Jörg Reimann

MHC Class I–Restricted CTL Responses to Exogenous Antigens

A number of different modified exogenous protein antigens can efficiently prime CTL responses. The biological significance of this for priming of normal CTL responses is unknown. Processing of exogenous antigen for MHC-I presentation seems an attractive possibility to bypass the necessity that all foreign antigens have to be translated within the cytosol of professional APC in order to prime CTL responses. Many microbes have a select host cell specificity that does not include APC. They kill their host cells or severely disturb the sophisticated cellular functions that are required in APC (84xNorkin, L.C. Clin. Microbiol. Rev. 1995; 8: 293–315PubMedSee all References, 45xHaywood, A.M. J. Virol. 1994; 68: 1–5PubMedSee all References). Another scenario for a virus-specific CTL response is thus that infected cells disintegrate (possibly as apoptotic bodies) and exogenous antigen from them is transferred to APC for further processing in different cellular compartments.There are many unresolved issues regarding processing of exogenous antigens for MHC-I presentation and the priming of CTL responses. First, which are the most important uptake mechanisms? Second, is there a special (MHC-II-like) compartment for “endosomal” processing, or do all APC “leak” antigen into the cytosol and thereby into the classical MHC-I processing pathway? Third, in a proposed endosomal-like compartment, which are the proteolytic enzymes involved in processing? Do they generate a spectrum of MHC-I-bound peptides distinct from those generated in the cytosolic pathway? Finally, how do MHC-I molecules gain access to the endosomal-like compartment, and how are trimeric peptide MHC-I–β2-microglobulin complexes transported to the cell surface and their membrane expression regulated?If exogenous antigens are instrumental in priming normal CTL responses, processing in noncytosolic, vesicular compartments, distinct from the endogenous classical class I processing pathway, might be important. If so, it may be relevant to reevaluate the endogenous/MHC-I and exogenous/MHC-II processing dogma and instead classify pathways in functional terms. In CTL activation, induction (by exogenous antigen) and tar- get cell recognition (by endogenous antigen) by class I–restricted peptide presentation would result from different processing pathways. This is an important issue in terms of vaccine development, as these should primarily be expressed in the most optimal processing compartment. If exogenous antigens are of major importance in generating CTL responses, vaccines based on live, replicating vectors (and nucleic acids) induce their protective effect in a nonviable form. In this transfer process, from the infected host cell to the professional APC cell, there might be a lesson to be learned in terms of formulations for direct uptake into APC cells. The potent clinical effect of the first human recombinant vaccine licensed for clinical use, which was based on self-assembly of the hepatitis B virus small surface antigen into 22 nm subviral particles, might be a “lesson of nature” in this context (Schirmbeck et al. 1996xSchirmbeck, R, Bohm, W, and Reimann, J. Intervirology, in press. 1996; See all ReferencesSchirmbeck et al. 1996).

Commensal Gut Flora Drives the Expansion of Proinflammatory CD4 T Cells in the Colonic Lamina Propria under Normal and Inflammatory Conditions

We tested in B6 mice whether the local expansion of CD4 T cells producing proinflammatory cytokines including IL-17 (Th17 cells) in the colonic lamina propria (cLP) depends on the commensal microflora. High numbers of CD4 Th17 cells were found in the lamina propria of the ileum and colon but not the duodenum, jejunum, mesenteric lymph nodes, spleen, or liver of specific pathogen-free (SPF) mice. The microflora is required for the accumulation of cytokine (IL-17, IFN-γ, TNF-α, IL-10)-producing CD4 T cells in the cLP because only low numbers of cytokine-producing cLP CD4 T cells were found in syngeneic (age- and sex-matched) germfree mice. The fraction of cLP Th17 cells was higher in (type I and type II) IFN- but not IL-4- or IL-12p40-deficient SPF congenics. cLP CD4 Th17 cells produce IL-17 but not IFN-γ, TNF-α, IL-4, or IL-10. cLP CD4 Th17 cells accumulate locally in colitis induced by adoptive transfer of IFN-γ+/+ or IFN-γ−/− CD4 T cells into congenic SPF (but not germfree) RAG−/− hosts. In this colitis model, cLP CD4 T cells that “spontaneously” produce IL-17 progressively increase in number in the inflamed cLP, and increasing serum IL-17 levels appear as the disease progresses. Commensal bacteria-driven, local expansion of cLP CD4 Th17 cells may contribute to the pathogenesis of this inflammatory bowel disease.

IL-12/IL-18-Dependent IFN-γ Release by Murine Dendritic Cells

Dendritic cells (DC) develop in GM-CSF-stimulated cultures from murine bone marrow progenitors in serum-free (or low serum) medium. CD11c+ myeloid DC from 7-day cultures stimulated with TNF-α, IFN-α, IFN-γ, or LPS up-regulated surface expression of CD40 and CD86 costimulator and MHC class II molecules, did not up-regulate the low “spontaneous” release of IL-18, and did not release IFN-γ. Stimulation of in vitro-generated DC with exogenous IL-12 and IL-18 (but not with IL-4 or LPS plus IL-18) induced IFN-γ expression and release in 15–20% of the DC (detectable by FACS analyses or ELISA). Endogenous IL-12 p70 produced by DC in response to ligation of CD40 stimulated IFN-γ release when exogenous IL-18 was supplied. In vivo-generated, splenic CD8α+ and CD8α− DC (from immunocompetent and immunodeficient H-2d and H-2b mice) cultured with IL-12 and IL-18 released IFN-γ. The presence of LPS during the stimulation of DC with IL-18 plus endogenous (CD40 ligation) or exogenous IL-12 did not affect their IFN-γ release. In contrast, splenic DC pretreated in vitro or in vivo by LPS strikingly down-regulated IFN-γ release in response to stimulation by IL-18 and (endogenous or exogenous) IL-12. Hence, DC are a source of early IFN-γ generated in response to a cascade of cytokine- and/or cell-derived signals that can be positively and negatively regulated.

A rapid colorimetric assay for the determination of IL-2-producing helper T cell frequencies

Interleukin 2 (IL-2) activity is tested in conditioned media by assessing its ability to support proliferation of selected IL-2 dependent T cell lines, conventionally measured by [3H]thymidine incorporation. Here, we compare this [3H]thymidine uptake test for measuring IL-2 activity with a rapid and sensitive colorimetric method which is based on the ability of viable cells to cleave 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The sensitivity of the colorimetric method was dependent on the indicator cell line used, being greatest with the cytotoxic T cell line 16 (CTLL-16). The colorimetric method is at least as sensitive as [3H]thymidine uptake tests, does not rely on radioactivity, and is ideally suited to screen large numbers of individual samples for IL-2 activity. The latter point was demonstrated by calculating IL-2-producing helper T cell frequencies in heterogeneous murine lymphocyte populations: in this assay, splenic T cells were clonally expanded under limiting dilution conditions and supernatants conditioned by these in vitro growing T cell clones were tested for IL-2 activity with the colorimetric method. This allowed us to obtain reliable estimates of the frequency of progenitor cells of IL-2-producing T cell clones in various populations.

NKT Cells Provide Help for Dendritic Cell-Dependent Priming of MHC Class I-Restricted CD8+ T Cells In Vivo

Dendritic cells (DC) are potent APCs for naive T cells in vivo. This is evident by inducing T cell responses through adoptive DC transfer. Priming specific CTL responses in vivo often requires “help”. We study alternative sources of help in DC-dependent priming of MHC class I-restricted CTL. Priming an anti-viral CTL response in naive B6 mice by adoptive transfer of antigenic peptide-pulsed DC required CD4+ T cell help. CTL priming was facilitated by providing MHC class II-dependent specific help. Furthermore, transfers of MHC class II-deficient pulsed DC into naive, normal hosts, or DC transfers into naive, CD4+ T cell-depleted hosts primed CTL inefficiently. Pretreatment of DC with immune-stimulating oligodeoxynucleotides rendered them more efficient for CD4+ T cell-independent priming of CTL. DC copresenting a Kb-binding antigenic peptide and the CD1d-binding glycolipid α-galactosyl-ceramide efficiently primed CTL in a class II-independent way. To obtain NKT cell-dependent help in CTL priming, the same DC had to present both the peptide and the glycolipid. CTL priming by adoptive DC transfer was largely NK cell-dependent. The requirement for NK cells was only partially overcome by recruiting NKT cell help into DC-dependent CTL priming. NKT cells thus are potent helper cells for DC-dependent CTL priming.

Comparison between hepatitis B surface antigen (HBsAg) particles derived from mammalian cells (CHO) and yeast cells (Hansenula polymorpha): Composition, structure and immunogenicity

The composition, structure and immunogenicity of hepatitis B surface antigen (HBsAg) particles derived from Chinese hamster ovary (CHO) cells and from cells of the yeast Hansenula polymorpha were compared. The particles were similar in size distribution (mean 20-33 nm), in shape (spherical), in gross composition (protein to lipid weight ratio of 60:40), and in types of lipids (phospholipids > > sterols = sterol esters = triacylglycerols). Differences related to genetic engineering and type of host cells were found in peptide and lipid compositions. CHO-HBsAg has three peptides: S, M and L, each in two forms of glycosylation, while the Hansenula-HBsAg has only the nonglycosylated S peptide. The electrical surface potential at the lipid/water interface of HBsAg derived from Hansenula is more negative than that of HBsAg derived from CHO, which was close to neutrality. Although the numbers of cysteine residues (all in the S peptides) are identical (14), 11 of them are free thiols in the CHO-HBsAg, compared with three to four in the Hansenula-HBsAg. The fact that 85% of the phospholipids are hydrolyzed by phospholipase C and that all the aminophospholipids react with trinitrobenzenesulfate suggests that the particles derived from both cell types are either leaky vesicles or have a lipoprotein-like structure. Subcutaneous injection into mice of fluorescein-isothiocyanate-labeled HBsAg particles from both sources resulted in their accumulation in the marginal sinus of lymph nodes. The humoral responses to subcutaneous injection into mice of CHO- and Hansenula-HBsAg were similar: however, the cytotoxic T lymphocyte response to CHO-HBsAg was lower.

Alternative pathways for processing exogenous and endogenous antigens that can generate peptides for MHC class I-restricted presentation.

Summary: The concept of distinct endogenous and exogenous pathways for generating peptides for MHC‐I and MHC‐II‐restricted presemation to CD4+ or CD8+ T cells fits well with the bulk of experimental data. Nevertheless, evidence is emerging for alternative processing pathways that generate peptides for MHC‐I‐restricted presentation. Using a well characterized, particulate viral antigen of prominent medical importance (the hepatitis B surface antigen), we summarize our evidence that the efficient, endolysosomal processing of exogenous antigens can lead to peptide‐loaded MHC‐I molecules. In addition, we describe evidence for endolysosomal processing of mutant, stress protein‐bound, endogenous antigens that liberate peptides binding to (and presented by) MHC‐I molecules. The putative biological role of alternative processing of antigens generating cytotoxic T lymphocyte‐stimulating epitopes is discussed.

Priming Th1 Immunity to Viral Core Particles Is Facilitated by Trace Amounts of RNA Bound to Its Arginine-Rich Domain

Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes. When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity. HBcAg contains 5–20 ng RNA/μg protein while nucleotide binding to HBeAg is not detectable. Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity. HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-γ release by nonimmune spleen cells. The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20–100 μg/mouse) are codelivered with the Ag. Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag. Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency. Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 μg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 μg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity. Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine. Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.

The Role of Up-Regulated Serine Proteases and Matrix Metalloproteinases in the Pathogenesis of a Murine Model of Colitis

Proteinases are important at several phases of physiological and pathological inflammation, mediating cellular infiltration, cytokine activation, tissue damage, remodeling, and repair. However, little is known of their role in the pathogenesis of inflammatory bowel disease. The aim of this study was to assess the role of tissue proteases in a mouse model of colitis. Proteolytic activity was analyzed, using gel and in situ zymography, in colonic tissues from severe combined immunodeficient mice with colitis induced by transfer of CD4(+) T lymphocytes. Serine proteinase levels increased in colitic tissue, with major species of 23 kd, 30 kd, and 45 kd. Co-migration and inhibition studies indicated that the 23-kd proteinase was pancreatic trypsin and that the 30-kd species was neutrophil elastase. Matrix metalloproteinase (MMP)-9 expression, and MMP-2 and MMP-9 activation, was elevated in colitic tissues. Proteinase levels followed a decreasing concentration gradient from proximal to distal colon. Proteolysis was localized to infiltrating leukocytes in diseased severe combined immunodeficient mice. Transmural inflammation was associated with serine proteinase and MMP activity in overlying epithelium and with marked subepithelial proteolytic activity. The results demonstrate a clear elevation in the levels and activation of proteases in colitis, potentially contributing to disease progression through loss of epithelial barrier function.

Enteric bacterial antigens activate CD4+ T cells from scid mice with inflammatory bowel disease

Scid mice transplanted with CD4+ T cells from congenic donor mice develop a chronic and lethal inflammatory bowel disease (IBD) 2–3 months post‐transplantation. In the present study we have investigated the response of CD4+ T cells from scid mice with colitis against fecal extracts. Our results show that in contrast to CD4+ T cells from normal BALB/c mice, CD4+ T cells from scid mice with colitis proliferate strongly in response to antigen‐presenting cells (APC) pulsed with fecal extracts. The IBD‐associated T cells did not respond to either extracts from food antigens or fecal extracts from germ‐free mice, which indicates that they recognize bacterial antigens in the fecal extracts. CD4+ T cells isolated from the colonic lamina propria of scid mice 3 weeks post transplantation also responded vigorously to fecal extracts, demonstrating that reactive CD4+ T cells are present in the gut mucosa of transplanted scid mice prior to clinical manifestations of IBD. CD4+ T cells activated by fecal extracts produced high amounts of IL‐2 and IFN‐γ, intermediate amounts of IL‐4 and low amounts of IL‐10, consistent with a Th1 profile. The proliferative reactivity towards fecal extracts was restricted by MHC class II molecules and dependent on antigen processing, as the response could be blocked by anti‐MHC class II antibodies or a short fixation of the APC. This study demonstrates that class II‐restricted CD4+ Th1 cells, which recognize enteric bacterial antigens, infiltrate the gut mucosa and spleen of transplanted scid mice prior to and during the course of colitis.