CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.

Multiple clinical trials have shown the efficacy of adoptively transferred allogeneic antigen-specific T cells for the treatment of viral infections and relapsed hematologic malignancies. In contrast, the therapeutic potential of autologous antigen-specific T cells has yet to be established since it has been technically difficult to generate sufficient numbers of these T cells, ex vivo. A major obstacle to the success of this objective derives from our inability to simply and rapidly isolate and/or expand large numbers of highly efficient antigen presenting cells (APCs) for repetitive stimulations of antigen-specific T cells in vitro. We show that autologous CD40-activated B cells represent a readily available source of highly efficient APC that appear to have several important advantages over other APCs for ex vivo T cell expansion including: (a) methodological simplicity necessary to generate continuously large numbers of APCs from just 50 cm3 of peripheral blood without loss of APC function; (b) capacity to induce high peak T cell proliferation and interferon-gamma production without IL-10 production; (c) ease in cryopreservation; and (d) markedly reduced cost. We, therefore, contend that CD40-activated B cells are an alternative source of highly efficient APCs with which to generate antigen-specific T cells ex vivo for autologous adoptive immunotherapy.     

Robust expansion of viral antigen-specific CD4+ and CD8+ T cells for adoptive T cell therapy using gene-modified activated T cells as antigen presenting cells

Cytomegalovirus (CMV) reactivation after stem cell transplantation can be treated with CMV-specific T cells, but current in vitro techniques using dendritic cells as antigen-presenting cells are time-consuming and expensive. To simplify the production of clinical grade CMV-specific T cells, we evaluated gene-modified activated T cells [antigen presenting T cells (T-APCs)] as a reliable and easily produced source of APCs to boost CD4+ and CD8+ T-cell responses against the immunodominant CMV antigen pp65. T-APCs expressing the full-length immunodominant CMV pp65 gene were used to stimulate the expansion of autologous T cells. After 10 to 14 days, the T cell lines were tested for antigen specificity by using the flow cytometric intracellular detection of interferon-gamma after stimulation for 6 hours with a pp65 peptide library of 15-mers, overlapping by 11 amino acids. Under optimal conditions, this technique induced a median 766-fold and a 652-fold expansion of pp65-specific CD4+ and CD8+ responder cells, respectively, in 15 T cell lines. In 13 of 15 T cell lines, over 10 antigen-specific CD4+ plus CD8+ T cells were generated starting with only 5x10 peripheral blood mononuclear cells, representing an over 3-log increase. These data indicate that T-APCs efficiently boost pp65-specific CD4+ and CD8+ T cell numbers to clinically useful levels. The approach has the advantage of using a single leukocyte collection from the donor to generate large numbers of CMV-specific T cells within a total 3-week culture period using only one stimulation of antigen.     

Correction: CpG incorporated DNA microparticles for elevated immune stimulation for antigen presenting cells

Correction for ‘CpG incorporated DNA microparticles for elevated immune stimulation for antigen presenting cells’ by Heejung Jung et al., RSC Adv., 2018, 8, 6608–6615.

In the published article there was an error in the primer DNA (22 nt) sequence in

CpG incorporated DNA microparticles for elevated immune stimulation for antigen presenting cells

As emerging evidence supports the immune stimulating capability of the CpG oligodeoxynucleotides (ODN), CpG-based adjuvants have been widely used. For efficient induction of immune responses, current issues affecting the use of nucleic acid-based adjuvants, e.g. stability in physiological conditions, delivery to immune cells, and successful release within the phagolysosome, should be addressed. Here, we present CpG-based DNA microparticles (DNA-MPs) fabricated by complementary rolling circle amplification (cRCA) as adjuvants for enhancing immune response and production of selective antibody production. Using cRCA method, the sizes of CpG-based DNA-MPs were finely controlled (0.5 and 1 μm) with superior and provided mismatched single stranded form of CpG ODN region for specific cleavage site by DNase II within the phagolysosome. Fabricated CpG-based 1 μm DNA-MPs (DNA-MP-1.0) were successfully internalized into primary macrophages and macrophage cell line (RAW264.7 cells), and elicited superior cytokine production e.g. TNF-α and IL-6, compared to conventional CpG ODNs. After in vivo administration of DNA-MP-1.0 with model antigen ovalbumin (OVA), significantly elevated OVA-specific antibody production was observed. With this in mind, DNA-MP-1.0 could serve as a novel type of adjuvant for the activation of macrophages and the following production of selective antibodies without any noticeable toxicity in vitro and in vivo.

As emerging evidence supports the immune stimulating capability of the CpG oligodeoxynucleotides (ODN), CpG-based adjuvants have been widely used.     

Effect of antigen stimulation on antigen-specific IgE-plaque-forming cells from peripheral-blood lymphocytes of atopics

In the present study, it was shown that allergen challenge in vitro produced an increase in the number of antigen-specific IgE-plaque-forming cells of peripheral-blood lymphocytes from grass- or ragweed-allergic patients. Thus, the blood lymphocytes of all twelve (four rye grass I and eight AgE) sensitive donors responded whereas the blood lymphocytes of five non-atopic controls were unresponsive to antigen challenge. Allergen challenge doses of 10(-10)-10(-12) g/ml were found to give the greatest number of plaque-forming cells whereas the number of plaque-forming cells at challenging doses between 10(-9) and 10(-7) g/ml were either the same or less than those obtained with unchallenged cells. The results are discussed as to whether this in vitro model system represents in vivo response to allergen of the allergic patient.     

The ontogeny of antigen-specific T cells

Although the fetal lamb is unable to form circulating antiovalbumin antibodies until about 120 days of gestation, ovalbumin-specific helper T cells can be stimulated to function at an earlier age. This would suggest that the critical event responsible for the precise sequential maturation of immunologic competence to different antigens at different developmental stages is not the first appearance of specific receptors on immunocytes. Alternative explanations of this phenomenon are discussed.     

Rapid re-expression of retrovirally introduced versus endogenous TCRs in engineered T cells after antigen-specific stimulation

To broaden the applicability of cellular immunotherapy, adoptive transfer of T-cell receptor (TCR) transferred T cells may be an attractive strategy. Using this approach, high numbers of defined antigen-specific T cells can be engineered. As the introduced TCR has to compete for cell surface expression with the endogenous TCR, the introduced TCR chains are under control of a strong viral promotor, which, in contrast to the endogenous promotor, is constitutively active. We examined whether this difference in regulation would result in differences in TCR internalization and re-expression of the introduced and endogenous TCR on dual TCR engineered T cells and the antigen-responsiveness of both the TCRs. We showed comparable TCR downregulation of TCRs expressed under regulation of a retroviral promotor or the endogenous promotor. However, the introduced TCRs were rapidly re-expressed on the cell surface after TCR stimulation. Despite rapid re-expression of the introduced TCR, T cells exerted similar antigen-sensitivity compared with control T cells, showing that cell mechanisms other than TCR cell surface expression are involved in antigen-sensitivity directly after antigen-specific stimulation. These results showed that TCR transduced T cells are functionally not different from nontransduced T cells and can potentially be used as an effective treatment strategy.     

Polyclonal stimulation of resting B lymphocytes by antigen-specific T lymphocytes

Resting B lymphocytes are activated, proliferate, and differentiate into antibody-secreting cells when cultured with long-term lines of major histocompatibility complex (MHC)-restricted, antigen-specific T cell in the presence of the antigen for which the T cells are specific. Under optimal conditions, essentially all B cells are activated and approximately 35% enter S phase in the absence of antigens for which the B cells are specific. Activation and proliferation are observed in cells from both normal mice and mice with the xid-determined immune defect. Highly purified B cells bearing Ia molecules for which the T cells are "cospecific" can present antigen to T cells with the resulting T cell stimulation leading to the activation and proliferation of the antigen-presenting B cells. However, B cells that do not bear Ia molecules for which the T cells are cospecific are also activated and proliferate if antigen and a source of antigen-presenting B cells or macrophage-rich cells of proper histocompatibility type are present. Thus, resting B cells, both normal and "xid", can be activated by non-MHC restricted factors without receptor cross-linkage. Experiments are presented that support the concept that local production and action of such unrestricted activating factors may be responsible for the MHC-restriction of T cell-B cell interaction seen in many circumstances.     

A requirement for antigen-specific helper T cells in the generation of cytotoxic T cells from thymocyte precursors

Thymocytes cultured with irradiated, allogeneic stimulator cells yield no cytotoxic effector cells after a period in culture. If, however, a population of irradiated spleen cells syngeneic to the responder cells are added to these cultures, cytotoxicity is generated. The helper activity present in the irradiated syngeneic spleen cells was found to be mediated by a cell bearing theta antigens. Furthermore, it was found to be antigen specific; helper cells which were tolerant of the stimulator cell antigens were unable to help the thymocyte responder cells, although these tolerant cells did contain helpers specific for a third party antigen. These experiments are consistent with a requirement for associative recognition of linked determinants in the induction of killer precursors which is thus strictly analogous to the induction of B-cell precursors via collaboration with helper T cells. In more extensive studies, it was found that histoincompatible helper cells (H-2b, H-2p, H-2q) were able to help a cytotoxic T cell (H-2k) response to a third party stimulator cell antigen (H-2d); that is, the helper T cells which interact with cytotoxic T-cell precursors are not strain specific. It seems likely that the histocompatible helper cells induce killer precursors in an antigen-specific cooperation event similar or identical to normal syngeneic cooperation.     

Characterization of apoptosis-resistant antigen-specific T cells in vivo

Clonal deletion via activation-induced apoptosis (AIA) of antigen- specific T cells (ASTC) plays a very important role in the induction of peripheral tolerance. However, none of the studies performed so far has shown a complete deletion of ASTC, a small population always persisting in the periphery. The mechanism by which this small population of ASTC escapes AIA has not been determined. Since the existence of these ASTC may influence the outcome of autoimmune diseases and long-term graft survival, we have characterized the properties of these residual ASTC in vivo with the objective of determining mechanisms that may contribute to their persistence. It was found that the resistance of the residual ASTC to AIA is not due to lack of activation or Fas/Fas-L expression. Compared to those susceptible to AIA, the residual ASTC express a high level of Th2-type cytokines that may help them to escape from AIA. Furthermore, they are able to suppress proliferation of other ASTC, suggesting they may, in fact, prolong tolerance in vivo.     

Nylon adherent antigen-specific rosette-forming T cells.

Shortly after intravenous immunization of mice with heterologous erythrocytes (RBC) antigen-specific Thy 1+ cells which form rosettes with the immunizing RBC (thymic-derived lymphocytes-forming rosettes [T-RFC]) appear in the spleen. These T-RFC are much less stable than Thy 1- RFC (non-thymic-derived [B-RFC]) although most if not all of both classes of RFC adhere to nylon. T-RFC are induced with low doses of antigen (which fail to induce B-RFC) and are inhibited by higher antigen doses which are optimal for induction of B-RFC. Pretreatment of mice with cyclophosphamide prevents the high dose inhibition of T-RFC. Although there are many parallels between the production of T-RFC and delayed-type hypersensitivity (DTH) it is unlikely that the T-RFC are essential for DTH reactions since DTH can be transferred with cells which pass through nylon, and such cells are almost totally depleted of T-RFC. Thus immunization can lead to the production of large numbers of antigen-specific T-RFC whose functional role in the immune response is unknown. However, the characteristics of the T-RFC suggest that they may play an important role in amplification of suppressor cell activity.     

An antigen-specific pathway for CD8 T cells across the blood-brain barrier

CD8 T cells are nature's foremost defense in encephalitis and brain tumors. Antigen-specific CD8 T cells need to enter the brain to exert their beneficial effects. On the other hand, traffic of CD8 T cells specific for neural antigen may trigger autoimmune diseases like multiple sclerosis. T cell traffic into the central nervous system is thought to occur when activated T cells cross the blood-brain barrier (BBB) regardless of their antigen specificity, but studies have focused on CD4 T cells. Here, we show that selective traffic of antigen-specific CD8 T cells into the brain occurs in vivo and is dependent on luminal expression of major histocompatibility complex (MHC) class I by cerebral endothelium. After intracerebral antigen injection, using a minimally invasive technique, transgenic CD8 T cells only infiltrated the brain when and where their cognate antigen was present. This was independent of antigen presentation by perivascular macrophages. Marked reduction of antigen-specific CD8 T cell infiltration was observed after intravenous injection of blocking anti-MHC class I antibody. These results expose a hitherto unappreciated route by which CD8 T cells home onto their cognate antigen behind the BBB: luminal MHC class I antigen presentation by cerebral endothelium to circulating CD8 T cells. This has implications for a variety of diseases in which antigen-specific CD8 T cell traffic into the brain is a beneficial or deleterious feature.     

Viral vectors for gene transfer into antigen presenting cells

Crucial insights for vaccine development have come from examining how the immune system responds to antimicrobial vaccines, as well as to viral vectors employed for gene therapy. The effectiveness of a vaccine depends upon both the method of antigen delivery and the presentation of antigen to lymphocytes. Much focus has turned to delivering antigens to dendritic cells, to promote clinically beneficial T- and B-cell responses. Recombinant viral vectors represent a powerful vehicle to deliver genes encoding microbial- or tumor-derived antigens to generate clinically beneficial immunity. Dendritic cell-based and viral vector-based vaccines are currently being evaluated in clinical trials as a means of inducing antitumor immunity.     

Selective modification of antigen-specific T cells by RNA electroporation

It has been observed that the efficient transfection of T cells by RNA electroporation requires prior activation of T cells with mitogens or by anti-CD3 antibody stimulation. We hypothesized that this requirement for T cell activation could be leveraged to express marker genes within activated T cells responding to antigen-pulsed dendritic cells and allow for the selective enrichment and modification of antigen-specific T cells. Using electroporation of mRNA encoding green fluorescent protein as a marker gene, we demonstrate that RNA electroporation can efficiently allow for the separation of cytomegalovirus-specific CD8+ and CD4+ T cells from bulk culture responding to cytomegalovirus pp65 antigen-pulsed dendritic cells. Furthermore, we demonstrate that cytomegalovirus-specific T cells can be functionally modified by RNA transfection of the C-X-C chemokine receptor, CXCR2, to migrate efficiently toward a variety of CXCR2-specific chemokines in vitro and in vivo. These studies demonstrate the utility of RNA transfection as a simple method by which to purify and selectively modify the function of antigen-specific T cells for use in adoptive immunotherapy, and importantly provide evidence that transient expression of proteins by RNA transfection is an efficient means of modulating the in vivo function of activated T cells.     

Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells

Tumor-promoted constraints negatively affect cytotoxic T lymphocyte (CTL) trafficking to the tumor core and, as a result, inhibit tumor killing. The production of reactive nitrogen species (RNS) within the tumor microenvironment has been reported in mouse and human cancers. We describe a novel RNS-dependent posttranslational modification of chemokines that has a profound impact on leukocyte recruitment to mouse and human tumors. Intratumoral RNS production induces CCL2 chemokine nitration and hinders T cell infiltration, resulting in the trapping of tumor-specific T cells in the stroma that surrounds cancer cells. Preconditioning of the tumor microenvironment with novel drugs that inhibit CCL2 modification facilitates CTL invasion of the tumor, suggesting that these drugs may be effective in cancer immunotherapy. Our results unveil an unexpected mechanism of tumor evasion and introduce new avenues for cancer immunotherapy.     

Expression of lymphotoxin-alphabeta on antigen-specific T cells is required for DC function

During an immune response, activated antigen (Ag)-specific T cells condition dendritic cells (DCs) to enhance DC function and survival within the inflamed draining lymph node (LN). It has been difficult to ascertain the role of the tumor necrosis factor (TNF) superfamily member lymphotoxin-alphabeta (LTalphabeta) in this process because signaling through the LTbeta-receptor (LTbetaR) controls multiple aspects of lymphoid tissue organization. To resolve this, we have used an in vivo system where the expression of TNF family ligands is manipulated only on the Ag-specific T cells that interact with and condition Ag-bearing DCs. We report that LTalphabeta is a critical participant required for optimal DC function, independent of its described role in maintaining lymphoid tissue organization. In the absence of LTalphabeta or CD40L on Ag-specific T cells, DC dysfunction could be rescued in vivo via CD40 or LTbetaR stimulation, respectively, suggesting that these two pathways cooperate for optimal DC conditioning.     

Estimating the precursor frequency of naive antigen-specific CD8 T cells

The constraint of fitting a diverse repertoire of antigen specificities in a limited total population of lymphocytes results in the frequency of naive cells specific for any given antigen (defined as the precursor frequency) being below the limit of detection by direct measurement. We have estimated this precursor frequency by titrating a known quantity of antigen-specific cells into naive recipients. Adoptive transfer of naive antigen-specific T cell receptor transgenic cells into syngeneic nontransgenic recipients, followed by stimulation with specific antigen, results in activation and expansion of both donor and endogenous antigen-specific cells in a dose-dependent manner. The precursor frequency is equal to the number of transferred cells when the transgenic and endogenous responses are of equal magnitude. Using this method we have estimated the precursor frequency of naive CD8 T cells specific for the H-2D(b)-restricted GP33-41 epitope of LCMV to be 1 in 2 x 10(5). Thus, in an uninfected mouse containing approximately 2-4 x 10(7) naive CD8 T cells we estimate there to be 100-200 epitope-specific cells. After LCMV infection these 100-200 GP33-specific naive CD8 T cells divide >14 times in 1 wk to reach a total of approximately 10(7) cells. Approximately 5% of these activated GP33-specific effector CD8 T cells survive to generate a memory pool consisting of approximately 5 x 10(5) cells. Thus, an acute LCMV infection results in a >1,000-fold increase in precursor frequency of D(b)GP33-specific CD8 T cells from 2 x 10(2) naive cells in uninfected mice to 5 x 10(5) memory cells in immunized mice.     

Induction of antigen-specific CD8+ cytotoxic T cells by dendritic cells co-electroporated with a dsRNA analogue and tumor antigen mRNA

The maturation state of dendritic cells (DCs) is an important determinant for the initiation and regulation of adaptive immune responses. In this study, we wanted to assess whether functional activation of human monocyte-derived DCs can be achieved by electroporation of an activation signal in the form of double-stranded (ds) RNA and whether simultaneous electroporation of the dsRNA with tumor antigen encoding mRNA can lead to the induction of a cytotoxic T-lymphocyte (CTL) response. Electroporation of immature DCs with poly(I:C(12)U), a dsRNA analogue, resulted in phenotypic as well as functional changes, indicative of DC maturation. Co-electroporation of DCs with both poly(I:C(12)U) and Melan-A/MART-1 encoding mRNA induced strong anti-Melan-A/MART-1 CD8(+) T-cell responses in vitro. Higher numbers of Melan-A/MART-1-specific CTLs were consistently obtained with poly(I:C(12)U)-activated DCs compared to DCs matured in the presence of an inflammatory cytokine cocktail. These results indicate that DC co-electroporation with both dsRNA and tumor antigen encoding mRNA induces fully activated and antigen-loaded DCs that promote antigen-specific CTL responses and may provide the basis for future immunotherapeutic strategies.     

Human dermal fibroblasts present tetanus toxoid antigen to antigen-specific T cell clones

Cultured human dermal fibroblasts treated with immune interferon express HLA-DR antigens. We report here that DR-positive fibroblasts present tetanus toxoid (TT) to autologous TT-specific monoclonal helper T cells vigorously depleted of monocytes by passage over Sephadex G10 columns followed by treatment with the monoclonal antibodies (mAb) OKM1 and Leu M1 plus complement. The extent of T cell proliferation in response to TT presented by DR-positive fibroblasts was similar to that elicited using monocytes as antigen-presenting cells. The proliferative response was TT dependent, antigen specific, depended upon DR expression by fibroblasts, appeared MHC restricted, and was completely blocked by mouse mAb to HLA-DR but not by mAb to HLA-A,B, or DQ. DR-positive fibroblasts pulsed with TT were similarly effective in antigen presentation. In summary, immune interferon-stimulated human dermal fibroblasts can substitute for classical antigen-presenting cells in antigen-specific proliferative responses. Since fibroblasts are a ubiquitous cell type in the body, they may play a significant role in the immunobiology of the host.     

Antiidiotype stimulation of antigen-specific antigen-independent antibody responses in vitro. I. Evidence for stimulation of helper T lymphocyte function

Antibodies specific for the idiotypes of B10 anti-(T,G)-A-L antibodies (anti-Id) induced in vivo (T,G)-A-L-primed lymphocytes to secrete anti- (T,G)-A-L antibodies in vitro in the absence of antigen. Responses to anti-Id were quantitatively and qualitatively similar to responses to antigen. Responses were specific in that: (a) only lymphocytes primed in vivo with (T,G)-A-L (but not other antigens or unprimed cells) were inducible; (b) only anti-Id (but not a variety of control antibodies) induced responses; and (c) only anti-(T,G)-A-L antibodies were secreted. Antigen-primed T lymphocytes mixed with unprimed B plus accessory cells also responded to anti-Id and the phenotype of the responding T cells was Lyt-1+, Lyt-2-. Supernatants obtained from antigen-primed T cells incubated for 3 d with anti-Id (but not when incubated with controls) provided help to unprimed B plus accessory cells in the presence of anti-Id, thus providing direct evidence for induction of T lymphocyte helper function by anti-Id. In contrast to responses to (T,G)-A-L, responses to anti-Id did not appear to be regulated by H-2-linked Ir genes. The system described is a powerful tool for delineation of the mechanisms whereby antiidiotype antibodies affect lymphocyte function.