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CD4 + T cells are central to adaptive immunity. Their role in cross-protection in viral infections such as influenza and severe acute respiratory syndrome (SARS) is well documented; however, molecular rules governing T cell receptor (TCR) engagement of peptide-human leukocyte antigen (pHLA) class II are less understood. Here, we exploit an aspect of HLA class II presentation, the peptide-flanking residues (PFRs), to "tune" CD4 + T cell responses within an in vivo model system of influenza. Using a recombinant virus containing targeted substitutions at immunodominant HLA-DR1 epitopes, we demonstrate limited weight loss and improved clinical scores after heterosubtypic re-challenge. We observe enhanced protection linked to lung-derived influenza-specific CD4 + and CD8 + T cells prior to re-infection. Structural analysis of the ternary TCR:pHLA complex identifies that flanking amino acids influence side chains in the core 9-mer peptide, increasing TCR affinity. Augmentation of CD4 + T cell immunity is achievable with a single mutation, representing a strategy to enhance adaptive immunity that is decoupled from vaccine modality., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8 + T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4 + T cells. Here, we investigate CD4 + T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4 + T cells in five HLA-DR1 + subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The tumor microenvironment comprises newly formed blood and lymphatic vessels which shape the influx, retention and departure of lymphocytes within the tumor mass. Thus, by influencing the intratumoral composition of lymphocytes, these vessels affect the manner in which the adaptive immune system responds to the tumor, either promoting or impairing effective antitumor immunity. In our study, we utilized a mouse model of carcinogen-induced fibrosarcoma to examine the composition of tumor-infiltrating lymphocytes during tumor progression. In particular, we sought to determine whether CD4(+) Foxp3(+) regulatory T cells (Tregs) became enriched during tumor progression thereby contributing to tumor-driven immunosuppression. This was not the case as the proportion of Tregs and effector CD4(+) T cells actually declined within the tumor owing to the unexpected accumulation of naïve T cells. However, we found no evidence for antigen-driven migration of these T cells or for their participation in an antitumor immune response. Our data support the notion that lymphocytes can enter tumors via aberrantly formed blood and lymphatic vessels. Such findings suggest that targeting both the tumor vasculature and lymphatics will alter the balance of lymphocyte subpopulations that enter the tumor mass. A consideration of this aspect of tumor immunology may be critical to the success of solid cancer immunotherapies., (© 2013 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.)

Balancing the generation of immune responses capable of controlling virus replication with those causing immunopathology is critical for the survival of the host and resolution of influenza-induced inflammation. Based on the capacity of interleukin-6 (IL-6) to govern both optimal T-cell responses and inflammatory resolution, we hypothesised that IL-6 plays an important role in maintaining this balance. Comparison of innate and adaptive immune responses in influenza-infected wild-type control and IL-6-deficient mice revealed striking differences in virus clearance, lung immunopathology and generation of heterosubtypic immunity. Mice lacking IL-6 displayed a profound defect in their ability to mount an anti-viral T-cell response. Failure to adequately control virus was further associated with an enhanced infiltration of inflammatory monocytes into the lung and an elevated production of the pro-inflammatory cytokines, IFN-α and TNF-α. These events were associated with severe lung damage, characterised by profound vascular leakage and death. Our data highlight an essential role for IL-6 in orchestrating anti-viral immunity through an ability to limit inflammation, promote protective adaptive immune responses and prevent fatal immunopathology., (© 2013 Cardiff University. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Natural killer (NK) cells provide an immediate first line of defence against viral infections. Memory responses, maintained by CD4(+) T cells, require exposure to viral antigen and provide long-term protection against future infections. It is known that NK cells can promote the development of the adaptive response through cytokine production and cross-talk with antigen-presenting cells. In this paper however, we summarize a series of recent publications, in mouse models and for the first time in man, with the unifying message that rapid viral antigen control by the innate immune system limits antigen exposure to CD4(+) cells thereby abrogating the development of a memory response. We discuss the significant implication of these studies on viral treatment strategies and immunization models., (© 2012 Blackwell Publishing Ltd.)

The ability to control HCV with IFN-α-based treatments provides an opportunity in humans to study how the rate of viral clearance in vivo impinges on the development of antiviral responses. Ex vivo (IFN-γ-producing) and cultured antiviral CD4(+) T cells, serum cytokines, and viral loads were measured repeatedly in a cohort of chronically HCV-infected subjects (n = 33) receiving IFN-α. Rapid control of virus indicated by an increased calculated rate of virus clearance, occurred in those subjects demonstrating absent/minimal T-cell responses (p < 0.0006). Surprisingly, in subjects who demonstrated the most robust T-cell responses (and reduced serum IL-10), there was actually a reduced rate of early virus clearance. A subsequent analysis of NK-cell function in available subjects (n = 8) revealed an inverse correlation between pretreatment NK-cell expression of NKp46 and the potential to upregulate cytotoxic function on exposure to IFN-α (p < 0.004), as well as the subsequent measured rate of viral clearance (p = 0.045). Thus, the CD4(+) T-cell response during IFN-α treatment appears to be shaped by the rate of innate virus suppression. These data suggest that individuals who respond most effectively to immune intervention may be most in need of subsequent vaccination to prevent reinfection., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Death receptor 3 (DR3, TNFRSF25), the closest family relative to tumor necrosis factor receptor 1, promotes CD4(+) T-cell-driven inflammatory disease. We investigated the in vivo role of DR3 and its ligand TL1A in viral infection, by challenging DR3-deficient (DR3(KO)) mice and their DR3(WT) littermates with the β-herpesvirus murine cytomegalovirus or the poxvirus vaccinia virus. The phenotype and function of splenic T-cells were analyzed using flow cytometry and molecular biological techniques. We report surface expression of DR3 by naive CD8(+) T cells, with TCR activation increasing its levels 4-fold and altering the ratio of DR3 splice variants. T-cell responses were reduced up to 90% in DR3(KO) mice during acute infection. Adoptive transfer experiments indicated this was dependent on T-cell-restricted expression of DR3. DR3-dependent CD8(+) T-cell expansion was NK and CD4 independent and due to proliferation, not decreased cell death. Notably, impaired immunity in DR3(KO) hosts on a C57BL/6 background was associated with 4- to 7-fold increases in viral loads during the acute phase of infection, and in mice with suboptimal NK responses was essential for survival (37.5%). This is the first description of DR3 regulating virus-specific T-cell function in vivo and uncovers a critical role for DR3 in mediating antiviral immunity.

Background: There is indirect evidence that T cell responses can control the metastatic spread of colorectal cancer (CRC). However, an enrichment of CD4(+)Foxp3(+) regulatory T cells (Tregs) has also been documented., Objective: To evaluate whether CRC promotes Treg activity and how this influences anti-tumour immune responses and disease progression., Methods: A longitudinal study of Treg activity on a cohort of patients was performed before and after tumour resection. Specific CD4(+) T cell responses were also measured to the tumour associated antigens carcinoembryonic antigen (CEA) and 5T4., Results: Tregs from 62 preoperative CRC patients expressed a highly significant increase in levels of Foxp3 compared to healthy age-matched controls (p=0.007), which returned to normal after surgery (p=0.0075). CD4(+) T cell responses to one or both of the tumour associated antigens, CEA and 5T4, were observed in approximately two-thirds of patients and one third of these responses were suppressed by Tregs. Strikingly, in all patients with tumour recurrence at 12 months, significant preoperative suppression was observed of tumour-specific (p=0.003) but not control CD4(+) T cell responses., Conclusion: These findings demonstrate that the presence of CRC drives the activity of Tregs and accompanying suppression of CD4(+) T cell responses to tumour-associated antigens. Suppression is associated with recurrence of tumour at 12 months, implying that Tregs contribute to disease progression. These findings offer a rationale for the manipulation of Tregs for therapeutic intervention.

Human CD4(+) αβ T cells are activated via T-cell receptor recognition of peptide epitopes presented by major histocompatibility complex (MHC) class II (MHC-II). The open ends of the MHC-II binding groove allow peptide epitopes to extend beyond a central nonamer core region at both the amino- and carboxy-terminus. We have previously found that these non-bound C-terminal residues can alter T cell activation in an MHC allele-transcending fashion, although the mechanism for this effect remained unclear. Here we show that modification of the C-terminal peptide-flanking region of an influenza hemagglutinin (HA(305-320)) epitope can alter T-cell receptor binding affinity, T-cell activation and repertoire selection of influenza-specific CD4(+) T cells expanded from peripheral blood. These data provide the first demonstration that changes in the C-terminus of the peptide-flanking region can substantially alter T-cell receptor binding affinity, and indicate a mechanism through which peptide flanking residues could influence repertoire selection.

Despite its use widely as a therapeutic agent, and proposed use as vaccine adjuvant, the effect of IFNalpha on T cell function is poorly understood. As a pleiotropic innate cytokine produced rapidly in response to pathogens, it is well placed to impinge on specific immune responses. The aim of this study was to examine the impact of IFNalpha on the function of human memory CD4(+) T cells using the recall Ags purified protein derivative, tetanus toxoid, and hemagglutinin. IFNalpha administered either in vivo or added exogenously in vitro tended to enhance proliferative responses of purified protein derivative-specific T cells in marked contrast to the other cognate populations whose responses were often diminished. Purifying the memory CD4(+)CD45RO(+) T cells confirmed IFNalpha acted directly on these cells and not via an intermediate. The T cells could be divided into two broad categories depending on how IFNalpha effected their responses to cognate Ag: 1) enhanced proliferation and a striking increase in IFNgamma-production compared with smaller increases in IL-10 (increased ratio of IFNgamma:IL-10), and 2) neutral or diminished proliferation coupled with a smaller increase in IFNgamma relative to the increase in IL-10 (reduced IFNgamma:IL-10 ratio). IFNalpha has a role in modifying memory T cell responses when they are exposed to cognate Ag and may be important in vaccination strategies designed to augment particular Th memory responses.

CD59, a broadly expressed GPI-anchored molecule, regulates formation of the membrane attack complex of the complement cascade. We previously demonstrated that mouse CD59 also down-modulates CD4(+) T cell activity in vivo. In this study, we explored the role of CD59 on human CD4(+) T cells. Our data demonstrate that CD59 is up-regulated on activated CD4(+) T cells and serves to down-modulate their activity in response to polyclonal and Ag-specific stimulation. The therapeutic potential of this finding was explored using T cells isolated from colorectal cancer patients. The findings were striking and indicated that blockade of CD59 significantly enhanced the CD4(+) T cell response to two different tumor Ags. These data highlight the potential for manipulating CD59 expression on T cells for boosting weak immune responses, such as those found in individuals with cancer.

Currently, our understanding of mechanisms underlying cell-mediated immunity and particularly of mechanisms that promote robust T cell memory to respiratory viruses is incomplete. Interleukin (IL)-6 has recently re-emerged as an important regulator of T cell proliferation and survival. Since IL-6 is abundant following infection with influenza virus, we analyzed virus-specific T cell activity in both wild type and IL-6 deficient mice. Studies outlined herein highlight a novel role for IL-6 in the development of T cell memory to influenza virus. Specifically, we find that CD4+ but not CD8+ T cell memory is critically dependent upon IL-6. This effect of IL-6 includes its ability to suppress CD4+CD25+ regulatory T cells (Treg). We demonstrate that influenza-induced IL-6 limits the activity of virus-specific Tregs, thereby facilitating the activity of virus-specific memory CD4+ T cells. These experiments reveal a critical role for IL-6 in ensuring, within the timeframe of an acute infection with a cytopathic virus, that antigen-specific Tregs have no opportunity to down-modulate the immune response, thereby favoring pathogen clearance and survival of the host.

CD59 blocks formation of the membrane attack complex of complement by inhibiting binding of C9 to the C5b-8 complex. To investigate a role for CD59 in promoting T cell responses, we compared T cell activation in CD59a-deficient (Cd59a-/-) and wild-type (WT) mice after in vitro stimulation and after infection with rVV. Virus-specific CD4+ T cell responses were significantly enhanced in Cd59a-/- mice compared with WT mice. Similarly, Cd59a-/- T cells responded more vigorously to in vitro stimulation with CD3-specific Abs compared with WT mice. This effect of CD59a on T cell proliferation was found to be complement-independent. Collectively, these results demonstrate that CD59a down-modulates CD4+ T cell activity in vitro and in vivo, thereby revealing another link between complement regulators and T cell activation.

In all but a small minority of cases, continued survival of solid organ grafts after transplantation depends on lifelong, nonselective immunosuppression that, although effective, results in increased rates of infection, cancer, and vascular disease. Therapeutic strategies that engage or mimic self-tolerance may allow prolonged allograft survival without the disadvantages of nonspecific immunotherapy. Pretreatment of recipient mice with donor alloantigen combined with transient modulation of the peripheral T cell pool with anti-CD4 Ab leads to the indefinite survival of MHC-incompatible cardiac allografts without further therapy. Tolerance is dependent on CD25+ CD4+ regulatory T cells that arise from naive CD25- precursors and regulate rejection via both IL-10 and CTLA-4. Although these cells are clearly effective at controlling rejection, the proven ability of recently activated CD25+ cells to mediate bystander regulation raises the possibility that tolerized individuals might also have a reduced capacity to respond to environmental pathogens. We have examined anti-influenza responses in tolerized primary heart recipients, secondary recipients following adoptive transfer of regulatory populations, and tolerized mice in which bystander regulation has been deliberately induced. Neither virus-specific CTL activity in vitro nor the clearance of virus in vivo was significantly diminished in any of these treatment groups compared with infected unmanipulated controls. The data suggest that the induction of dominant allograft tolerance dependent on regulatory T cells does not necessarily result in attenuated responses to pathogens providing further support for the development of tolerance induction protocols in clinical transplantation.

Treatment with monoclonal antibodies (mAbs) specific for CD25 (anti-CD25 mAb) has been shown to suppress growth of a variety of different tumours in mice. These studies did not however determine whether or not anti-CD25 mAbs facilitate tumour rejection by depletion of regulatory T cells or by binding to tumour-specific effector cells. Using a murine model of melanoma we have found that treatment of mice with anti-CD25 mAb facilitates long-term CD4+ T cell-mediated tumour immunity through depletion of CD25+ regulatory cells. We further show that the effector CD4+ T cells confer long-term tumour immunity even in the presence of CD25+ regulatory cells and do not require CD8+ T cells for tumour rejection. The inhibitory impact of anti-CD25 mAb treatment on tumour growth may be the result of depleting CD25+ regulatory cells that normally inhibit the generation of immune responses to self-antigens that are shared by the tumour. We have performed experiments to determine whether or not immune responses to melanocyte antigens are generated in anti-CD25 mAb-treated, melanoma-immune mice. The results of the experiments indicate that a T cell response to the melanocyte antigen tyrosinase accompanies suppression of tumour growth in mice lacking CD25+ regulatory cells.