The involvement of class Ib molecules in the host response to infection with Salmonella and its relevance to autoimmunity.

Class I molecules with limited polymorphism have been implicated in the host response to infectious agents. Following infection with Salmonella typhimurium, mice develop a CD8+ CTL response that specifically recognizes bacteria infected cells. An immunodominant component of the CTL response recognizes a peptide epitope derived from the Salmonella GroEL molecule that is presented by the non-polymorphic MHC class Ib molecule Qa-1. T cells recognizing the bacterial peptide also cross-recognize a homologous peptide from the mammalian hsp60 molecule. Since Qa-1 has a functional equivalent in humans, this observation may be relevant not only to the host response involved in clearing infection but also in understanding the link between infection with Gram-negative pathogens and autoimmune disease.     

T cell recognition of QA-1b antigens on cells lacking a functional Tap-2 transporter.

MHC class Ia H chains and beta 2-microglobulin assemble with appropriate peptides to form stable cell surface molecules that serve as targets for Ag-specific CTL. The structural similarities of class Ia and the less polymorphic Q/T/M (class Ib) molecules suggest that class Ib molecules also play a role in antigen presentation, although the origin of the peptides they present remains mostly unclear. The cell line RMA-S has a defect in class I Ag presentation, presumably due to a mutation in a peptide transporter gene. This defect can be overcome by transfection of RMA-S cells with the Tap-2 gene (formerly Ham-2) that encodes an ATP-binding transporter protein. We now show that a substantial portion of alloreactive CTL specific for Qa-1 class Ib molecules recognize Qa-1b on RMA-S cells and thus differ from most class Ia specific CTL. Those anti-Qa-1b CTL that do not recognize untransfected RMA-S do lyse RMA-S transfected with Tap-2. We also examine the effects of Qdm, a gene that maps to the D region and alters recognition of Qa-1. Qdm(k) strains lack an epitope(s) recognized by some (Qdm dependent) anti-Qa-1 CTL whereas Qdm+ strains express this epitope. Thus, Qdm-dependent CTL do not recognize Qa-1 on Qdm(k) targets whereas Qdm-independent CTL recognize Qa-1 epitopes in all strains. Although Qdm-independent CTL varied as to whether they recognized RMA-S vs RMA, all nine Qdm-dependent clones only recognized Qa-1b on RMA and not RMA-S. This result is consistent with Qdm encoding a peptide dependent upon the TAP transporter for cell membrane expression.     

A peptide from heat shock protein 60 is the dominant peptide bound to Qa-1 in the absence of the MHC class Ia leader sequence peptide Qdm

The MHC class Ib molecule Qa-1 binds specifically and predominantly to a single 9-aa peptide (AMAPRTLLL) derived from the leader sequence of many MHC class Ia proteins. This peptide is referred to as Qdm. In this study, we report the isolation and sequencing of a heat shock protein 60-derived peptide (GMKFDRGYI) from Qa-1. This peptide is the dominant peptide bound to Qa-1 in the absence of Qdm. A Qa-1-restricted CTL clone recognizes this heat shock protein 60 peptide, further verifying that it binds to Qa-1 and a peptide from the homologous Salmonella typhimurium protein GroEL (GMQFDRGYL). These observations have implications for how Qa-1 can influence NK cell and T cell effector function via the TCR and CD94/NKG2 family members, and how this effect can change under conditions that cause the peptides bound to Qa-1 to change.     

Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens

The development of many autoimmune diseases has been etiologically linked to exposure to infectious agents. For example, a subset of patients with a history of Salmonella infection develop reactive arthritis. The persistence of bacterial antigen in arthritic tissue and the isolation of Salmonella or Yersinia reactive CD8+ T cells from the joints of patients with reactive arthritis support the etiological link between Gram-negative bacterial infection and autoimmune disease. Models proposed to account for the link between infection and autoimmunity include inflammation-induced presentation of cryptic self-epitopes, antigen persistence and molecular mimicry. Several studies support molecular mimicry as a mechanism for the involvement of class II epitopes in infectious disease-induced self-reactivity. Here, we have identified an immunodominant epitope derived from the S. typhimurium GroEL molecule. This epitope is presented by the mouse H2-T23-encoded class Ib molecule Qa-1 and was recognized by CD8+ cytotoxic T lymphocytes induced after natural infection. S. typhimurium-stimulated cytotoxic T lymphocytes recognizing the GroEL epitope cross-reacted with a peptide derived from mouse heat shock protein 60 and recognized stressed macrophages. Our results indicate involvement of MHC class Ib molecules in infection-induced autoimmune recognition and indicate a mechanism for the etiological link between Gram-negative bacterial infection and autoimmunity.     

Differential requirement for tapasin in the presentation of leader- and insulin-derived peptide antigens to Qa-1b-restricted CTLs

The loading of MHC class I molecules with peptides involves a variety of accessory proteins, including TAP-associated glycoprotein (tapasin), which tethers empty MHC class I molecules to the TAP peptide transporter. We have evaluated the role of tapasin for the assembly of peptides with the class Ib molecule Qa-1b. In normal cells, Qa-1b is predominantly bound by a peptide, the Qa-1 determinant modifier (Qdm), derived from the signal sequence of class Ia molecules. Our results show that tapasin links Qa-1b to the TAP peptide transporter, and that tapasin facilitates the delivery of Qa-1b molecules to the cell surface. Tapasin was also required for the presentation of endogenous Qdm peptides to Qdm-specific, Qa-1b-restricted CTLs. In sharp contrast, tapasin expression was dispensable for the presentation of an insulin peptide to insulin-specific, Qa-1b-restricted CTL isolated from TCR transgenic mice. However, tapasin deficiency significantly impaired the positive selection of these insulin-specific, Qa-1b-restricted transgenic CD8+ T cells. These findings reveal that tapasin plays a differential role in the loading of Qdm and insulin peptides onto Qa-1b molecules, and that tapasin is dispensable for retention of empty Qa-1b molecules in the endoplasmic reticulum, and are consistent with the proposed peptide-editing function of tapasin.     

A cyclophilin B gene encodes antigenic epitopes recognized by HLA-A24-restricted and tumor-specific CTLs.

We have studied Ags recognized by HLA class I-restricted CTLs established from tumor site to better understand the molecular basis of tumor immunology. HLA-A24-restricted and tumor-specific CTLs established from T cells infiltrating into lung adenocarcinoma recognized the two antigenic peptides encoded by a cyclophilin B gene, a family of genes for cyclophilins involved in T cell activation. These two cyclophilin B peptides at positions 84-92 and 91-99 induced HLA-A24-restricted CTL activity against tumor cells in PBMCs of leukemia patients, but not in epithelial cancer patients or in healthy donors. In contrast, the modified peptides at position 2 from phenylalanine to tyrosine, which had more than 10 times higher binding affinities to HLA-A24 molecules, could induce HLA-A24-restricted CTL activity against tumor cells in PBMCs from leukemia patients, epithelial cancer patients, or healthy donors. PHA-activated normal T cells were resistant to lysis by the CTL line or by these peptide-induced CTLs. These results indicate that a cyclophilin B gene encodes antigenic epitopes recognized by CTLs at the tumor site, although T cells in peripheral blood (except for those from leukemia patients) are immunologically tolerant to the cyclophilin B. These peptides might be applicable for use in specific immunotherapy of leukemia patients or that of epithelial cancer patients.     

H2-M3-restricted T cells participate in the priming of antigen-specific CD4+ T cells

H2-M3-restricted CD8+ T cells provide early protection against bacterial infections. In this study, we demonstrate that activated H2-M3-restricted T cells provide early signals for efficient CD4+ T cell priming. C57BL/6 mice immunized with dendritic cells coated with the MHC class II-restricted listeriolysin O peptide LLO(190-201) (LLO) generated CD4+ T cells capable of responding to Listeria monocytogenes (LM) infection. Inclusion of a H2-M3-restricted formylated peptide fMIGWII (fMIG), but not MHC class Ia-restricted peptides, during immunization with LLO significantly increased IFN-gamma-producing CD4+ T cell numbers, which was associated with increased protection against LM infection. Studies with a CD4+ T cell-depleting mAb indicate that the reduction in bacterial load in fMIG plus LLO immunized mice is likely due to augmented numbers of LLO-specific CD4+ T cells, generated with the help of H2-M3-restricted CD8+ T cells. We also found that augmentation of LLO-specific CD4+ T lymphocytes with H2-M3-restricted T cells requires presentation of LLO and fMIG by the same dendritic cells. Interestingly, the augmented CD4+ T cell response generated with fMIG also increased primary LM-specific responses by MHC class Ia-restricted CD8 T cells. Coimmunization with LLO and fMIG also increases the number of memory Ag-specific CD4+ T cells. We also demonstrate that CD8 T cells restricted to another MHC class Ib molecule, Qa-1, whose human equivalent is HLA-E, are also able to enhance Ag-specific CD4+ T cell responses. These results reveal a novel function for H2-M3- and Qa-1-restricted T cells; provision of help to CD4+ Th cells during the primary response.     

New epitope peptides derived from hepatitis C virus (HCV) 2a which have the capacity to induce cytotoxic T lymphocytes in HLA-A2+ HCV-infected patients

Because cytotoxic T lymphocytes (CTLs) play an important role in the specific immunotherapy of hepatitis C virus (HCV) infection, a series of CTL epitopes has been defined from HCV genotype 1a or 1b protein. Here, we attempted to identify HCV2a-derived epitopes that are capable of inducing HLA-A2-restricted and peptide-specific CTLs. Peripheral blood mononuclear cells (PBMCs) of HLA-A2+ HCV2ainfected patients or healthy donors were stimulated in vitro with each of the HCV2a-derived peptides, which were prepared based on the HLA-A2-binding motif, and their peptide-specific and HLA-A2-restricted cytotoxicities were examined. The HCV2a 432-441, HCV2a 716-724, and HCV2a 2251-2260 peptides were found to efficiently induce peptide-specific CTLs from the PBMCs of HLA-A2+ HCV2ainfected patients. Cytotoxicity was mainly mediated by CD8+ T cells in a HLA class I-restricted manner. These results indicate that the HCV2a 432-441, HCV2a 716-724, and HCV2a 2251-2260 peptides might be applicable for peptide-based immunotherapy of HLA-A2+ HCV2a-infected patients.     

Estimating the effectiveness of simian immunodeficiency virus-specific CD8+ T cells from the dynamics of viral immune escape

Antiviral CD8(+) T cells are thought to play a significant role in limiting the viremia of human and simian immunodeficiency virus (HIV and SIV, respectively) infections. However, it has not been possible to measure the in vivo effectiveness of cytotoxic T cells (CTLs), and hence their contribution to the death rate of CD4(+) T cells is unknown. Here, we estimated the ability of a prototypic antigen-specific CTL response against a well-characterized epitope to recognize and kill infected target cells by monitoring the immunodominant Mamu-A*01-restricted Tat SL8 epitope for escape from Tat-specific CTLs in SIVmac239-infected macaques. Fitting a mathematical model that incorporates the temporal kinetics of specific CTLs to the frequency of Tat SL8 escape mutants during acute SIV infection allowed us to estimate the in vivo killing rate constant per Tat SL8-specific CTL. Using this unique data set, we show that at least during acute SIV infection, certain antiviral CD8(+) T cells can have a significant impact on shortening the longevity of infected CD4(+) T cells and hence on suppressing virus replication. Unfortunately, due to viral escape from immune pressure and a dependency of the effectiveness of antiviral CD8(+) T-cell responses on the availability of sufficient CD4(+) T cells, the impressive early potency of the CTL response may wane in the transition to the chronic stage of the infection.     

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.     

Induction of HLA class I-restricted CD8+ CTLs specific for the major outer membrane protein of Chlamydia trachomatis in human genital tract infections.

HLA class I-restricted CD8+ CTLs specific for the major outer membrane protein (MOMP) of Chlamydia trachomatis are present in the peripheral blood of humans who acquired genital tract infections with the organism. Three HLA-A2-restricted epitopes and two HLA-B51-restricted epitopes were identified in serovar E-MOMP. One of the five epitopes spans a variable segment of MOMP and is likely a serovar E-specific epitope. The other four epitopes are localized in constant segments and are C. trachomatis species specific. CTL populations specific for one or more of the four constant segment epitopes were isolated from all 10 infected subjects tested, regardless of infecting serovars, but from only one of seven uninfected subjects tested. The CTLs failed to recognize corresponding peptides derived from Chlamydia pneumoniae MOMP, further suggesting that they indeed resulted from genital tract infections with C. trachomatis. Significantly, ME180 human cervical epithelial cells productively infected with C. trachomatis were killed by the MOMP peptide-specific CTLs. Further investigations of the ability of such CTLs to lyse normal infected epithelial cells and their presence at inflamed sites in the genital tract will help understand the protective or pathological role of CTLs in chlamydial infections. The MOMP CTL epitopes may be explored as potential components of a subunit vaccine against sexually transmitted diseases caused by C. trachomatis. Moreover, the knowledge provided here will facilitate studies of HLA class I pathways of chlamydial Ag processing and presentation in physiologically relevant human APCs.     

Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

Promiscuity of MHC class Ib-restricted T cell responses

Murine infection with the Gram-positive intracellular bacterium Listeria monocytogenes activates CD8(+) T cells that recognize bacterially derived N-formyl methionine peptides in the context of H2-M3 MHC class Ib molecules. Three peptides, fMIGWII, fMIVIL, and fMIVTLF, are targets of L. monocytogenes-specific CD8(+) T cells. To investigate epitope cross-recognition by H2-M3-restricted CD8(+) T cells, we deleted the sequence encoding fMIGWII from a virulent strain of L. monocytogenes. Infection with fMIGWII-deficient L. monocytogenes unexpectedly primed CD8(+) T cells that stain with fMIGWII/H2-M3 tetramers and lyse fMIGWII-coated target cells in vivo. Because the fMIGWII sequence is nonredundant, we speculated that other bacterially derived Ags are priming these responses. HPLC peptide fractionation of bacterial culture supernatants revealed several distinct L. monocytogenes-derived peptides that are recognized by fMIGWII-specific T cells. Our results demonstrate that the dominant H2-M3-restricted CD8(+) T cell population, although reactive with fMIGWII, is primed by other, non-fMIGWII peptides derived from L. monocytogenes. Although this degree of Ag receptor promiscuity is unusual for the adaptive immune system, it may be a more common feature of T cell responses restricted by nonpolymorphic MHC class Ib molecules.     

Class I MHC-restricted cytotoxic T lymphocyte recognition of cells infected with human cytomegalovirus does not require endogenous viral gene expression.

The human pathogen CMV, is a major cause of morbidity and mortality in immunocompromised hosts. The CD8+ class I-restricted CTL response to CMV assists in preventing progression of CMV infection to life-threatening disease; however, the viral Ag recognized by CD8+ CTL are not well characterized. In general, virus-specific CTL recognize endogenously synthesized viral proteins processed and presented associated with class I MHC molecules. Although proteins or inactivated virions have been experimentally delivered to the cytoplasm to result in class I MHC presentation, this mode of Ag delivery to the class I processing pathway after natural viral entry has not been documented in humans. Our data demonstrate that the CMV-specific class I-restricted CTL response in individuals latently infected with CMV is predominantly specific for selected structural virion proteins introduced into the cell after viral penetration and efficient recognition occurs in the absence of de novo viral gene expression. This CTL response may provide a biological advantage for limiting the spread of infection after CMV reactivation because infected cells are lysed before viral assembly.     

Correlation of Qa-1 determinants defined by antisera and by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) activated in H-2 identical, Qa-1 disparate mixed leukocyte cultures recognize H-2-nonrestricted target antigens indistinguishable by strain or tissue distribution from serologically defined Qa-1 antigens. Cloned Qa-l-specific CTL define determinants encoded by four Qa-1 genotypes; we used anti-Qa-1 sera in antibody blocking experiments to determine if these determinants reside on molecules recognized by Qa-1-specific antibodies. Antisera containing Qa-1.1-specific and TL-specific antibodies blocked recognition of two CTL-defined determinants associated with Qa-1 ^a . Although both Qa-1 and TL molecules are expressed on activated T cells from appropriate strains, our studies indicated that the CTL recognized Qa-1, not TL. In addition, anti-Qa-1.2 serum inhibited CTL recognition of Qa-1^b- and Qa-1^c-encoded determinants. Qa-1 ^d target cells are unique in that they express determinants recognized by anti-Qa-1^a CTL and by anti-Qa-1^b CTL. Killing of Qa-1 ^d targets by anti-Qa-1^a CTL was not inhibited by anti-Qa-1.1 serum, but was partially inhibited by anti-Qa-1.2 serum. Cytotoxicity of Qa-1 ^d cells by one anti-Qa-1^b CTL clone was inhibited by both anti-Qa-1.2 and anti-Qa-1.1 sera, indicating close association of both serological determinants with the determinants recognized by the CTL. Thus, all of the CTL-defined Qa-1 determinants resided on molecules recognized by Qa-1-specific antibodies, but anti-Qa-1^a CTL and Qa-1.1-specific antibodies did not have identical specificities.Abbreviations used in this paper B6 C57BL/6J - CAB concanavalin A stimulated lymphoblasts - CML cell-mediated lympholysis - CTL cytotoxic T lymphocyte - NMS normal mouse serum - MHC major histocompatibility complex - MLC mixed leukocyte culture - MR maximum release - SMDM supplemented Mishell-Dutton medium - SR spontaneous release     

Oligosaccharide-dependent and independent Qa-1 determinants

A contribution of N-linked oligosaccharides to determinants recognized by alloreactive cytotoxic T lymphocytes has not been demonstrated. Employing cloned CTL and tunicamycin, an inhibitor of protein glycosylation, we found that carbohydrate addition was required for the formation of two of six Qa-1 determinants. The other determinants were detectable on nonglycosylated Qa-1 molecules, similar to observations in most reports that allodeterminants on class I molecules are not dependent on glycosylation for serologic detection. Examination of TM-treated, Con A-activated lymphoblasts revealed a direct correlation between the determinants defined by the reactivity of CTL clones with target cells from four Qa-1 genotypes and their dependence on carbohydrate side chains for expression. Most anti-Qa-1b CTL clones recognized either a glycosylation-dependent determinant found only on Qa-1b cells or glycosylation-independent determinants on both Qa-1b and Qa-1c cells. Similarly, clones that killed only Qa-1a cells recognized a glycosylation-independent determinant. However, clones reactive with both Qa-1a and Qa-1d cells recognized a glycosylation-dependent determinant on Qa-1a molecules and a glycosylation-independent determinant on Qa-1d molecules. This result indicates that such clones recognize cross-reactive conformational determinants, not carbohydrate itself. Thus, N-linked oligosaccharides serve to stabilize the conformation of some Qa-1 determinants, but others remain intact on nonglycosylated molecules. The absence of similar data for H-2K/D/L molecules suggest that a reexamination of other class I antigens with cloned CTL is in order to determine whether Qa-1 molecules are unique.     

CD8+ T cell protective immunity against Chlamydia pneumoniae includes an H2-M3-restricted response that is largely CD4+ T cell-independent

CD8+ T cells are important for immunity to the intracellular bacterial pathogen Chlamydia pneumoniae (Cpn). Recently, we reported that type 1 CD8+ (Tc1) from Cpn-infected B6 mice recognize peptides from multiple Cpn Ags in a classical MHC class Ia-restricted fashion. In this study, we show that Cpn infection also induces nonclassical MHC class Ib-(H2-M3)-restricted CD8+ T cell responses. H2-M3-binding peptides representing the N-terminal formylated sequences from five Cpn Ags sensitized target cells for lysis by cytolytic effectors from the spleens of infected B6 mice. Of these, only peptides fMFFAPL (P1) and fMLYWFL (P4) stimulated IFN-gamma production by infection-primed splenic and pulmonary CD8+ T cells. Studies with Cpn-infected Kb-/-/Db-/- mice confirmed the Tc1 cytokine profile of P1- and P4-specific CD8+ T cells and revealed the capacity of these effectors to exert in vitro H2-M3-restricted lysis of Cpn-infected macrophages and in vivo pulmonary killing of P1- and P4-coated splenocytes. Furthermore, adoptive transfer of P1- and P4-specific CD8+ T cells into naive Kb-/-/Db-/- mice reduced lung Cpn loads following challenge. Finally, we show that in the absence of MHC class Ia-restricted CD8+ T cell responses, CD4+ T cells are largely expendable for the control of Cpn growth, and for the generation, memory maintenance, and secondary expansion of P1- and P4-specific CD8+ T cells. These results suggest that H2-M3-restricted CD8+ T cells contribute to protective immunity against Cpn, and that chlamydial Ags presented by MHC class Ib molecules may represent novel targets for inclusion in anti-Cpn vaccines.     

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression,but non-specifically enhanced on induction of Tax expression

Background

Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4⁺ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.

Results

Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ_26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.

Conclusions

HTLV-1 gene expression in primary CD4⁺ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.

     

Retrovirus D/New England and its relation to Mason-Pfizer monkey virus.

We have generated lymphocytic choriomeningitis virus-specific, H-2-restricted cytotoxic thymus-derived lymphocyte (CTL) clones. By using these reagents in several in vitro assays with infected target cells, we show that CTLs by themselves prevent the release of infectious virus into culture fluids and significantly lower the titers of infectious virus previously produced. This ability of cloned CTLs is not influenced by monensin. However, monensin does abrogate the ability of CTLs from spleens of mice primed 6 to 8 days previously with virus to kill virus-infected syngeneic targets. When tested for the participation of lymphokines in this system, the CTLs proliferate when reacted with syngeneic lymphocytic choriomeningitis virus-infected macrophages but fail to make interleukin-2. These CTLs make gamma interferon when reacted with syngeneic virus-infected targets. However, the production of interferon does not directly correlate with CTL-mediated killing. The number of H-2K and D molecules expressed on the target cell surface is not altered during the course of lymphocytic choriomeningitis virus infection. Electron microscopy shows finger-like projections of the CTL clone thrust into the infected cell and lesions bearing an internal diameter of approximately 15 nm in those membranes, illustrating the lytic process.     

Class I restricted interaction between suppressor and cytolytic cells in the response to minor histocompatibility antigens

Inoculation of 10(8) unirradiated, minor H antigen-incompatible spleen cells into recipients leads to a failure of the induction of cytolytic T lymphocytes (CTL) specific for these antigens. In contrast, a strong CTL response against minor H antigens is obtained when the inoculated cells are irradiated or treated with Thy-1-, Lyt-1- or Lyt-2-specific antibody and complement. Thus the failure of CTL induction is probably due to suppression mediated by radiosensitive, Lyt-1+2+ T cells in the immunizing inoculum. We demonstrate here that the inoculated cells must share class I MHC loci with the recipients for the suppression to occur. Thus, the interaction between the suppressor T (Ts) cells and their targets (presumably the CTL precursors) is restricted by class I molecules. A disparity at class II loci between the inoculated cells and the recipients overrides the class I-restricted suppression, possibly through a positive allogeneic effect. The simplest interpretation of the class I restriction of Ts cell-target cell interaction is that the CTL precursors recognize minor H antigens in the context of class I molecules on the surface of the Ts cells themselves.