MHC class I restricted T cell responses tolisteria monocytogenes, an intracellular bacterial pathogen

Studies of the murine immune response to infection with the intracellular bacterial pathogenListeria monocytogenes have provided a wealth of information about innate and acquired immune defenses in the setting of an infectious disease. Our studies have focused on the MHC class I restricted, CD8⁺ T cell responses of Balb/c mice toL. monocytogenes infection. Four peptides that derive from proteins thatL. monocytogenes secretes into the cytosol of infected cells are presented to cytotoxic T lymphocyte (CTL) by the H2-K^d major histocompatibility complex (MHC) class I molecule. We have found that bacterially secreted proteins are rapidly degraded in the host cell cytosol by proteasomes that utilize, at least in part, the N-end rule to determine the rate of degradation. The MHC class I antigen processing pathway is remarkably efficient at generating peptides that bind to MHC class I molecules. The magnitude of in vivo T cell responses, however, is influenced to only a small degree by the amount of antigen or the efficiency of antigen presentation. Measurements of in vivo T cell expansion followingL. monocytogenes infection indicate that differences in the sizes of peptide-specific T cell responses are more likely owing to differences in the repertoire of naive T cells than to differences in peptide presentation. This notion is supported by our additional finding that dominant T cell populations express a more diverse T cell receptor (TCR) repertoire than do subdominant T cell populations.     

Invariant chain as a vehicle to load antigenic peptides on human MHC class I for cytotoxic T‐cell activation

Protective T‐cell responses depend on efficient presentation of antigen (Ag) in the context of major histocompatibility complex class I (MHCI) and class II (MHCII) molecules. Invariant chain (Ii) serves as a chaperone for MHCII molecules and mediates trafficking to the endosomal pathway. The genetic exchange of the class II‐associated Ii peptide (CLIP) with antigenic peptides has proven efficient for loading of MHCII and activation of specific CD4⁺ T cells. Here, we investigated if Ii could similarly activate human CD8⁺ T cells when used as a vehicle for cytotoxic T‐cell (CTL) epitopes. The results show that wild type Ii, and Ii in which CLIP was replaced by known CTL epitopes from the cancer targets MART‐1 or CD20, coprecipitated with HLA‐A*02:01 and mediated colocalization in the endosomal pathway. Furthermore, HLA‐A*02:01‐positive cells expressing CLIP‐replaced Ii efficiently activated Ag‐specific CD8⁺ T cells in a TAP‐ and proteasome‐independent manner. Finally, dendritic cells transfected with mRNA encoding IiMART‐1 or IiCD20 primed naïve CD8⁺ T cells. The results show that Ii carrying antigenic peptides in the CLIP region can promote efficient presentation of the epitopes to CTLs independently of the classical MHCI peptide loading machinery, facilitating novel vaccination strategies against cancer.     

DC‐induced CD8+ T‐cell response is inhibited by MHC class II‐dependent DX5+CD4+ Treg

CD4⁺ T cells are important for CD8⁺ T‐cell priming by providing cognate signals for DC maturation. We analyzed the capacity of CD4⁺ T cells to influence CD8⁺ T‐cell responses induced by activated DC. Surprisingly, mice depleted for CD4⁺ cells were able to generate stronger antigen‐specific CD8⁺ T‐cell responses after DC vaccination than non‐depleted mice. The same observation was made when mice were vaccinated with MHC class II^?/? DC, indicating the presence of a MHC class II‐dependent CD4⁺ T‐cell population inhibiting CD8⁺ T‐cell responses. Recently we described the expansion of DX5⁺CD4⁺ T cells, a T‐cell population displaying immune regulatory properties, upon vaccination with DC. Intriguingly, we now observe an inverse correlation between CD8⁺ T‐cell induction and expansion of DX5⁺CD4⁺ T cells as the latter cells did not expand after vaccination with MHC class II^?/? DC. In vitro, DX5⁺CD4⁺ T cells were able to limit proliferation, modulate cytokine production and induce Foxp3⁺ expression in OVA‐specific CD8⁺ T cells. Together, our data show an inhibitory role of CD4⁺ T cells on the induction of CD8⁺ T‐cell responses by activated DC and indicate the involvement of DX5⁺CD4⁺, but not CD4⁺CD25⁺, T cells in this process.     

Complement opsonization of HIV‐1 results in a different intracellular processing pattern and enhanced MHC class I presentation by dendritic cells

Induction of optimal HIV‐1‐specific T‐cell responses, which can contribute to controlling viral infection in vivo, depends on antigen processing and presentation processes occurring in DCs. Opsonization can influence the routing of antigen processing and pathways used for presentation. We studied antigen proteolysis and the role of endocytic receptors in MHC class I (MHCI) and II (MHCII) presentation of antigens derived from HIV‐1 in human monocyte‐derived immature DCs (IDCs) and mature DCs, comparing free and complement opsonized HIV‐1 particles. Opsonization of virions promoted MHCI presentation by DCs, indicating that complement opsonization routes more virions toward the MHCI presentation pathway. Blockade of macrophage mannose receptor (MMR) and β7‐integrin enhanced MHCI and MHCII presentation by IDCs and mature DCs, whereas the block of complement receptor 3 decreased MHCI and MHCII presentation. In addition, we found that IDC and MDC proteolytic activities were modulated by HIV‐1 exposure; complement‐opsonized HIV‐1 induced an increased proteasome activity in IDCs. Taken together, these findings indicate that endocytic receptors such as MMR, complement receptor 3, and β7‐integrin can promote or disfavor antigen presentation probably by routing HIV‐1 into different endosomal compartments with distinct efficiencies for degradation of viral antigens and MHCI and MHCII presentation, and that HIV‐1 affects the antigen‐processing machinery.     

Mature proteins derived from Epstein–Barr virus fail to feed into the MHC class I antigenic pool

The immediate presentation of peptide epitopes on MHC class I (MHC I) after antigen expression has led to the concept that MHC I ligands are mostly derived from defective ribosomal products (DRiPs), a subset of newly synthesized proteins that are rapidly degraded by the proteasome. Whether and to what extent mature proteins contribute to the antigenic pool, however, has remained elusive. Here, we developed a conditional antigen expression system that allows studying antigen presentation from mature proteins by inducing their rapid proteasomal degradation in the absence of further antigen synthesis. Target cells in which expression of two Epstein–Barr virus (EBV) antigens was induced were rapidly recognized by antigen‐specific CD8⁺ T cells in a time‐ and dosage‐dependent manner, demonstrating that antigen presentation was linked to antigen synthesis. By contrast, T cells failed to recognize target cells containing large amounts of mature protein even after induction of their rapid proteasomal degradation. Thus, the presentation of these antigens proved to be strictly dependent on protein synthesis whereas mature proteins failed to furnish the antigenic pool. These results have implications for the design of immunotherapeutic strategies that aim at targeting proteins with increased half‐lives and are hence overexpressed in tumors.     

Reassessing the role of HLA‐DRB3 T‐cell responses: Evidence for significant expression and complementary antigen presentation

In humans, several HLA‐DRB loci (DRB1/3/4/5) encode diverse β‐chains that pair with α‐chains to form DR molecules on the surface of APC. While DRB1 and DRB5 have been extensively studied, the role of DRB3/4 products of DR52/DR53 haplotypes has been largely neglected. To clarify the relative expression of DRB3, we quantified DRB3 mRNA levels in comparison with DRB1 mRNA from the same haplotype in both B cells and monocytes, observing quantitatively significant DRB3 synthesis. In CD19⁺ cells, DRB1*03/11/13 was 3.5‐fold more abundant than DRB3, but in CD14⁺ this difference was only two‐fold. Monocytes also had lower overall levels of DR mRNA compared with B cells, which was confirmed by cell surface staining of DRB1 and DRB3. To evaluate the functional role of DRB3, tetramer‐guided epitope mapping was used to detect T cells against tetanus toxin and several influenza antigens presented by DRB3*0101/0202 or DRB1*03/11/13. None of the epitopes discovered were shared among any of the DR molecules. Quantitative assessment of DRB3‐tetanus toxin specific T cells revealed that they are present at similar frequencies as those observed for DRB1. These results suggest that DRB3 plays a significant role in antigen presentation with different epitopic preferences to DRB1. Therefore, DRB3, like DRB5, serves to extend and complement the peptide repertoire of DRB1 in antigen presentation.     

MHC class II alleles associated with Th1 rather than Th17 type immunity drive the onset of early arthritis in a rat model of rheumatoid arthritis

Polymorphisms in the MHC class II (MHCII) genes are strongly associated with rheumatoid arthritis, supporting the importance of autoreactive T helper (Th) cells for the development of this disease. Here, we used pristane‐induced arthritis (PIA), induced by the non‐antigenic hydrocarbon pristane, to study the impact of different MHCII alleles on T‐cell activation and differentiation. In MHCII‐congenic rats with disease‐promoting MHCII alleles, pristane primarily induced activation of Th1 cells, whereas activated T cells were Th17 biased in rats with protective MHCII alleles. Neutralization of IFN‐γ during T‐cell activation abrogated the development of disease, suggesting that Th1 immunity is important for disease induction. Neutralization of IL‐17, by contrast, suppressed arthritis only when performed in rats with established disease. Adoptive T‐cell transfers showed that T cells acquired arthritogenic capacity earlier in strains with a prevailing Th1 response. Moreover, upon pristane injection, these strains exhibited more Ag‐primed OX40+ and proliferating T cells of polyclonal origin. These data show that T cells are polarized upon the first encounter with peptide‐MHCII complexes in an allele‐dependent fashion. In PIA, the polyclonal expansion of autoreactive Th1 cells was necessary for the onset of arthritis, while IL‐17 mediated immunity contributed to the progression to chronic disease.     

Essential differences in ligand presentation and T cell epitope recognition among HLA molecules of the HLA-B44 supertype

Human leukocyte antigens (HLA) have long been grouped into supertypes to facilitate peptide-based immunotherapy. Analysis of several hundreds of peptides presented by all nine antigens of the HLA-B44 supertype (HLA-B*18, B*37, B*40, B*41, B*44, B*45, B*47, B*49 and B*50) revealed unique peptide motifs for each of them. Taking all supertype members into consideration only 25 out of 670 natural ligands were found on more than one HLA molecule. Further direct comparisons by two mass spectrometric methods--isotope labeling as well as a label-free approach--consistently demonstrated only minute overlaps of below 3% between the ligandomes of different HLA antigens. In addition, T cell reactions of healthy donors against immunodominant HLA-B*44 and HLA-B*40 epitopes from EBV lacked promiscuous T-cell recognition within the HLA-B44 supertype. Taken together, these results challenge the common paradigm of broadly presented epitopes within this supertype.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

ICOS controls Foxp3+ regulatory T‐cell expansion,maintenance and IL‐10 production during helminth infection

Foxp3⁺ regulatory T (Treg) cells are key immune regulators during helminth infections, and identifying the mechanisms governing their induction is of principal importance for the design of treatments for helminth infections, allergies and autoimmunity. Little is yet known regarding the co‐stimulatory environment that favours the development of Foxp3⁺ Treg‐cell responses during helminth infections. As recent evidence implicates the co‐stimulatory receptor ICOS in defining Foxp3⁺ Treg‐cell functions, we investigated the role of ICOS in helminth‐induced Foxp3⁺ Treg‐cell responses. Infection of ICOS^?/? mice with Heligmosomoides polygyrus or Schistosoma mansoni led to a reduced expansion and maintenance of Foxp3⁺ Treg cells. Moreover, during H. polygyrus infection, ICOS deficiency resulted in increased Foxp3⁺ Treg‐cell apoptosis, a Foxp3⁺ Treg‐cell specific impairment in IL‐10 production, and a failure to mount putatively adaptive Helios^?Foxp3⁺ Treg‐cell responses within the intestinal lamina propria. Impaired lamina propria Foxp3⁺ Treg‐cell responses were associated with increased production of IL‐4 and IL‐13 by CD4⁺ T cells, demonstrating that ICOS dominantly downregulates Type 2 responses at the infection site, sharply contrasting with its Type 2‐promoting effects within lymphoid tissue. Thus, ICOS regulates Type 2 immunity in a tissue‐specific manner, and plays a key role in driving Foxp3⁺ Treg‐cell expansion and function during helminth infections.     

Cis association of leukocyte Ig‐like receptor 1 with MHC class I modulates accessibility to antibodies and HCMV UL18

Leukocyte Ig‐like receptor (LIR) 1 (CD85j/ILT2/LILRB1) is an inhibitory receptor with broad specificity for MHC class I (MHC‐I) and the human CMV MHC‐I homologue UL18. LIR‐1 can inhibit NK cells through the conventional interaction with MHC‐I expressed on a target cell (in trans) but the nature and the effects of LIR‐1 interactions with MHC‐I in cis are not well understood. Here we show that MHC‐I expressed in cis has an impact on the detection of LIR‐1 with various antibodies. We found the cis interaction alters recognition by only one of two antibodies known to block functional trans recognition by LIR‐1 on NK cells. Specifically, we observed an enhancement of recognition with GHI/75 in the presence of various MHC‐I alleles on 721.221 cells. We found that blocking the LIR‐1 contact site with anti‐MHC‐I antibodies decreased detection of LIR‐1 with GHI/75. We also observed a decrease in GHI/75 following acid denaturation of MHC‐I. Finally, disruption of LIR‐1 cis interactions with MHC‐I significantly enhanced UL18‐Fc binding to NK92 cells and enhanced the relative inhibition of NK92 cells by HLA‐G. These results have implications for LIR‐1 function in scenarios such as infection when MHC‐I levels on effector cells may be increased by IFNs.     

Expansion of Foxp3+ T‐cell populations by Candida albicans enhances both Th17‐cell responses and fungal dissemination after intravenous challenge

Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3⁺ regulatory T (Treg) cells remains largely unexplored. Our aims were to characterize Foxp3⁺ Treg‐cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4⁺Foxp3⁺ population that correlated positively with fungal burden. Depletion from Foxp3^hCD2 reporter mice in vivo confirmed that Foxp3⁺ cells exacerbated fungal burden and inflammatory renal disease. The CD4⁺Foxp3⁺ population expanded further after in vitro stimulation with C. albicans antigens (Ags), and included at least three cell types. These arose from proliferation of the natural Treg‐cell subset, together with conversion of Foxp3^? cells to the induced Treg‐cell form, and to a cell type sharing effector Th17‐cell characteristics, expressing ROR‐γt, and secreting IL‐17A. The expanded Foxp3⁺ T cells inhibited Th1 and Th2 responses, but enhanced Th17‐cell responses to C. albicans Ags in vitro, and in vivo depletion confirmed their ability to enhance the Th17‐cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3⁺ T cells promotes Th17‐cell responses that drive pathology.     

Partial and transient modulation of the CD3–T‐cell receptor complex,elicited by low‐dose regimens of monoclonal anti‐CD3, is sufficient to induce disease remission in non‐obese diabetic mice

It has been established that a total of 250 μg of monoclonal anti‐mouse CD3 F(ab′)₂ fragments, administered daily (50 μg per dose), induces remission of diabetes in the non‐obese diabetic (NOD) mouse model of autoimmune diabetes by preventing β cells from undergoing further autoimmune attack. We evaluated lower‐dose regimens of monoclonal anti‐CD3 F(ab′)₂ in diabetic NOD mice for their efficacy and associated pharmacodynamic (PD) effects, including CD3–T‐cell receptor (TCR) complex modulation, complete blood counts and proportions of circulating CD4⁺, CD8⁺ and CD4⁺ FoxP3⁺ T cells. Four doses of 2 μg (total dose 8 μg) induced 53% remission of diabetes, similarly to the 250 μg dose regimen, whereas four doses of 1 μg induced only 16% remission. While the 250 μg dose regimen produced nearly complete and sustained modulation of the CD3 –TCR complex, lower doses, spaced 3 days apart, which induced similar remission rates, elicited patterns of transient and partial modulation. In treated mice, the proportions of circulating CD4⁺ and CD8⁺ T cells decreased, whereas the proportions of CD4⁺ FoxP3⁺ T cells increased; these effects were transient. Mice with greater residual β‐cell function, estimated using blood glucose and C‐peptide levels at the initiation of treatment, were more likely to enter remission than mice with more advanced disease. Thus, lower doses of monoclonal anti‐CD3 that produced only partial and transient modulation of the CD3–TCR complex induced remission rates comparable to higher doses of monoclonal anti‐CD3. Accordingly, in a clinical setting, lower‐dose regimens may be efficacious and may also improve the safety profile of therapy with monoclonal anti‐CD3, potentially including reductions in cytokine release‐related syndromes and maintenance of pathogen‐specific immunosurveillance during treatment.     

Analysis of human class II antigens by cloned cytolytic T cell reagents: a study using HLA loss mutant lymphoblastoid cell lines and monoclonal antibodies detecting the HLA-DP product(s)

We have utilized cloned T cell reagents and ionizing radiation-induced mutants of an HLA heterozygous lymphoblastoid cell line (LCL) to investigate the determinants detected by the cell-mediated lympholysis (CML) assay. Cells of an LCL clone, 721.501, an HLA haplotype loss mutant expressing the HLA-A2-Cw1-Bw51-DR1-Dw1-DQw1-DPw2-GLO haplotype were used as sensitizing cells for responder cells in vitro. "Cloned" reagents were generated by single-cell deposition of cells of a bulk reagent primed against 721.501 cells. Those clones were screened for cytolytic activity against HLA loss mutant targets (derived from LCL 721) of four different categories; HLA-A2 loss only, A2-Cw1-Bw51 loss, A2-Cw1-Bw51-DR1-DQw1 loss, and the entire HLA haplotype loss. Of 196 clones tested, 36 were cytolytic, including three anti-A2, five anti-Bw51/Cw1, 12 anti-DR1/DQw1, 13 anti-DP region associated with DPw2, and three of undetermined specificity, based on cytolytic patterns against the HLA loss mutant targets. Of 25 anti-HLA class II lytic clones, 23 (92%) fitted the characteristics of helper cell-independent cytolytic T cells (HITc), whereas only two of eight (25%) anti-class I clones were HITc. The 13 anti-DP region clones were divided into three subgroups defined by blocking by anti-FA and not Tü39 monoclonal antibodies (MoAb), by Tü39 and not anti-FA, and by both MoAbs.