Trypanosoma cruzi infection does not impair major histocompatibility complex class I presentation of antigen to cytotoxic T lymphocytes

Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas' disease, lives free within the cytoplasm of infected host cells. This intracellular niche suggests that parasite antigens may be processed and presented on major histocompatibility complex (MHC) class I molecules for recognition by CD8⁺ T cells. However, the parasite persists indefinitely in the mammalian host, indicating its success at evading immune clearance. It has been shown that T. cruzi interferes with processing and presentation of antigenic peptides in the MHC class II pathway. This investigation sought to determine whether interference in MHC class I processing and presentation occurs with T. cruzi infection. Surface expression of MHC class I molecules was found to be unaffected or up-regulated by T. cruzi infection in vitro. A model system employing a β-galactosidase (β-gal)-specific murine cytotoxic T lymphocyte (CTL) line (0805B) showed: (i) in vitro infection of mouse peritoneal macrophages or J774 cells with T. cruzi did not inhibit MHC class I presentation of exogenous peptide (a nine-amino acid epitope of β-gal) to the CTL line, (ii) in vitro infection of a β-gal-expressing 3T3 cell line (LZEJ) with T. cruzi did not inhibit MHC class I presentation of the endogenous protein to the CTL line and (iii) mouse renal adenocarcinoma cells infected with T. cruzi and subsequently infected with adenovirus expressing β-gal were able to present antigen to the β-gal-specific CTL line. These findings indicate that the failure of the immune response to clear T. cruzi does not result from global interference by the parasite with MHC class I processing and presentation. Parasites engineered to express β-gal were unable to sensitize infected antigen-presenting cells in vitro to lysis by the CTL 0805B line. This was probably due to the intracellular localization of the β-gal within the parasite and its inaccessibility to the host cell cytoplasm.     

Major histocompatibility complex class I presentation of exogenously acquired minor alloantigens initiates skin allograft rejection

Major histocompatibility complex (MHC) class I molecules present peptides from endogenous proteins. However, in some cases class I-restricted peptides can also derive from exogenous antigens. This MHC class I exogenous presentation could be involved in minor histocompatibility antigen (mHAg)-disparate allograft rejection when donor alloantigens are not expressed in graft antigen-presenting cells (APC) that initiate the rejection mechanism. Here we addressed this question by using a skin graft experimental model where donors (H-2^b or H-2^d Tgβ-gal mice) expressed the mHAg like β-galactosidase (β-gal) in keratinocytes but not in Langerhans' cells (LC) which have an APC function. Rejection of Tgβ-gal skin by a β-gal-specific CD8 cytotoxic T lymphocyte (CTL) effector mechanism should require presentation by donor and/or recipient LC of MHC class I-restricted peptides of exogenous β-gal shed by keratinocytes. Indeed, our results showed that 1) H-2^b Tgβ-gal skin was rejected by H-2^bxs and H-2^bxd recipients; 2) rejection was mediated by β-gal-specific CD8⁺ CTL effectors; and 3) H-2^bxd mice having rejected H-2^b Tgβ-gal skin generated β-gal-specific CTL restricted by H-2^b and H-2^d class I molecules and rejected subsequently grafted H-2^d Tgβ-gal skin in an accelerated fashion, demonstrating that recipient LC have presented exogenous β-gal-derived MHC class I epitopes. These results lead to the conclusion that MHC class I exogenous presentation of donor mHAg can initiate allograft rejection.     

Dendritic cells process exogenous viral proteins and virus-like particles for class I presentation to CD8+ cytotoxic T lymphocytes

Previous reports have indicated that both dendritic cells and macrophages have the ability to induce cytotoxic T lymphocyte (CTL) and T helper (Th) cell responses in vivo. Dendritic cells process exogenous antigens conventionally for presentation on major histocompatibility complex (MHC) class II molecules. However, unconventional processing of exogenous antigens in vitro for presentation on MHC class I molecules is still an open question. In this study, we report that a cloned dendritic cell line (D2SC/1) is able to present cell debris-associated exogenous viral proteins to MHC class I-restricted CTL in vitro The dendritic cell line was very efficient in processing recombinant lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) and presenting the class I-restricted epitope to CTL primed in vivo. Peritoneal macrophages could also process the recombinant LCMV NP for subsequent MHC class I presentation, but were less efficient compared to the dendritic cells. Furthermore, recombinant yeast-derived virus-like particles carrying the HIV-1 V3 loop (V3-VLP), which are protenaceous and do not contain any lipid, were also found to be efficiently processed by the dendritic cell line for presentation of the class I-restricted epitope. These results clearly indicate that viral proteins, in particulate form or associated with cell debris, are processed by dendritic cells for CTL induction.     

Ovalbumin injected with complete Freund's adjuvant stimulates cytolytic responses

CD8⁺ cytotoxic T lymphocytes (CTL) recognize antigens (Ag) associated with class I major histocompatibility complex (MHC) molecules. Endogenously synthesized protein Ag are processed into peptides in the cytoplasm and transported to the endoplasmic reticulum where they are bound by class I proteins. Exogenous Ag do not enter the class I processing pathway of most cells and thus do not activate CD8⁺ CTL. Nevertheless, several investigators have reported that immunization with exogenous Ag can activate CD8⁺ T cells that have immunoregulatory activity. To determine how exogenous Ag entered the class I pathway in vivo and whether immunosuppressive CD8⁺ T cells were cytolytic, we have shown in this report that injection with OVA emulsified in the complete Freund's adjuvant (CFA) primed CD8⁺, class I MHC-restricted, OVA-specific CTL in mice. These CTL recognize the OVA_257–264 epitope, produce tumor necrosis factor-α and interferon-γ upon activation. Both oil and mycobacteria components in CFA were required for inducing CTL responses. Priming was not attributed to direct sensitization of class I-bearing cells by contaminating peptides. Rather, phagocytic cells, but not CD4⁺ helper T cells, were required for priming CD8⁺ CTL by OVA-CFA. Thus, OVA in CFA is taken up by antigen-presenting cells and processed into the class I pathway by phagocytic cells in vivo. In addition, CTL induced by OVA-CFA suppressed the antibody response to OVA in adoptive recipients. These results suggest that CD8⁺ CTL specific for exogenous proteins might be routinely stimulated by injecting proteins in conventional adjuvants and that such cells have the potential to regulate immune responses in vivo.     

MHC class I antigen processing of Listeria monocytogenes proteins: implications for dominant and subdominant CTL responses

Summary: Listeria monocytogenes (L. monocytogenes) secretes proteins associated with its virulence into the cytosol of infected cells. These secreted proteins are degraded by host cell proteasomes and processed into peptides that are bound by MHC class I molecules in the endoplasmic reticulum. We have found that the MHC class I antigen-processing pathway is very efficient at generating the epitopes that are presented to cytolytic T lymphocytes (CTL). Depending on which antigen is investigated, from 3 to 30 % of degraded antigens are processed into nonamer peptides that are bound by MHC class I molecules. Surprisingly, neither the efficiency of epitope generation nor the absolute number of epitopes per infected cell determines the magnitude of the in vivo CTL response. One of the least prevalent epitopes, derived from an antigen that is virtually undetectable in infected cells, primes the immunodominant CTL response in L. monocytogenes -infected mice. Our studies suggest that immunodominant and subdominant T-cell responses cannot be predicted by the prevalence of antigens or epitopes alone, and that additional factors, yet to be determined, are involved.     

Antigen presentation by Leishmania mexicana-infected macrophages: Activation of helper T cells specific for amastigote cysteine proteinases requires intracellular killing of the parasites

Leishmania mexicana amastigotes proliferate in the phagolysosomes of mammalian macrophages. The parasites abundantly synthesize lysosomal cysteine proteinases, which are encoded by the lmcpb gene family. One of these genes was overexpressed in Escherichia coli, and the purified recombinant protein was used as an antigen to induce and establish a T helper 1 (T_h1) cell line. The T cells recognize epitopes shared by the native cysteine proteinases and the recombinant protein. Infected bone marrow-derived macrophages induced to express major histocompatibility complex class II molecules by interferon (IFN)-γ do not affect parasite viability. These macrophages fail to stimulate the proliferation of the T cell line. In contrast, strong T cell stimulation is observed after the parasites are killed by treatment with L-leucine methylester, or after activation of macrophages by IFN-γ and tumor necrosis factor-α. It is concluded that infected macrophages efficiently present this lysosomal Leishmania antigen once the parasites are inactivated and degraded. This observation may be of considerable relevance for the outcome of Leishmania infections provided that it can be extended to other parasite antigens.     

Direct and indirect T cell priming by dendritic cell vaccines

The mechanisms by which dendritic cell (DC) vaccines prime host T cells in vivo was analyzed. Mice were immunized with syngeneic bone marrow-derived DC and as surrogate antigen β-galactosidase (β-gal) was used. DC either pulsed with peptide, loaded with β-gal antigen or gene-modified induced β-gal-specific cytotoxic T lymphocytes (CTL) and moderate rejection of an in vivo challenge with β-gal expressing tumors. In addition, β-gal-specific CTL lysed the syngeneic DC that were used as vaccines. Using SCID mice reconstituted with F1 lymphocytes, direct priming by gene-modified DC vaccines was demonstrated by the presence of β-gal-specific CTL of the haplotype exclusively expressed by DC while indirect priming by host antigen-presenting cells (APC) was shown by the detection of CTL of the haplo type exclusively present on host APC and absent on DC vaccines. Since DC immunization in syngeneic mice was associated with an increase in NK1.1⁺/Ly49C⁻ cells and detectable lysis of DC in vitro by lymphokine-activated killer cells, DC vaccines appear to interact with host natural killer cells as well as with antigen-specific T cells. These effector cells in turn may lyse DC vaccines thereby leading to the release of antigens that can be taken up by host APC.     

The role of macrophages in the induction and regulation of immunity elicited by exogenous antigens

Different delivery vehicles may target to different antigen presenting cells (APC) because of their composition, size and/or physical properties. In this study, we examined the priming of cytotoxic T lymphocyte (CTL) responses to a soluble exogenous protein in vivo, using various delivery vehicles. In addition, we determined the role of macrophages as APC in vivo for each of these delivery vehicles by comparing the induction of antigen-specific CTL and serum antibodies in normal and macrophage-depleted mice. Influenza A virus-derived virosomes, liposomes and monophosphoryl lipid A/squalene (MPL/SQ) efficiently induced antigen-specific CTL as well as antibody responses, of which virosomes proved to be the most efficient inducers. In mice that were immunized with cell-associated antigen, strong CTL responses but no antigen-specific antibodies were detectable, while aluminium hydroxide and aluminium phosphate elicited antigen-specific antibodies but no CTL responses. Eli mination of macrophages in vivo before immunization abrogated CTL responses induced with liposomes and MPL/SQ, but did not affect induction of antigen-specific CTL with virosomes or cell-associated antigen. Importantly, serum antibody levels were not altered after macrophage depletion, regardless of the delivery vehicle used, suggesting that in the absence of macrophages, other APC may phagocytose the exogenous antigens for major histocompatibility complex (MHC) class II processing and presentation. These results suggest that soluble exogenous antigens delivered in different carrier systems may be processed differently by different APC in vivo for MHC class I- or class II-restricted presentation.     

Cross‐Binding Between Plasmodium falciparum CTL Epitopes and HLA Class I Molecules

Plasmodium falciparum CTL epitope minigenes containing HLA‐A2 and HLA‐B7 subtype supermotifs were cloned into a plasmid expression vector; this expression was measured in eight human HLA class I molecule specific cell lines. Three assays for in vitro antigen presentation analysis were developed to examine the cross‐binding between CTL epitopes and HLA class I molecules, including cell surface peptide‐MHC class I binding assay, binding stabilization assay and MHC class I assembling assay. The results demonstrated that the HLA‐B51 restricted CTL epitope of Plasmodium falciparum could be presented by other HLA class I molecules; however, no other presentation was found for HLA‐A2.1 CTL epitope. This work suggests the possibility for improved vaccine‐coverage rates by development of a CTL vaccine which contains epitopes capable of cross‐binding among different MHC class I alleles.     

Selective stimulation of murine cytotoxic T cell and antibody responses by particulate or monomeric hepatitis B virus surface (S) antigen

In the murine system, we tested in vivo the immunogenicity of different preparations of the yeast-derived surface antigen (S-antigen or S-protein) of hepatitis B virus (HBV). Native S-protein molecules self-assemble into stable 22-nm particles. BALB/c mice immunized with low doses of native S-particles without adjuvants efficiently generated an H-2 class I-restricted CD8⁺ cytotoxic T lymphocyte (CTL) response, and developed easily detectable serum antibody titers against conformational determinants of the native S-particle or linear epitopes of the denatured S-protein. Disruption of S-particles with sodium dodecyl sulfate and β-2-mercaptoethanol generated p24 S-monomers. Injection of an equal dose of S-monomers into mice efficiently primed CTL, but did not stimulate an antibody response against conformational or linear epitopes of the native or denatured S-protein. In vivo priming of CTL by S-particles or S-monomers required “endogenous” processing of the antigen because the injection of an equimolar (or higher) dose of an antigenic, S-derived 12-mer peptide into mice did not prime CTL. Native (particulate) or denatured (monomeric) S-antigen injected with mineral oil (incomplete Freund's adjuvant) or aluminum hydroxide failed to stimulate a CTL response. Hence, different preparations can be produced from a small protein antigen which specifically stimulate selected compartments of the immune system.     

Down-regulation of MHC class II molecules and inability to up-regulate class I molecules in murine macrophages after infection with Toxoplasma gondii

Toxoplasma gondii is able to invade phagocytic cells of the monocyte-macrophage lineage and replicates within a parasitophorous vacuole. Since macrophages may activate specific T lymphocytes by presenting pathogen-derived antigens in association with molecules of the MHC, we investigated the in vitro expression of host cell molecules involved in antigen processing and presentation before and during infection of murine bone marrow-derived macrophages (BMM) with T. gondii. Fifty-one hours after addition of T. gondii tachyzoites at different parasite-to-host ratios, up-regulation of total MHC class II molecules by interferon-gamma (IFN-γ) was dose-dependently abrogated in up to 50% of macrophages compared with uninfected control cultures. Quantitative analyses by flow cytometry revealed that the IFN-γ-induced surface expression of class II antigens as well as the IFN-γ-induced up-regulation of class I molecules was significantly decreased in T. gondii-infected macrophage cultures compared with uninfected controls. However, the constitutive expression of MHC class I antigens was not altered after parasitic infection, and infected BMM remained clearly positive for these molecules. After infection of macrophages preactivated with IFN-γ for 48 h, T. gondii also actively down-regulated an already established expression of MHC class II molecules. Furthermore, kinetic analysis revealed that the reduction in intracellular and plasma membrane-bound class II molecules started ≈ 20 h after infection. While MHC class II antigens were most prominently reduced in parasite-positive host cells, culture supernatant from T. gondii-infected BMM cultures also significantly inhibited expression of these molecules in uninfected macrophages. However, down-regulation of MHC class II molecules was not mediated by an increased production of prostaglandin E₂, IL-10, transforming growth factor-beta or nitric oxide by infected BMM compared with uninfected controls. Our data indicate that intracellular T. gondii interferes with the MHC class I and class II antigen presentation pathway of murine macrophages and this may be an important strategy for evasion from the host's immune response and for intracellular survival of the parasite.     

CTL induction by cross‐priming is restricted to immunodominant epitopes

CTL are induced by two pathways, i.e. direct priming, where tumor cells present tumor antigens to naïve specific CTL, and cross‐priming, where professional APC cross‐present captured tumor antigens to CTL. Here, we examined direct priming versus cross‐priming after immunizing (H‐2^b×H‐2^d) F1 mice with either H‐2^b or H‐2^d positive tumor cells transfected with the GP or nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV). Cross‐priming was observed for the immunodominant epitopes LCMV‐gp33 and ‐np118, although direct induction resulted in higher CTL frequencies. In contrast, CTL specific for the subdominant epitopes LCMV‐gp283 or ‐np396 were induced only if epitopes were presented directly on MHC class I molecules of the immunizing cell. The broader repertoire and the higher CTL frequencies induced after vaccination with haplotype‐matched tumor cells resulted in more efficient anti‐tumor and antiviral protection. Firstly, our results indicate that certain virus and tumor antigens may not be detected by CD8⁺ T cells because of impaired cross‐priming. Secondly, efficient cross‐priming contributes to the immunodominant nature of a tumor‐specific CTL epitope. Thirdly, vaccine strategies using autologous or syngenic antigen‐expressing cells induce a broader repertoire of tumor‐specific CTL and higher CTL frequencies.     

In vitro priming of cytotoxic T lymphocytes against poorly immunogenic epitopes by engineered antigen-presenting cells

Cytotoxic T lymphocytes (CTL) recognize antigenic peptides presented by major histocompatibility complex class I (MHC-I) molecules on the surface of target cells. Optimal induction of CD8⁺ CTL depends on the amount of relevant peptide/MHC-I complexes and the presence of co-stimulatory molecules on antigen-presenting cells (APC). The antigen-processing defective mutant cell line RMA-S, when cultured at low temperature, expresses high amounts of MHC-I molecules that do not contain endogenously derived peptides. These “empty” MHC-I molecules can be stabilized by addition of MHC-binding peptides. RMA-S cultured at low temperatures with selected peptides have been used for in vitro CTL induction with conflicting results. RMA-S cells do not express detectable amounts of B7 co-stimulatory molecule. This could explain their unpredictable efficiency as APC. We have evaluated whether RMA-S cells, stably transfected with cDNA encoding for the human B7.1 molecule could provide effective co-stimulation for CD8⁺ T lymphocytes. RMA-S/B7 cells, loaded with different synthetic peptides, demonstrated a high and sometimes unique efficiency for in vitro primary CTL induction, even when “sub-optimal” antigen peptides were used. Most importantly, RMA-S/B7 cells pulsed with naturally processed peptides extracted from the poorly immunogenic B16 melanoma cells were able to prime CD8⁺ cells against B16 melanoma. We conclude that the use of RMA-S/B7 cells as APC represents an ideal strategy for in vitro CTL immunization without prior in vivo priming. This system may also help to address the issue of the different contributions of co-stimulation and relative occupancy of MHC-I by single peptide epitopes in CTL priming.     

Cytotoxic T lymphocyte responses to wild-type and mutant mouse p53 peptides

Cytotoxic T lymphocytes (CTL) recognize peptides presented at the cell surface in association with major histocompatibility complex (MHC) class I molecules. The finding that peptides binding to MHC class I molecules share common amino acid motifs renders feasible the selection of antigenic peptides by simply scanning protein sequences, and thus, provides the possibility of inducing CTL to pre-defined specificities. Tumor cells possess antigens known to generate MHC class I-restricted CD8⁺ CTL responses. Thus, these antigens represent good targets to induce tumor-specific immunity. Among these antigens, the p53 tumor suppressor gene product is an attractive candidate for cancer immunotherapy. Mutations in the p53 gene have been found to be very frequently associated with a malignant transformation and often lead to p53 protein overexpression. Thus, we investigated the possibility of inducing CTL to wild-type or mutant p53 peptides in a BALB/c (H-2^d) mouse model. Peptides possessing the H2-K^d binding motif were selected and tested for binding to the H-2K^d molecules in vitro. Synthetic peptides p53_122–130 wild-type or “mutant” (Lys → Glu substitution at position 129) were shown to be the best binder peptides and were tested for their immunogenicity in mice. H-2K^d-restricted p53-specific CD8⁺ CTL were generated following immunization of mice with either wild-type (wt) p53_122–130 or mutant (mut) p53_122–130 (E129) peptides. Only low-affinity CTL can be obtained by immunization with the wt sequence. In contrast, CTL elicited with the mut peptide recognized the mut sequence at a 10–100-fold lower concentration. This indicates that CTL elicited with the mut peptide recognized the mut sequence very efficiently, whereas the wt sequence is poorly recognized, if at all. Taken together, these results thus suggest that p53-specific tumor immunotherapy may be successful only if the mutated protein is taken into consideration.     

TAP1-independent loading of class I molecules by exogenous viral proteins

Presentation of peptides derived from endogenous proteins on class I molecules needs functional TAP peptide transporters. To reveal whether class I-associated presentation of exogenous proteins also required the presence of TAP transporters, we assessed in vitro the ability of spleen cells and macrophages from TAP1-deficient mice (TAP1?/?) to present peptides derived from exogenous recombinant viral proteins on their class I molecules. We found that recombinant glycoand nucleoprotein from lymphocytic choriomeningitis virus and nucleoprotein of vesicular stomatitis virus were presented as efficiently by TAP1 ?/? cells as by control cells. Peptide regurgitation was not involved. Since particulate, nonreplicating antigens can efficiently prime anti-viral cytotoxic T cells in vivo, this new, TAP-independent pathway of class I-associated antigen presentation may be applicable for vaccine strategies.     

Listeriolysin generates a route for the presentation of exogenous antigens by major histocompatibility complex class I

We have exploited the pore forming activity of listeriolysin, the hemolysin of Listeria monocytogenes, to activate CD8⁺ T cells with soluble proteins in vivo and in vitro. Immunization with soluble, hemolytically active listeriolysin induces both cytotoxic CD8⁺ T cells and CD4⁺ T cells, and the CD8⁺ T cells can be propagated with soluble listeriolysin in vitro. Moreover, conventional antigens like ovalbumin mixed together with listeriolysin are also efficiently introduced into the MHC class I pathway in vitro and in vivo. Hence, listeriolysin effectively directs itself and passenger molecules into the intracellular compartment that leads to the cytotoxic T cell response. In this way, we circumvent the bias of CD8⁺ T cells to recognize intracellular antigens presented by major histocompatibility complex class I molecules. As cytotoxic CD8⁺ T cells are of pivotal importance in eliminating viral and microbial pathogens, the findings reported here could prove to be useful in vaccine development.     

MUC1 peptide epitopes associated with five different H-2 class I molecules

We have previously described the induction of murine CD8⁺ major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) recognizing the 20-amino acid repeat region of the human mucin 1 (MUC1) variable number of tandem repeats region (VNTR), a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. In that study, CTL could detect MUC1 peptides associated with the MHC of all nine strains examined, and we now report the different epitopes presented by five different MHC class I molecules. The epitopes were defined in CTL assays using peptide-pulsed phytohemagglutinin blasts or MHC class I-transfected L cells as targets; in addition, peptide binding assays and T cell proliferation studies were performed. Within the 20-amino acid VNTR, nine potential epitopes could be defined. The epitopes for the four MHC class I molecules [K^b (three epitopes), D^d, L^d and K^k] were closely related, all containing the amino acids PDTRPAP. For D^b, three epitopes were identified, all containing APGSTAP. Most of the epitopes did not contain a consensus motif for the particular MHC class I allele, and bound with low ‘affinity’, compared with known high-affinity peptides. CD8⁺ T cell proliferation also occurred to the same MHC class I-presented epitopes. Finally, when conventional anchor residues were introduced into the peptides, peptide binding increased, whereas CTL recognition was either retained (K^b) or lost (D^b) depending on the epitope.     

Phagocyte-induced antigen-specific activation of unprimed CD8+ T cells in vitro

The strict segregation of the major histocompatibility complex (MHC) class I and class II loading pathways has been challenged by recent reports indicating that MHC class I molecules can acquire antigen in the phagocytic pathway. We now show that this alternative peptide loading pathway can be used efficiently to generate macrophages able to activate unprimed antigen-specific cytotoxic T cells in vitro. Short peptides (8–11 residues), administered in the phagocytic pathway at nanomolar concentrations, were found to be effective in specifically activating naïve cytotoxic T lymphocytes (CTL) in vitro, but longer peptides or whole protein antigen were not. Whole protein antigen coated on beads did, however, render macrophages susceptible to lysis by an antigen-specific CTL clone. This indicates that proteolysis in the phagocytic pathway has limited capability for class I-restricted presentation. We propose a model for class I loading in the phagocytic pathway consisting of direct trafficking of nascent MHC class I from the trans-Golgi network to the phagosome, prior to appearance at the cell surface, and the use of the narrow cavity between bead and phagosomal membrane as a peptide exchange/loading compartment. Targeting immunogenic class I-binding peptide to the phagocytic pathway of macrophages facilitates presentation in association with class I. This is a useful tool for CTL response induction in vitro.     

Clearance of Sendai virus by CD8+ T cells requires direct targeting to virus-infected epithelium

Minimal numbers of CD8⁺ T cells are found in bronchoalveolar lavage (BAL) populations recovered from Sendai virus-infected mice that are homozygous (?/?) for β2-microglobulin (β2-m) gene disruption. The prevalence of the CD8⁺ set was substantially increased in the pneumonic lungs of 8?12-week radiation chimeras made using substantially class I major histocompatibility complex (MHC) glycoprotein-negative β2-m (?/?) recipients and normal β2-m (+/+) bone marrow. Even so, the CD8⁺ (but not the CD4⁺) lymphocyte counts were still much lower than in the (+/+)→(+/+) controls. The (+/+)→(+/+) and (+/+)→(?/?) chimeras cleared Sendai virus and potent virus-immune CD8⁺ cytotoxic T lymphocytes (CTL) specific for H-2K^b + viral nucleoprotein peptide were found in the BAL from both groups. However, following in vivo depletion of the CD4⁺ population, only the (+/+)→(+/+) mice were able to deal with the infection. Similarly, adoptively transferred, H-2K^b-restricted CD8⁺ T cells from previously-primed (+/+) mice also failed to clear virus from the lungs of (+/+)→(?/?) chimeras infected within 2 weeks of reconstitution with bone marrow, though they were effective in the (+/+)→(+/+) controls. Sendai virus-immune CD8⁺ T cells are thus unable to eliminate virus-infected β2-m (?/?) lung epithelial cells that might be thought to be expressing very small amounts of either isolated class I heavy chain, or class I MHC glycoprotein that has bound β2-m derived from β2-m (+/+) T cells or macrophages present in the pneumonic lung. Furthermore, the CD8⁺ CTL that are being exposed to β2-m (+/+) stimulators in the BAL population cannot operate in some bystander mode to clear virus from respiratory epithelium.     

The outcome of cross‐priming during virus infection is not directly linked to the ability of the antigen to be cross‐presented

The initiation of CD8⁺ T cell (CTL) immune responses can occur via cross‐priming. Recent data suggested a relationship between cross‐presentation and immunodominance of epitope‐specific T cells. To test this association, we evaluated the efficacy of cross‐presentation for several virus epitopes in vitro and examined if this can be extrapolated in vivo. Employing lymphocytic choriomeningitis virus (LCMV), we demonstrate that the cross‐presentation and cross‐priming of LCMV antigens were dominated by NP396, but not NP205 when analyzing the LCMV‐NP. Although with LCMV‐GP, cross‐presentation was dominated by GP276, and cross‐priming was dominated by GP33. Importantly, although NP396 was significantly more efficient than GP33 in cross‐presentation, cross‐priming of their specific CTL was comparable. In a subsequent virus challenge after cross‐priming, GP33‐specific CTL dominated the response. Accordingly, based on our data, the ability of viral epitopes to be cross‐presented in vitro does not entirely reflect what would occur in cross‐priming. Thus, weak cross‐presenting antigens may still cross‐prime an efficient CTL response depending on other in vivo elements such as the naïve T‐cell precursor frequencies.