Characterization of polarization states of canine monocyte derived macrophages.

Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immune­related diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs.

Mouse IgG2a Isotype Therapeutic Antibodies Elicit Superior Tumor Growth Control Compared with mIgG1 or mIgE.

In the last decades, antibody-based tumor therapy has fundamentally improved the efficacy of treatment for patients with cancer. Currently, almost all tumor antigen-targeting antibodies approved for clinical application are of IgG1 Fc isotype. Similarly, the mouse homolog mIgG2a is the most commonly used in tumor mouse models. However, in mice, the efficacy of antibody-based tumor therapy is largely restricted to a prophylactic application. Direct isotype comparison studies in mice in a therapeutic setting are scarce. In this study, we assessed the efficacy of mouse tumor-targeting antibodies of different isotypes in a therapeutic setting using a highly systematic approach. To this end, we engineered and expressed antibodies of the same specificity but different isotypes, targeting the artificial tumor antigen CD90.1/Thy1.1 expressed by B16 melanoma cells. Our experiments revealed that in a therapeutic setting mIgG2a was superior to both mIgE and mIgG1 in controlling tumor growth. Furthermore, the observed mIgG2a antitumor effect was entirely Fc mediated as the protection was lost when an Fc-silenced mIgG2a isotype (LALA-PG mutations) was used. These data confirm mIgG2a superiority in a therapeutic tumor model. Significance: Direct comparisons of different antibody isotypes of the same specificity in cancer settings are still scarce. Here, it is shown that mIgG2a has a greater effect compared with mIgG1 and mIgE in controlling tumor growth in a therapeutic setting.

Shortened Hinge Design of Fab x sdAb-Fc Bispecific Antibodies Enhances Redirected T-Cell Killing of Tumor Cells.

T cell engager (TCE) antibodies have emerged as promising cancer therapeutics that link cytotoxic T-cells to tumor cells by simultaneously binding to CD3E on T-cells and to a tumor-associated antigen (TAA) expressed by tumor cells. We previously reported a novel bispecific format, the IgG-like Fab x sdAb-Fc (also known as half-IG_VH-h-CH2-CH3), combining a conventional antigen-binding fragment (Fab) with a single domain antibody (sdAb). Here, we evaluated this Fab x sdAb-Fc format as a T-cell redirecting bispecific antibody (TbsAbs) by targeting mEGFR on tumor cells and mCD3E on T cells. We focused our attention specifically on the hinge design of the sdAb arm of the bispecific antibody. Our data show that a TbsAb with a shorter hinge of 23 amino acids (TbsAb.short) showed a significantly better T cell redirected tumor cell elimination than the TbsAb with a longer, classical antibody hinge of 39 amino acids (TbsAb.long). Moreover, the TbsAb.short form mediated better T cell-tumor cell aggregation and increased CD69 and CD25 expression levels on T cells more than the TbsAb.long form. Taken together, our results indicate that already minor changes in the hinge design of TbsAbs can have significant impact on the anti-tumor activity of TbsAbs and may provide a new means to improve their potency.

Generation and characterization of novel co-stimulatory anti-mouse TNFR2 antibodies.

Tumor necrosis factor receptor 2 (TNFR2) has gained much research interest in recent years because of its potential pivotal role in autoimmune disease and cancer. However, its function in regulating different immune cells is not well understood. There is a need for well-characterized reagents to selectively modulate TNFR2 function, thereby enabling definition of TNFR2-dependent biology in human and mouse surrogate models. Here, we describe the generation, production, purification, and characterization of a panel of novel antibodies targeting mouse TNFR2. The antibodies display functional differences in binding affinity and potency to block TNFα. Furthermore, epitope binding showed that the anti-mTNFR2 antibodies target different domains on the TNFR2 protein, associated with varying capacity to enhance CD8+ T-cell activation and costimulation. Moreover, the anti-TNFR2 antibodies demonstrate binding to isolated splenic mouse Tregs ex vivo and activated CD8+ cells, reinforcing their potential use to establish TNFR2-dependent immune modulation in translational models of autoimmunity and cancer.

Bispecific antibodies targeting dual tumor-associated antigens in cancer therapy.

PURPOSE: Bispecific antibodies (BsAbs) have emerged as a leading drug class for cancer therapy and are becoming increasingly of interest for therapeutic applications. As of April 2020, over 123 BsAbs are under clinical evaluation for use in oncology (including the two marketed BsAbs Blinatumomab and Catumaxomab). The majority (82 of 123) of BsAbs under clinical evaluation can be categorized as bispecific immune cell engager whereas a second less well-discussed subclass of BsAbs targets two tumor-associated antigens (TAAs). In this review, we summarize the clinical development of dual TAAs targeting BsAbs and provide an overview of critical considerations when designing dual TAA targeting BsAbs. METHODS: Herein the relevant literature and clinical trials published in English until April 1st 2020 were searched using PubMed and ClinicalTrials.gov database. BsAbs were considered to be active in clinic if their clinical trials were not terminated, withdrawn or completed before 2018 without reporting results. Data missed by searching ClinicalTrials.gov was manually curated. RESULTS: Dual TAAs targeting BsAbs offer several advantages including increased tumor selectivity, potential to concurrently modulate two functional pathways in the tumor cell and may yield improved payload delivery. CONCLUSIONS: Dual TAAs targeting BsAbs represent a valuable class of biologics and early stage clinical studies have demonstrated promising anti-tumor efficacy in both hematologic malignancies and solid tumors.

Hsp70 and NF-kB Mediated Control of Innate Inflammatory Responses in a Canine Macrophage Cell Line.

The pathogenesis of many inflammatory diseases is associated with the uncontrolled activation of nuclear factor kappa B (NF-κB) in macrophages. Previous studies have shown that in various cell types, heat shock protein 70 (Hsp70) plays a crucial role in controlling NF-κB activity. So far, little is known about the role of Hsp70 in canine inflammatory processes. In this study we investigated the potential anti-inflammatory effects of Hsp70 in canine macrophages as well as the mechanisms underlying these effects. To this end, a canine macrophage cell line was stressed with arsenite, a chemical stressor, which upregulated Hsp70 expression as detected by flow cytometry and qPCR. A gene-edited version of this macrophage cell line lacking inducible Hsp70 was generated using CRISPR-Cas9 technology. To determine the effects of Hsp70 on macrophage inflammatory properties, arsenite-stressed wild-type and Hsp70 knockout macrophages were exposed to lipopolysaccharide (LPS), and the expression of the inflammatory cytokines IL-6, IL-1ß and tumor necrosis factor-α (TNF-α) and levels of phosphorylated NF-κB were determined by qPCR and Western Blotting, respectively. Our results show that non-toxic concentrations of arsenite induced Hsp70 expression in canine macrophages; Hsp70 upregulation significantly inhibited the LPS-induced expression of the pro-inflammatory mediators TNF-α and IL-6, as well as NF-κB activation in canine macrophages. Furthermore, the gene editing of inducible Hsp70 by CRISPR-Cas9-mediated gene editing neutralized this inhibitory effect of cell stress on NF-κB activation and pro-inflammatory cytokine expression. Collectively, our study reveals that Hsp70 may regulate inflammatory responses through NF-κB activation and cytokine expression in canine macrophages.

A novel efficient bispecific antibody format, combining a conventional antigen-binding fragment with a single domain antibody, avoids potential heavy-light chain mis-pairing.

Due to the technical innovations in generating bispecific antibodies (BsAbs) in recent years, BsAbs have become important reagents for diagnostic and therapeutic applications. However, the difficulty of producing a heterodimer consisting of two different arms with high yield and purity constituted a major limitation for their application in academic and clinical settings. Here, we describe a novel Fc-containing BsAb format (Fab × sdAb-Fc) composed of a conventional antigen-binding fragment (Fab), and a single domain antibody (sdAb), which avoids heavy-light chain mis-pairing during antibody assembly. In this study, the Fab x sdAb-Fc BsAbs were efficiently produced by three widely used heavy-heavy chain heterodimerization methods: Knobs-into-holes (KIH), Charge-pairs (CP) and controlled Fab-arm exchange (cFAE), respectively. The novel Fab x sdAb-Fc format provided a rapid and efficient strategy to generate BsAb with high purity and a unique possibility to further purify desired BsAbs from undesired antibodies based on molecular weight (MW). Compared to conventional BsAb formats, the advantages of Fab x sdAb-Fc format may thus provide a straightforward opportunity to apply bispecific antibody principles to research and development of novel targets and pathways in diseases such as cancer and autoimmunity.

Leucinostatin acts as a co-inducer for heat shock protein 70 in cultured canine retinal pigment epithelial cells.

Dysregulation of retinal pigment epithelium (RPE) cells is the main cause of a variety of ocular diseases. Potentially heat shock proteins, by preventing molecular and cellular damage and modulating inflammatory disease, may exert a protective role in eye disease. In particular, the inducible form of heat shock protein 70 (Hsp70) is widely upregulated in inflamed tissues, and in vivo upregulation of Hsp70 expression by HSP co-inducing compounds has been shown to be a potential therapeutic strategy for inflammatory diseases. In order to gain further understanding of the potential protective effects of Hsp70 in RPE cells, we developed a method for isolation and culture of canine RPE cells. Identity of RPE cells was confirmed by detection of its specific marker, RPE65, in qPCR, flow cytometry, and immunocytochemistry analysis. The ability of RPE cells to express Hsp70 upon experimental induction of cell stress, by arsenite, was analyzed by flow cytometry. Finally, in search of a potential Hsp70 co-inducer, we investigated whether the compound leucinostatin could enhance Hsp70 expression in stressed RPE cells. Canine RPE cells were isolated and cultured successfully. Purity of cells that strongly expressed RPE65 was over 90%. Arsenite-induced stress led to a time- and dose-dependent increase in Hsp70 expression in canine RPE cells in vitro. In addition, leucinostatin, which enhanced heat shock factor-1-induced transcription from the heat shock promoter in DNAJB1-luc-O23 reporter cell line, also enhanced Hsp70 expression in arsenite-stressed RPE cells, in a dose-dependent fashion. These findings demonstrate that leucinostatin can boost Hsp70 expression in canine RPE cells, most likely by activating heat shock factor-1, suggesting that leucinostatin might be applied as a new co-inducer for Hsp70 expression.

CD8(+) T cells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products.

CD8(+) T cells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide. Mass spectrometry identified a known H-2K(b) -presented linear epitope (LLO296-304 ) in the digests, as well as four spliced peptides that were trimmed by ERAP into peptides with in silico predicted H-2K(b) binding affinity. These spliced peptides, which displayed sequence similarity with LLO296-304 , bound to H-2K(b) molecules in cellular assays and one of the peptides was recognized by CD8(+) T cells of infected mice. This spliced epitope differed by one amino acid from LLO296-304 and double staining with LLO296-304 - and spliced peptide-folded MHC multimers showed that LLO296-304 and its spliced variant were recognized by the same CD8(+) T cells. Thus, PCPS multiplies the variety of peptides that is processed from an antigen and leads to the production of epitope variants that can be recognized by cross-reacting pathogen-specific CD8(+) T cells. Such mechanism may reduce the chances for pathogen immune evasion.

Surface coating of siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers: enhanced gene silencing and reduced adverse effects in vitro.

Cationic vectors have demonstrated the potential to facilitate intracellular delivery of therapeutic oligonucleotides. However, enhanced transfection efficiency is usually associated with adverse effects, which also proves to be a challenge for vectors based on cationic peptides. In this study a series of proteolytically stable palmitoylated α-peptide/ß-peptoid peptidomimetics with a systematically varied number of repeating lysine and homoarginine residues was shown to self-assemble with small interfering RNA (siRNA). The resulting well-defined nanocomplexes were coated with anionic lipids giving rise to net anionic liposomes. These complexes and the corresponding liposomes were optimized towards efficient gene silencing and low adverse effects. The optimal anionic liposomes mediated a high silencing effect, which was comparable to that of the control (cationic Lipofectamine 2000), and did not display any noticeable cytotoxicity and immunogenicity in vitro. In contrast, the corresponding nanocomplexes mediated a reduced silencing effect with a more narrow safety window. The surface coating with anionic lipid bilayers led to partial decomplexation of the siRNA-peptidomimetic nanocomplex core of the liposomes, which facilitated siRNA release. Additionally, the optimal anionic liposomes showed efficient intracellular uptake and endosomal escape. Therefore, these findings suggest that a more efficacious and safe formulation can be achieved by surface coating of the siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers.

Basophil-derived amphiregulin is essential for UVB irradiation-induced immune suppression.

UVB irradiation (290-320 nm) is used to treat skin diseases like psoriasis and atopic dermatitis, and is known to suppress contact hypersensitivity (CHS) reactions in mouse models. Regulatory T cells (Treg cells) have been shown to be responsible for this UVB-induced suppression of CHS. The epidermal growth factor (EGF)-like growth factor amphiregulin (AREG) engages EGFR on Treg cells and, in different disease models, it was shown that mast cell-derived AREG is essential for optimal Treg cell function in vivo. Here we determined whether AREG has a role in UVB-induced, Treg cell-mediated suppression of CHS reactions in the skin. Our data show that AREG is essential for UVB-induced CHS suppression. In contrast to the general assumption, however, mast cells were dispensable for UVB-induced immune suppression, whereas basophil-derived AREG was essential. These data reveal, to our knowledge, a previously unreported function for basophils in the homeostasis of immune responses in the skin. Basophils thus fulfill a dual function: they contribute to the initiation of effective type 2 immune responses and, by enhancing the suppressive capacity of local Treg cell populations, also to local immune regulation in the skin.

Amphiregulin enhances regulatory T cell-suppressive function via the epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) is known to be critically involved in tissue development and homeostasis as well as in the pathogenesis of cancer. Here we showed that Foxp3(+) regulatory T (Treg) cells express EGFR under inflammatory conditions. Stimulation with the EGF-like growth factor Amphiregulin (AREG) markedly enhanced Treg cell function in vitro, and in a colitis and tumor vaccination model we showed that AREG was critical for efficient Treg cell function in vivo. In addition, mast cell-derived AREG fully restored optimal Treg cell function. These findings reveal EGFR as a component in the regulation of local immune responses and establish a link between mast cells and Treg cells. Targeting of this immune regulatory mechanism may contribute to the therapeutic successes of EGFR-targeting treatments in cancer patients.

CCR2 defines a distinct population of NK cells and mediates their migration during influenza virus infection in mice.

Natural killer (NK) cells are innate lymphocytes that play an important role in control of viral infections. We recently showed that intranasal infection of mice with influenza virus induced the accumulation of NK cells in the airways. NK cells however did not proliferate in the airways or in the draining lymph node, but in the bone marrow mainly. As also monocyte-precursors undergo vigorous proliferation in the bone marrow (BM) during infections and then egress CCR2-dependently, we decided to determine the role of CCR2 in NK cell migration during intranasal influenza virus infection. We show that a unique population of NK cells in the BM expressed CCR2 and that monocyte chemotactic protein-1 (MCP-1), one of the CCR2 ligands, was produced in the airways of influenza virus infected mice. Analysis of BM chimeric mice reconstituted with a mix of wild-type (wt) and CCR2-deficient BM cells showed that upon influenza virus infection, a significantly lower proportion of CCR2-deficient than wt NK cells was recovered from the bronchoalveolar lavage (BAL). Taken together, our data demonstrate that during influenza virus infection a proportion of NK cells migrate in a CCR2-dependent fashion.

The bone marrow functions as the central site of proliferation for long-lived NK cells.

NK cells play an important role in the early defense against invading pathogens. Although it is well established that infection leads to a substantial, local increase in NK cell numbers, little is known about the mechanisms that trigger their proliferation and migration. In this study, we investigated the dynamics of NK cell responses after intranasal respiratory virus infection. We show that NK cell numbers increased in the airways after influenza virus infection but find no evidence of proliferation either at the site of infection or in the draining lymph nodes. Instead, we find that the bone marrow (BM) is the primary site of proliferation of both immature and mature NK cells during infection. Using an adoptive transfer model, we demonstrate that peripheral, long-lived and phenotypically mature NK cells migrate back to the BM and proliferate there, both homeostatically and in response to infection. Thus, the BM is not only a site of NK cell development but also an important site for proliferation of long-lived mature NK cells.

Identification of novel avian influenza virus derived CD8+ T-cell epitopes.

Avian influenza virus (AIV) infection is a continuing threat to both humans and poultry. Influenza virus specific CD8+ T cells are associated with protection against homologous and heterologous influenza strains. In contrast to what has been described for humans and mice, knowledge on epitope-specific CD8+ T cells in chickens is limited. Therefore, we set out to identify AIV-specific CD8+ T-cell epitopes. Epitope predictions based on anchor residues resulted in 33 candidate epitopes. MHC I inbred chickens were infected with a low pathogenic AIV strain and sacrificed at 5, 7, 10 and 14 days post infection (dpi). Lymphocytes isolated from lung, spleen and blood were stimulated ex vivo with AIV-specific pooled or individual peptides and the production of IFNγ was determined by ELIspot. This resulted in the identification of 12 MHC B12-restricted, 3 B4-restricted and 1 B19-restricted AIV- specific CD8+ T-cell epitopes. In conclusion, we have identified novel AIV-derived CD8+ T-cell epitopes for several inbred chicken strains. This knowledge can be used to study the role of CD8+ T cells against AIV infection in a natural host for influenza, and may be important for vaccine development.

Immunoproteasome-deficiency has no effects on NK cell education, but confers lymphocytes into targets for NK cells in infected wild-type mice.

Natural killer (NK) cells are part of the innate immune system and contribute to the eradication of virus infected cells and tumors. NK cells express inhibitory and activating receptors and their decision to kill a target cell is based on the balance of signals received through these receptors. MHC class I molecules are recognized by inhibitory receptors, and their presence during NK cell education influences the responsiveness of peripheral NK cells. We here demonstrate that mice with reduced MHC class I cell surface expression, due to deficiency of immunoproteasomes, have responsive NK cells in the periphery, indicating that the lower MHC class I levels do not alter NK cell education. Following adoptive transfer into wild-type (wt) recipients, immunoproteasome-deficient splenocytes are tolerated in naive but rejected in virus-infected recipients, in an NK cell dependent fashion. These results indicate that the relatively low MHC class I levels are sufficient to protect these cells from rejection by wt NK cells, but that this tolerance is broken in infection, inducing an NK cell-dependent rejection of immunoproteasome-deficient cells.

Proteasome immunosubunits protect against the development of CD8 T cell-mediated autoimmune diseases.

Exposure of cells to inflammatory cytokines induces the expression of three proteasome immunosubunits, two of which are encoded in the MHC class II region. The induced subunits replace their constitutive homologs in newly formed "so-called" immunoproteasomes. Immunosubunit incorporation enhances the proteasome's proteolytic activity and modifies the proteasome's cleavage-site preferences, which improves the generation of many MHC class I-presented peptides and shapes the fine specificity of pathogen-specific CD8 T cell responses. In this article, we report on a second effect of immunoproteasome formation on CD8 T cell responses. We show that mice deficient for the immunosubunits ß5i/low molecular mass polypeptide (LMP7) and ß2i/multicatalytic endopeptidase complex-like-1 develop early-stage multiorgan autoimmunity following irradiation and bone marrow transplantation. Disease symptoms are caused by CD8 T cells and are transferable into immunosubunit-deficient, RAG1-deficient mice. Moreover, using the human Type 1 Diabetes Genetics Consortium MHC dataset, we identified two single nucleotide polymorphisms within the ß5i/LMP7-encoding gene sequences, which were in strong linkage disequilibrium, as independent genetic risk factors for type 1 diabetes development in humans. Strikingly, these single nucleotide polymorphisms significantly enhanced the risk conferred by HLA haplotypes that were previously shown to predispose for type 1 diabetes. These data suggested that inflammation-induced immunosubunit expression in peripheral tissues constitutes a mechanism that prevents the development of CD8 T cell-mediated autoimmune diseases.

PA28 and the proteasome immunosubunits play a central and independent role in the production of MHC class I-binding peptides in vivo.

Proteasomes play a fundamental role in the processing of intracellular antigens into peptides that bind to MHC class I molecules for the presentation of CD8(+) T cells. Three IFN-γ-inducible catalytic proteasome (immuno)subunits as well as the IFN-γ-inducible proteasome activator PA28 dramatically accelerate the generation of a subset of MHC class I-presented antigenic peptides. To determine whether these IFN-γ-inducible proteasome components play a compounded role in antigen processing, we generated mice lacking both PA28 and immunosubunits ß5i/LMP7 and ß2i/MECL-1. Analyses of MHC class I cell-surface levels ex vivo demonstrated that PA28 deficiency reduced the production of MHC class I-binding peptides both in cells with and without immunosubunits, in the latter cells further decreasing an already diminished production of MHC ligands in the absence of immunoproteasomes. In contrast, the immunosubunits but not PA28 appeared to be of critical importance for the induction of CD8(+) T-cell responses to multiple dominant Influenza and Listeria-derived epitopes. Taken together, our data demonstrate that PA28 and the proteasome immunosubunits use fundamentally different mechanisms to enhance the supply of MHC class I-binding peptides; however, only the immunosubunit-imposed effects on proteolytic epitope processing appear to have substantial influence on the specificity of pathogen-specific CD8(+) T-cell responses.

Enumeration of cytotoxic CD8 T cells ex vivo during the response to Listeria monocytogenes infection.

Cytotoxicity is a key effector function of CD8 T cells. However, what proportion of antigen-specific CD8 T cells in vivo exert cytotoxic activity during a functional CD8 T-cell response to infection still remains unknown. We used the Lysispot assay to directly enumerate cytotoxic CD8 T cells from the spleen ex vivo during the immune response to infection with the intracellular bacterium Listeria monocytogenes. We demonstrate that not all antigen-responsive gamma interferon (IFN-gamma)-secreting T cells display cytotoxic activity. Most CD8 T cells detected at early time points of the response were cytotoxic. This percentage continuously declined during both the expansion and contraction phases to about 50% at the peak and to <10% of IFN-gamma-producing cells in the memory phase. As described for clonal expansion, this elaboration of a program of differentiation after an initial stimulus was not affected by antigen or CD4 help but, like proliferation, could be influenced by later reinfection. These data indicate that cytotoxic effector function during the response to infection is regulated independently from IFN-gamma secretion or expansion or contraction of the overall CD8 T-cell response.

The proteasome immunosubunit multicatalytic endopeptidase complex-like 1 is a T-cell-intrinsic factor influencing homeostatic expansion.

Homeostatic regulatory mechanisms maintain the constant ratios between different lymphocyte subsets in the secondary lymphoid organs. How this dynamic equilibrium is achieved, in particular following the clonal expansion and subsequent contraction of different cells after infection, remains poorly understood. Expression of the proteasome immunosubunits has been shown to influence not only major histocompatibility complex class I (MHC-I) antigen processing and thereby T-cell responses, but also the CD4/CD8 T-cell ratios in lymphoid organs. We examined the relationships between these different immunosubunit-mediated effects in mice of various proteasome subunit compositions during infection with Listeria monocytogenes. Mice that lacked the immunosubunit multicatalytic endopeptidase complex-like 1 (MECL-1) maintained enhanced CD4/CD8 T-cell ratios during infection, while MHC-I surface levels resembled those in wild-type (wt) mice. LMP7 gene-deficient mice, on the other hand, showed reduced MHC-I expression, while their splenic CD4/CD8 ratios were similar to those in wt mice. Remarkably, analysis of bone marrow-chimeric immunosubunit gene-deficient mice, reconstituted with a mixture of wt and LMP7- plus MECL-1-deficient bone marrow, revealed that the LMP7- plus MECL-1-deficient T-cell population maintained a higher CD4/CD8 T-cell ratio than the wt T-cell population before, during, and after infection and T-cell memory formation. Since in these mice the immunosubunit-positive and immunosubunit-negative T-cell populations were selected in the same thymus and expanded in the same lymphoid environments, our findings indicate that MECL-1 influences the homeostatic equilibrium between T-cell subsets, not through indirect extracellular signals, such as MHC-I expression or the cytokine milieu, but through direct effects on T-cell-intrinsic processes.