IL-2 Mediates CD4+ T Cell Help in the Breakdown of Memory-Like CD8+ T Cell Tolerance under Lymphopenic Conditions

Background

Lymphopenia results in the proliferation and differentiation of naïve T cells into memory-like cells in the apparent absence of antigenic stimulation. This response, at least in part due to a greater availability of cytokines, is thought to promote anti-self responses. Although potentially autoreactive memory-like CD8⁺ T cells generated in a lymphopenic environment are subject to the mechanisms of peripheral tolerance, they can induce autoimmunity in the presence of antigen-specific memory-like CD4⁺ T helper cells.

Methodology/Principal Findings

Here, we studied the mechanisms underlying CD4 help under lymphopenic conditions in transgenic mice expressing a model antigen in the beta cells of the pancreas. Surprisingly, we found that the self-reactivity mediated by the cooperation of memory-like CD8⁺ and CD4⁺ T cells was not abrogated by CD40L blockade. In contrast, treatment with anti-IL-2 antibodies inhibited the onset of autoimmunity. IL-2 neutralization prevented the CD4-mediated differentiation of memory-like CD8⁺ T cells into pathogenic effectors in response to self-antigen cross-presentation. Furthermore, in the absence of helper cells, induction of IL-2 signaling by an IL-2 immune complex was sufficient to promote memory-like CD8⁺ T cell self-reactivity.

Conclusions/Significance

IL-2 mediates the cooperation of memory-like CD4⁺ and CD8⁺ T cells in the breakdown of cross-tolerance, resulting in effector cytotoxic T lymphocyte differentiation and the induction of autoimmune disease.     

In Situ Patrolling of Regulatory T Cells Is Essential for Protecting Autoimmune Exocrinopathy

Background

Migration of T cells, including regulatory T (Treg) cells, into the secondary lymph organs is critically controlled by chemokines and adhesion molecules. However, the mechanisms by which Treg cells regulate organ-specific autoimmunity via these molecules remain unclear. Although we previously reported autoimmune exocrinopathy resembling Sjögren''s syndrome (SS) in the lacrimal and salivary glands from C-C chemokine receptor 7 (CCR7)-deficient mice, it is still unclear whether CCR7 signaling might specifically affect the dynamics and functions of Treg cells in vivo. We therefore investigated the cellular mechanism for suppressive function of Treg cells via CCR7 in autoimmunity using mouse models and human samples.

Methods and Findings

Patrolling Treg cells were detected in the exocrine organs such as lacrimal and salivary glands from normal mice that tend to be targets for autoimmunity while the Treg cells were almost undetectable in the exocrine glands of CCR7 ^−/− mice. In addition, we found the significantly increased retention of CD4⁺CD25⁺Foxp3⁺ Treg cells in the lymph nodes of CCR7 ^−/− mice with aging. Although Treg cell egress requires sphingosine 1-phosphate (S1P), chemotactic function to S1P of CCR7−/− Treg cells was impaired compared with that of WT Treg cells. Moreover, the in vivo suppression activity was remarkably diminished in CCR7 ^−/− Treg cells in the model where Treg cells were co-transferred with CCR7 ^−/− CD25^-CD4⁺ T cells into Rag2 ^−/− mice. Finally, confocal analysis showed that CCR7⁺Treg cells were detectable in normal salivary glands while the number of CCR7⁺Treg cells was extremely decreased in the tissues from patients with Sjögren''s syndrome.

Conclusions

These results indicate that CCR7 essentially governs the patrolling functions of Treg cells by controlling the traffic to the exocrine organs for protecting autoimmunity. Characterization of this cellular mechanism could have clinical implications by supporting development of new diagnosis or treatments for the organ-specific autoimmune diseases such as Sjögren''s syndrome and clarifying how the local immune system regulates autoimmunity.     

A Novel Mechanism of Soluble HLA-G Mediated Immune Modulation: Downregulation of T Cell Chemokine Receptor Expression and Impairment of Chemotaxis

Background

In recent years, many immunoregulatory functions have been ascribed to soluble HLA-G (sHLA-G). Since chemotaxis is crucial for an efficient immune response, we have investigated for the first time the effects of sHLA-G on chemokine receptor expression and function in different human T cell populations.

Methodology/Principal Findings

T cell populations isolated from peripheral blood were stimulated in the presence or absence of sHLA-G. Chemokine receptors expression was evaluated by flow cytometry. sHLA-G downregulated expression of i) CCR2, CXCR3 and CXCR5 in CD4⁺ T cells, ii) CXCR3 in CD8⁺ T cells, iii) CXCR3 in Th1 clones iv) CXCR3 in TCR Vδ2γ9 T cells, and upregulated CXCR4 expression in TCR Vδ2γ9 T cells. sHLA-G inhibited in vitro chemotaxis of i) CD4⁺ T cells towards CCL2, CCL8, CXCL10 and CXCL11, ii) CD8⁺ T cells towards CXCL10 and CXCL11, iii) Th1 clones towards CXCL10, and iv) TCR Vδ2γ9 T cells towards CXCL10 and CXCL11. Downregulation of CXCR3 expression on CD4+ T cells by sHLA-G was partially reverted by adding a blocking antibody against ILT2/CD85j, a receptor for sHLA-G, suggesting that sHLA-G downregulated chemokine receptor expression mainly through the interaction with ILT2/CD85j. Follicular helper T cells (T_FH) were isolated from human tonsils and stimulated as described above. sHLA-G impaired CXCR5 expression in T_FH and chemotaxis of the latter cells towards CXCL13. Moreover, sHLA-G expression was detected in tonsils by immunohistochemistry, suggesting a role of sHLA-G in local control of T_FH cell chemotaxis. Intracellular pathways were investigated by Western Blot analysis on total extracts from CD4+ T cells. Phosphorylation of Stat5, p70 s6k, β-arrestin and SHP2 was modulated by sHLA-G treatment.

Conclusions/Significance

Our data demonstrated that sHLA-G impairs expression and functionality of different chemokine receptors in T cells. These findings delineate a novel mechanism whereby sHLA-G modulates T cell recruitment in physiological and pathological conditions.     

Dendritic Cells in Chronic Mycobacterial Granulomas Restrict Local Anti-Bacterial T Cell Response in a Murine Model

Background

Mycobacterium-induced granulomas are the interface between bacteria and host immune response. During acute infection dendritic cells (DCs) are critical for mycobacterial dissemination and activation of protective T cells. However, their role during chronic infection in the granuloma is poorly understood.

Methodology/Principal Findings

We report that an inflammatory subset of murine DCs are present in granulomas induced by Mycobacteria bovis strain Bacillus Calmette-guerin (BCG), and both their location in granulomas and costimulatory molecule expression changes throughout infection. By flow cytometric analysis, we found that CD11c⁺ cells in chronic granulomas had lower expression of MHCII and co-stimulatory molecules CD40, CD80 and CD86, and higher expression of inhibitory molecules PD-L1 and PD-L2 compared to CD11c⁺ cells from acute granulomas. As a consequence of their phenotype, CD11c⁺ cells from chronic lesions were unable to support the reactivation of newly-recruited, antigen 85B-specific CD4⁺IFNγ⁺ T cells or induce an IFNγ response from naïve T cells in vivo and ex vivo. The mechanism of this inhibition involves the PD-1:PD-L signaling pathway, as ex vivo blockade of PD-L1 and PD-L2 restored the ability of isolated CD11c⁺ cells from chronic lesions to stimulate a protective IFNγ T cell response.

Conclusions/Significance

Our data suggest that DCs in chronic lesions may facilitate latent infection by down-regulating protective T cell responses, ultimately acting as a shield that promotes mycobacterium survival. This DC shield may explain why mycobacteria are adapted for long-term survival in granulomatous lesions.     

A Decreased Frequency of Regulatory T Cells in Patients with Common Variable Immunodeficiency

Introduction

Common variable immunodeficiency disorder (CVID) is a heterogeneous syndrome, characterized by deficient antibody production and recurrent bacterial infections in addition abnormalities in T cells. CD4⁺CD25^high regulatory T cells (Treg) are essential modulators of immune responses, including down-modulation of immune response to pathogens, allergens, cancer cells and self-antigens.

Objective

In this study we set out to investigate the frequency of Treg cells in CVID patients and correlate with their immune activation status.

Materials and Methods

Sixteen patients (6 males and 10 females) with CVID who had been treated with regular intravenous immunoglobulin and 14 controls were enrolled. Quantitative analyses of peripheral blood mononuclear cells (PBMC) were performed by multiparametric flow cytometry using the following cell markers: CD38, HLA-DR, CCR5 (immune activation); CD4, CD25, FOXP3, CD127, and OX40 (Treg cells); Ki-67 and IFN-γ (intracellular cytokine).

Results

A significantly lower proportion of CD4⁺CD25^highFOXP3 T cells was observed in CVID patients compared with healthy controls (P<0.05). In addition to a higher proportion of CD8⁺ T cells from CVID patients expressing the activation markers, CD38⁺ and HLA-DR⁺ (P<0.05), we observed no significant correlation between Tregs and immune activation.

Conclusion

Our results demonstrate that a reduction in Treg cells could have impaired immune function in CVID patients.     

Role of 4-1BB Receptor in the Control Played by CD8+ T Cells on IFN-γ Production by Mycobacterium tuberculosis Antigen-Specific CD4+ T Cells

Background

Antigen-specific IFN-γ producing CD4⁺ T cells are the main mediators of protection against Mycobacterium tuberculosis infection both under natural conditions and following vaccination. However these cells are responsible for lung damage and poor vaccine efficacy when not tightly controlled. Discovering new tools to control nonprotective antigen-specific IFN-γ production without affecting protective IFN-γ is a challenge in tuberculosis research.

Methods and Findings

Immunization with DNA encoding Ag85B, a candidate vaccine antigen of Mycobacterium tuberculosis, elicited in mice a low but protective CD4⁺ T cell-mediated IFN-γ response, while in mice primed with DNA and boosted with Ag85B protein a massive increase in IFN-γ response was associated with loss of protection. Both protective and non-protective Ag85B-immunization generated antigen-specific CD8⁺ T cells which suppressed IFN-γ-secreting CD4⁺ T cells. However, ex vivo ligation of 4-1BB, a member of TNF-receptor super-family, reduced the massive, non-protective IFN-γ responses by CD4⁺ T cells in protein-boosted mice without affecting the low protective IFN-γ-secretion in mice immunized with DNA. This selective inhibition was due to the induction of 4-1BB exclusively on CD8⁺ T cells of DNA-primed and protein-boosted mice following Ag85B protein stimulation. The 4-1BB-mediated IFN-γ inhibition did not require soluble IL-10, TGF-β, XCL-1 and MIP-1β. In vivo Ag85B stimulation induced 4-1BB expression on CD8⁺ T cells and in vivo 4-1BB ligation reduced the activation, IFN-γ production and expansion of Ag85B-specific CD4⁺ T cells of DNA-primed and protein-boosted mice.

Conclusion/Significance

Antigen-specific suppressor CD8⁺ T cells are elicited through immunization with the mycobacterial antigen Ag85B. Ligation of 4-1BB receptor further enhanced their suppressive activity on IFN-γ-secreting CD4⁺ T cells. The selective expression of 4-1BB only on CD8⁺ T cells in mice developing a massive, non-protective IFN-γ response opens novel strategies for intervention in tuberculosis pathology and vaccination through T-cell co-stimulatory-based molecular targeting.     

Heterosexual and Homosexual Partners Practising Unprotected Sex May Develop Allogeneic Immunity and to a Lesser Extent Tolerance

Background

Epidemiological studies suggest that allogeneic immunity may inhibit HIV-1 transmission from mother to baby and is less frequent in multiparous than uniparous women. Alloimmune responses may also be elicited during unprotected heterosexual intercourse, which is associated ex vivo with resistance to HIV infection.

Methodology/Principal Findings

The investigation was carried out in well-defined heterosexual and homosexual monogamous partners, practising unprotected sex and a heterosexual cohort practising protected sex. Allogeneic CD4⁺ and CD8⁺ T cell proliferative responses were elicited by stimulating PBMC with the partners'' irradiated monocytes and compared with 3^rd party unrelated monocytes, using the CFSE method. Significant increase in allogeneic proliferative responses was found in the CD4⁺ and CD8⁺ T cells to the partners'' irradiated monocytes, as compared with 3^rd party unrelated monocytes (p≤0.001). However, a significant decrease in proliferative responses, especially of CD8⁺ T cells to the partners'' compared with 3^rd party monocytes was consistent with tolerization, in both the heterosexual and homosexual partners (p<0.01). Examination of CD4⁺CD25⁺FoxP3⁺ regulatory T cells by flow cytometry revealed a significantly greater proportion of these cells in the homosexual than heterosexual partners practising unprotected sex (p<0.05). Ex vivo studies of infectivity of PBMC with HIV-1 showed significantly greater inhibition of infectivity of PBMC from heterosexual subjects practising unprotected compared with those practising protected sex (p = 0.02).

Conclusions/Significance

Both heterosexual and homosexual monogamous partners practising unprotected sex develop allogeneic CD4⁺ and CD8⁺ T cell proliferative responses to the partners'' unmatched cells and a minority may be tolerized. However, a greater proportion of homosexual rather than heterosexual partners developed CD4⁺CD25FoxP3⁺ regulatory T cells. These results, in addition to finding greater inhibition of HIV-1 infectivity in PBMC ex vivo in heterosexual partners practising unprotected, compared with those practising protected sex, suggest that allogeneic immunity may play a significant role in the immuno-pathogenesis of HIV-1 infection.     

Human Cord Blood Stem Cell-Modulated Regulatory T Lymphocytes Reverse the Autoimmune-Caused Type 1 Diabetes in Nonobese Diabetic (NOD) Mice

Background

The deficit of pancreatic islet β cells caused by autoimmune destruction is a crucial issue in type 1 diabetes (T1D). It is essential to fundamentally control the autoimmunity for treatment of T1D. Regulatory T cells (Tregs) play a pivotal role in maintaining self-tolerance through their inhibitory impact on autoreactive effector T cells. An abnormality of Tregs is associated with initiation of progression of T1D.

Methodology/Principal Findings

Here, we report that treatment of established autoimmune-caused diabetes in NOD mice with purified autologous CD4⁺CD62L⁺ Tregs co-cultured with human cord blood stem cells (CB-SC) can eliminate hyperglycemia, promote islet β-cell regeneration to increase β-cell mass and insulin production, and reconstitute islet architecture. Correspondingly, treatment with CB-SC-modulated CD4⁺CD62L⁺ Tregs (mCD4CD62L Tregs) resulted in a marked reduction of insulitis, restored Th1/Th2 cytokine balance in blood, and induced apoptosis of infiltrated leukocytes in pancreatic islets.

Conclusions/Significance

These data demonstrate that treatment with mCD4CD62L Tregs can reverse overt diabetes, providing a novel strategy for the treatment of type 1 diabetes as well as other autoimmune diseases.     

Preserved MHC Class II Antigen Processing in Monocytes from HIV-Infected Individuals

Background

MHC-II restricted CD4+ T cells are dependent on antigen presenting cells (APC) for their activation. APC dysfunction in HIV-infected individuals could accelerate or exacerbate CD4+ T cell dysfunction and may contribute to increased levels of immunodeficiency seen in some patients regardless of their CD4+ T cell numbers. Here we test the hypothesis that APC from HIV-infected individuals have diminished antigen processing and presentation capacity.

Methodology/Principal Findings

Monocytes (MN) were purified by immuno-magnetic bead isolation techniques from HLA-DR1.01+ or DR15.01+ HIV-infected and uninfected individuals. MN were analyzed for surface MHC-II expression and for antigen processing and presentation capacity after overnight incubation with soluble antigen or peptide and HLA-DR matched T cell hybridomas. Surface expression of HLA-DR was 20% reduced (p<0.03) on MN from HIV-infected individuals. In spite of this, there was no significant difference in antigen processing and presentation by MN from 14 HIV-infected donors (8 HLA-DR1.01+ and 6 HLA-DR15.01+) compared to 24 HIV-uninfected HLA-matched subjects.

Conclusions/Significance

We demonstrated that MHC class II antigen processing and presentation is preserved in MN from HIV-infected individuals. This further supports the concept that this aspect of APC function does not further contribute to CD4+ T cell dysfunction in HIV disease.     

APC Activation Restores Functional CD4+CD25+ Regulatory T Cells in NOD Mice that Can Prevent Diabetes Development

Background

Defects in APC and regulatory cells are associated with diabetes development in NOD mice. We have shown previously that NOD APC are not effective at stimulating CD4⁺CD25⁺ regulatory cell function in vitro. We hypothesize that failure of NOD APC to properly activate CD4⁺CD25⁺ regulatory cells in vivo could compromise their ability to control pathogenic cells, and activation of NOD APC could restore this defect, thereby preventing disease.

Methodology/Principal Findings

To test these hypotheses, we used the well-documented ability of complete Freund''s adjuvant (CFA), an APC activator, to prevent disease in NOD mice. Phenotype and function of CD4⁺CD25⁺ regulatory cells from untreated and CFA-treated NOD mice were determined by FACS, and in vitro and in vivo assays. APC from these mice were also evaluated for their ability to activate regulatory cells in vitro. We have found that sick NOD CD4⁺CD25⁺ cells expressed Foxp3 at the same percentages, but decreased levels per cell, compared to young NOD or non-NOD controls. Treatment with CFA increased Foxp3 expression in NOD cells, and also increased the percentages of CD4⁺CD25⁺Foxp3⁺ cells infiltrating the pancreas compared to untreated NOD mice. Moreover, CD4⁺CD25⁺ cells from pancreatic LN of CFA-treated, but not untreated, NOD mice transferred protection from diabetes. Finally, APC isolated from CFA-treated mice increased Foxp3 and granzyme B expression as well as regulatory function by NOD CD4⁺CD25⁺ cells in vitro compared to APC from untreated NOD mice.

Conclusions/Significance

These data suggest that regulatory T cell function and ability to control pathogenic cells can be enhanced in NOD mice by activating NOD APC.     

Distinct Differences in the Expansion and Phenotype of TB10.4 Specific CD8 and CD4 T Cells after Infection with Mycobacterium tuberculosis

Background

Recently we and others have identified CD8 and CD4 T cell epitopes within the highly expressed M. tuberculosis protein TB10.4. This has enabled, for the first time, a comparative study of the dynamics and function of CD4 and CD8 T cells specific for epitopes within the same protein in various stages of TB infection.

Methods and Findings

We focused on T cells directed to two epitopes in TB10.4; the MHC class I restricted epitope TB10.4 _3–11 (CD8/10.4 T cells) and the MHC class II restricted epitope TB10.4 _74–88 (CD4/10.4 T cells). CD4/10.4 and CD8/10.4 T cells displayed marked differences in terms of expansion and contraction in a mouse TB model. CD4/10.4 T cells dominated in the early phase of infection whereas CD8/10.4 T cells were expanded after week 16 and reached 5–8 fold higher numbers in the late phase of infection. In the early phase of infection both CD4/10.4 and CD8/10.4 T cells were characterized by 20–25% polyfunctional cells (IL-2⁺, IFN-γ⁺, TNF-α⁺), but whereas the majority of CD4/10.4 T cells were maintained as polyfunctional T cells throughout infection, CD8/10.4 T cells differentiated almost exclusively into effector cells (IFN-γ⁺, TNF-α⁺). Both CD4/10.4 and CD8/10.4 T cells exhibited cytotoxicity in vivo in the early phase of infection, but whereas CD4/10.4 cell mediated cytotoxicity waned during the infection, CD8/10.4 T cells exhibited increasing cytotoxic potential throughout the infection.

Conclusions/Significance

Our results show that CD4 and CD8 T cells directed to epitopes in the same antigen differ both in their kinetics and functional characteristics throughout an infection with M. tuberculosis. In addition, the observed strong expansion of CD8 T cells in the late stages of infection could have implications for the development of post exposure vaccines against latent TB.     

Enhancement of Tumour-Specific Immune Responses In Vivo by ‘MHC Loading-Enhancer’ (MLE)

Background

Class II MHC molecules (MHC II) are cell surface receptors displaying short protein fragments for the surveillance by CD4+ T cells. Antigens therefore have to be loaded onto this receptor in order to induce productive immune responses. On the cell surface, most MHC II molecules are either occupied by ligands or their binding cleft has been blocked by the acquisition of a non-receptive state. Direct loading with antigens, as required during peptide vaccinations, is therefore hindered.

Principal Findings

Here we show, that the in vivo response of CD4+ T cells can be improved, when the antigens are administered together with ‘MHC-loading enhancer’ (MLE). MLE are small catalytic compounds able to open up the MHC binding site by triggering ligand-release and stabilizing the receptive state. Their enhancing effect on the immune response was demonstrated here with an antigen from the influenza virus and tumour associated antigens (TAA) derived from the NY-ESO-1 protein. The application of these antigens in combination with adamantane ethanol (AdEtOH), an MLE compound active on human HLA-DR molecules, significantly increased the frequency of antigen-specific CD4+ T cells in mice transgenic for the human MHC II molecule. Notably, the effect was evident only with the MLE-susceptible HLA-DR molecule and not with murine MHC II molecules non-susceptible for the catalytic effect of the MLE.

Conclusion

MLE can specifically increase the potency of a vaccine by facilitating the efficient transfer of the antigen onto the MHC molecule. They may therefore open a new way to improve vaccination efficacy and tumour-immunotherapy.     

Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

Background

Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique.

Methodology/Principal Findings

After transplantation with CD34⁺CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2^−/−IL-2Rγc^−/− mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19⁺CD27⁺ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively.

Conclusion/Significance

This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.     

Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Background

The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4⁺ CD25^highFoxp3⁺ regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-γ chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.

Methodology/Principal Findings

CD4⁺CD25⁺ and CD4⁺CD25^neg T cells were isolated from PBMC of normal controls (n = 21) using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4⁺CD25^high and CD4⁺CD25^neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4⁺CD25^neg or CD8⁺CD25^neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4⁺CD25⁺ T cells in the presence of 1–100 nM RAPA (p<0.001). RAPA-expanded Treg were largely CD4⁺CD25^highFoxp3⁺ cells and were resistant to apoptosis, while CD4⁺CD25^neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4⁺CD25^neg cells. Activated Treg±RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/mTOR pathway.

Conclusions/Significance

RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation.     

CD8+ T Cells Cause Disability and Axon Loss in a Mouse Model of Multiple Sclerosis

Background

The objective of this study was to test the hypothesis that CD8⁺ T cells directly mediate motor disability and axon injury in the demyelinated central nervous system. We have previously observed that genetic deletion of the CD8⁺ T cell effector molecule perforin leads to preservation of motor function and preservation of spinal axons in chronically demyelinated mice.

Methodology/Principal Findings

To determine if CD8⁺ T cells are necessary and sufficient to directly injure demyelinated axons, we adoptively transferred purified perforin-competent CD8⁺ spinal cord-infiltrating T cells into profoundly demyelinated but functionally preserved perforin-deficient host mice. Transfer of CD8⁺ spinal cord-infiltrating T cells rapidly and irreversibly impaired motor function, disrupted spinal cord motor conduction, and reduced the number of medium- and large-caliber spinal axons. Likewise, immunodepletion of CD8⁺ T cells from chronically demyelinated wildtype mice preserved motor function and limited axon loss without altering other disease parameters.

Conclusions/Significance

In multiple sclerosis patients, CD8⁺ T cells outnumber CD4⁺ T cells in active lesions and the number of CD8⁺ T cells correlates with the extent of ongoing axon injury and functional disability. Our findings suggest that CD8⁺ T cells may directly injure demyelinated axons and are therefore a viable therapeutic target to protect axons and motor function in patients with multiple sclerosis.     

Human CD4+ Memory T Cells Can Become CD4+IL-9+ T Cells

Background

IL-9 is a growth factor for T- and mast-cells that is secreted by human Th2 cells. We recently reported that IL-4+TGF-β directs mouse CD4⁺CD25⁻CD62L⁺ T cells to commit to inflammatory IL-9 producing CD4⁺ T cells.

Methodology/Principal Findings

Here we show that human inducible regulatory T cells (iTregs) also express IL-9. IL-4+TGF-β induced higher levels of IL-9 expression in plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) activated human resting memory CD4⁺CD25⁻CD45RO⁺ T cells as compared to naïve CD4⁺CD25⁻CD45RA⁺ T cells. In addition, as compared to pbCD3/sCD28 plus TGF-β stimulation, IL-4+TGF-β stimulated memory CD4⁺CD25⁻CD45RO⁺ T cells expressed reduced FOXP3 protein. As analyzed by pre-amplification boosted single-cell real-time PCR, human CD4⁺IL-9⁺ T cells expressed GATA3 and RORC, but not IL-10, IL-13, IFNγ or IL-17A/F. Attempts to optimize IL-9 production by pbCD3/sCD28 and IL-4+TGF-β stimulated resting memory CD4⁺ T cells demonstrated that the addition of IL-1β, IL-12, and IL-21 further enhance IL-9 production.

Conclusions/Significance

Taken together these data show both the differences and similarities between mouse and human CD4⁺IL9⁺ T cells and reaffirm the powerful influence of inflammatory cytokines to shape the response of activated CD4⁺ T cells to antigen.     

Non-Redundant Role for IL-12 and IL-27 in Modulating Th2 Polarization of Carcinoembryonic Antigen Specific CD4 T Cells from Pancreatic Cancer Patients

Background

Pancreatic cancer is a very aggressive disease with dismal prognosis; peculiar is the tumor microenvironment characterized by an extensive fibrotic stroma, which favors rapid tumor progression. We previously reported that pancreatic cancer patients have a selective Th2 skew in the anti-carcinoembryonic antigen (CEA) CD4⁺ T cell immunity, which correlates with the presence of a predominant GATA-3⁺ tumor lymphoid infiltrate. This has negative effects in both effective anti-tumor immunity and further favoring fibrinogenesis. Aim of this study was to evaluate whether the Th2 polarization of CEA-specific CD4⁺ T cells from pancreatic cancer patients is stable or can be reverted by immunomodulating cytokines.

Methodology/Principal Findings

We first evaluated the influence of IL-12 and IL-27, as single agents and in association, on the polarization of CEA-specific Th2 CD4⁺ T cell clones from a pancreatic cancer patient. We found that only the combination of IL-12 and IL-27 modified the polarization of Th2 effectors by both reduction of IL-5, GM-CSF and IL-13 and induction of IFN-γ production, which lasted after cytokine removal. Second, we evaluated the effect of the combined treatment on polyclonal CEA-specific CD4⁺ T cells in short-time re-stimulation assays. In agreement with the data obtained with the clones, we found that the combined treatment functionally modulated the Th2 polarization of CEA-specific CD4⁺ T cells and enhanced pre-existing Th1 type immunity.

Conclusions/Significance

Collectively, our results demonstrate that tumor antigen specific Th2 CD4⁺ T cells in pancreatic cancer are endowed with functional plasticity. Hence, loco-regional cytokines delivery or targeted therapy based on antibodies or molecules directed to the tumor stroma might improve anti-tumor immunity and ameliorate fibrosis, without systemic toxicity.     

Prophylactic Efficacy of TcVac2 against Trypanosoma cruzi in Mice

Background

Chagas disease is a major health problem in Latin America, and an emerging infectious disease in the US. Previously, we have screened the Trypanosoma cruzi sequence database by a computational/bioinformatics approach, and identified antigens that exhibited the characteristics of vaccine candidates.

Methodology

We investigated the protective efficacy of a multi-component DNA-prime/protein-boost vaccine (TcVac2) constituted of the selected candidates and cytokine (IL-12 and GM-CSF) expression plasmids in a murine model. C57BL/6 mice were immunized with antigen-encoding plasmids plus cytokine adjuvants, followed by recombinant proteins; and two-weeks later, challenged with T. cruzi trypomastigotes. ELISA and flow cytometry were employed to measure humoral (antibody isotypes) and cellular (lymphocyte proliferation, CD4⁺ and CD8⁺ T cell phenotype and cytokines) responses. Myocardial pathology was evaluated by H&E and Masson''s trichrome staining.

Principal Findings

TcVac2 induced a strong antigen-specific antibody response (IgG2b>IgG1) and a moderate level of lymphocyte proliferation in mice. Upon challenge infection, TcVac2-vaccinated mice expanded the IgG2b/IgG1 antibodies and elicited a substantial CD8⁺ T cell response associated with type 1 cytokines (IFN-γ and TNF-α) that resulted in control of acute parasite burden. During chronic phase, antibody response persisted, splenic activation of CD8⁺ T cells and IFN-γ/TNF-α cytokines subsided, and IL-4/IL-10 cytokines became dominant in vaccinated mice. The tissue parasitism, inflammation, and fibrosis in heart and skeletal muscle of TcVac2-vaccinated chronic mice were undetectable by histological techniques. In comparison, mice injected with vector or cytokines only responded to T. cruzi by elicitation of a mixed (type 1/type 2) antibody, T cell and cytokine response, and exhibited persistent parasite burden and immunopathology in the myocardium.

Conclusion

TcVac2-induced activation of type 1 antibody and lymphocyte responses provided resistance to acute T. cruzi infection, and consequently, prevented the evolution of chronic immunopathology associated with parasite persistence in chagasic hearts.     

Trafficking of high avidity HER-2/neu-specific T cells into HER-2/neu-expressing tumors after depletion of effector/memory-like regulatory T cells

Background

Cancer vaccines are designed to activate and enhance cancer-antigen-targeted T cells that are suppressed through multiple mechanisms of immune tolerance in cancer-bearing hosts. T regulatory cell (Treg) suppression of tumor-specific T cells is one barrier to effective immunization. A second mechanism is the deletion of high avidity tumor-specific T cells, which leaves a less effective low avidity tumor specific T cell repertoire available for activation by vaccines. Treg depleting agents including low dose cyclophosphamide (Cy) and antibodies that deplete CD25-expressing Tregs have been used with limited success to enhance the potency of tumor-specific vaccines. In addition, few studies have evaluated mechanisms that activate low avidity cancer antigen-specific T cells. Therefore, we developed high and low avidity HER-2/neu-specific TCR transgenic mouse colonies specific for the same HER-2/neu epitope to define the tolerance mechanisms that specifically affect high versus low avidity tumor-specific T cells.

Methodology/Principal Findings

High and low avidity CD8⁺ T cell receptor (TCR) transgenic mice specific for the breast cancer antigen HER-2/neu (neu) were developed to provide a purified source of naïve, tumor-specific T cells that can be used to study tolerance mechanisms. Adoptive transfer studies into tolerant FVB/N-derived HER-2/neu transgenic (neu-N) mice demonstrated that high avidity, but not low avidity, neu-specific T cells are inhibited by Tregs as the dominant tolerizing mechanism. High avidity T cells persisted, produced IFNγ, trafficked into tumors, and lysed tumors after adoptive transfer into mice treated with a neu-specific vaccine and low dose Cy to deplete Tregs. Analysis of Treg subsets revealed a Cy-sensitive CD4⁺Foxp3⁺CD25^low tumor-seeking migratory phenotype, characteristic of effector/memory Tregs, and capable of high avidity T cell suppression.

Conclusion/Significance

Depletion of CD25^low Tregs allows activation of tumor-clearing high avidity T cells. Thus, the development of agents that specifically deplete Treg subsets should translate into more effective immunotherapies while avoiding autoimmunity.