Hapten-specific naïve B cells are biomarkers of vaccine efficacy against drugs of abuse.

Vaccination against drugs of abuse shows efficacy in animal models, yet few subjects achieve effective serum antibody titers in clinical studies. A barrier to translation is the lack of pre-vaccination screening assays that predict the most effective conjugate vaccines or subjects amenable to vaccination. To address this obstacle, we developed a fluorescent antigen-based enrichment method paired with flow cytometry to characterize hapten-specific B cells. Using this approach, we studied naïve and activated B cells specific for structurally-related model haptens based on derivatization of the morphinan structure at the C6 position on oxycodone or at the C8 position on hydrocodone, and showing different pre-clinical efficacy against the prescription opioid oxycodone. Prior to vaccination, naïve B cells exhibited relatively higher affinity for the more effective C6-derivatized oxycodone-based hapten (6OXY) and the 6OXY-specific naïve B cell population contained a higher number of B cells with greater affinity for free oxycodone. Higher affinity of naïve B cells for hapten or oxycodone reflected greater efficacy of vaccination in blocking oxycodone distribution to brain in mice. Shortly after immunization, activated hapten-specific B cells were detected prior to oxycodone-specific serum antibodies and provided earlier evidence of vaccine failure or success. Analysis of hapten-specific naïve and activated B cells may aid rational vaccine design and provide screening tools to predict vaccine clinical efficacy against drugs of abuse or other small molecules.

CD154+ graft antigen-specific CD4+ T cells are sufficient for chronic rejection of minor antigen incompatible heart grafts.

We used a defined model system to address the role of minor histocompatibility antigen-specific CD4+ T cells in chronic rejection. The coronary arteries of vascularized heart grafts expressing the model antigen ovalbumin developed intimal hyperplasia in normal recipients and those lacking CD8+ T cells but not in those lacking CD4+ T cells. Furthermore, purified ovalbumin-specific CD4+ T cells from T-cell antigen receptor transgenic mice caused intimal hyperplasia in ovalbumin-expressing heart grafts in lymphocyte-deficient mice. The graft antigen-specific CD4+ T cells only caused intimal hyperplasia when expressing CD154 and were found in the intima of the affected coronary arteries along with CD40+ cells, CD11c+ dendritic cells and CD11b+, Gr-1+ monocytes. These results show that minor histocompatibility antigen-specific CD4+ T cells are required to cause the classical vascular changes of chronic rejection. They are capable of doing so without contributions from other lymphocytes, and may cause intimal hyperplasia by using CD154 to stimulate other non-lymphoid cells in the intima.

Single-cell analysis of signal transduction in CD4 T cells stimulated by antigen in vivo.

Flow cytometry was used to study signaling events in individual CD4 T cells after antigen recognition in the body. Phosphorylation of c-jun and p38 mitogen-activated protein kinase was detected within minutes in all antigen-specific CD4 T cells in secondary lymphoid tissues after injection of peptide antigen into the bloodstream. The remarkable rapidity of this response correlated with the finding that most naive T cells are in constant contact with dendritic antigen-presenting cells. Contrary to predictions from in vitro experiments, antigen-induced c-jun and p38 mitogen-activated protein kinase phosphorylation did not depend on CD28 signals and was insensitive to inhibition by cyclosporin A. Our results highlight the efficiency of the in vivo immune response and underscore the need to verify which signaling pathways identified in vitro actually operate under physiological conditions.

Cutting edge: in vivo identification of TCR redistribution and polarized IL-2 production by naive CD4 T cells.

TCR aggregation at the point of contact with an APC is thought to play an important role in T cell signal transduction. However, this potentially important phenomenon has never been documented during an immune response in vivo. Here we used immunohistology to show that the TCR on naive Ag-specific CD4 T cells in the lymph nodes of mice injected with Ag redistributed to one side of the cell. In cases where the APC could be identified, the TCR was concentrated on the side of the T cell facing the APC. In those T cells that produced IL-2, the TCR and IL-2 localized to the same side of the cell. In vivo IL-2 production depended on costimulatory signaling through CD28, whereas TCR redistribution did not. These results show that Ag-stimulated CD4 T cells produce IL-2 in a polarized fashion and undergo CD28-independent TCR redistribution in vivo.

Visualizing the generation of memory CD4 T cells in the whole body.

It is thought that immunity depends on naive CD4 T cells that proliferate in response to microbial antigens, differentiate into memory cells that produce anti-microbial lymphokines, and migrate to sites of infection. Here we use immunohistology to enumerate individual naive CD4 T cells, specific for a model antigen, in the whole bodies of adult mice. The cells resided exclusively in secondary lymphoid tissues, such as the spleen and lymph nodes, in mice that were not exposed to antigen. After injection of antigen alone into the blood, the T cells proliferated, migrated to the lungs, liver, gut and salivary glands, and then disappeared from these organs. If antigen was injected with the microbial product lipopolysaccharide, proliferation and migration were enhanced, and two populations of memory cells survived for months: one in the lymph nodes that produced the growth factor interleukin-2, and a larger one in the non-lymphoid tissues that produced the anti-microbial lymphokine interferon-gamma. These results show that antigen recognition in the context of infection generates memory cells that are specialized to proliferate in the secondary lymphoid tissues or to fight infection at the site of microbial entry.

In vivo activation of antigen-specific CD4 T cells.

Physical detection of antigen-specific CD4 T cells has revealed features of the in vivo immune response that were not appreciated from in vitro studies. In vivo, antigen is initially presented to naïve CD4 T cells exclusively by dendritic cells within the T cell areas of secondary lymphoid tissues. Anatomic constraints make it likely that these dendritic cells acquire the antigen at the site where it enters the body. Inflammation enhances in vivo T cell activation by stimulating dendritic cells to migrate to the T cell areas and display stable peptide-MHC complexes and costimulatory ligands. Once stimulated by a dendritic cell, antigen-specific CD4 T cells produce IL-2 but proliferate in an IL-2--independent fashion. Inflammatory signals induce chemokine receptors on activated T cells that direct their migration into the B cell areas to interact with antigen-specific B cells. Most of the activated T cells then die within the lymphoid tissues. However, in the presence of inflammation, a population of memory T cells survives. This population is composed of two functional classes. One recirculates through nonlymphoid tissues and is capable of immediate effector lymphokine production. The other recirculates through lymph nodes and quickly acquires the capacity to produce effector lymphokines if stimulated. Therefore, antigenic stimulation in the presence of inflammation produces an increased number of specific T cells capable of producing effector lymphokines throughout the body.

Dendritic cell longevity and T cell persistence is controlled by CD154-CD40 interactions.

Inflammatory mediators facilitate the maturation of dendritic cells (DC), enabling them to induce the activation, proliferation and differentiation of cognate T cells. The role of CD40 on DC and CD154 on T cells has been studied by the co-adoptive transfer of antigen-pulsed DC and TCR-transgenic (Tg) T cells in vivo. It is shown that in the absence of CD40-CD154 interactions, initial Tg T cell expansion occurs in vivo, but over time, T cell expansion cannot be sustained. The basis for the demise of the T cell population is likely due to the disappearance of the antigen-pulsed DC in the draining lymph nodes when CD154-CD40 interactions are interrupted. These findings show that both T cell and DC persistence in vivo is dependent on CD40-CD154 interactions. In addition to the physical persistence of the DC, CD40 triggering of DC also greatly increases the period for which they can productively present antigen to Tg T cells. Hence DC persistence and antigen-presenting cell capacity are both dependent on CD40 signaling. While TNF-alpha can mature DC as measured by a variety of criteria, the unique capacity of CD40 signaling to sustain T cell responses and induce DC maturation is underscored by the inability of TNF-alpha to rescue the immune deficiency of CD40(-/-) DC. Hence, the profound impact of CD154 deficiency on cell-mediated immunity may be due to its ability to limit the duration of antigen presentation in vivo and cause the premature demise of antigen-specific T cells.

Antibody is required for protection against virulent but not attenuated Salmonella enterica serovar typhimurium.

Resolution of infection with attenuated Salmonella is an active process that requires CD4(+) T cells. Here, we demonstrate that costimulation via the surface molecule CD28, but not antibody production by B cells, is required for clearance of attenuated aroA Salmonella enterica serovar typhimurium. In contrast, specific antibody is critical for vaccine-induced protection against virulent bacteria. Therefore, CD28(+) CD4(+) T cells are sufficient for clearance of avirulent Salmonella in naive hosts, whereas CD4(+) T cells and specific antibodies are required for protection from virulent Salmonella in immune hosts.

Antigen-experienced CD4 T cells display a reduced capacity for clonal expansion in vivo that is imposed by factors present in the immune host.

It is thought that protective immunity is mediated in part by Ag-experienced T cells that respond more quickly and vigorously than naive T cells. Using adoptive transfer of OVA-specific CD4 T cells from TCR transgenic mice as a model system, we show that Ag-experienced CD4 T cells accumulate in lymph nodes more rapidly than naive T cells after in vivo challenge with Ag. However, the magnitude of clonal expansion by Ag-experienced T cells was much less than that of naive T cells, particularly at early times after primary immunization. Ag-experienced CD4 T cells quickly reverted to the slower but more robust clonal expansion behavior of naive T cells after transfer into a naive environment. Conversely, the capacity for rapid clonal expansion was acquired by naive CD4 T cells after transfer into passively immunized recipients. These results indicate that rapid in vivo response by Ag-experienced T cells is facilitated by Ag-specific Abs, whereas the limited capacity for clonal expansion is imposed by some other factor in the immune environment, perhaps residual Ag.

Characterization of CD4+ T cell responses during natural infection with Salmonella typhimurium.

CD4+ T cells are important for resistance to infection with Salmonella typhimurium. However, the Ag specificity of this T cell response is unknown. Here, we demonstrate that a significant fraction of Salmonella-specific CD4+ T cells respond to the flagellar filament protein, FliC, and that this Ag has the capacity to protect naive mice from lethal Salmonella infection. To characterize this Ag-specific response further, we generated FliC-specific CD4+ T cell clones from mice that had resolved infection with an attenuated strain of Salmonella. These clones were found to respond to an epitope from a constant region of FliC, enabling them to cross-react with flagellar proteins expressed by a number of distinct Salmonella serovars.

Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells.

The effects of inflammatory cytokines on naive T cells have been studied using MHC protein/peptide complexes on microspheres, thus avoiding the use of APCs whose functions may be affected by the cytokines. IL-1, but not IL-12, increased proliferation of CD4+ T cells in response to Ag and IL-2, which is consistent with effects on in vivo priming of CD4+ cells. In contrast, proliferation of CD8+ T cells to Ag and IL-2 required IL-12, and IL-12 replaced adjuvant in stimulating an in vivo response to peptide. These results support a model in which distinct inflammatory cytokines act directly on naive CD4+ and CD8+ T cells to provide a third signal, along with Ag and IL-2, to optimally activate differentiation and clonal expansion.

Clonal expansion of antigen-specific CD4 T cells following infection with Salmonella typhimurium is similar in susceptible (Itys) and resistant (Ityr) BALB/c mice.

The results show that CD4 T cells specific for a recombinant antigen expressed in Salmonella typhimurium proliferate normally in mice that express the susceptible form of the Ity gene at early times after infection but do not retain the capacity to produce gamma interferon later in the infection.

Antigen-specific CD4+ T cells that survive after the induction of peripheral tolerance possess an intrinsic lymphokine production defect.

Injection of soluble foreign antigen without an adjuvant induces a state of antigen-specific immunological unresponsiveness. We investigated the cellular mechanisms that underlie this form of peripheral tolerance by physically tracking a small population of ovalbumin (OVA) peptide/I-Ad-specific, CD4+ T cell receptor (TCR) transgenic T cells following adoptive transfer into normal recipients. Injection of OVA peptide in the absence of adjuvant caused the antigen-specific T cells to proliferate for a brief period after which most of the T cells disappeared. The remaining OVA-specific T cells had converted to a memory phenotype but were poorly responsive in vivo as evidenced by a failure to accumulate in the draining lymph nodes following immunization with OVA peptide in adjuvant. These surviving T cells possessed a long-lasting, but reversible, defect in Il-2 and TNF-alpha production and in vivo proliferation, but did not gain capacity to produce Th2-type cytokines or suppress the clonal expansion of T cells specific for another antigen. Therefore, some antigen-specific T cells survive this peripheral tolerance protocol but are functionally unresponsive due to an intrinsic activation defect.

Peripheral immune tolerance blocks clonal expansion but fails to prevent the differentiation of Th1 cells.

Clonal anergy in Ag-specific CD4+ T cells is shown in these experiments to inhibit IL-2 production and clonal expansion in vivo. We also demonstrate that the defect in IL-2 gene inducibility can be achieved in both naive and Th1-like memory T cells when repeatedly exposed to aqueous peptide Ag. Nevertheless, this induction of clonal anergy did not interfere with the capacity of naive T cells to differentiate into Th1-like effector cells, nor did it prevent such helper cells from participating in T-dependent IgG2a anti-hapten responses and delayed-type hypersensitivity reactions. Thus, clonal anergy can contribute to the development of Ag-specific immune tolerance by limiting the size of a Th cell population, but not by disrupting its effector function.

Self-reactive B lymphocytes overexpressing Bcl-xL escape negative selection and are tolerized by clonal anergy and receptor editing.

Self-reactive B cells Tg for both a bcl-xL death inhibitory gene and an Ig receptor recognizing hen egg lysozyme (HEL-Ig) efficiently escaped developmental arrest and deletion in mice expressing membrane-bound self-antigen (mHEL). In response to the same antigen, Tg HEL-Ig B cells not expressing bcl-xL were deleted, while cells expressing bcl-2 were arrested at the immature B stage. Bcl-xL Tg B cells escaping negative selection were anergic in both in vitro and in vivo assays and showed some evidence for receptor editing. These studies suggest that Bcl-x may have a distinct role in controlling survival at the immature stage of B cell development and demonstrate that tolerance is preserved when self-reactive B cells escape central deletion.

Visualization of specific B and T lymphocyte interactions in the lymph node.

Early events in the humoral immune response were visualized in lymph nodes by simultaneous tracking of antigen-specific CD4 T and B cells after immunization. The T cells were initially activated in the T cell areas when the B cells were still randomly dispersed in the B cell-rich follicles. Both populations then migrated to the edges of the follicles and interacted there, resulting in CD154-dependent B cell proliferation and germinal center formation. These results provide visual documentation of cognate T-B cell interactions and localize them to the follicular border.

Cutting edge: antigen-dependent regulation of telomerase activity in murine T cells.

Telomeres, structures on the ends of linear chromosomes, function to maintain chromosomal integrity. Telomere shortening occurs with cell division and provides a mechanism for limiting the replicative potential of normal human somatic cells. Telomerase, a ribonucleoprotein enzyme, synthesizes telomeric repeats on chromosomal termini, potentially extending the capacity for cell division. The present study demonstrates that resting T cells express little/no activity, and optimal Ag-specific induction of telomerase activity in vitro requires both TCR and CD28-B7 costimulatory signals. Regulation of telomerase in T cells during in vivo Ag-dependent activation was also assessed by adoptive transfer of TCR transgenic T cells and subsequent Ag challenge. Under these conditions, telomerase was induced in transgenic T cells coincident with a phase of extensive clonal expansion. These findings suggest that telomerase may represent an adoptive response that functions to preserve replicative potential in Ag-reactive lymphocytes.

Direct evidence that functionally impaired CD4+ T cells persist in vivo following induction of peripheral tolerance.

A small population of CD4+ OVA-specific TCR transgenic T cells was tracked following the induction of peripheral tolerance by soluble Ag to address whether functionally unresponsive, or anergic T cells, persist in vivo for extended periods of time. Although injection of OVA peptide in the absence of adjuvant caused a transient expansion and deletion of the Ag-specific T cells, a population that showed signs of prior activation persisted in the lymphoid tissues for several months. These surviving OVA-specific T cells had long-lasting, but reversible defects in their ability to proliferate in lymph nodes and secrete IL-2 and TNF-alpha in vivo following an antigenic challenge. These defects were not associated with the production of Th2-type cytokines or the capacity to suppress the clonal expansion of a bystander population of T cells present in the same lymph nodes. Therefore, our results provide direct evidence that a long-lived population of functionally impaired Ag-specific CD4+ T cells is generated in vivo after exposure to soluble Ag.

Revealing the in vivo behavior of CD4+ T cells specific for an antigen expressed in Escherichia coli.

The clonal expansion and anatomic location of microbe-specific CD4+ Th cells was studied by tracking the fate of adoptively transferred DO11.10 TCR transgenic T cells specific for OVA peptide 323-339/I-Ad in BALB/c mice infected s.c. with Escherichia coli expressing a MalE-OVA fusion protein. After infection, the DO11.10 T cells accumulated in the T cell-rich paracortical regions of the draining lymph nodes, proliferated there for several days, and then moved into the B cell-rich follicles before they slowly disappeared from the lymph nodes. These changes occurred despite the fact that viable organisms were never found in the lymph nodes. The DO11.10 T cells also accumulated in the s.c. infection site, but about 1 day later than in the draining lymph nodes. Injection of purified MalE-OVA fusion protein alone induced a transient accumulation of DO11.10 T cells in the paracortical regions, but these T cells never entered follicles and the mice did not produce anti-OVA antibodies. The DO11.10 T cells that survived in animals injected with MalE-OVA alone were hyporesponsive to in vitro Ag restimulation and did not produce IL-2 and IFN-gamma, whereas DO11.10 T cells from mice infected with MalE-OVA-expressing bacteria produced both lymphokines. These results suggest that Ag-specific T cells are first activated in secondary lymphoid organs following primary bacterial infection and then migrate to the infection site. Furthermore, productive activation of the T cells during the primary response is dependent on bacterial components other than the Ag itself.

A natural immunological adjuvant enhances T cell clonal expansion through a CD28-dependent, interleukin (IL)-2-independent mechanism.

The adoptive transfer of naive CD4(+) T cell receptor (TCR) transgenic T cells was used to investigate the mechanisms by which the adjuvant lipopolysaccharide (LPS) enhance T cell clonal expansion in vivo. Subcutaneous administration of soluble antigen (Ag) resulted in rapid and transient accumulation of the Ag-specific T cells in the draining lymph nodes (LNs), which was preceded by the production of interleukin (IL)-2. CD28-deficient, Ag-specific T cells produced only small amounts of IL-2 in response to soluble Ag and did not accumulate in the LN to the same extent as wild-type T cells. Injection of Ag and LPS, a natural immunological adjuvant, enhanced IL-2 production and LN accumulation of wild-type, Ag-specific T cells but had no significant effect on CD28-deficient, Ag-specific T cells. Therefore, CD28 is critical for Ag-driven IL-2 production and T cell proliferation in vivo, and is essential for the LPS-mediated enhancement of these events. However, enhancement of IL-2 production could not explain the LPS-dependent increase of T cell accumulation because IL-2-deficient, Ag-specific T cells accumulated to a greater extent in the LN than wild-type T cells in response to Ag plus LPS. These results indicate that adjuvants improve T cell proliferation in vivo via a CD28-dependent signal that can operate in the absence of IL-2.