the genotypes of transmitted viruses and prognostic markers of disease progression and show that infection with HLA-B*57/B*5801 escape mutants is associated with lower viral load and higher CD4+ counts.     

L3T4+ cytotoxic T lymphocytes specific for class I H-2 antigens are activated in primary mixed lymphocyte reactions

Thy-1+, L3T4+, Ly-2- cytotoxic lymphocytes (CTL) are generated in a primary anti-H-2d mixed lymphocyte reaction, by using responders depleted of Ly-2+ cells. In addition to expressing the L3T4 marker, as detected by anti-L3T4 antibody and complement-mediated elimination, the L3T4+ CTL are inhibited by L3T4 antibody. The observation of these L3T4+ CTL in cells recovered from primary mixed lymphocyte reactions confirms the previous reports. However it is demonstrated for the first time that a subpopulation of these are class I-specific by their specific inhibition with an antiserum to class I antigens. The class I specificity of the CTL was further shown by their ability to kill class II antigen negative P815 tumor cells. The lysis of this target cell by L3T4+ CTL was also specifically blocked by the class I antiserum. The data is consistent with the presence also of a class II-specific population of L3T4+ cytotoxic cells. The fact that a level of L3T4+ cell-mediated cytotoxic activity comparable to Ly-2+ cytolytic activity is generated in a primary mixed lymphocyte response, even though the precursor frequency of L3T4+ killer cells is 10 times lower than for Ly-2+ killers, is suggestive of their physiologic significance. It was also shown that the activation of these cells is not dependent on the presence of xenogeneic serum components or exogenous helper or mitogenic factors in the culture medium. The findings provide further evidence against both the phenotype-function and phenotype-major histocompatibility complex antigen specificity models of T cell diversity.     

Phenotypic and functional heterogeneity of thymic and splenic lymphokine activated killer cells relative to ornithine sensitivity

We have previously reported the selective inhibition of cytotoxic T lymphocytes (CTL) by 10 mM ornithine (ORN) relative to natural killer (NK) cell-derived lymphokine activated killer cells (LAK). To determine if this were due to differences in the progenitor cells or the type of stimulus, we used cortisone-resistant thymocytes (CRT) as a source of mature T cells for induction of LAK and CTL, and compared the results with spleen. Thymic and splenic CTL precursors (CTLp) from C57B1/6 (B6) mice were CD8+, ASGM1-, ORN sensitive. Splenic LAK precursors (LAKp) were CD8-, ASGM1+, ORN resistant when assayed against both YAC-1 and P815 tumor targets. In contrast, CRT-derived LAKp were CD8-, ASGM1+, ORN resistant against YAC-1, whereas LAKp against P815 were CD8+, ASGM1+, ORN sensitive. ORN sensitivity was also observed among CTL and LAK in DBA/2 mice and was associated with CD8+ phenotype. Therefore, our initial observation of differential ORN sensitivity in CTL vs LAK was a function of the progenitor cells; furthermore, CD8+ cytolytic cells are ORN sensitive whether activated by antigen (CTL) or IL-2 (T-LAK).     

T helper cells in cytotoxic T lymphocyte development: role of L3T4(+)-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1-2+) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1+2- Th cells because: (i) cultures depleted by negative selection of Lyt-1+ T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1+2- T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4+ Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4+ cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.     

Stimulation with allogeneic Epstein-Barr virus-transformed lymphoblastoid cell lines generates HLA class I-specific CTLs with different target cell avidity.

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCL) are potent inducers of cytotoxic T-lymphocytes (CTL) in allogeneic mixed lymphocyte cultures (MLC). The contribution of EBV antigens to the induction of cytotoxic responses was investigated by comparing CTL clones derived from allogeneic MLCs of lymphocytes from one EBV seropositive and one seronegative donor for their capacity to lyse paired EBV positive and negative targets. The majority of the clones showed a conventional "HLA-specific" cytotoxicity and lysed equally well HLA-matched LCLs and mitogen-induced T- or B-blasts. A minority of the clones from both donors exhibited an "LCL-selective" killing potential as they lysed poorly T- and B-blasts. The LCL-selective clones did not recognize EBV antigens because they could not discriminate between EBV negative Burkitt lymphoma (BL) lines and their in vitro EBV-converted sublines. MAbs to CD3, CD8, and MHC class I antigens blocked the lysis of LCLs by HLA-specific and LCL-selective CTLs with comparable efficiency suggesting that the two effector types express T-cell receptors of similar affinity. T-blasts were unable to inhibit the lysis of LCLs in cross competition assays. This correlated with a significantly lower expression of the cell adhesion molecules ICAM-1 and LFA-3. The results suggest that stimulation with allogeneic LCLs activates HLA class I-specific CTLs with variable target cell avidity. Only CTLs that act independently of the enhancing effect of cell adhesion molecules are able to lyse mitogen-induced T- and B-blasts.     

H2-restricted recognition of cloned HLA class I gene products expressed in mouse cells

Long-term syngeneic mouse cytolytic T lymphocyte (CTL) clones were obtained from DBA/2 (H2d) mice immunized with P815 (H2d) cells transfected with cloned human class I histocompatibility genes, HLA-CW3 or HLA-A24. Three distinct patterns of specificity were defined on P815 HLA transfectant target cells. One clone lysed HLA-CW3 but not -A24 transfectants, and a second lysed HLA-A24 but not -CW3 transfectant target cells. The third clone lysed P815 targets transfected with either HLA gene. None of the CTL clones lysed L cells (H2k) transfected with the same HLA genes or human targets that expressed these HLA specificities. Several lines of evidence indicated that recognition of HLA transfectants by these CTL clones was H2 restricted. First, lysis of P815 HLA transfectants could be inhibited by anti-H2Kd monoclonal antibody. In addition, the anti-P815-HLA CTL clones could lyse a (human X mouse) hybrid target that expressed both HLA class I and H2Kd antigens, but not a clonal derivative that no longer expressed H2Kd. The most direct evidence for H2-restricted recognition of P815-HLA transfectants by the syngeneic CTL clones was obtained by double transfection of mouse L cells (H2k) with both HLA and H2 class I genes. L cells transfected with HLA and H2Kd genes were susceptible to lysis by the same CTL clones that lysed the corresponding P815-HLA transfectant targets. Thus under certain conditions, CTL recognition of xenogeneic class I histocompatibility gene products can be restricted by other class I gene products.     

Prognostic significance of tumor-infiltrating T-lymphocytes in primary and metastatic lesions of advanced stage ovarian cancer

Purpose  Ovarian cancer patients with intra-tumoral CD3⁺ T-lymphocytes in primary tumor tissue have a better prognosis. This study aims to analyze the presence and relative influence of three important T-lymphocyte subsets, tumor-infiltrating CD8⁺ cytotoxic T-lymphocytes (CTL), CD45R0⁺ memory T-lymphocytes, and FoxP3⁺ regulatory T-lymphocytes (Treg), in primary tumor tissue and omental metastases of patients with ovarian cancer. Experimental design  The number of CD8⁺, CD45R0⁺, and FoxP3⁺ T-lymphocytes was determined by immunohistochemistry on a tissue micro array containing ovarian tumor tissue and/or omental metastases obtained at primary debulking surgery from 306 FIGO stage I–IV ovarian cancer patients. Immunohistochemistry data were correlated to clinicopathological parameters and survival data. Results  High number of CD8⁺ CTL and a high CD8⁺/FoxP3⁺ ratio in ovarian-derived tumor tissue were associated with increased disease-specific survival and proved to be independent prognostic factors in multivariate analyses. In advanced stage patients, the presence of CD8⁺ CTL, CD45R0⁺ memory T-lymphocytes, FoxP3⁺ Treg or a high CD8⁺/FoxP3⁺ ratio in ovarian-derived tumor tissue was associated with an increased disease specific survival in univariate analysis, as was the presence of CD45R0⁺ memory T-lymphocytes and FoxP3⁺ Treg in omental metastases. Furthermore, in advanced stage patients CD8⁺ cytotoxic and FoxP3⁺ regulatory T-lymphocytes infiltrating ovarian-derived tumor tissue were independent predictors of increased prognosis. Conclusions  T-lymphocytes infiltrating primary and metastatic ovarian cancer sites are associated with improved prognosis. These associations are especially distinct in advanced stage patients, underlining the potential for immunotherapy as a broadly applicable therapeutic strategy. Ninke Leffers and Marloes J. M. Gooden contributed equally.     

Fas-independent cytotoxicity mediated by human CD4+ CTL directed against herpes simplex virus-infected cells

The present study was undertaken to clarify the mechanisms of cytotoxicity mediated by virus-specific human CD4+ CTLs using the lymphocytes of family members with a Fas gene mutation. CD4+ CTL bulk lines and clones directed against HSV-infected cells were established from lymphocytes of a patient with a homozygous Fas gene mutation and of the patient's mother. HSV-specific CD4+ CTLs generated from lymphocytes of the patient and her mother exerted cytotoxicity against HSV-infected cells from the patient (Fas-/-) and from her mother (Fas+/-) to almost the same degree in an HLA class II-restricted manner. mRNAs for the major mediators of CTL cytotoxicity, Fas ligand, perforin, and granzyme B, were detected in these CD4+ CTLs using the RT-PCR and flow cytometry. The cytotoxicity of the HSV-specific CD4+ CTLs appeared to be Ca2+-dependent and was almost completely inhibited by concanamycin A, a potent inhibitor of the perforin-based cytotoxic pathway. Although the Fas/Fas ligand system has been reported to be the most important mechanism for CD4+ CTL-mediated cytotoxicity in the murine system, the present findings strongly suggest that granule exocytosis, not the Fas/Fas ligand system, is the main pathway for the cytotoxicity mediated by HSV-specific human CD4+ CTLs.     

Inefficient cytotoxic T lymphocyte-mediated killing of HIV-1-infected cells in vivo

Understanding the role of cytotoxic T lymphocytes (CTLs) in controlling HIV-1 infection is vital for vaccine design. However, it is difficult to assess the importance of CTLs in natural infection. Different human leukocyte antigen (HLA) class I alleles are associated with different rates of progression to AIDS, indicating that CTLs play a protective role. Yet virus clearance rates following antiretroviral therapy are not impaired in individuals with advanced HIV disease, suggesting that weakening of the CTL response is not the major underlying cause of disease progression and that CTLs do not have an important protective role. Here we reconcile these apparently conflicting studies. We estimate the selection pressure exerted by CTL responses that drive the emergence of immune escape variants, thereby directly quantifying the efficiency of HIV-1-specific CTLs in vivo. We estimate that only 2% of productively infected CD4+ cell death is attributable to CTLs recognising a single epitope. We suggest that CTLs kill a large number of infected cells (about 10(7)) per day but are not responsible for the majority of infected cell death.     

Generation and ex vivo expansion of HTLV-1 specific CD8+ cytotoxic T-lymphocytes for adoptive immunotherapy

CD8(+) cytotoxic T-lymphocytes (CTLs) have been proven, in multiple animal models, to be the most powerful antiviral and antitumor components of the immune system. We have developed a protocol to activate and expand tumor and virus peptide-specific CD8(+) T-lymphocytes from the peripheral blood of healthy, human trophic leukemia virus-1 (HTLV-1) seronegative human leucocyte antigen (HLA)-A*0201 individuals. A combination of density-based separation and culture conditions was employed to isolate dendritic cells (DCs), which are the most potent antigen-presenting cells (APCs), and T-lymphocytes. The DCs were pulsed with HLA-A*0201 binding peptides and cultured with autologous T-lymphocytes to generate peptide-specific CTLs. The CTLs were generated against a nine-amino-acid peptide from the Tax protein of HTLV-1. The CTLs were expanded according to a restimulation schedule employing peptide-pulsed autologous monocytes and low-dose interleukin-2 (IL-2) to numbers in excess of 100 x 10(6) cells following 5 weeks of culture. Expanded cells contained primarily CD3(+) T-cells, of which CD8(+) T-lymphocytes constituted greater than two-thirds of the cell population. Obtained CTLs exhibited potent antigen-specific lysis of peptide-pulsed target cells in a dose-dependent fashion in in vitro (51)Cr release cytotoxicity assay. This antigen-specific killing was shown to be HLA class I restricted and mediated by CD8(+) T-lymphocytes. Since the T-lymphocytes were obtained from HTLV-1 seronegative donors, the generation of peptide-specific CTLs represents reliable and reproducible elicitation of a primary immune response in vitro against naive antigens and subsequent expansion of generated CTLs for adoptive immunotherapy. (c) 1996 John Wiley & Sons, Inc.