Exclusive Th2 primary effector function in spleens but mixed Th1/Th2 function in lymph nodes of murine neonates

Recent studies have shown that neonatal mice are competent to develop mature, Ag-specific Th1 function in situ. However, under many conditions, Th2 responses dominate in the neonate, while Th1 responses are more prevalent in adults. To compare further the immune responses of neonates and adults, we used the enzyme-linked immunospot method to measure the frequencies of primary Th1/Th2 effectors generated in situ in the spleens and lymph nodes. As assessed by the detection of IFN-gamma- or IL-4-producing cells, adults developed mixed Th1/Th2 responses in both organs. Neonatal lymph nodes contained mature frequencies of IFN-gamma- and IL-4-producing cells. In striking contrast, while mature frequencies of Th2 cells developed in neonatal spleens, virtually no IFN-gamma-secreting cells were detected. Exclusive Th2 function was observed in both BALB/c and C57BL/6 neonates, strains in which the Th2 and Th1 lineages, respectively, are favored in adults. Although Th1 effectors were virtually undetectable, the addition of rIL-12 boosted the frequency of IFN-gamma-secreting cells to adult levels. Therefore, Th1 effectors apparently developed in situ, but Th1 effector function either was not promoted or was inhibited upon subsequent exposure to the Ag in culture. Together, these results indicate that the quality of a primary Th response in neonates is strongly dependent on the site of initial Ag exposure; responses initiated in the lymph nodes are mixed Th1/Th2, whereas responses occurring in the spleen are heavily Th2 biased.     

Tissue-level regulation of Th1 and Th2 primary and memory CD4 T cells in response to Listeria infection

Ag-specific Th1 and Th2 cytokine-producing CD4 T cells were quantitated in secondary lymphoid and tertiary tissues following oral Listeria monocytogenes infection. Although the response to Listeria was previously believed to be predominantly Th1 like, CD4 T cells producing IL-4 or IL-5 comprised a substantial proportion of the overall primary and memory response. The frequency of IFN-gamma-, IL-4-, or IL-5-producing primary effector or memory CD4 T cells was significantly higher in lung, liver, and intestinal lamina propria (LP) as compared with spleen and lymph node. However, maximum numbers of IL-4- and IL-5-producing cells were detected in the LP several days after the peak of the Th1 response, and IL-5 production was skewed toward the mucosal tissues. Remarkably, the recall response resulted in sustained Th1 and Th2 responses in tertiary, but not lymphoid tissues and long-term retention of Th1 and Th2 memory cells in equal proportions in the LP. Finally, CD40 ligand was essential for induction of IFN-gamma in the spleen and LP, but not in the liver and lung, while the IL-4 response required CD40 ligand only in the spleen. Therefore, the rules governing the effector phenotype, and the overall magnitude of the CD4 response, are regulated at the level of individual tissues.     

Murine neonatal CD4+ lymph node cells are highly deficient in the development of antigen-specific Th1 function in adoptive adult hosts

It is well established that murine neonates are biased toward Th2 responses. Th2-dominant responses are observed following immunization with a variety of Ags, using different carrier/adjuvant systems, and are seen in both BALB/c and C57BL/6 mice. Therefore, Th2 skewing appears to be a universal phenomenon unique to the neonatal period. One important question about this phenomenon is whether these responses are due to T cell intrinsic properties or are regulated by the neonatal environment. Here we have addressed this issue by transferring neonatal or adult CD4(+) lymph node cells to adoptive adult recombinase-activating gene 2(-/-) hosts and studied the development of Th responses. Neonatal CD4(+) cells were highly deficient in the development of both primary and secondary Ag-specific Th1 responses. This did not appear to be due to anergy of a developed population, since exogenous IL-2 only marginally increased production of the Th1 cytokine IFN-gamma. This profound Th1 deficiency was observed despite similar proliferation by neonatal and adult cells within the recombinase-activating gene 2(-/-) hosts. Moreover, neonatal CD4(+) cells up-regulated activation markers in a manner similar to adult CD4(+) cells. Therefore, although their proliferation and phenotypic maturation proceeded normally, neonatal CD4(+) cells appeared to be intrinsically deficient in the functional maturation of Th1 lineage cells. These results offer a candidate explanation for the reduced graft-vs-host responses observed following transplantation of cord blood cells or murine neonatal lymphoid cells to allogeneic adult hosts.     

A pool of central memory-like CD4 T cells contains effector memory precursors

The L51S mutation in the D10.G4.1 TCR alpha-chain reduces the affinity of the TCR to its ligand by affecting the interactions among the TCR, the beta-chain of I-A(k), and the bound peptide. We show that this mutation drives the generation of a pool of memory CD44(high)CD62L(neg)CD45RB(neg) CD4 TCR transgenic T cells. Their activation threshold is low, such that they proliferate in response to lower concentrations of agonist peptides than naive L51S CD4 T cells. Unlike effector memory CD4 T cells, however, they lack immediate effector function in response to TCR stimulation. These cells express IL-2R alpha only after culture with specific peptide. Although they can be recovered from lymph nodes, the majority lack the expression of the lymph node homing receptor CCR7. When these cells receive a second TCR stimulation in vitro, they differentiate into potent Th2-like effector cells, producing high levels of IL-4 at doses of agonist peptide too low to stimulate cytokine release from similarly differentiated naive L51S CD4 T cells. Having these properties, the L51S TCR transgenic memory CD4 T cells cannot be classified as either strict central memory or effector memory, but, rather, as a pool of memory T cells containing effector memory precursors.     

Autoimmune intervention by CD154 blockade prevents T cell retention and effector function in the target organ

The CD40-CD154 interaction is an attractive target for therapeutic intervention in many autoimmune disorders, including multiple sclerosis. Previously, we showed that CD154 blockade both inhibited the onset of experimental autoimmune encephalomyelitis and blocked clinical disease progression (relapses) in mice with established disease. The mechanism of this protection is poorly understood. Because CD154 plays a role in Th1 development, its blockade has been thought to promote anti-inflammatory Th2 responses. However, these conclusions have primarily been based on extrapolated data from in vitro experiments, which may not accurately reflect the more complex events occurring in vivo. In this paper we determine how the immune response develops under the influence of therapeutic CD154 blockade in vivo. We demonstrate that anti-CD154 treatment does not alter the early expansion of Ag-specific T cells in secondary lymphoid organs or result in deviation to a Th2-dominant response. Interestingly, the late expansion and retention of Th1 cells in the lymph nodes were markedly reduced following immunization of Ab-treated mice, and this coincided with a recompartmentalization of these cells to the spleen. Most importantly, anti-CD154 treatment eliminated the retention/expansion of encephalitogenic Th1 cells, but not their entry into the CNS. These data indicate that a major mechanism by which CD154 blockade protects against autoimmune disease is by controlling the amplitude of acute phase Th1 responses in the draining lymph nodes and by preventing the sustained expansion of effector cells within the target organ.     

Long-lived Th2 memory in experimental allergic asthma

Although life-long immunity against pathogens is beneficial, immunological memory responses directed against allergens are potentially harmful. Because there is a paucity of information about Th2 memory cells in allergic disease, we established a model of allergic asthma in BALB/c mice to explore the generation and maintenance of Th2 memory. We induced disease without the use of adjuvants, thus avoiding Ag depots, and found that unlike allergic asthma in mice immunized with adjuvant, immunizing with soluble and aerosol OVA resulted in pathological lung lesions resembling human disease. To test memory responses we allowed mice with acute disease to recover and then re-exposed them to aerosol OVA a second time. Over 400 days later these mice developed OVA-dependent eosinophilic lung inflammation, airway hyperresponsiveness, mucus hypersecretion, and IgE. Over 1 year after recuperating from acute disease, mice had persistent lymphocytic lung infiltrates, Ag-specific production of IL-4 and IL-5 from spleen and lung cells in vitro, and elevated IgG1. Moreover, when recuperated mice were briefly aerosol challenged, we detected early expression of Th2 cytokine RNA in lungs. Taken together, these data demonstrate the presence of long-lived Th2 memory cells in spleen and lungs involved in the generation of allergic asthma upon Ag re-exposure.     

Stability and function of secondary Th1 memory cells are dependent on the nature of the secondary stimulus

Following acute infection in some mouse models, CD4(+) memory T cells steadily decline over time. Conversely, in humans, CD4(+) memory T cells can be maintained for many years at levels similar to CD8(+) T cells. Because we previously observed that the longevity of Th1 memory cell survival corresponded to their functional avidity, we hypothesized that secondary challenge, which enriches for high functional avidity Th1 responders, would result in more stable Th1 memory populations. We found that following a heterologous secondary challenge, Th1 memory cells were maintained at stable levels compared with primary Th1 memory cells, showing little to no decline after day 75 postinfection. The improved stability of secondary Th1 memory T cells corresponded to enhanced homeostatic turnover; enhanced trafficking of effector memory Th1 cells to tissue sites of infection, such as the liver; and acquisition or maintenance of high functional avidity following secondary challenge. Conversely, a weaker homologous rechallenge failed to induce a stable secondary Th1 memory population. Additionally, homologous secondary challenge resulted in a transient loss of functional avidity by Th1 memory cells recruited into the secondary response. Our findings suggest that the longevity of Th1 memory T cells is dependent, at least in part, on the combined effects of primary and secondary Ag-driven differentiation. Furthermore, they demonstrate that the quality of the secondary challenge can have profound effects on the longevity and function of the ensuing secondary Th1 memory population.     

CD8 T cell recall responses are regulated by the tissue tropism of the memory cell and pathogen

Whether memory CD8 T cells can be reactivated in nonlymphoid tissues is unclear. Using mice lacking the spleen, lymph nodes, or both, we show that the secondary T cell response, but not homeostatic maintenance of memory cells, required lymphoid tissue. Whereas primary and secondary CD8 T cell responses to vesicular stomatitis virus infection were lymph node dependent, responses to Listeria monocytogenes infection were driven primarily in the spleen. Memory cell subset reactivation was also regulated by location of the responding population and the pathogen. Thus, CD62Llow effector memory T cells (TEM) cells responded nearly as well as CD62Lhigh central memory T cells (TCM) and TCM cells after L. monocytogenes infection, and both subsets generated equivalent populations of secondary memory cells. In contrast, TCM cells, but not TEM cells, mounted a robust response to vesicular stomatitis virus infection. TCM and TEM cells also required lymphoid tissue to mount recall responses, and the bone marrow did not contribute significantly to the response of either subset. Our findings indicated that characteristics of the infectious agent and the migratory preferences of memory cells dictated the secondary lymphoid tissue requirement for the recall response to infection.     

Enhanced type 1 immunity after secondary viral challenge in mice primed as neonates

The goal of infant immunization against viral infection is to develop protective long term memory responses. Priming neonatal mice with a low dose of Cas-Br-E murine leukemia virus (Cas) results in adult-like, type 1 protective responses. However, other studies suggest that Ag priming of neonates leads to an increase in type 2 secondary responses even when primary responses were type 1. We assessed whether type 1 CD8+ T cell-mediated responses developed in murine neonates are maintained after secondary challenge with Cas in adulthood. Despite the induction of significant anti-viral CD8+-mediated cytotoxic T lymphocyte and IFN-gamma responses, initial neonatal priming led to a lower frequency of virus-specific T cells compared with adult priming. Adult frequencies were reached in mice primed as neonates only after secondary challenge in adulthood. A nonspecific and transient CD4+-mediated IL-4 response was present in all groups after secondary challenge with Cas or medium, indicating that this rise in type 2 cytokine production was not unique to mice that had been primed as neonates. Rather, type 1 anti-viral memory CD8+ T cell responses developed in neonatal mice are stable, protective, and enhanced after secondary challenge.     

Schnurri-2 controls memory Th1 and Th2 cell numbers in vivo

Schnurri-2 (Shn-2) is a large zinc-finger containing protein, and it plays a critical role in cell growth, signal transduction and lymphocyte development. In Shn-2-deficient CD4 T cells, the activation of NF-kappaB was up-regulated and their ability to differentiate into Th2 cells was enhanced. We herein demonstrate that Th1 and Th2 memory cells are not properly generated from Shn-2-deficient effector Th1/Th2 cells. Even a week after the transfer of effector Th1/Th2 cells into syngeneic mice, a dramatic decrease in the number of Shn-2-deficient donor T cells was detected particularly in the lymphoid organs. The transferred Shn-2-deficient Th1/Th2 cells express higher levels of the activation marker CD69. No significant defect in the BrdU incorporation in the Shn-2-deficient transferred CD4 T cells was observed. The numbers of apoptotic cells were selectively higher in Shn-2-deficient donor Th1/Th2 cell population. Moreover, Shn-2-deficient effector Th1 and Th2 cells showed an increased susceptibility to cell death in in vitro cultures with increased expression of FasL. Transfer of Th2 effector cells over-expressing the p65 subunit of NF-kappaB resulted in a decreased number of p65-expressing cells in the lymphoid organs. As expected, T cell-dependent Ab responses after in vivo immunization of Shn-2-deficient mice were significantly reduced. Thus, Shn-2 appears to control the generation of memory Th1/Th2 cells through a change in their susceptibility to cell death.     

Induction of protective immunity to Listeria monocytogenes in neonates

Neonates suffer unduly from infections and also respond suboptimally to most commonly used vaccines. However, a CD8 T cell response can be elicited in neonates if the Ag is introduced into the cytoplasm of APCs. Listeria monocytogenes (Lm) targets the cytoplasm of APC and is a strong CD8 and CD4 Th1-promoting vaccine vehicle in adult mice. We hypothesized that an attenuated strain of Lm would be safe and induce long-lasting protective immunity, even in neonates. We found that neonatal mice immunized only once with the attenuated strain DeltaactA-Lm developed robust primary and secondary CD8 and CD4 Th1 responses and were fully protected from lethal challenge with virulent wild-type Lm without the need for a booster immunization. Furthermore, DeltaactA-Lm expressing a heterologous recombinant Ag induced a strong CD8 and Th1 memory response to that Ag. Based on these data, we propose that DeltaactA-Lm or derivatives thereof might serve as a vaccine vehicle for neonatal immunization.     

Selective accumulation of Th2-skewing immature erythroid cells in developing neonatal mouse spleen

Environmental factors likely regulate neonatal immunity and self-tolerance. However, evidence that the neonatal immune system is suppressed or deviated is varied depending on the antigen and the timing of antigen exposure relative to birth. These disparate findings may be related to the availability of the appropriate antigen presenting cells but also point to the possibility of homeostatic changes in non-lymphoid cells in the relevant lymphoid tissues. Here we show that, while leukocytes are the most abundant cell population present in spleen during the first 4-5 days after birth, a massive accumulation of nucleated immature erythroid population in the spleen takes places on day 6 after birth. Although the relative frequency of these immature erythorid cells slowly decreases during the development of neonates, they remain one of the most predominant populations up to three weeks of age. Importantly, we show that the immature erythroid cells from neonate spleen have the capacity to modulate the differentiation of CD4 T cells into effector cells and provide a bias towards a Th2 type instead of Th1 type. These nucleated erythroid cells can produce cytokines that participate in the Th2/Th1 balance, an important one being IL-6. Thus, the selective accumulation of immature erythroid cells in the spleen during a specific period of neonatal development may explain the apparent differences observed in the type(s) of immune responses generated in infants and neonates. These findings are potentially relevant to the better management of immune deficiency in and to the design of vaccination strategies for the young.     

Skewed Th1/Th2 immune response to Sarcoptes scabiei

Scabies is a contagious skin disease of humans and many other species of mammals. Previous studies suggested that the balance between the Th1 and Th2 immune responses may influence the outcome of a scabies infestation in a sensitized host. Therefore, in this study, we examined the T-helper cell cytokine profiles of splenocytes and lymph node cells in BALB/c mice that were immunized with scabies extract (primary response), infested with scabies mites (primary response), or immunized and then infested (secondary response). Lymphocyte cytokine expression was analyzed by flow cytometry after staining for intracellular cytokines. Immunization with scabies extract induced production of interferon-gamma (IFNgamma) (Th1 response) by both spleen and lymph node cells. Mice that were infested with scabies increased production of interleukin-4 by lymph node cells and of IFNgamma by splenocytes. Mice that were first immunized and then infested with mites increased production of IFNgamma by both spleen and lymph node cells. However, this increased level of IFNgamma was only about half of that induced by immunization alone. These results suggest that live scabies mites produced something that inhibited IFNgamma production in the lymph nodes of scabies-immunized mice. Our data also indicate that lymphocytes in the spleen and lymph nodes can present different cytokine response profiles.     

Activation of CD4 T cells by somatic transgenesis induces generalized immunity of uncommitted T cells and immunologic memory

Cellular immune responses were analyzed in vivo after a single intraspleen inoculation of DNA coding for a 12-residue Th cell determinant associated with a 12-residue B cell epitope, a process termed somatic transgene immunization. We show that CD4 T cells are readily activated and produce IL-2, IFN-gamma and IL-4, characteristics of an uncommitted phenotype. Linked recognition of the two epitopes coded in the same transgene promoted IgM-IgG1 switch and enhanced the total Ab response but had no effect on IgG2a Abs. Although originating in the spleen, T cell responsiveness was found to spread immediately and with similar characteristics to all lymph nodes in the body. A single inoculation was also effective in establishing long term immunologic memory as determined by limiting dilution analysis, with memory T cells displaying a cytokine profile different from that of primary effector T cells. These studies provide evidence that by initiating immunity directly in secondary lymphoid organs, an immune response is generated with characteristics that differ from those using vaccines of conventional DNA or protein in adjuvant administered in peripheral sites. Somatic transgene immunization can therefore be used to probe T cell responsiveness in vivo and represents a tool to further understanding of the nature of the adaptive immune response.     

CCR4 participation in Th type 1 (mycobacterial) and Th type 2 (schistosomal) anamnestic pulmonary granulomatous responses

CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+ CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-gamma and IL-4 producers. Studies of CCR4 knockout (CCR4(-/-)) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-gamma production during secondary but not primary challenge. Adoptive transfer indicated CCR4(-/-)CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4(+/+)) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4(+/+)CD4+ T cells and CCR4(-/-) dendritic cells revealed intact IL-2 but impaired IFN-gamma production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.     

Distinct regulation of H2-M3-restricted memory T cell responses in lymph node and spleen

CD8 T cell populations restricted by H2-M3 MHC class Ib molecules expand rapidly during primary Listeria monocytogenes infection but only minimally upon reinfection. In contrast, CD8 T cells restricted by MHC class Ia molecules undergo more delayed expansion during primary infection but rapid and robust expansion following reinfection. In this study we demonstrate that primary H2-M3-restricted CD8 T cell responses are unaffected by the frequency of naive MHC class Ia-restricted T cells during L. monocytogenes infection. The magnitude of H2-M3-restricted memory responses, in contrast, is down-modulated by increasing frequencies of MHC class Ia-restricted effector T cells following secondary systemic infection. Suppression by MHC class Ia-restricted T cells, however, is not a universal feature of MHC class Ib-restricted memory responses. Primary systemic L. monocytogenes infection followed by secondary tissue infection, for example, results in robust expansion of H2-M3-restricted memory T cells in draining lymph nodes, despite the activation of MHC class Ia-restricted memory T cell responses. Thus, whereas MHC class Ia-restricted memory T cell populations predominate in spleens following systemic reinfection, H2-M3-restricted memory T cell responses remain prominent in lymph nodes draining localized infections. Our studies demonstrate that interactions between CD8 T cell populations can differ, depending on the status of the responding T cells (naive vs memory) and the route of reinfection. These results may have important implications for prime-boost vaccination strategies.     

TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.     

Functional responses and costimulator dependence of memory CD4+ T cells

To examine the functional characteristics of memory CD4+ T cells, we used an adoptive transfer system to generate a stable population of Ag-specific memory cells in vivo and compared their responses to Ag with those of a similar population of Ag-specific naive cells. Memory cells localized to the spleen and lymph nodes of mice and exhibited extremely rapid recall responses to Ag in vivo, leaving the spleen within 3-5 days of Ag encounter. Unlike their naive counterparts, memory cells produced effector cytokines (IFN-gamma, IL-4, IL-5) within 12-24 h of Ag exposure and did not require multiple cycles of cell division to do so. Memory cells proliferated at lower Ag concentrations than did naive cells, were less dependent on costimulation by B7 molecules, and independent of costimulation by CD40. Furthermore, effector cytokine production by memory cells also occurred in the absence of either B7 or CD40 costimulation. Lastly, memory cells were resistant to tolerance induction. Together, these findings suggest that the threshold for activation of memory CD4+ cells is lower than that of naive cells. This would permit memory cells to rapidly express their effector functions in vivo earlier in the course of a secondary immune response, when the levels of Ag and the availability of costimulation may be relatively low.