MHC-restricted Cytotoxic T Lymphocytes Research Articles

Two class I genes of the chicken MHC have different functions: BF1 is recognized by NK cells while BF2 is recognized by CTLs.

The function of the chicken's major histocompatibility complex (MHC or B complex) class I major (BF2) and minor (BF1) glycoproteins is compared for their expression, ability to present viral antigens to cytotoxic T lymphocytes (CTLs), and interaction with natural killer (NK) cells. MHC-restricted CTLs recognized virus antigen in the context of the BF2*21 major glycoprotein but not the BF1*21 minor glycoprotein. Marek's disease virus (MDV), a large DNA virus known to reduce the cell surface expression of class I glycoprotein, reduced the expression of BF2 glycoprotein while BF1glycoprotein expressions are remained as no change or slight increase. In addition, the expression of BF1*21 class I glycoprotein protected target cells from NK cell lysis while the expression of the BF2*21 class I glycoprotein enhanced NK cell lysis of target cells. Therefore, BF1 and BF2 provide two different cellular immune functions; BF1 negatively regulates the NK cell killing activity and BF2 restricts the antigen specific CTL immune response.

Immediate-early protein of equid herpesvirus type 1 as a target for cytotoxic T-lymphocytes in the Thoroughbred horse.

Cytotoxic T-lymphocytes (CTLs) are associated with protective immunity against disease caused by equid herpesvirus type 1 (EHV-1). However, the EHV-1 target proteins for CTLs are poorly defined. This limits the development of vaccine candidates designed to stimulate strong CTL immunity. Here, classical CTL assays using lymphocytes from horses of three defined MHC class I types that experienced natural infection with EHV-1 and a modified vaccinia virus construct containing an EHV-1 gene encoding the immediate-early (IE) protein are reported. Horses homozygous for the equine leukocyte antigen (ELA)-A2 haplotype, but not the ELA-A5 haplotype, produced MHC-restricted CTL responses against the IE protein. Previously, horses homozygous for the ELA-A3 haplotype also mounted CTL responses against the IE protein. Both haplotypes are common in major horse breeds, including the Thoroughbred. Thus, the IE protein is an attractive candidate molecule for future studies of T-cell immunity to EHV-1 in the horse.

Epitope specificity appears to be an important determinant of in vivo killing ability of simian immunodeficiency virus (SIV)-specific CD8+ T cells (105.43)

Abstract CD8+ cytotoxic T lymphocytes (CTL) are a critical component of antiviral immunity and play an important role in the control of lentiviruses. We have developed an in vivo CTL assay to directly measure the killing capacity of MHC-restricted SIV-specific CTL in rhesus macaques (RM) vaccinated with a recombinant herpes simplex virus prime/ DNA boost SIV vaccine regimen. Tetramer frequencies of Mamu-A*02-restricted Nef YY9-specific CD8+ T-cells were at least 10-fold higher than Mamu-A*02-restricted Gag GY9-specific CD8+ T-cells in individual animals both pre- and post-challenge. Prior to SIV challenge, both CTL populations showed poor and incomplete killing (22-35%) of target cells over 18 hours. Seven weeks post-challenge, there was a marked increase in the CTL killing capacity of both Nef YY9 and Gag GY9-specific CTL with 26-38% killing occurring over the first two hours and up to 100% killing over 18 hours. Surprisingly, Gag GY9-specific CTL consistently showed equivalent or greater in vivo killing when compared to Nef YY9-specific CTL even though the percentages of IFN-γ secreting and degranulating cells upon peptide stimulation were comparable. These data suggest that epitope specificity rather than tetramer frequency determines the ability of SIV-specific CTL to kill infected cells. Taken together, these data may have important implications for the development of a successful HIV vaccine.

A Virosomal Immunization Strategy against Cervical Cancer and Pre-Malignant Cervical Disease

In this study, we demonstrate that fusion-active virosomes, containing recombinant human papillomavirus type 16 (HPV16) E7 protein antigen, are capable of inducing a robust class I MHC-restricted cytotoxic T-lymphocyte (CTL) response against HPV-transformed tumour cells in a murine model system. Virosomes are reconstituted viral envelopes, which do not contain the genetic material of the native virus. During the reconstitution process, protein antigens can be encapsulated within the virosomes. In the present study, we used virosomes derived from influenza virus. These virosomes retain the cell binding and membrane fusion characteristics of native influenza virus, and have the capacity to deliver encapsulated antigens to the cytosol of antigen-presenting cells through fusion from within acidic endosomes. After immunization of mice with virosomes containing encapsulated HPV16 E7 protein, the animals developed a strong E7-specific CTL response as assessed by 51Cr release measurements and MHC tetramer staining of spleen cells. Immunization with E7-containing virosomes also resulted in E7-specific antibody responses. In tumour challenge experiments, immunization of mice with E7-containing virosomes prevented tumour outgrowth in >70% of the animals. Thus, influenza-derived virosomes with encapsulated HPV E7 protein antigen act as an excellent vaccine delivery system for induction of cellular immunity against HPV-transformed cells and represent a promising immunotherapeutic vaccine for the treatment of (precursor lesions of) cervical cancer.

Identification of an H-2D b-restricted CD8+ cytotoxic T lymphocyte epitope in the matrix protein of respiratory syncytial virus

Cytotoxic T lymphocytes (CTL) play a significant role in the clearance of respiratory syncytial virus (RSV) infection in humans and mice. Identification of class I MHC-restricted CTL epitopes is critical in elucidating mechanisms of CTL responses against viral infections. However, only four H-2 d-restricted epitopes have been reported in mice. Because of the diversity of transgenic and knockout mice available to study immune responses, new epitopes in additional strains of mice must be identified. We therefore attempted to discover novel CTL epitopes in C57Bl/6 mice. Our efforts revealed a new H-2D b-restricted CTL epitope from the RSV M protein, corresponding to aa 187–195 (NAITNAKII). Also, M187–195-specific CTLs were activated with kinetics similar to the immunodominant BALB/c epitope, M2 82–90. This is the first RSV-specific CTL epitope described in a strain of mice other than BALB/c. Furthermore, identification of this H-2 b-restricted CTL epitope provides access to genetically modified H-2 b mice for more detailed studies of CTL mechanisms in RSV infection.

Antigen-specific tumor vaccine efficacy in vivo against prostate cancer with low class I MHC requires competent class II MHC.

Cancers can escape immune recognition by means of evading class I major histocompatibility complex (MHC) -mediated recognition by cytotoxic T lymphocytes. However, immunization strategies targeting defined tumor-associated antigens have not been extensively characterized in murine prostate cancer models. Therefore, we evaluated antigen-specific, antitumor immunity after antigen-encoding vaccinia immunization against mouse prostate cancer cells expressing a model tumor-associated antigen (beta-galactosidase) and exhibiting partially deficient class I MHC. Low class I MHC expression in beta-galactosidase-expressing D7RM-1 prostate cancer cells was shown by fluorescence activated cell sorting, and deficient class I MHC-mediated antigen presentation was shown in resistance of D7RM-1 to cytolysis by beta-galactosidase-specific cytotoxic T lymphocytes (CTL). Despite partially deficient class I MHC presenting function, immunization with vaccinia encoding beta-galactosidase conferred antigen-specific protection against D7RM-1 cancer. Antigen-specific immunity was recapitulated in beta(2)m knockout mice (with deficient class I MHC and CTL function), confirming that class I MHC antigen presentation was not required for immunity against tumor partially deficient in class I MHC. Conversely, antigen-specific antitumor immunity was abrogated in A(b)beta knockout mice (with deficient class II MHC and helper T cell function), demonstrating a requirement for functional class II MHC. Resistant tumors from the otherwise effectively immunized beta(2)m knockout mice (among which tumor progression had been reduced or delayed) showed reduced target antigen expression, corroborating antigen-specificity (and showing an alternative immune escape mechanism), whereas antigen expression (like tumor growth) was unaffected among A(b)beta knockout mice. Our results demonstrate that class I MHC-restricted antigen presentation and CTL activity is neither necessary nor sufficient for antigen-encoding vaccinia immunization to induce protective immunity against class I MHC-low tumors, whereas host class II MHC-mediated antigen presentation facilitates antigen-specific immunity against prostate cancer in vivo. Reduced expression of the target antigen developed rapidly in vivo as an immune escape mechanism for such cancers.

Vaccination to Treat Persistent Viral Infection

Persistent infections caused by such agents as the human immunodeficiency virus, hepatitis B virus, Epstein–Barr virus, etc., present formidable medical problems. A defining characteristic of these infections is that anti-viral cytotoxic T lymphocytes (CTL) may be lost or, if present, fail to clear the infection. Here we report a vaccination strategy which was successful in generating lytic CTL in persistently infected mice. Vaccination with an immunodominant CTL epitope derived from the nucleoprotein of lymphocytic choriomeningitis virus (LCMV) delivered in the form of a lipopeptide incorporating a universal CD4 helper epitope successfully induced lytic MHC-restricted CTL in mice persistently infected with LCMV since birth. However, induction of such CTL did not eliminate the virus, most likely because the CTL were generated at low frequencies and had 2 to 3 logs lower affinity than CTL generated in uninfected mice inoculated with the vaccine. Both CTL populations from either uninfected or persistently infected mice produced significant and similar amounts of interferon-γ and IL-6. Vaccine-induced low-affinity CTL were still inadequate at complete removal of the virus when combined with LCMV-specific CD4 helper T lymphocytes. Thus, our results establish that CTL can be generated in persistently infected mice and that a crucial factor for clearing viral infection is the affinity of the CTL.

Novel breast-tumor-associated MUC1-derived peptides: Characterization in Db−/− × β2 microglobulin (β2m) null mice transgenic for a chimeric HLA-A2.1/Db-β2 microglobulin single chain

The MUC1 protein was found to be up-regulated in a spectrum of malignant tumors. T-cell responses to the MUC1 extracellular tandem repeat array (TRA) were observed in murine models as well as in breast-carcinoma patients. In the present study, we evaluated the anti-tumor potential of HLA-A2.1-motif-selected peptides from non-TRA domains of the molecule. Peptide immunogenicity was examined in the Db−/− × β2 microglobulin (β2m) null mice transgenic for a modified HLA-A2.1/Db-β2 microglobulin single chain (HHD mice). Our results show the existence of 3 novel HLA-A2.1-restricted MUC1-derived cytotoxic T-lymphocyte (CTL) epitopes. These peptides are processed and presented by the HHD-transfected breast-tumor cell line MDA-MB-157. Moreover, CTL induced by these 3 peptides show higher lysis of target cells pulsed with breast-carcinoma-derived peptides than of targets pulsed with normal breast-tissue-derived peptides. These data suggest an important role for non-TRA MUC1-derived peptides as inducers of a MHC-restricted CTL reaction to a breast-carcinoma cell line and patient-derived tumor extracts. Int. J. Cancer 85:391–397, 2000. ©2000 Wiley-Liss, Inc.

Novel breast-tumor-associated MUC1-derived peptides: Characterization in Db−/− × β2 microglobulin (β2m) null mice transgenic for a chimeric HLA-A2.1/Db-β2 microglobulin single chain

The MUC1 protein was found to be up-regulated in a spectrum of malignant tumors. T-cell responses to the MUC1 extracellular tandem repeat array (TRA) were observed in murine models as well as in breast-carcinoma patients. In the present study, we evaluated the anti-tumor potential of HLA-A2.1-motif-selected peptides from non-TRA domains of the molecule. Peptide immunogenicity was examined in the Db-/- x beta2 microglobulin (beta2m) null mice transgenic for a modified HLA-A2.1/Db-beta2 microglobulin single chain (HHD mice). Our results show the existence of 3 novel HLA-A2.1-restricted MUC1-derived cytotoxic T-lymphocyte (CTL) epitopes. These peptides are processed and presented by the HHD-transfected breast-tumor cell line MDA-MB-157. Moreover, CTL induced by these 3 peptides show higher lysis of target cells pulsed with breast-carcinoma-derived peptides than of targets pulsed with normal breast-tissue-derived peptides. These data suggest an important role for non-TRA MUC1-derived peptides as inducers of a MHC-restricted CTL reaction to a breast-carcinoma cell line and patient-derived tumor extracts.

B7 costimulation is critical for antibody class switching and CD8(+) cytotoxic T-lymphocyte generation in the host response to vesicular stomatitis virus.

Antibody and cytotoxic T-lymphocyte (CTL) responses have critical roles in eliminating many viral infections. In addition to stimulation of the T-cell receptor, T cells require costimulatory signals to respond optimally. We evaluated the role of B7 costimulatory molecules (B7-1 and B7-2) in the immune response to viral infection using vesicular stomatitis virus (VSV) and mice lacking either B7-1 or B7-2 or both molecules. Mice lacking both B7-1 and B7-2 had essentially no anti-VSV immunoglobulin G1 (IgG1) response, decreased IgG2a responses, and normal IgM responses, while mice lacking either B7-1 or B7-2 had unaltered anti-VSV antibody responses compared to wild-type mice. Depletion of CD4(+) cells further reduced the IgG2a response in mice lacking both B7 molecules, suggesting that CD4(-) cells may supply help for IgG2a in the absence of B7 costimulation. The absence of both B7 molecules profoundly reduced generation of both primary and secondary VSV-specific class I major histocompatibility complex (MHC)-restricted CTL, whereas VSV-specific CTL responses in mice lacking either B7-1 or B7-2 were similar to those of wild-type animals. Class I MHC-restricted CTL in wild-type mice were not dependent on CD4(+) cells, suggesting that the failure of CTL in the absence of B7s is due to a lack of B7 costimulation directly to the CD8(+) CTL. These data demonstrate that B7-1 and B7-2 have critical, overlapping functions in the antibody and CTL responses to this viral infection.

Induction of cytotoxic T lymphocyte activity by fusion-active peptide-containing virosomes

Priming of cytotoxic T lymphocyte (CTL) activity with exogenous antigen requires introduction of the antigen into the MHC class I presentation pathway of antigen-presenting cells. In the present study, we used fusogenic reconstituted envelopes (virosomes), derived from influenza virus, as a carrier system for delivery of a synthetic soluble peptide corresponding to a major murine CTL epitope of the influenza virus nucleoprotein (NP). Virosomes containing encapsulated NP-peptide efficiently sensitized target cells for recognition by influenza-specific CTLs generated through priming of mice with infectious virus. Intramuscular immunization of mice with peptide-containing virosomes induced a potent class I MHC-restricted CTL response against influenza-infected target cells. By contrast, an equal dose of NP-peptide encapsulated in fusion-inactivated virosomes did not induce CTL activity, indicating an essential role of the membrane fusion activity of the virosomes in the induction of the response. Likewise, NP-peptide encapsulated in liposomes, NP-peptide mixed with empty virosomes and NP-peptide in IFA failed to induce a CTL response. These results demonstrate that fusion-active virosomes represent a promising delivery system for induction of class I MHC-restricted CTL activity with non-replicating viral antigens.

Alloreactive cytotoxic T-cell function, peptide nonspecific.

The recognition requirements necessary for murine alloreactive cytotoxic T-cells to carry out their effector function has been investigated using target cells that express a unique class I major histocompatibility complex (MHC)-peptide pair. The human cell line T2 and the murine cell line RMA-S are defective in peptide transport components needed to effectively express stable MHC class I molecules at the cell surface. When T2 cells were infected with a vaccinia virus that encoded the Kd gene and provided with a Kd-motif peptide from the nucleoprotein of influenza virus (NPP), these cells could be lysed by polyclonal allo Kd-reactive cytotoxic T-lymphocytes (CTL). Similar results were obtained with the murine RMA-S-Kd cell line, transfected with cDNA able to express some 'empty' Kd that is heat-labile. Adding another Kd-motif peptide from influenza virus haemagglutinin (HAP) stabilized the surface expression of Kd and allowed the RMA-S-Kd cells to be lysed before or after heat shock. At 27 degrees C anti-Kd alloreactive CTL-lysed target cells in the presence and absence of HAP peptide. Alloreactive CTL appear to have a more stringent requirement for a high density of MHC class I on cell surfaces relative to peptide-specific MHC-restricted CTL. We conclude that while Kd-restricted CTL activity is strictly peptide-specific, anti-Kd-specific alloreactivity is MHC allele-specific, but peptide-nonspecific. This conclusion is at odds with the Standard Model of T-cell receptor (TCR) function, but consistent with the predictions of a Competing Model of TCR function.

Rotavirus-Specific Cytotoxic T Lymphocytes Recognize Overlapping Epitopes in the Amino-Terminal Region of the VP7 Glycoprotein

Rotavirus-specific cytotoxic T lymphocytes (CTL) play an important role in the resolution of rotavirus infection. The outer capsid glycoprotein, VP7, elicits a class I MHC-restricted CTL response. Vaccinia virus recombinants expressing the VP7 genes from simian rotavirus SA11 (serotype G3) and from the RF strain of bovine rotavirus (serotype G6) were used to analyze the CTL activity to this antigen in BALB/c (H-2d) and C57BL/6 (H-2b) mice neonatally infected with homologous and heterologous rotaviruses. A vaccinia virus recombinant expressing the first amino-terminal 88 amino acids of VP7 was constructed and used to search for cross-reactive CTL against this region of the protein. By using synthetic Kb, Db, and Kdmotif-fitting peptides two overlapping CTL epitopes have been identified located in the first hydrophobic domain (H1) of VP7. Splenocytes obtained from rotavirus SA11-infected C57BL/6 mice induced the strongest CTL response against target cells sensitized with a peptide containing a Kb-restricted CTL epitope (amino acids 8–16). A second Kd-restricted epitope (residues 5–13) was recognized by splenocytes derived from rotavirus-infected BALB/c mice. These findings reveal the existence of CTL epitopes in the H1 signal sequence of the VP7 glycoprotein that coexist with a CTL epitope (residues 31–40) previously described within the H2 region.

Simian immunodeficiency virus (SIV)-specific CD8 + cytotoxic T lymphocyte responses of naive and vaccinated cynomolgus macaques infected with SIVmac32H(J5): quantitative analysis by in vitro antigenic stimulation

Detailed analyses of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte (CTL) responses in vaccinated and infected macaques may help to clarify the role of CTL immunity in protection against lentiviruses. Here, the optimal conditions for the measurement of SIV Gag-specific CTL were investigated by bulk and limiting dilution assays of peripheral blood mononuclear cells (PBMC) from naive and vaccinated cynomolgus macaques ( Macaca fascicularis) infected with SIVmac32H(J5). In vitro restimulation was generally required for CTL detection. Selective activation of CD8 + and MHC-restricted SIV Gag-specific CTL was induced by stimulation with autologous para-formaldehyde-fixed B-lymphoblastoid cell lines infected with a recombinant vaccinia virus expressing SIV Gag. Applied to limiting dilution assays, antigenic stimulation reproducibly demonstrated SIV Gag-specific CTL precursors (CTLp) in PBMC of all animals studied, including those lacking significant responses in standard bulk CTL assays.

Different roles for CD4+ and CD8+ T lymphocytes and macrophage subsets in the control of a generalized virus infection.

The importance of T-lymphocyte subsets in the control of poxvirus infections is controversial. To determine the relative contribution of lymphocyte subsets important for recovery from infection with ectromelia virus (EV), a natural murine poxvirus pathogen, C57BL/6 (B6) mice lacking functional CD8+ T cells because of disruption of the beta2-microglobulin gene or lacking functional CD4+ T cells because of disruption of the I-(A)beta gene, acutely depleted of CD8+ or CD4+ T cells with monoclonal antibody, or depleted of macrophage subsets by the macrophage suicide technique were used. Recovery from infection was strictly dependent on the effector functions of CD8+ T cells, in the absence of which 100% mortality resulted. This lymphocyte population had demonstrable antiviral activity early in the infection process even before class I major histocompatibility complex (MHC)-restricted CD8+ cytotoxic T-lymphocyte (CTL) activity was detectable. CD4+ T cells were found to be necessary for the generation of an optimal virus-specific, class I MHC-restricted CD8+ CTL response and contributed to virus clearance not involving cytolytic mechanisms. In both models of CD4+ T-cell deficiency, virus clearance was incomplete and persisted at low levels in most organs and at very high levels in the skin, but the animals did not die. The elimination of macrophage subpopulations impeded virus clearance, impaired the generation of class I MHC-restricted antiviral CTL response, and resulted in 100% mortality. These findings establish an absolute requirement for CD8+ and CD4+ T lymphocytes and macrophage subsets in the elimination of a natural murine poxvirus infection and support the idea that macrophages may be essential accessory cells for the generation of class I MHC-restricted antiviral CTL responses.

Tumor associated antigens in human renal cell carcinoma: MHC restricted recognition by cytotoxic T lymphocytes.

Based on previous studies in human melanoma leading to the molecular cloning of genes encoding peptide antigens recognized by MHC-restricted cytotoxic T lymphocytes (CTL) we extended our efforts to renal cancer systems established in tissue culture. In two patients we obtained stable CD8+ CTL clones with high cytolytic activity for the corresponding autologous tumor cell line in vitro. Neither autologous EBV-transformed B lymphocytes or PHA-activated PBL nor natural killer targets K562 were lysed by these CTL clones. MZ1257-RCC CTL5-30 lysed autologous tumor cells as well as normal kidney cell cultures in an HLA-A2 restricted fashion. Further specificity analysis showed cross reactivity with HLA-A2 matched renal cancer cell lines, melanoma cell lines and tumor cell lines of other origins. HLA-A2 negative target cells were not lysed. The restriction element for T cell recognition in another renal cancer system, MZ1973-RCC, appeared to be HLA-B8 as tested with CTL 5-10. Crossreactivity of CTL 5-10 was documented with one HLA-B8 positive RCC line. Tumor cell lines of other origin were not lysed by CTL 5-10. For further study of CTL-defined epitopes, peptides were extracted from tumor cell lines. Peptides from cultured cell lines were acid-eluted and fractionated by reversed phase HPLC. The peptide fractions were tested in cytotoxicity assays for their ability to reconstitute RCC epitopes by addition to the HLA-A2.1 positive antigen processing mutant cell line 721.124xCEM.T2. One HPLC peak was identified containing an epitope for the HLA-A2 restricted MZ1257-RCC CTL 5-30. These peptide epitopes were present in the autologous renal cancer cell line MZ1257-RCC as well as in an allogeneic HLA-A2 positive RCC cell line MZ1973 and melanoma cell line SK29-MEL, but were absent in peptides eluted from autologous EBV-B cells. We conclude that renal cell carcinoma may induce autologous CTL. Epitopes recognized by MHC-restricted CTL on RCC may be shared by HLA-matched allogeneic renal cancer or even tumors of other origin, such as melanoma. The current challenge is the determination of the amino acid sequences of these CTL-defined peptides as a step to further characterize the nature of these tumor associated antigens.

Consequences of cytotoxic T lymphocyte interaction with major histocompatibility complex class I-expressing neurons in vivo.

Neurons have evolved strategies to evade immune surveillance that include an inability to synthesize the heavy chain of the class I major histocompatibility complex (MHC), proteins that are necessary for cytotoxic T lymphocyte (CTL) recognition of target cells. Multiple viruses have taken advantage of the lack of CTL-mediated recognition and killing of neurons by establishing persistent neuronal infections and thereby escaping attack by antiviral CTL. We have expressed a class I MHC molecule (Db) in neurons of transgenic mice using the neuron-specific enolase (NSE) promoter to determine the pathogenic consequences of CTL recognition of virally infected, MHC-expressing central nervous system (CNS) neurons. The NSE-Db transgene was expressed in H-2b founder mice, and transgene-derived messenger RNA was detected by reverse transcriptase-polymerase chain reaction in transgenic brains from several lines. Purified primary neurons from transgenic but not from nontransgenic mice adhered to coverslips coated with a conformation-dependent monoclonal antibody directed against the Dv molecule and presented viral peptide to CTL in an MHC-restricted manner, indicating that the Db molecule was expressed on transgenic neurons in a functional form. Transgenic mice infected with the neurotropic lymphocytic choriomeningitis virus (LCMV) and given anti-LCMV, MHC-restricted CTL displayed a high morbidity and mortality when compared with controls receiving MHC-mismatched CTL or expressing alternative transgenes. After CTL transfer, transgenic brains showed an increased number of CD8+ cells compared with nontransgenic controls as well as an increased rate of clearance of infectious virus from the CNS. Additionally, an increase in blood-brain barrier permeability was detected during viral clearance in NSE-Db transgenic mice and lasted several months after clearance of virus from neurons. In contrast, LCMV-infected, nontransgenic littermates and mice expressing other gene products from the NSE promoter showed no CNS disease, no increased intraparenchymal CTL, and no blood-brain barrier damage after the adoptive transfer of antiviral CTL. Our study indicates that viral infections and CTL-CNS interactions may induce blood-brain barrier disruptions and neurologic disease by a "hit-and-run" mechanism, triggering a cascade of pathogenic events that proceeds in the absence of continual viral stimulation.

Focal expression of interleukin-2 does not break unresponsiveness to "self" (viral) antigen expressed in beta cells but enhances development of autoimmune disease (diabetes) after initiation of an anti-self immune response.

The participation of IL-2 in insulin-dependent (type 1) diabetes (IDDM) was analyzed in transgenic (tg) mice expressing the nucleoprotein (NP) of lymphocytic choriomeningitis virus and IL-2 under control of the rat insulin promoter focally in beta cells of the islets of Langerhans. Insertion and expression of the viral (self) gene or of the IL-2 gene alone did not lead to IDDM. Infiltration primarily of CD4 and B lymphocytes and increased expression of MHC class I and II molecules occurred in islets where IL-2 was expressed. By contrast, neither cellular infiltrates nor expression of MHC class I or II glycoproteins above base levels was noted in tgs expressing the viral protein alone. Double tg mice expressing both the viral protein and IL-2 in their islets displayed a modest increase in incidence of spontaneous diabetes compared with that of single transgenic mice expressing IL-2 alone. Breaking of immunological unresponsiveness or sensitization to self antigens did not occur. Neither cytotoxic T lymphocytes (CTL) nor antibodies directed against the viral tg (NP) were generated. However, after challenge with lymphocytic choriomeningitis virus, double tg mice developed anti-self (viral) CTL and IDDM (incidence > 95%) within 2 mo. The generation of virus ("self")-specific MHC-restricted CTL was dependent on CD4+ help. In contrast, viral inoculum to single tg mice expressing either the viral protein or IL-2 failed to enhance the incidence of IDDM over 30% for viral protein or 10% for IL-2 after an 8-mo observation period. Hence, in this autoimmune model in situ expression of IL-2 did not break unresponsiveness but markedly enhanced ongoing disease.

Induction of HIV-1 envelope (gp120)-specific cytotoxic T lymphocyte responses in mice by recombinant CHO cell-derived gp120 is enhanced by enzymatic removal of N-linked glycans.

Priming of CD8+ cytotoxic T lymphocyte (CTL) responses with recombinant proteins has been facilitated by the development of novel adjuvants that deliver antigens into the class I major histocompatibility complex (MHC) pathway. However, the extent to which secondary structure or glycosylation of these proteins prevents priming of class I MHC-restricted CTL responses is not clear. To address this issue, recombinant HIV-1 gp120 envelope proteins produced in yeast insect, or mammalian cells were compared for the ability to elicit CD8+ CTL activity in mice. Envelope-specific CD8+ T lymphocytes were detected in BALB/c mice immunized with env 2-3, a 55-kDa yeast-derived envelope protein that is not glycosylated and lacks a native conformation. This response was directed against a previously described epitope in the V3 region of gp120, as well as a newly identified epitope located near the carboxy-terminus of the molecule. Similar levels of V3-directed CTL activity were observed in mice immunized with recombinant gp120 produced in insect (Spodoptera fugiperda) cells using a baculovirus expression system (gp120BAC). In contrast, induction of CTL responses was considerably less efficient when mice were immunized with gp120CHO, a native, fully glycosylated envelope protein produced in mammalian CHO cells. Denaturation of gp120CHO prior to immunization was not sufficient to prime CTL responses. However, envelope-specific CD8+ CTL activity was elicited when N-linked glycans were removed by treatment with an endoglycosidase. Possible mechanisms by which N-linked glycans influence delivery or processing of recombinant proteins for class I MHC presentation, and the implications of these findings for the design of subunit vaccines, are discussed.

Tumor specific cytolysis by tumor infiltrating lymphocytes in breast cancer.

In vitro studies have demonstrated that exposure of tumor infiltrating lymphocytes (TIL) to human recombinant interleukin-2 (rIL-2) will generate activated T-lymphocytes with major histocompatibility complex (MHC)-restricted and non-MHC-restricted cytotoxicity toward a panel of tumor target cells. In melanoma and ovarian carcinoma, TIL display MHC-restricted and autologous tumor-specific cytotoxicity. Such tumor-reactive cytotoxic T-lymphocytes (CTL) represent important material for understanding cellular immunity in cancer and developing specific immunotherapeutic approaches in melanoma and ovarian cancer. In breast cancer, some TIL have been demonstrated to secrete cytokines upon interaction with autologous tumor cells, indicating that autologous tumor-reactive lymphocytes may also exist among TIL in breast cancer. Therefore, the authors conducted a study to investigate the cytotoxic profile of rIL-2-activated lymphocytes in breast cancer. Lymphocytes were isolated from primary solid tumors (TIL) of breast carcinomas (10 patients) and from peritoneal effusions (effusion-associated mononuclear cells [EAMNC]) from 2 patients with newly diagnosed metastatic breast carcinoma. Tumor infiltrating lymphocytes or EAMNC were cultured with rIL-2 in long term cultures whereby their expansion index, phenotype, and cytotoxic potential were studied. Both TIL and EAMNC proliferated by greater than 300-fold (370-3650; mean, 1656) after 23-82 days in cultures containing mixtures of TIL or EAMNC, autologous tumor cells, and rIL-2. By fluorescence analysis, freshly isolated TIL and EAMNC were found to consist of 77.5 plus or minus 10.7% CD3+ T-cells, 33.2 plus or minus 8.9% CD4+, and 47.2 plus or minus 16.8% CD8+ cells. Their CD4 to CD8 ratio was 0.70. After expansion of lymphocytes with rIL-2 in the majority of patients (9 of 12), CD3+CD8+ T-lymphocytes were present in greater numbers than CD3+CD4+ T-lymphocytes. Recombinant interleukin-2-activated CD3+CD8+ cells exhibited preferential cytolytic activity against autologous tumor cells. The cytolytic activity of CD3+CD8+ cells was inhibited either by anti-T-cell receptor (TCR)-alpha/-beta and anti-CD3 monoclonal antibodies (MoAb) or after pretreatment of tumor target cells with MoAb against the class I MHC antigens. Recombinant interleukin-2-activated CD3+CD4+ cells demonstrated potent cytolytic activity against both autologous and allogeneic tumor cells. CD3+CD8+ T-cell clones isolated from representative TIL exhibited preferential autologous tumor-specific cytotoxicity whereas the cytolytic activity of CD3+CD4+ T-cell clones was mostly (12 of 14 clones) nonrestricted to the autologous tumor. To the authors' knowledge, this is the first report to demonstrate that TIL from primary tumors of breast carcinomas and EAMNC from metastatic disease can be propagated in large numbers in vitro with rIL-2 while retaining autologous tumor specific and MHC-restricted CTL activity. These findings may be of importance to ongoing clinical trials using TIL or EAMNC in the immunotherapy of patients with advanced breast cancer.