Karoline W. Schjetne

Novel APC-like properties of human NK cells directly regulate T cell activation

Initiation of the adaptive immune response is dependent on the priming of naive T cells by APCs. Proteomic analysis of unactivated and activated human NK cell membrane-enriched fractions demonstrated that activated NK cells can efficiently stimulate T cells, since they upregulate MHC class II molecules and multiple ligands for TCR costimulatory molecules. Furthermore, by manipulating antigen administration, we show that NK cells possess multiple independent unique pathways for antigen uptake. These results highlight NK cell-mediated cytotoxicity and specific ligand recognition by cell surface-activating receptors on NK cells as unique mechanisms for antigen capturing and presentation. In addition, we analyzed the T cell-activating potential of human NK cells derived from different clinical conditions, such as inflamed tonsils and noninfected and CMV-infected uterine decidual samples, and from transporter-associated processing antigen 2-deficient patients. This in vivo analysis revealed that proinflammatory, but not immune-suppressive, microenvironmental requirements can selectively dictate upregulation of T cell-activating molecules on NK cells. Taken together, these observations offer new and unexpected insights into the direct interactions between NK and T cells and suggest novel APC-like activating functions for human NK cells.

Cutting Edge: Link Between Innate and Adaptive Immunity: Toll-Like Receptor 2 Internalizes Antigen for Presentation to CD4+ T Cells and Could Be an Efficient Vaccine Target

An ideal vaccine for induction of CD4+ T cell responses should induce local inflammation, maturation of APC, and peptide loading of MHC class II molecules. Ligation of Toll-like receptor (TLR) 2 provides the first two of these three criteria. We have studied whether targeting of TLR2 results in loading of MHC class II molecules and enhancement of CD4+ T cell responses. To dissociate MHC class II presentation from APC maturation, we have used an antagonistic, mouse anti-human TLR2 mAb (TL2.1) as ligand and measured proliferation of a mouse Cκ-specific human CD4+ T cell clone. TL2.1 mAb was 100-1000 times more efficiently presented by APC compared with isotype-matched control mAb. Moreover, TL2.1 mAb was internalized into endosomes and processed by the conventional MHC class II pathway. This novel function of TLR2 represents a link between innate and adaptive immunity and indicates that TLR2 could be a promising target for vaccines.

Delivery of Antigen to CD40 Induces Protective Immune Responses against Tumors

Ligation of CD40 induces maturation of dendritic cells (DC) and could be a useful target for vaccines. In this study, we have constructed two types of Ab-based vaccine constructs that target mouse CD40. One type is a recombinant Ab with V regions specific for CD40 and has defined T cell epitopes inserted into its C region. The other type is a homodimer, each chain of which is composed of a targeting unit (single-chain fragment variable targeting CD40), a dimerization motif, and an antigenic unit. Such proteins bound CD40, stimulated maturation of DC, and enhanced primary and memory T cell responses. When delivered i.m. as naked DNA followed by electroporation, the vaccines induced T cell responses against MHC class II-restricted epitopes, Ab responses, and protection in two tumor models (myeloma and lymphoma). Two factors apparently contributed to these results: 1) agonistic ligation of CD40 and induction of DC maturation, and 2) delivery of Ag to APC and presentation on MHC class II molecules. These results highlight the importance of agonistic targeting of Ag to CD40 for induction of long-lasting and protective immune responses.

Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement.

The manufacture of clinical grade cellular products for adoptive immunotherapy requires ex vivo culture and expansion of human T cells. One of the key components in manufacturing of T cell therapies is human serum (HS) or fetal bovine serum (FBS), which can potentially expose immunotherapy recipient to adventitious infectious pathogens and are thus considered as non‐cGMP compliant for adoptive therapy. Here we describe a novel xeno‐free serum replacement (SR) with defined components that can be reproducibly used for the production of clinical grade T‐cell therapies in combination with several different cell culture media. Dynabeads CD3/CD28 Cell Therapy System (CTS)‐activated or antigen‐specific T cells expanded using the xeno‐free SR, CTS Immune Cell SR, showed comparable growth kinetics observed with cell culture media supplemented with HS or FBS. Importantly the xeno‐free SR supplemented medium supported the optimal expansion of T cells specific for subdominant tumour‐associated antigens and promoted expansion of T cells with central memory T‐cell phenotype, which is favourable for in vivo survival and persistence following adoptive transfer. Furthermore, T cells expanded using xeno‐free SR medium were highly amenable to lentivirus‐mediated gene transduction for potential application for gene‐modified T cells. Taken together, the CTS Immune Cell SR provides a novel platform strategy for the manufacture of clinical grade adoptive cellular therapies.

Human CD14 is an efficient target for recombinant immunoglobulin vaccine constructs that deliver T cell epitopes.

It has been shown in the mouse that recombinant immunoglobulin (Ig) molecules with T cell epitopes inserted into the constant domain (Troybodies) can target antigen‐presenting cells (APC) for efficient delivery of T cell epitopes. Here, we have extended the Troybody concept to human applications. Moreover, we show that a receptor of innate immunity, CD14, which is a part of the lipopolysaccharide receptor complex on monocyte APC, is an efficient target. For construction of CD14‐specific Troybodies, we used rearranged variable(diversity)joining regions cloned from the 3C10 mouse B cell hybridoma. As a model T cell epitope, amino acids 40–48 of mouse Cκ, presented on human leukocyte antigen‐DR4, were inserted into a loop connecting β‐strands in CH1 of human γ3. In the presence of monocytes, CD14‐specific Troybodies were >100 times as efficient as a nontargeting control antibody (Ab) at stimulating Cκ40–48‐specific/DR4‐restricted T cells. Presentation was dependent on the conventional processing pathway for presentation on major histocompatibility complex (MHC) class II molecules. Enhanced presentation of the Cκ epitope was most likely a result of increased loading of MHC class II molecules, as the CD14‐specific monoclonal Ab 3C10 did not induce maturation of the APC. The results show that CD14, a receptor of innate immunity, may be a promising target of recombinant Ig‐based vaccines for elicitation of T cell responses in humans.

Induction of central T cell tolerance: Recombinant antibodies deliver peptides for deletion of antigen‐specific CD4+8+ thymocytes

In order to prevent or ameliorate autoimmune disease, it would be desirable to induce central tolerance to peripheral self‐antigens. We have investigated whether recombinant antibodies (Ab) that deliver T cell epitopes to antigen‐presenting cells (APC) in the thymus can be used to induce thymocyte deletion. Troybodies are recombinant Ab with V regions specific for APC surface molecules that have T cell epitopes genetically introduced in their C domains. When MHC class II‐specific Troybodies with the λ2315 T cell epitope were injected into λ2315‐specific TCR transgenic mice, a profound deletion of CD4+8+ thymocytes was observed. MHC class II‐specific Troybodies were 10–100‐fold more efficient than non‐targeting peptide Ab, and 500‐fold more efficient than synthetic peptide at inducing deletion. Similar findings were observed when MHC class II‐specific Troybodies with the OVA323–339 T cell epitope were injected into OVA‐specific TCR transgenic mice. Although deletion was transient after a single injection, newborn mice repeatedly injected with MHC class II‐specific Troybodies for 4 weeks, had reduced antigen‐specific T cells in peripheral lymphoid tissues and reduced T cell responses. These experiments suggest that Troybodies constructed to target specifically thymic APC could be useful tools for induction and maintenance of central T cell tolerance in autoimmune diseases.

Naive Idiotope-Specific B and T Cells Collaborate Efficiently in the Absence of Dendritic Cells

Anti-idiotope (anti-Id) Abs have a role in therapy against B cell lymphomas, as inhibitors of pathogenic autoantibodies, and as surrogate Ags for immunization. Despite these observations, the mechanism by which Id+ Ig generates anti-Id Abs is essentially unknown. To address this issue, we generated a double knock-in mouse that expresses V regions of a somatically mutated anti-Id mAb with intermediate affinity (affinity constant [Ka] = 0.77 × 107 M−1) for the myeloma protein M315. The anti-Id mice have normal peripheral B cell populations, and allelic exclusion is efficient. Anti-Id B cells from BCR knock-in mice, together with Id-specific CD4+ T cells from previously established TCR-transgenic mice, enabled us to study Id-specific T cell–B cell collaboration by dilution of transferred cells into syngeneic BALB/c recipients. We show that previously unstimulated (naive) Id-specific B and T cells collaborate efficiently in vivo, even at low frequencies and in the presence of low amounts of Id+ Ig, resulting in germinal center formation, plasma cell development, and secretion of isotype-switched anti-Id Abs. We further demonstrate that Id-specific T cell–B cell collaboration occurs readily in the absence of adjuvant and is not dependent on Id-presentation by dendritic cells. The results underscore the potency of anti-Id B cells in MHC class II–restricted presentation of Id+ Ig and suggest that Id-specific T cell–B cell collaboration is of physiological relevance.

Recombinant antibodies for delivery of antigen : a single loop between β-strands in the constant region can accommodate long, complex and tandem T cell epitopes

Recombinant antibodies are increasingly used for efficient delivery of T cell epitopes to antigen-presenting cells (APCs), both for vaccination purposes and for immune modulation. We have previously shown that recombinant antibodies can accommodate single T cell epitopes inserted into loops between beta-strands in constant (C) domains. Such recombinant antibodies have in addition been equipped with variable regions that target APCs for increased delivery of C region T cell epitopes. We here show that loop 6 (loop FG) in C(H)1 of human gamma 3 can be exchanged with (i) long T cell epitopes up to 37 amino acids, (ii) epitopes with complex secondary structure such as gluten epitopes with a type II polyproline helical confirmation and (iii) two tandemly linked T cell epitopes. T cell responses increased with T cell epitope elongation, presumably due to a positive influence of flanking residues. Recombinant antibodies targeted to either CD14 on monocytes or HLA-DP on monocytes and dendritic cells gave similar results and were 2-4 logs more efficient at stimulating human T cells than were non-targeted controls. Thus, single loops in C regions of recombinant antibodies seem versatile and may be used for delivery of lengthy, complex and multiple T cell epitopes to human APCs.