"Negative vaccination" by specific CD4 T cell tolerisation enhances virus-specific protective antibody responses

Background

Cooperation of CD4⁺ T helper cells with specific B cells is crucial for protective vaccination against pathogens by inducing long-lived neutralizing antibody responses. During infection with persistence-prone viruses, prolonged virus replication correlates with low neutralizing antibody responses. We recently described that a viral mutant of lymphocytic choriomeningitis virus (LCMV), which lacks a T helper epitope, counterintuitively induced an enhanced protective antibody response. Likewise, partial depletion of the CD4⁺ T cell compartment by using anti-CD4 antibodies enhanced protective antibodies.

Principal Findings

Here we have developed a protocol to selectively reduce the CD4⁺ T cell response against viral CD4⁺ T cell epitopes. We demonstrate that in vivo treatment with LCMV-derived MHC-II peptides induced non-responsiveness of specific CD4⁺ T cells without affecting CD4⁺ T cell reactivity towards other antigens. This was associated with accelerated virus-specific neutralizing IgG-antibody responses. In contrast to a complete absence of CD4⁺ T cell help, tolerisation did not impair CD8⁺ T cell responses.

Conclusions

This result reveals a novel “negative vaccination” strategy where specific CD4⁺ T cell unresponsiveness may be used to enhance the delayed protective antibody responses in chronic virus infections.     

Screening of human tumor antigens for CD4 T cell epitopes by combination of HLA-transgenic mice, recombinant adenovirus and antigen peptide libraries

Background

As tumor antigen-specific CD4⁺ T cells can mediate strong therapeutic anti-tumor responses in melanoma patients we set out to establish a comprehensive screening strategy for the identification of tumor-specific CD4⁺ T cell epitopes suitable for detection, isolation and expansion of tumor-reactive T cells from patients.

Methods and Findings

To scan the human melanoma differentiation antigens TRP-1 and TRP-2 for HLA-DRB1*0301-restricted CD4⁺ T cell epitopes we applied the following methodology: Splenocytes of HLA-DRB1*0301-transgenic mice immunized with recombinant adenovirus encoding TRP-1 (Ad5.TRP-1) or TRP-2 (Ad5.TRP-2) were tested for their T cell reactivity against combinatorial TRP-1- and TRP-2-specific peptide libraries. CD4⁺ T cell epitopes thus identified were validated in the human system by stimulation of peripheral blood mononuclear cells (PBMC) from healthy donors and melanoma patients. Using this strategy we observed that recombinant Ad5 induced strong CD4⁺ T cell responses against the heterologous tumor antigens. In Ad5.TRP-2-immunized mice CD4⁺ T cell reactivity was detected against the known HLA-DRB1*0301-restricted TRP-2_60–74 epitope and against the new epitope TRP-2_149–163. Importantly, human T cells specifically recognizing target cells loaded with the TRP-2_149–163-containing library peptide or infected with Ad5.TRP-2 were obtained from healthy individuals, and short term in vitro stimulation of PBMC revealed the presence of epitope-reactive CD4⁺ T cells in melanoma patients. Similarly, immunization of mice with Ad5.TRP-1 induced CD4⁺ T cell responses against TRP-1-derived peptides that turned out to be recognized also by human T cells, resulting in the identification of TRP-1_284–298 as a new HLA-DRB1*0301-restricted CD4⁺ T cell epitope.

Conclusions

Our screening approach identified new HLA-DRB1*0301-restricted CD4⁺ T cell epitopes derived from melanoma antigens. This strategy is generally applicable to target antigens of other tumor entities and to different HLA class II molecules even without prior characterization of their peptide binding motives.     

Correlation of memory T cell responses against TRAP with protection from clinical malaria, and CD4 CD25 high T cells with susceptibility in Kenyans

Background

Immunity to malaria develops naturally in endemic regions, but the protective immune mechanisms are poorly understood. Many vaccination strategies aim to induce T cells against diverse pre-erythrocytic antigens, but correlates of protection in the field have been limited. The objective of this study was to investigate cell-mediated immune correlates of protection in natural malaria. Memory T cells reactive against thrombospondin-related adhesive protein (TRAP) and circumsporozoite (CS) protein, major vaccine candidate antigens, were measured, as were frequencies of CD4⁺ CD25^high T cells, which may suppress immunity, and CD56⁺ NK cells and γδ T cells, which may be effectors or may modulate immunity.

Methodology and Principal Findings

112 healthy volunteers living in rural Kenya were entered in the study. Memory T cells reactive against TRAP and CS were measured using a cultured IFNγ ELISPOT approach, whilst CD4⁺ CD25^high T cells, CD56⁺ NK cells, and γδ T cells were measured by flow cytometry. We found that T cell responses against TRAP were established early in life (<5 years) in contrast to CS, and cultured ELISPOT memory T cell responses did not correlate with ex-vivo IFNγ ELISPOT effector responses. Data was examined for associations with risk of clinical malaria for a period of 300 days. Multivariate logistic analysis incorporating age and CS response showed that cultured memory T cell responses against TRAP were associated with a significantly reduced incidence of malaria (p = 0.028). This was not seen for CS responses. Higher numbers of CD4⁺ CD25^high T cells, potentially regulatory T cells, were associated with a significantly increased risk of clinical malaria (p = 0.039).

Conclusions

These data demonstrate a role for central memory T cells in natural malarial immunity and support current vaccination strategies aimed at inducing durable protective T cell responses against the TRAP antigen. They also suggest that CD4⁺ CD25^high T cells may negatively affect naturally acquired malarial immunity.     

Cell-to-cell interactions and signals involved in the reconstitution of peripheral CD8 T(CM) and T(EM) cell pools

We here describe novel aspects of CD8⁺ and CD4⁺ T cell subset interactions that may be clinically relevant and provide new tools for regulating the reconstitution of the peripheral CD8⁺ T cell pools in immune-deficient states. We show that the reconstitution capacity of transferred isolated naïve CD8⁺ T cells and their differentiation of effector functions is limited, but both dramatically increase upon the co-transfer of CD4⁺ T cells. This helper effect is complex and determined by multiple factors. It was directly correlated to the number of helper cells, required the continuous presence of the CD4⁺ T cells, dependent on host antigen-presenting cells (APCs) expressing CD40 and on the formation of CD4/CD8/APC cell clusters. By comparing the recovery of (CD44⁺CD62L^high) T_CM and (CD44⁺CD62L^low) T_EM CD8⁺ T cells, we found that the accumulation of T_CM and T_EM subsets is differentially regulated. T_CM-cell accumulation depended mainly on type I interferons, interleukin (IL)-6, and IL-15, but was independent of CD4⁺ T-cell help. In contrast, T_EM-cell expansion was mainly determined by CD4⁺ T-cell help and dependent on the expression of IL-2Rβ by CD8 cells, on IL-2 produced by CD4⁺ T-cells, on IL-15 and to a minor extent on IL-6.     

Impaired CD4 T cell memory response to Streptococcus pneumoniae precedes CD4 T cell depletion in HIV-infected Malawian adults

Objective

Invasive pneumococcal disease (IPD) is a leading cause of morbidity and mortality in HIV-infected African adults. CD4 T cell depletion may partially explain this high disease burden but those with relatively preserved T cell numbers are still at increased risk of IPD. This study evaluated the extent of pneumococcal-specific T cell memory dysfunction in asymptomatic HIV infection early on in the evolution of the disease.

Methods

Peripheral blood mononuclear cells were isolated from asymptomatic HIV-infected and HIV-uninfected Malawian adults and stained to characterize the underlying degree of CD4 T cell immune activation, senescence and regulation. Pneumococcal-specific T cell proliferation, IFN-γ, IL-17 production and CD154 expression was assessed using flow cytometry and ELISpot.

Results

We find that in asymptomatic HIV-infected Malawian adults, there is considerable immune disruption with an increase in activated and senescent CD4⁺CD38⁺PD-1⁺ and CD4⁺CD25^highFoxp3⁺ Treg cells. In the context of high pneumococcal exposure and therefore immune stimulation, show a failure in pneumococcal-specific memory T cell proliferation, skewing of T cell cytokine production with preservation of interleukin-17 but decreased interferon-gamma responses, and failure of activated T cells to express the co-stimulatory molecule CD154.

Conclusion

Asymptomatic HIV-infected Malawian adults show early signs of pneumococcal- specific immune dysregulation with a shift in the balance of CD4 memory, T helper 17 cells and Treg. Together these data offer a mechanistic understanding of how antigen-specific T cell dysfunction occurs prior to T cell depletion and may explain the early susceptibility to IPD in those with relatively preserved CD4 T cell numbers.     

Inefficient Nef-mediated downmodulation of CD3 and MHC-I correlates with loss of CD4+T cells in natural SIV infection

Recent data suggest that Nef-mediated downmodulation of TCR-CD3 may protect SIVsmm-infected sooty mangabeys (SMs) against the loss of CD4⁺ T cells. However, the mechanisms underlying this protective effect remain unclear. To further assess the role of Nef in nonpathogenic SIV infection, we cloned nef alleles from 11 SIVsmm-infected SMs with high (>500) and 15 animals with low (<500) CD4⁺ T-cells/µl in bulk into proviral HIV-1 IRES/eGFP constructs and analyzed their effects on the phenotype, activation, and apoptosis of primary T cells. We found that not only efficient Nef-mediated downmodulation of TCR-CD3 but also of MHC-I correlated with preserved CD4⁺ T cell counts, as well as with high numbers of Ki67⁺CD4⁺ and CD8⁺CD28⁺ T cells and reduced CD95 expression by CD4⁺ T cells. Moreover, effective MHC-I downregulation correlated with low proportions of effector and high percentages of naïve and memory CD8⁺ T cells. We found that T cells infected with viruses expressing Nef alleles from the CD4low SM group expressed significantly higher levels of the CD69, interleukin (IL)-2 and programmed death (PD)-1 receptors than those expressing Nefs from the CD4high group. SIVsmm Nef alleles that were less active in downmodulating TCR-CD3 were also less potent in suppressing the activation of virally infected T cells and subsequent cell death. However, only nef alleles from a single animal with very low CD4⁺ T cell counts rendered T cells hyper-responsive to activation, similar to those of HIV-1. Our data suggest that Nef may protect the natural hosts of SIV against the loss of CD4⁺ T cells by at least two mechanisms: (i) downmodulation of TCR-CD3 to prevent activation-induced cell death and to suppress the induction of PD-1 that may impair T cell function and survival, and (ii) downmodulation of MHC-I to reduce CTL lysis of virally infected CD4⁺ T cells and/or bystander CD8⁺ T cell activation.     

Negative Selection by an Endogenous Retrovirus Promotes a Higher-Avidity CD4+ T Cell Response to Retroviral Infection

Effective T cell responses can decisively influence the outcome of retroviral infection. However, what constitutes protective T cell responses or determines the ability of the host to mount such responses is incompletely understood. Here we studied the requirements for development and induction of CD4⁺ T cells that were essential for immunity to Friend virus (FV) infection of mice, according to their TCR avidity for an FV-derived epitope. We showed that a self peptide, encoded by an endogenous retrovirus, negatively selected a significant fraction of polyclonal FV-specific CD4⁺ T cells and diminished the response to FV infection. Surprisingly, however, CD4⁺ T cell-mediated antiviral activity was fully preserved. Detailed repertoire analysis revealed that clones with low avidity for FV-derived peptides were more cross-reactive with self peptides and were consequently preferentially deleted. Negative selection of low-avidity FV-reactive CD4⁺ T cells was responsible for the dominance of high-avidity clones in the response to FV infection, suggesting that protection against the primary infecting virus was mediated exclusively by high-avidity CD4⁺ T cells. Thus, although negative selection reduced the size and cross-reactivity of the available FV-reactive naïve CD4⁺ T cell repertoire, it increased the overall avidity of the repertoire that responded to infection. These findings demonstrate that self proteins expressed by replication-defective endogenous retroviruses can heavily influence the formation of the TCR repertoire reactive with exogenous retroviruses and determine the avidity of the response to retroviral infection. Given the overabundance of endogenous retroviruses in the human genome, these findings also suggest that endogenous retroviral proteins, presented by products of highly polymorphic HLA alleles, may shape the human TCR repertoire that reacts with exogenous retroviruses or other infecting pathogens, leading to interindividual heterogeneity.     

Cord blood derived CD4+ CD25(high) T cells become functional regulatory T cells upon antigen encounter

Background: Upon antigen exposure, cord blood derived T cells respond to ubiquitous environmental antigens by high proliferation. To date it remains unclear whether these “excessive” responses relate to different regulatory properties of the putative T regulatory cell (Treg) compartment or even expansion of the Treg compartment itself.Methods: Cord blood (>37 week of gestation) and peripheral blood (healthy controls) were obtained and different Treg cell subsets were isolated. The suppressive potential of Treg populations after antigen exposure was evaluated via functional inhibition assays ([³H]thymidine incorporation assay and CFSE staining) with or without allergen stimulation. The frequency and markers of CD4⁺CD25^highFoxP3⁺ T cells were characterized by mRNA analysis and flow cytometry.Results: Cord blood derived CD4⁺CD25^high cells did not show substantial suppressor capacity upon TCR activation, in contrast to CD4⁺CD25^high cells freshly purified from adult blood. This could not be explained by a lower frequency of FoxP3⁺CD4⁺CD25^highcells or FOXP3 mRNA expression. However, after antigen-specific stimulation in vitro, these cells showed strong proliferation and expansion and gained potent suppressive properties. The efficiency of their suppressive capacity can be enhanced in the presence of endotoxins. If T-cells were sorted according to their CD127 expression, a tiny subset of Treg cells (CD4⁺CD25⁺CD127^low) is highly suppressive even without prior antigen exposure.Conclusion: Cord blood harbors a very small subset of CD4⁺CD25^high Treg cells that requires antigen-stimulation to show expansion and become functional suppressive Tregs.     

CD4+ T cells modulate expansion and survival but not functional properties of effector and memory CD8+ T cells induced by malaria sporozoites

CD4⁺ helper T cells are critical orchestrators of immune responses to infection and vaccination. During primary responses, naïve CD8⁺ T cells may need “CD4 help” for optimal development of memory populations. The immunological factors attributed to CD4 help depend on the context of immunization and vary depending on the priming system. In response to immunization with radiation-attenuated Plasmodium yoelii sporozoites, CD8⁺ T cells in BALB/c mice fail to generate large numbers of effector cells without help from CD4⁺ T cells – a defect not observed in most systems. Given this unique early dependence on CD4 help, we evaluated the effects of CD4⁺ cells on the development of functional properties of CD8⁺ T cells and on their ability to abolish infection. First, we determined that this effect was not mediated by CD4⁺ non-T cells and did not involve CD1d-restricted NKT cells. We found that CD8⁺ T cells induced by sporozoites without CD4 help formed memory populations severely reduced in magnitude that could not limit parasite development in the liver. The inability of these “helpless” memory T cells to protect is not a result of defects in effector function, as their capacity to produce cytokines and undergo cytotoxic degranulation was indistinguishable from control memory T cells. These data indicate that CD4⁺ T help may not be necessary to develop the functional attributes of CD8⁺ T cells; however they are crucial to ensure the survival of effector and memory cells induced in primary responses.     

Local induction of immunosuppressive CD8+ T cells in the gut-associated lymphoid tissues

Background

In contrast to intestinal CD4⁺ regulatory T cells (T_regs), the generation and function of immunomodulatory intestinal CD8⁺ T cells is less well defined. To dissect the immunologic mechanisms of CD8⁺ T cell function in the mucosa, reactivity against hemagglutinin (HA) expressed in intestinal epithelial cells of mice bearing a MHC class-I-restricted T-cell-receptor specific for HA was studied.

Methodology and Principal Findings

HA-specific CD8⁺ T cells were isolated from gut-associated tissues and phenotypically and functionally characterized for the expression of Foxp3⁺ and their suppressive capacity. We demonstrate that intestinal HA expression led to peripheral induction of HA-specific CD8⁺Foxp3⁺ T cells. Antigen-experienced CD8⁺ T cells in this transgenic mouse model suppressed the proliferation of CD8⁺ and CD4⁺ T cells in vitro. Gene expression analysis of suppressive HA-specific CD8⁺ T cells revealed a specific up-regulation of CD103, Nrp1, Tnfrsf9 and Pdcd1, molecules also expressed on CD4⁺ T_reg subsets. Finally, gut-associated dendritic cells were able to induce HA-specific CD8⁺Foxp3⁺ T cells.

Conclusion and Significance

We demonstrate that gut specific antigen presentation is sufficient to induce CD8⁺ T_regs in vivo which may maintain intestinal homeostasis by down-modulating effector functions of T cells.     

Concurrent detection of circulating minor histocompatibility antigen-specific CD8+ T cells in SCT recipients by combinatorial encoding MHC multimers

Allogeneic stem cell transplantation (SCT) is a potentially curative treatment for patients with hematologic malignancies. Its therapeutic effect is largely dependent on recognition of minor histocompatibility antigens (MiHA) by donor-derived CD8⁺ T cells. Therefore, monitoring of multiple MiHA-specific CD8⁺ T cell responses may prove to be valuable for evaluating the efficacy of allogeneic SCT. In this study, we investigated the use of the combinatorial encoding MHC multimer technique to simultaneously detect MiHA-specific CD8⁺ T cells in peripheral blood of SCT recipients. Feasibility of this approach was demonstrated by applying dual-color encoding MHC multimers for a set of 10 known MiHA. Interestingly, single staining using a fluorochrome- and Qdot-based five-color combination showed comparable results to dual-color staining for most MiHA-specific CD8⁺ T cell responses. In addition, we determined the potential value of combinatorial encoding MHC multimers in MiHA identification. Therefore, a set of 75 candidate MiHA peptides was predicted from polymorphic genes with a hematopoietic expression profile and further selected for high and intermediate binding affinity for HLA-A2. Screening of a large cohort of SCT recipients resulted in the detection of dual-color encoded CD8⁺ T cells following MHC multimer-based T cell enrichment and short ex vivo expansion. Interestingly, candidate MiHA-specific CD8⁺ T cell responses for LAG3 and TLR10 derived polymorphic peptides could be confirmed by genotyping of the respective SNPs. These findings demonstrate the potency of the combinatorial MHC multimer approach in the monitoring of CD8⁺ T cell responses to known and potential MiHA in limited amounts of peripheral blood from allogeneic SCT recipients.     

Monitoring of regulatory T cell frequencies and expression of CTLA-4 on T cells, before and after DC vaccination, can predict survival in GBM patients

Purpose

Dendritic cell (DC) vaccines have recently emerged as an innovative therapeutic option for glioblastoma patients. To identify novel surrogates of anti-tumor immune responsiveness, we studied the dynamic expression of activation and inhibitory markers on peripheral blood lymphocyte (PBL) subsets in glioblastoma patients treated with DC vaccination at UCLA.

Experimental Design

Pre-treatment and post-treatment PBL from 24 patients enrolled in two Phase I clinical trials of dendritic cell immunotherapy were stained and analyzed using flow cytometry. A univariate Cox proportional hazards model was utilized to investigate the association between continuous immune monitoring variables and survival. Finally, the immune monitoring variables were dichotomized and a recursive partitioning survival tree was built to obtain cut-off values predictive of survival.

Results

The change in regulatory T cell (CD3⁺CD4⁺CD25⁺CD127^low) frequency in PBL was significantly associated with survival (p = 0.0228; hazard ratio = 3.623) after DC vaccination. Furthermore, the dynamic expression of the negative co-stimulatory molecule, CTLA-4, was also significantly associated with survival on CD3⁺CD4⁺ T cells (p = 0.0191; hazard ratio = 2.840) and CD3⁺CD8⁺ T cells (p = 0.0273; hazard ratio = 2.690), while that of activation markers (CD25, CD69) was not. Finally, a recursive partitioning tree algorithm was utilized to dichotomize the post/pre fold change immune monitoring variables. The resultant cut-off values from these immune monitoring variables could effectively segregate these patients into groups with significantly different overall survival curves.

Conclusions

Our results suggest that monitoring the change in regulatory T cell frequencies and dynamic expression of the negative co-stimulatory molecules on peripheral blood T cells, before and after DC vaccination, may predict survival. The cut-off point generated from these data can be utilized in future prospective immunotherapy trials to further evaluate its predictive validity.     

Deficient regulatory T cell activity and low frequency of IL-17-producing T cells correlate with the extent of cardiomyopathy in human Chagas' disease

Background

Myocardium damage during Chagas'' disease results from the immunological imbalance between pro- and production of anti-inflammatory cytokines and has been explained based on the Th1–Th2 dichotomy and regulatory T cell activity. Recently, we demonstrated that IL-17 produced during experimental T. cruzi infection regulates Th1 cells differentiation and parasite induced myocarditis. Here, we investigated the role of IL-17 and regulatory T cell during human Chagas'' disease.

Methodology/Principal Findings

First, we observed CD4⁺IL-17⁺ T cells in culture of peripheral blood mononuclear cells (PBMC) from Chagas'' disease patients and we evaluated Th1, Th2, Th17 cytokine profile production in the PBMC cells from Chagas'' disease patients (cardiomyopathy-free, and with mild, moderate or severe cardiomyopathy) cultured with T. cruzi antigen. Cultures of PBMC from patients with moderate and severe cardiomyopathy produced high levels of TNF-α, IFN-γ and low levels of IL-10, when compared to mild cardiomyopathy or cardiomyopathy-free patients. Flow cytometry analysis showed higher CD4⁺IL-17⁺ cells in PBMC cultured from patients without or with mild cardiomyopathy, in comparison to patients with moderate or severe cardiomyopathy. We then analyzed the presence and function of regulatory T cells in all patients. All groups of Chagas'' disease patients presented the same frequency of CD4⁺CD25⁺ regulatory T cells. However, CD4⁺CD25⁺ T cells from patients with mild cardiomyopathy or cardiomyopathy-free showed higher suppressive activity than those with moderate and severe cardiomyopathy. IFN-γ levels during chronic Chagas'' disease are inversely correlated to the LVEF (P = 0.007, r = −0.614), while regulatory T cell activity is directly correlated with LVEF (P = 0.022, r = 0.500).

Conclusion/Significance

These results indicate that reduced production of the cytokines IL-10 and IL-17 in association with high levels of IFN-γ and TNF-α is correlated with the severity of the Chagas'' disease cardiomyopathy, and the immunological imbalance observed may be causally related with deficient suppressor activity of regulatory T cells that controls myocardial inflammation.     

Antigen-specific B cells reactivate an effective cytotoxic T cell response against phagocytosed Salmonella through cross-presentation

Background

The eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8⁺ T cell response. Dendritic cells (DCs) are considered to orchestrate the cytotoxic CD8⁺ T cell response via cross-presentation of bacterial antigens onto MHC class I molecules. Cross-presentation of Salmonella by DCs however, is accompanied by the induction of apoptosis in the DCs. Besides antibody production, B cells are required to clear Salmonella infection for other unknown reasons.

Methodology/Principal Findings

Here we show that Salmonella-specific B cells that phagocytose Salmonella upon BCR-ligation reactivate human memory CD8⁺ T cells via cross-presentation yielding a Salmonella-specific cytotoxic T cell response. The reactivation of CD8⁺ T cells is dependent on CD4⁺ T cell help. Unlike the DCs, B cell-mediated cross-presentation of Salmonella does not coincide with apoptosis.

Conclusions/Significance

B cells form a new player in the activation of the cytotoxic effector arm of the immune response and the generation of effective adaptive immunity in Salmonella infection.     

Physiological induction of regulatory Qa-1-restricted CD8+ T cells triggered by endogenous CD4+ T cell responses

T cell-dependent autoimmune diseases are characterized by the expansion of T cell clones that recognize immunodominant epitopes on the target antigen. As a consequence, for a given autoimmune disorder, pathogenic T cell clones express T cell receptors with a limited number of variable regions that define antigenic specificity. Qa-1, a MHC class I-like molecule, presents peptides from the variable region of TCRs to Qa-1-restricted CD8+ T cells. The induction of Vß-specific CD8+ T cells has been harnessed in an immunotherapeutic strategy known as the “T cell vaccination” (TCV) that comprises the injection of activated and attenuated CD4+ T cell clones so as to induce protective CD8+ T cells. We hypothesized that Qa-1-restricted CD8+ regulatory T cells could also constitute a physiologic regulatory arm of lymphocyte responses upon expansion of endogenous CD4+ T cells, in the absence of deliberate exogenous T cell vaccination. We immunized mice with two types of antigenic challenges in order to sequentially expand antigen-specific endogenous CD4+ T cells with distinct antigenic specificities but characterized by a common Vß chain in their TCR. The first immunization was performed with a non-self antigen while the second challenge was performed with a myelin-derived peptide known to drive experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We show that regulatory Vß-specific Qa-1-restricted CD8+ T cells induced during the first endogenous CD4+ T cell responses are able to control the expansion of subsequently mobilized pathogenic autoreactive CD4+ T cells. In conclusion, apart from the immunotherapeutic TCV, Qa-1-restricted specialized CD8+ regulatory T cells can also be induced during endogenous CD4+ T cell responses. At variance with other regulatory T cell subsets, the action of these Qa-1-restricted T cells seems to be restricted to the immediate re-activation of CD4+ T cells.     

TIM-3 expression characterizes regulatory T cells in tumor tissues and is associated with lung cancer progression

Background

T cell immunoglobulin-3 (TIM-3) has been established as a negative regulatory molecule and plays a critical role in immune tolerance. TIM-3 is upregulated in exhausted CD8⁺ T cells in both chronic infection and tumor. However, the nature of TIM-3⁺CD4⁺ T cells in the tumor microenvironment is unclear. This study is to characterize TIM-3 expressing lymphocytes within human lung cancer tissues and establish clinical significance of TIM-3 expression in lung cancer progression.

Methodology

A total of 51 human lung cancer tissue specimens were obtained from pathologically confirmed and newly diagnosed non-small cell lung cancer (NSCLC) patients. Leukocytes from tumor tissues, distal normal lung tissues, and peripheral blood mononuclear cells (PBMC) were analyzed for TIM-3 surface expression by flow cytometry. TIM-3 expression on tumor-infiltrating lymphocytes (TILs) was correlated with clinicopathological parameters.

Conclusions

TIM-3 is highly upregulated on both CD4⁺ and CD8⁺ TILs from human lung cancer tissues but negligibly expressed on T cells from patients'' peripheral blood. Frequencies of IFN-γ⁺ cells were reduced in TIM-3⁺CD8⁺ TILs compared to TIM-3⁻CD8⁺ TILs. However, the level of TIM-3 expression on CD8⁺ TILs failed to associate with any clinical pathological parameter. Interestingly, we found that approximately 70% of TIM-3⁺CD4⁺ TILs expressed FOXP3 and about 60% of FOXP3⁺ TILs were TIM-3⁺. Importantly, TIM-3 expression on CD4⁺ T cells correlated with poor clinicopathological parameters of NSCLC such as nodal metastasis and advanced cancer stages. Our study reveals a new role of TIM-3 as an important immune regulator in the tumor microenvironment via its predominant expression in regulatory T cells.     

Profile of Central and Effector Memory T Cells in the Progression of Chronic Human Chagas Disease

Background

Chronic Chagas disease presents several different clinical manifestations ranging from asymptomatic to severe cardiac and/or digestive clinical forms. Several studies have demonstrated that immunoregulatory mechanisms are important processes for the control of the intense immune activity observed in the chronic phase. T cells play a critical role in parasite specific and non-specific immune response elicited by the host against Trypanosoma cruzi. Specifically, memory T cells, which are basically classified as central and effector memory cells, might have a distinct migratory activity, role and function during the human Chagas disease.

Methodology/Principal Findings

Based on the hypothesis that the disease severity in humans is correlated to the quality of immune responses against T. cruzi, we evaluated the memory profile of peripheral CD4⁺ and CD8⁺ T lymphocytes as well as its cytokine secretion before and after in vitro antigenic stimulation. We evaluated cellular response from non-infected individuals (NI), patients with indeterminate (IND) or cardiac (CARD) clinical forms of Chagas disease. The expression of CD45RA, CD45RO and CCR7 surface molecules was determined on CD4⁺ and CD8⁺ T lymphocytes; the pattern of intracellular cytokines (IFN-γ, IL-10) synthesized by naive and memory cells was determined by flow cytometry. Our results revealed that IND and CARD patients have relatively lower percentages of naive (CD45RA^high) CD4⁺ and CD8⁺ T cells. However, statistical analysis of ex-vivo profiles of CD4⁺ T cells showed that IND have lower percentage of CD45RA^high in relation to non-infected individuals, but not in relation to CARD. Elevated percentages of memory (CD45RO^high) CD4⁺ T cells were also demonstrated in infected individuals, although statistically significant differences were only observed between IND and NI groups. Furthermore, when we analyzed the profile of secreted cytokines, we observed that CARD patients presented a significantly higher percentage of CD8⁺CD45RA^high IFN-γ-producing cells in control cultures and after antigen pulsing with soluble epimastigote antigens.

Conclusions

Based on a correlation between the frequency of IFN-γ producing CD8+ T cells in the T cell memory compartment and the chronic chagasic myocarditis, we propose that memory T cells can be involved in the induction of the development of the severe clinical forms of the Chagas disease by mechanisms modulated by IFN-γ. Furthermore, we showed that individuals from IND group presented more T_CM CD4⁺ T cells, which may induce a regulatory mechanism to protect the host against the exacerbated inflammatory response elicited by the infection.     

Caspase-3 is transiently activated without cell death during early antigen driven expansion of CD8(+) T cells in vivo

Background

CD8⁺ T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8⁺ T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8⁺ T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8⁺ T cell responses has yet to be examined.

Methods and Findings

We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8⁺ T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8⁺ T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3^hi and caspase-3^low CD8⁺ T cells. The expression of active caspase-3 peaked before effector phenotype (CD62L^low) CD8⁺ T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8⁺ cells remained active caspase-3^low throughout the contraction phase.

Conclusions

Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8⁺ T cells. Furthermore, the contraction of CD8⁺ T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3.     

IgE-Mediated Enhancement of CD4+ T Cell Responses in Mice Requires Antigen Presentation by CD11c+ Cells and Not by B Cells

IgE antibodies, administered to mice together with their specific antigen, enhance antibody and CD4⁺ T cell responses to this antigen. The effect is dependent on the low affinity receptor for IgE, CD23, and the receptor must be expressed on B cells. In vitro, IgE-antigen complexes are endocytosed via CD23 on B cells, which subsequently present the antigen to CD4⁺ T cells. This mechanism has been suggested to explain also IgE-mediated enhancement of immune responses in vivo. We recently found that CD23⁺ B cells capture IgE-antigen complexes in peripheral blood and rapidly transport them to B cell follicles in the spleen. This provides an alternative explanation for the requirement for CD23⁺ B cells. The aim of the present study was to determine whether B-cell mediated antigen presentation of IgE-antigen complexes explains the enhancing effect of IgE on immune responses in vivo. The ability of spleen cells, taken from mice 1–4 h after immunization with IgE-antigen, to present antigen to specific CD4⁺ T cells was analyzed. Antigen presentation was intact when spleens were depleted of CD19⁺ cells (i.e., primarily B cells) but was severely impaired after depletion of CD11c⁺ cells (i.e., primarily dendritic cells). In agreement with this, the ability of IgE to enhance proliferation of CD4⁺ T cells was abolished in CD11c-DTR mice conditionally depleted of CD11c⁺ cells. Finally, the lack of IgE-mediated enhancemen of CD4⁺ T cell responses in CD23^-/- mice could be rescued by transfer of MHC-II-compatible as well as by MHC-II-incompatible CD23⁺ B cells. These findings argue against the idea that IgE-mediated enhancement of specific CD4⁺ T cell responses in vivo is caused by increased antigen presentation by B cells. A model where CD23⁺ B cells act as antigen transporting cells, delivering antigen to CD11c⁺ cells for presentation to T cells is consistent with available experimental data.