Antigen-specific CD4 effector T cells: Analysis of factors regulating clonal expansion and cytokine production: Clonal expansion and cytokine production by CD4 effector T cells

In order to fully understand T cell-mediated immunity, the mechanisms that regulate clonal expansion and cytokine production by CD4⁺ antigen-specific effector T cells in response to a wide range of antigenic stimulation needs clarification. For this purpose, panels of antigen-specific CD4⁺ T cell clones with different thresholds for antigen-induced proliferation were generated by repeated stimulation with high- or low-dose antigen. Differences in antigen sensitivities did not correlate with expression of TCR, CD4, adhesion or costimulatory molecules. There was no significant difference in antigen-dependent cytokine production by TG40 cells transfected with TCR obtained from either high- or low-dose-responding T cell clones, suggesting that the affinity of TCRs for their ligands is not primary determinant of T cell antigen reactivity. The proliferative responses of all T cell clones to both peptide stimulation and to TCRβ crosslinking revealed parallel dose-response curves. These results suggest that the TCR signal strength of effector T cells and threshold of antigen reactivity is determined by an intrinsic property, such as the TCR signalosome and/or intracellular signaling machinery. Finally, the antigen responses of high- and low-peptide-responding T cell clones reveal that clonal expansion and cytokine production of effector T cells occur independently of antigen concentration. Based on these results, the mechanisms underlying selection of high “avidity” effector and memory T cells in response to pathogen are discussed.     

Modulation of MOG 37-50-specific CD8+ T cell activation and expansion by CD43

Several recent reports have described an effector role for CD8(+) T cells during EAE. We have previously demonstrated reduced disease incidence and severity in CD43(-/-) mice following MOG immunization, and attributed this attenuation in disease progression to the effects of CD43 deficiency on CD4+ T cells. Here, we extend those studies to examine the effects of the loss of CD43 on MOG-specific CD8+ T cells. A reduced frequency of MOG-specific CD8+ T cells following immunization was observed in CD43(-/-) mice relative to wild-type controls, as demonstrated by intracellular cytokine and MHC tetramer staining. In addition, adoptive transfer of CD8+ MOG 35-55-primed LN cells from CD43(-/-) mice resulted in significantly attenuated EAE induction as compared to recipients of wild-type CD8+ MOG-primed cells. Analysis of intracellular signaling intermediates revealed a deficiency in the ability of MOG-specific CD8+ T cells to phosphorylate ERK in response to antigen. These results characterize an important role for CD43 during the activation and expansion of autoreactive MOG-specific CD8+ T cells.     

Activation via TCR or IL-2 receptor of a CD8+ suppressor T cell clone: Effect on IL-10 production and on proliferation of the suppressor T cell

In a previous study, we established CD8⁺ suppressor T cell (Ts) clone 13G2 which produced the suppressive lymphokine, interleukin-10 (IL-10). In this study, we examined what physiological activator could induce both production of IL-10 from 13G2 and the proliferation of 13G2. Both the antigenic stimulation mimicked by the anti-CD3 antibody and the T cell growth factor interleukin-2 (IL-2) induced IL-10 production from the 13G2 clone equally well. 13G2 cells proliferated remarkably with IL-2 stimulation, while anti-CD3 only slightly induced proliferation of the clone. 13G2 cells also produced IL-10 in the presence of hydroxyurea which blocked transit of cells from G₁ to S phase. However, cycloheximide blocked the production of IL-10 from the Ts clone. The study demonstrates that both the anti-CD3 antibody and IL-2 induced IL-10 synthesis of the Ts clone equally well, and the proliferative response of Ts cells was induced more by IL-2 than by anti-CD3. IL-2 proved to be a good stimulator for Ts cells to produce suppressive lymphokine and to multiply their population.Abbreviation Ts suppressor T cell - Th helper T cell - Ag antigen - APC antigen presenting cell - IL interleukin - TCR T cell receptor - mAb monoclonal antibody     

GITR expression on T-cell receptor-stimulated human CD8 T cell in a JNK-dependent pathway

Glucocorticoid-induced tumor necrosis factor receptor (TNFR) (GITR) family-related gene is a member of the TNFR super family. GITR works as one of the immunoregulatory molecule on CD4(+) regulatory T cells and has an important role on cell survival or cell death in CD4(+) T cells. Little is known about the expression of GITR on human CD8(+) T cells on antigen-specific and non-specific activation. Here, we report that expression of GITR on human CD8(+) T cells on T-cell receptor (TCR) (anti-CD3)-mediated stimulation is dependent on the JNK pathway. The activation of CD8(+) T cells was measured by the expression of IL-2 receptor-α (CD25), GITR and by IFN-γ production upon re-stimulation with anti-CD3 antibody. We studied the signaling pathway of such inducible expression of GITR on CD8(+) T cells. We found that a known JNK-specific inhibitor, SP600125, significantly down-regulates GITR expression on anti-CD3 antibody-mediated activated CD8(+) T cells by limiting JNK phosphorylation. Subsequently, after stimulation of the CD8(+) cells, we tested for the production of IFN-γ by the activated cells following restimulation with the same stimulus. It appears that the expression of GITR on activated human CD8(+) T cells might also be regulated through the JNK pathway when the activation is through TCR stimulation. Therefore, GITR serves as an activation marker on activated CD8(+) cells and interference with JNK phosphorylation, partially or completely, by varying the doses of SP600125 might have implications in CD8(+) cytotoxic T cell response in translational research.     

Intracellular CD154 expression reflects antigen-specific CD8+ t cells but shows less sensitivity than intracellular cytokine and MHC tetramer staining

A recent report showed that analysis of CD154 expression in the presence of the secretion inhibitor Brefeldin A (Bref A) could be used to assess the entire repertoire of antigen-specific CD4(+) T helper cells. However, the capacity of intracellular CD154 expression to identify antigen-specific CD8(+) T cells has yet to be investigated. In this study, we compared the ability of intracellular CD154 expression to assess antigen-specific CD8(+) T cells with that of accepted standard assays, namely intracellular cytokine IFN-gamma staining (ICS) and MHC class I tetramer staining. The detection of intracellular CD154 molecules in the presence of Bref A reflected the kinetic trend of antigen-specific CD8+ T cell number, but unfortunately showed less sensitivity than ICS and tetramer staining. However, ICS levels peaked and saturated 8 h after antigenic stimulation in the presence of Bref A and then declined, whereas intracellular CD154 expression peaked by 8 h and maintained the saturated level up to 24 h post-stimulation. Moreover, intracellular CD154 expression in antigen-specific CD8+ T cells developed in the absence of CD4(+) T cells changed little, whereas the number of IFN-gamma-producing CD8(+) T cells decreased abruptly. These results suggest that intracellular CD154 could aid the assessment of antigen-specific CD8(+) T cells, but does not have as much ability to identify heterogeneous CD4(+) T helper cells. Therefore, the combined analytical techniques of ICS and tetramer staining together with intracellular CD154 assays may be able to provide useful information on the accurate phenotype and functionality of antigen-specific CD8(+) T cells.     

4-1BB costimulation enhances HSV-1-specific CD8+ T cell responses by the induction of CD11c+CD8+ T cells

Since 4-1BB plays a predominant role in CD8+ T cell responses, we investigated the effects of 4-1BB triggering on the primary and memory CD8+ T responses to HSV-1 infection. 4-1BB was detected on 10-15% of CD4+ and CD8+ T cells following the infection. 4-1BB-positive T cells were in the proliferative mode and showed the enhanced expression of anti-apoptotic proteins. Agonistic anti-4-1BB treatment exerted preferential expansion of CD8+ T cells and gB/H-2Kb-positive CD8+ T cells, and enhanced cytotoxicity against HSV-1 that was mainly mediated by CD11c+CD8+ T cells. CD11c+CD8+ T cells were re-expanded following re-challenge with HSV-1 at post-infection day 50, indicating that CD11c+CD8+ phenotype was maintained in memory CD8+ T cell pool. Our studies demonstrated that 4-1BB stimulation enhanced both primary and memory anti-HSV-1 CD8+ T cell responses, which was mediated by a massive expansion of antigen-specific CD11c+CD8+ T cells.     

Development of a transient CD4(+)CD8(+) T cell subset in the cervical lymph nodes following intratracheal instillation with an adenovirus vector

Past studies have described the serendipitous appearance of peripheral CD4(+)CD8(+) double-positive (DP) T cells in both humans and nonhuman primates usually following a viral infection or resulting from a malignancy. However, understanding the role of DP T cells has been hampered by the lack of their reproducible generation. Herein, we describe DP T cells produced after a single intratracheal or intranasal dose of recombinant adenovirus 2 or 5 vector into mice. In a time-dependent fashion, DP T cells localized only in the deep cervical lymph nodes but not in the lungs or in any of the respiratory lymph nodes. These DP T cells were TCR(alpha)beta(+) and CD8(alpha)beta(+), but not TCR(gamma)delta(+) nor CD8(alpha)alpha(+), suggesting that these cells are unrelated to intestinally derived DP T cells. Upon co-stimulation with anti-CD3 and anti-CD28, DP T cells showed increased expression of VLA-1, VLA-2, and CD69, and were more effective than CD4(+) T cells in T helper cell activity, as evidenced by increased IgA, IgG, and IgM production. Such co-stimulation also favored the production of IFN-gamma and IL-10 where CD4(+) T cells were more inclined to produce IFN-gamma and IL-2.     

The protective efficacy of chlamydial protease-like activity factor vaccination is dependent upon CD4+ T cells

We have previously determined the protective efficacy of intranasal vaccination with chlamydial protease-like activity factor (CPAF) against genital chlamydial infection. Since T-helper 1 (Th1) responses are important for anti-chlamydial immunity, we examined the contribution of CD4(+) T cells in CPAF mediated immunity against intravaginal (i.vag.) Chlamydia muridarum infection in C57BL/6 mice. CPAF+IL-12 vaccination induced antigen-specific CD4(+) T cells that secreted elevated levels of IFN-gamma, and generated strong humoral responses. The protective effects of CPAF vaccination against genital chlamydial challenge were abrogated by anti-CD4 neutralizing antibody treatment. Moreover, anti-chlamydial immunity could be adoptively transferred to na?ve recipients using CPAF-specific CD4(+) T cells. Therefore, CPAF mediated anti-chlamydial immunity is highly dependent upon antigen-specific CD4(+) T cells.     

Differential requirements for proliferation of CD4+ and gammadelta+ T cells to spirochetal antigens

Alphabeta+ and gammadelta+ T cells have different mechanisms of epitope recognition and are stimulated by antigens of different chemical nature. An immunization model with antigens from the spirochete Brachyspira hyodysenteriae was used to examine the requirements for proliferation of circulating porcine CD4+ and gammadelta+ T cells in mixed lymphocyte cultures. CD4+ T cells only responded to stimulation with B. hyodysenteriae antigens, whereas gammadelta+ T cells proliferated when cultures were stimulated with either spirochetal antigens or interleukin-2 (IL-2). T cells that had proliferated expressed high levels of IL-2-receptor-alpha (IL-2Ralpha). Furthermore, neutralization of IL-2 at the beginning of the culture period was more efficient in blocking gammadelta+ than CD4+ T cell proliferation. Immunization induced interferon-gamma (IFN-gamma) production by CD4+ T cells, whereas only a small fraction of the antigen-stimulated gammadelta+ T cells produced this cytokine. Our results indicate that, under the same environmental conditions, CD4+ T cell functions are more tightly regulated when compared to gammadelta+ T cells. We conclude that these differences are due, in part, to the enhanced gammadelta+ T cell responsiveness to IL-2.     

Anti-idiotype responses abrogate anti-CD4–induced tolerance to a tumor-specific antigen and promote systemic tumor immunity

Purpose: Immunologic-based cancer treatment modalities represent an active area of investigation. Included in these strategies are passive administration of monoclonal antibodies which recognize tumor-associated antigens and active vaccination with identified tumor antigens. However, several problems associated with these types of treatment strategies have been identified. Methods: In this report, we address certain issues by employing a murine model for experimental pulmonary metastasis and a tumor antigen vaccination strategy that induces complete tumor immunity in this system. Utilizing this model, we attempt to address issues related to unresponsiveness to tumor antigen immunization induced by passive administration of a rat monoclonal anti-CD4 and the induction of anti-idiotype responses to a passively administered monoclonal antibody and the effects on the induction of tumor immunity. Results: The results presented indicate that passive administration of rat monoclonal anti-CD4 exhibits immunosuppressive effects that inhibit the production of antibodies to the tumor antigen immunization and abolishes tumor immunity. Repeated administration of the rat monoclonal anti-CD4 results in an anti-idiotype response that can abrogate unresponsiveness to tumor antigen immunization and promote systemic tumor immunity. Conclusions: The data examine a number of potential problems associated with immunologic-based treatments for cancer. These problems include the potential for tolerance to the tumor antigen and establishing an immunocompromised state where immunization with a tumor antigen failed to generate tumor immunity. Approaches to eliminate tolerant T cells by targeting anti-CD4 via anti-idiotype responses that could be generated in vivo without CD4⁺ T cells allowed for recovery of nontolerant T cells, and an antibody response to the tumor antigen that results in tumor immunity.Abbreviations CTL Cytotoxic T lymphocyte - FITC Fluorescein isothiocyanate - OD Optical density - PBS Phosphate-buffered saline - SV40 Simian virus 40     

Ex vivo activated OVA specific and non-specific CD4+CD25+ regulatory T cells exhibit comparable suppression to OVA mediated T cell responses

CD4+CD25+ regulatory T cells (Tr) are important in maintaining immune tolerance to self-antigen (Ag) and preventing autoimmunity. Reduced number and inadequate function of Tr are observed in chronic autoimmune diseases. Adoptively transferred Tr effectively suppress ongoing autoimmune disease in multiple animal models. Therefore, strategies to modulate Tr have become an attractive approach to control autoimmunity. Activation of Tr is necessary for their optimal immune regulatory function. However, due to the low ratio of Tr to any given antigen (Ag) and the unknown nature of Ag in many autoimmune diseases, specific activation is not practical for potential therapeutic intervention. It has been shown in animal models that once activated, Tr can exhibit immune suppression in a bystander Ag-non-specific fashion, suggesting the effector phase of Tr is Ag independent. To investigate whether the immune suppression by activated bystander Tr is as potent as that of the Ag specific Tr, Tr cells were isolated from BALB/c or ovalbumin (OVA) specific T cell receptor (TCR) transgenic mice (DO11.10) and their immune suppression of an OVA specific T cell response was compared. We found that once activated ex vivo, Tr from BALB/c and DO11.10 mice exhibited comparable inhibition on OVA specific T cell responses as determined by T cell proliferation and cytokine production. Furthermore, their immune suppression function was compared in a delayed type hypersensitivity (DTH) model induced by OVA specific T cells. Again, OVA specific and non-specific Tr exhibited similar inhibition of the DTH response. Taken together, the results indicate that ex vivo activated Ag-non-specific Tr are as efficient as Ag specific Tr in immune suppression, therefore our study provides additional evidence suggesting the possibility of applying ex vivo activated Tr therapy for the control of autoimmunity.     

Actin integrity is indispensable for CD95/Fas-induced apoptosis of HIV-specific CD8<Superscript>+</Superscript> T cells

We have recently provided data suggesting a potential role for mitochondria and Bcl-2-family molecules in apoptosis sensitivity of HIV-specific CD8⁺ T cells. Here, we report on the role of filamentous (F) actin in this process. Disruption of actin by cytochalasin D (cytD) or lantrunculin A remarkably reduced CD95/Fas-induced apoptosis of HIV-specific CD8⁺ T cells while their spontaneous apoptosis was unaffected. This inhibition cannot be attributed to changes of CD95/Fas distribution or levels in these cells. Furthermore, cytD treatment reduced CD95/Fas-induced apoptosis of CD8⁺ T cells from HIV⁺ patients independently of their differentiation status. CD95/Fas-induced apoptosis of both CD38⁺ and CD38⁻ HIV-specific CD8⁺ T cells was inhibited by cytD treatment indicating that actin mediates this apoptotic process independently of the activation level of these cells. CytD was found to reduce the activation of caspase-8 induced by short treatment of purified CD8⁺ T cells from HIV⁺ patients with anti-CD95/Fas. Our data reveal actin as a critical mediator of HIV-specific CD8⁺ T cell apoptosis; further analysis of the molecular mechanisms governing this process may potentially contribute to design new therapies targeting the enhancement of the immune system in HIV infection.     

Apoptosis of CD4<Superscript>+</Superscript>CD25<Superscript>high</Superscript> T cells in response to Sirolimus requires activation of T cell receptor and is modulated by IL-2

Targeted molecular therapies inhibit proliferation and survival of cancer cells but may also affect immune cells. We have evaluated the effects of Sirolimus and Sorafenib on proliferation and survival of lymphoid cell subsets. Both drugs were cytotoxic to CD4⁺CD25^high T cells, and were growth inhibitory for CD4⁺ and CD8⁺ T cells. Cytotoxicity depended on CD3/CD28 stimulation and was detectable within 12 h, with 80–90% of CD4⁺CD25^high cells killed by 72 h. Cell death was due to apoptosis, based on Annexin V and 7AAD staining. Addition of IL-2 prevented the apoptotic response to Sirolimus, potentially accounting for reports that Sirolimus can enhance proliferation of CD4⁺CD25^high cells. These results predict that Sirolimus or Sorafenib would reduce CD4⁺CD25^high cells if administered prior to antigenic stimulation in an immunotherapy protocol. However, administration of IL-2 protects CD4⁺CD25^high T cells from cytotoxic effects of Sirolimus, a response that may be considered in design of therapeutic protocols.     

HLA-A0201 positive pancreatic cell lines: new findings and discrepancies

Pancreatic cancer is being pursued as an immunotherapy target using antigen-specific vaccine approaches activating CD8(+) CTL and CD4(+) T-helper cells. CD8(+) CTL exert their anti-tumor effects in an HLA-restricted manner and only tumor cells carrying a matched HLA class I sub-type are targets for antigen-specific CTL. In the process of characterizing CD8(+) T cell responses against pancreatic cancer, we screened a number of human pancreatic tumor cell lines for HLA-A0201 positive (HLA-A2(+)) cell lines to be used in the evaluation of CTL function. This analysis revealed some new findings and discrepancies in the literature on the HLA sub-type of some commonly used pancreatic cell lines. We found that Capan-1 cells, originally reported to be HLA-A0201(+), actually only express HLA-A010101 and HLA-A300101 and were targets for HLA-A0201-restricted CTL only after transduction with an HLA-A0201-expressing lentivirus. Panc-1 cells were found to be HLA-A0201 positive, in agreement with published reports, while CF-Pac-1 cells were found to express both HLA-A020101 and HLA-A030101. We also found a normal human pancreatic ductal epithelial cell line, HPDE, to be HLA-A0201 positive. Our findings were verified with two different sequence-based typing methods, antibody staining followed by flow cytometry analysis, and functional analysis using an HLA-A0201-restricted peptide-specific T cell response.     

Successful elimination of memory-type CD8+ T cell subsets by the administration of anti-Gr-1 monoclonal antibody in vivo

During investigating the expression of Gr-1 antigen on various subsets of mouse spleen cells, we found that Gr-1 was expressed on memory-type CD8(+)CD44(high)CD62L(high) T cells in addition to granulocytes. Intraperitoneal administration of anti-Gr-1 mAb caused almost complete elimination of Ly-6C(+) memory-type CD8(+) T cells as well as Ly-6G(+) granulocytes. Anti-Gr-1 mAb-treated mouse spleen cells exhibited greatly reduced IFN-gamma production in response to anti-CD3 mAb both in vitro and in vivo. This reduced cytokine production appeared to be derived from elimination of IFN-gamma-producing Gr-1(+)CD8(+) T cells. Indeed, CD8(+) T cells with IFN-gamma-producing activity and cytotoxicity were generated from isolated Gr-1(+)CD8(+) cells but not from Gr-1(-)CD8(+) T cells. We also demonstrated that therapeutic effect of MBL-2 tumor-immunized spleen cells was greatly reduced by anti-Gr-1 mAb-treatment. Thus, we initially demonstrated that anti-Gr-1 mAb might become a good tool to investigate a precise role for memory-type CD8(+) T cells in vivo.     

Simultaneous detection of DNA synthesis, activation and cytokine secretion in collagen II (250-270)-activated T lymphocytes by flow cytometry

T cell activation and secretion of cytokines from activated peripheral blood mononuclear cells (PBMC) in culture have traditionally been measured by 3H-thymidine incorporation for assessment of cell proliferation. However, this method has many disadvantages that limit its usage in analyzing antigen-specific T responses, because of the low specific frequencies of the cells. Collagen II (250-270) may be an important autoantigen involved in the pathology of rheumatoid arthritis (RA). To further study the specific T cells response to CII 250-270, we developed an improved method for measuring lymphocyte proliferation and activation, and intracellular cytokine production, by flow cytometry at the single cell level. BrdU, an analog of thymidine, was incorporated into cellular DNA as a marker of individual cell proliferation. The cells were fixed and permeabilized, and a monoclonal antibody against BrdU conjugated with a fluorescent dye was used to measure BrdU incorporation. A Tris staining technique for the simultaneous determination of cell surface activation markers (CD69 or CD25) and intracellular cytokine production was also used and the parameters were assessed by 3-color flow cytometry. Optimal conditions were selected to improve the sensitivity and specificity of the assays. This method allowed simultaneous detection of lymphocytic DNA synthesis, phenotype analysis and cytokine production at the single cell level, and thus it may be a useful tool for analyzing immune responses.     

In vivo functional efficacy of tumor-specific T cells expanded using HLA-Ig based artificial antigen presenting cells (aAPC)

Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and in the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic T cells (CTL). This has stimulated interest in development of standardized artificial antigen presenting cells (aAPC) to reliably expand antigen specific CTL. However, for successful immunotherapy the aAPC ex vivo generated CTL must have anti-tumor activity in vivo. Here, we demonstrate that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity in a human/SCID mouse melanoma model. HLA-Ig based aAPC expanded CTL were detected in the peripheral blood up to 15 days after transfer. Non-invasive bioluminescence imaging of tumor bearing mice demonstrated antigen dependent localization of transferred CTL to the tumor site. Moreover, adoptive transfer of HLA-Ig based aAPC generated CTL inhibited the tumor growth both in prevention and treatment modes of therapy and was comparable to that achieved by dendritic cell expanded CTL. Thus, our data demonstrate potential therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL to control tumor growth.     

Molecular mechanisms of extracellular adenine nucleotides-mediated inhibition of human Cd4<Superscript>+</Superscript> T lymphocytes activation

We have previously reported that ATPγS, a slowly hydrolyzed analog of ATP, inhibits the activation of human CD4⁺ T lymphocytes by anti-CD3 and anti-CD28 mAb. In this report we have partially characterized the signaling mechanisms involved in this immunosuppressive effect. ATPγS had no inhibitory effect on CD4⁺ T-cell activation induced by PMA and anti-CD28, indicating that it acts proximally to the TCR. It had no effect on the calcium rise induced by CD3/CD28 stimulation, but inhibited the phosphorylation of three kinases, ERK2, p38 MAPK and PKB, that play a key role in the activation of T cells. The receptor involved in these actions remains unidentified.