Existence of MHC class I-restricted alloreactive CD4+ T cells reacting with peptide transporter-deficient cells

It is generally accepted that as the result of positive thymic selection, CD8-expressing T cells recognize peptide antigens presented in the context of MHC class I molecules and CD4-expressing T cells interact with peptide antigens presented by MHC class II molecules. Here we report the generation of TCRalpha/beta(+), CD3(+), CD4(+), CD8(-), MHC class I-restricted alloreactive T-cell clones which were induced using peripheral blood mononuclear cells from healthy individuals following in vitro stimulation with transporter associated with antigen processing (TAP)-deficient cell lines T2. The CD4(+) T-cell clones showed an HLA-A2.1-specific proliferative response against T2 cells which was inhibited by anti-CD3 and anti-CD4 monoclonal antibodies. These results suggest that interaction of the TCR with peptide-bound HLA class I molecules contributes to antigen-specific activation of these co-receptor-mismatched T-cell clones. Antigen recognition by alloreactive MHC class I-restricted CD4(+) T cells was inhibited by removing peptides bound to HLA molecules on T2 cells suggesting that the alloreactive CD4(+) T cells recognize peptides that bind in a TAP-independent manner to HLA-A2 molecules. The existence of such MHC class I-restricted CD4(+) T cells which can recognize HLA-A2 molecules in the absence of TAP function may provide a basis for the development of immunotherapy against TAP-deficient tumor variants which would be tolerant to immunosurveillance by conventional MHC class I-restricted cytotoxic lymphocytes.     

Age-associated changes in interferon-γ and interleukin-4 secretion by purified human CD4+ and CD8+ T cells

Aging is associated with a decline in immune function. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), two important immune deviation-related cytokines, are mainly produced by type 1 and type 2 T cells, respectively. To investigate the age-associated changes in the secretion of these two cytokines, 20 elderly and 20 young subjects fulfilling the SENIEUR protocol were enrolled. The ratios of CD4+ to CD8+ T cells were not different between the two age groups. The CD4+ and CD8+ T cells were purified by a magnetic cell sorting system, and then activated by concurrent anti-CD3 and anti-CD28 stimulation. The released cytokines were determined by ELISA. Both the CD4+ and the CD8+ T cells of the elderly individuals secreted a significantly larger amount of IFN-gamma after activation. Profound IL-4 production by CD8+ T cells was observed in the older subjects compared with that of the young subjects. These data suggested that age-associated decrease in immunity may be related to an imbalance in the secretion of immune deviation cytokines. The number of IL-4-secreting CD8+ T cells (T cytotoxic 2) rose significantly in the older individuals. Our design also provided a useful way to differentiate the T cell subsets secreting the same cytokine, such as IFN-gamma-producing T helper 1 and T cytotoxic 1 cells.     

Effective modulation of CD4+CD25+high regulatory T and NK cells in malignant patients by combination of interferon-α and interleukin-2

Overinduced CD4⁺CD25^+high regulatory T cells (Treg) and downregulated NK cells contribute to tumor-relevant immune tolerance and interfere with tumor immunity. In this study, we aimed to design a novel strategy with cytokine combination to correct the dysregulated Treg and NK cells in malignant patients. Initially, a total of 58 healthy individuals and 561 malignant patients were analyzed for their cellular immunity by flow cytometry. The average percentages of CD4⁺CD25^+high/lymphocyte were 1.30?±?1.19?% ( $ bar{x} $ ?±?SD) in normal adults and 3.274?±?4.835?% in malignant patients (p?<?0.001). The ratio of CD4⁺CD25^+high to CD4⁺ was 3.58?±?3.19?% in normal adults and 6.01?±?5.89?% to 13.50?±?23.60?% in different kinds of malignancies (p?<?0.001). Of normal adults, 15.52?% had >3?% Treg and 12.07?% had <10?% NK cells. In contrast, the Treg (>3?%) and NK (<10?%) percentages were 40.82 and 34.94?% in malignant patients, respectively. One hundred and ten patients received the immunomodulation therapy with IFN-?? and/or IL-2. The overinduced Treg in 86.3?% and the reduced NK cells in 71.17?% of the patients were successfully modulated. In comparison, other lymphocyte subpopulations in most patients were much less affected by this treatment. No other treatment-relevant complications except slight pyrexia, fatigue, headache, and myalgia were observed. In conclusion, dysregulated Treg and/or NK cells were common in malignant patients. Different from any regimens ever reported, this strategy was simple and effective without severe complications and will become a basic regimen for other cancer therapies.     

IFN-γ-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain

It is well established that IFN-γ is required for the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the temporal and tissue-specific cellular sources of IFN-γ during P. berghei ANKA infection have not been investigated, and it is not known whether IFN-γ production by a single cell type in isolation can induce cerebral pathology. In this study, using IFN-γ reporter mice, we show that NK cells dominate the IFN-γ response during the early stages of infection in the brain, but not in the spleen, before being replaced by CD4(+) and CD8(+) T cells. Importantly, we demonstrate that IFN-γ-producing CD4(+) T cells, but not innate or CD8(+) T cells, can promote the development of ECM in normally resistant IFN-γ(-/-) mice infected with P. berghei ANKA. Adoptively transferred wild-type CD4(+) T cells accumulate within the spleen, lung, and brain of IFN-γ(-/-) mice and induce ECM through active IFN-γ secretion, which increases the accumulation of endogenous IFN-γ(-/-) CD8(+) T cells within the brain. Depletion of endogenous IFN-γ(-/-) CD8(+) T cells abrogates the ability of wild-type CD4(+) T cells to promote ECM. Finally, we show that IFN-γ production, specifically by CD4(+) T cells, is sufficient to induce expression of CXCL9 and CXCL10 within the brain, providing a mechanistic basis for the enhanced CD8(+) T cell accumulation. To our knowledge, these observations demonstrate, for the first time, the importance of and pathways by which IFN-γ-producing CD4(+) T cells promote the development of ECM during P. berghei ANKA infection.     

Protection against Mycobacterium tuberculosis Infection Offered by a New Multistage Subunit Vaccine Correlates with Increased Number of IFN-γ+IL-2+ CD4+ and IFN-γ+ CD8+ T Cells

Protein subunit vaccines present a compelling new area of research for control of tuberculosis (TB). Based on the interaction between Mycobacterium tuberculosis and its host, five stage-specific antigens of M. tuberculosis that participate in TB pathogenesis—Rv1813, Rv2660c, Ag85B, Rv2623, and HspX—were selected. These antigens were verified to be recognized by T cells from a total of 42 whole blood samples obtained from active TB patients, patients with latent TB infections (LTBIs), and healthy control donors. The multistage polyprotein A1D4 was developed using the selected five antigens as a potentially more effective novel subunit vaccine. The immunogenicity and protective efficacy of A1D4 emulsified in the adjuvant MTO [monophosphoryl lipid A (MPL), trehalose-6,6′-dibehenate (TDB), components of MF59] was compared with Bacillus Calmette-Guerin (BCG) in C57BL/6 mice. Our results demonstrated that A1D4/MTO could provide more significant protection against M. tuberculosis infection than the PBS control or MTO adjuvant alone judging from the A1D4-specific Th1-type immune response; however, its efficacy was inferior to BCG as demonstrated by the bacterial load in the lung and spleen, and by the pathological changes in the lung. Antigen-specific single IL-2-secreting cells and different combinations with IL-2-secreting CD4⁺ T cells were beneficial and correlated with BCG vaccine-induced protection against TB. Antigen-specific IFN-γ⁺IL-2⁺ CD4⁺ T cells were the only effective biomarker significantly induced by A1D4/MTO. Among all groups, A1D4/MTO immunization also conferred the highest number of antigen-specific single IFN-γ⁺ and IFN-γ⁺TNF-α⁺ CD4⁺ T cells, which might be related to the antigen load in vivo, and single IFN-γ⁺ CD8⁺ T cells by mimicking the immune patterns of LTBIs or curable TB patients. Our strategy seems promising for the development of a TB vaccine based on multistage antigens, and subunit antigen A1D4 suspended in MTO adjuvant warrants preclinical evaluation in animal models of latent infection and may boost BCG vaccination.     

HSP70 Enhances Immunosuppressive Function of CD4+CD25+FoxP3+ T Regulatory Cells and Cytotoxicity in CD4+CD25? T Cells

Human CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) control effector T cells and play a central role in peripheral tolerance and immune homeostasis. Heat shock protein 70 (HSP70) is a major immunomodulatory molecule, but its effect on the functions of Tregs is not well understood. To investigate target-dependent and –independent Treg functions, we studied cytokine expression, regulation of proliferation and cytotoxicity after exposure of Tregs to HSP70. HSP70-treated Tregs significantly inhibited proliferation of CD4⁺CD25⁻ target cells and downregulated the secretion of the proinflammatory cytokines IFN-γ and TNF-α. By contrast, HSP70 increased the secretion of Treg suppressor cytokines IL-10 and TGF-β. Treatment with HSP70 enhanced the cytotoxic properties of Tregs only to a minor extent (4-fold), but led to stronger responses in CD4⁺CD25⁻ cells (42-fold). HSP70-induced modulation of T-cell responses was further enhanced by combined treatment with HSP70 plus IL-2. Treatment of Tregs with HSP70 led to phosphorylation of PI3K/AKT and the MAPKs JNK and p38, but not that of ERK1/2. Exposure of Tregs to specific inhibitors of PI3K/AKT and the MAPKs JNK and p38 reduced the immunosuppressive function of HSP70-treated Tregs as indicated by the modified secretion of specific target cell (IFN-γ, TNF-α) and suppressor cytokines (IL-10, TGF-β). Taken together, the data show that HSP70 enhances the suppressive capacity of Tregs to neutralize target immune cells. Thus HSP70-enhanced suppression of Tregs may prevent exaggerated immune responses and may play a major role in maintaining immune homeostasis.     

Unprimed CD4+ and CD8+ T cells can be rapidly activated by a CD3×CD19 bispecific antibody to proliferate and become cytotoxic

We previously reported that a CD3×CD19 bispecific antibody (bsAb) can induce efficient killing of tumour cells by preactivated T cells isolated from patients with B cell malignancy. For future intravenous application we investigated whether resting T cells from peripheral blood can be stimulated to proliferate and become cytotoxic with the CD3×CD19 bsAb alone. Indeed peripheral blood mononuclear cells, isolated from healthy donors or patients with B cell malignancy, started to proliferate within 1 day in response to CD3×CD19 bsAb. Within the same time spaancytotoxic activity against CD19-positive tumour cells was already detectable. Maintenance of cytotoxic activity was seen during 3 days of culture but optimal lysis of the target cells then required fresh CD3×CD19 bsAb in the cytotoxicity assay. Essentially the same results for proliferation and cytotoxicity were found when separated CD4-positive and CD8-positive T cells were activated by the bsAb in the presence of autologous monocytes. These results may be relevant for the in vivo application of the bsAb when used as immunotherapy in patients with B cell malignancy.This work was supported by grant IKMN 90-10 from the Dutch Cancer Society. M.C. was supported by a grant from the UK Medical Research Couneil     

Detection of circulating tumor lysate–reactive CD4+ T cells in melanoma patients

Purpose: We wanted to study whether an allogeneic melanoma lysate would be a feasible stimulatory antigen source for detection of a peripheral CD4⁺ T-cell immune response in patients with medically untreated malignant melanoma. The lysate was produced from a melanoma cell line (FM3.29) which expresses high amounts of melanoma antigens. Methods: Fresh peripheral blood was incubated with and without lysate for 6 h in the presence of anti-CD28/anti-CD49d MoAb (for costimulation). After flow cytometric estimation of the frequency of CD69⁺/IFN-⁺ cells in the CD4⁺ population, the response to lysate was calculated as the difference between the number of activated IFN--producing CD4⁺ cells in the lysate-stimulated and the nonstimulated sample. Results: An immune response to lysate was observed in blood samples from 11 of 15 patients (73%) with metastatic melanoma. A weak response was found in 1 of 4 patients radically operated for localized disease, whereas no responders were seen among 7 healthy donors. The fraction of circulating lysate-activated T cells ranged from 0.0037% to 0.080% of the CD4⁺ population. A negative result of the assay was found occasionally, especially in donors with high background levels of spontaneous IFN- production, indicating an inhibitory effect of the lysate. Conclusions: This method for detection of a peripheral T-cell immune response in melanoma patients has several advantages for clinical use. The tumor lysate preparations may contain large numbers of stimulating antigens (known, as well as unknown) and are easily prepared and handled. Potentially, the assay might be useful as a diagnostic tool, a marker of residual or recurrent disease, a prognostic factor, or a predictor or monitor of the effect of antineoplastic therapy including immune-modulating therapy.     

Murine Peyer's patch dendritic cells prime naïve CD4+ T cells to produce interferon-γ

We investigated the role of Peyer's patch (PP) dendritic cells (DCs) in the production of interferon (IFN)-γ from naïve CD4⁺ T cells of T cell receptor transgenic mice. PP DCs were found to prime naïve CD4⁺ T cells for the production of higher levels of IFN-γ, when compared to spleen (SP) DCs. However, a similar level of interleukin-12 (IL-12) production was observed for PP and SP DCs stimulated via the CD40 molecule. In addition, PP DCs expressed slightly higher levels of B7.2 (CD86) compared to SP DCs. This data demonstrates that PP DCs have a distinct function in the induction of IFN-γs and suggests that PP DCs may enhance IFN-γ production via another cytokine or costimulatory molecule, in addition to IL-12.     

Abnormally High Levels of Virus-Infected IFN-γ+CCR4+CD4+CD25+ T Cells in a Retrovirus-Associated Neuroinflammatory Disorder

Background

Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL). The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified.

Principal Findings

Here, we demonstrate that CD4⁺CD25⁺CCR4⁺ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2), Th17, and regulatory T (Treg) cells in healthy individuals, we demonstrate that IFN-γ production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4⁺CD25⁺CCR4⁺ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-γ-producing CD4⁺CD25⁺CCR4⁺Foxp3⁻ T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity.

Conclusions

We have defined a unique T cell subset—IFN-γ⁺CCR4⁺CD4⁺CD25⁺ T cells—that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.     

Fortification of a protein-free cell culture medium with plant peptones improves cultivation and productivity of an interferon-γ-producing CHO cell line

A strong tendency is currently emerging to remove not only serum but also any product of animal origin from animal cell culture media during production of recombinant proteins. This should facilitate downstream processing and improve biosafety. One way consists in the fortification of protein-free nutritive media with plant protein hydrolysates. To investigate the effects of plant peptones on mammalian cell cultivation and productivity, CHO 320 cells, a clone of CHO K1 cells genetically modified to secrete human interferon-gamma (IFN-gamma), were first adapted to cultivation in suspension in a protein-free medium. Both cell growth and IFN-gamma secretion were found to be equivalent to those reached in serum-containing medium. Eight plant peptones, selected on the basis of their content in free amino acids and oligopeptides, as well as molecular weight distribution of oligopeptides, were tested for their ability to improve culture parameters. These were improved in the presence of three peptones, all having an important fraction of oligopeptides ranging from 1 to 10 kDa and a small proportion of peptides higher than 10 kDa. These peptones do not seem to add significantly to the nutritive potential to basal protein-free nutritive medium. Nevertheless, supplementation of an oligopeptide-enriched wheat peptone improved cell growth by up to 30% and IFN-gamma production by up to 60% in shake-flask experiments. These results suggest that the use of plant peptones with potential growth factor-like or antiapoptotic bioactivities could improve mammalian cell cultivation in protein-free media while increasing the product biosafety.     

Effects of Sirolimus treatment on patients with β-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency.     

Relative deficiency in interferon-γ-secreting CD4+ T cells is strongly associated with poorer COVID-19 vaccination responses in older adults

Although the two-dose mRNA vaccination regime provides protection against SARS-CoV-2, older adults have been shown to exhibit poorer vaccination responses. In addition, the role of vaccine-induced T-cell responses is not well characterised. We aim to assess the impact of age on immune responses after two doses of the BNT162b2 mRNA vaccine, focussing on antigen-specific T-cells. A prospective 3-month study was conducted on 15 young (median age 31 years, interquartile range (IQR) 25–35 years) and 14 older adults (median age 72 years, IQR 70–73 years). We assessed functional, neutralising antibody responses against SARS-CoV-2 variants using ACE-2 inhibition assays, and changes in B and T-cell subsets by high-dimensional flow cytometry. Antigen-specific T-cell responses were also quantified by intracellular cytokine staining and flow cytometry. Older adults had attenuated T-helper (Th) response to vaccination, which was associated with weaker antibody responses and decreased SARS-CoV-2 neutralisation. Antigen-specific interferon-γ (IFNγ)-secreting CD4+ T-cells to wild-type and Omicron antigens increased in young adults, which was strongly positively correlated with their neutralising antibody responses. Conversely, this relationship was negative in older adults. Hence, older adults' relative IFNγ-secreting CD4+ T cell deficiency might explain their poorer COVID-19 vaccination responses. Further exploration into the aetiology is needed and would be integral in developing novel vaccination strategies and improving infection outcomes in older adults.     

T cell receptor-mediated activation of CD4+CD44hi T cells bypasses Bcl10: an implication of differential NF-kappaB dependence of naïve and memory T cells during T cell receptor-mediated responses

Previous studies have demonstrated that Bcl10 (B-cell leukemia/lymphoma 10) is essential for T cell receptor-mediated NF-kappaB activation and subsequent proliferation and interleukin 2 (IL2) production. However, here we demonstrate that, contrary to expectations, Bcl10 is differentially required for T cell activation, including for both proliferation and cytokine production. When CD4+ and CD8+ T cells were divided based on expression levels of CD44, which distinguishes na?ve cells (CD44lo) versus those that are antigen-experienced (CD44hi), IL2 production by and proliferation of CD4+CD44lo na?ve cells and both subpopulations of CD8+ T cells were clearly Bcl10-dependent, whereas these same functional properties of CD4+CD44hi T cells occurred largely independent of Bcl10. As with the other subpopulations of T cells, CD4+CD44hi T cells did not activate the NF-kappaB pathway in the absence of Bcl10; nevertheless, these CD4+CD44hi antigen-experienced T cells efficiently secreted IL2 after T cell receptor stimulation. Strikingly, therefore, T cell receptor-mediated IL2 production in these cells is NF-kappaB-independent. Our studies suggest that antigen-experienced CD4+ T cells differ from their na?ve counterparts and from CD8+ T cells in their ability to achieve activation independent of the Bcl10/NF-kappaB pathway.     

Oral administration of antigen does not influence the proliferation and IFN-γ production of responsive CD8+ T cells but enables to establish T cell clones with different lymphokine production profile.

Feeding of a whole casein diet, which abolished the α_s1-casein-specific proliferation and IFN-γ productivity of CD⁴⁺ T cells, did not affect the proliferative response of CD8⁺ T cells with regard to the antigen dose response, cell dose response, kinetics of the proliferation and epitope specificity, as well as IFN-γ production. To assess the characteristics of the CD8⁺ T cells, we established α_s1-casein-specific CD8⁺ T cell clones from both casein-fed and control mice. The established clones produced different amount of IFN-γ and IL-10, and one clone derived from the casein-fed mice produced a remarkable amount of IL-10. The clones from casein-fed mice produced considerable amounts of TGF-β, while those from control mice produced only small amounts. The possible role of CD8⁺ T cells in oral tolerance is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Maintenance of CMV-specific CD8+ T cell responses and the relationship of IL-27 to IFN-γ levels with aging

We investigated whether healthy young (age ? 40) and elderly (age ? 65) people infected with cytomegalovirus (CMV) had similar levels of CD8⁺ T cell cytokine production and proliferation in response to an immunodominant CMV pp65 peptide pool given the role of CD8⁺ T cells in controlling viral infection and the association of CMV with immunosenescence. Plus, we determined the effects of aging and CMV-infectious status on plasma levels of IL-27, an innate immune cytokine with pro- and anti-inflammatory properties, as well as on its relationship to IFN-γ in that IL-27 can promote the production of IFN-γ. The results of our study show that young and elderly people had similar levels of CD8⁺ T cell proliferation, and IFN-γ and TNF-α production in response to CMV pp65 peptides. Plasma levels of IL-27 were similar between the two groups although CMV-infected young and elderly people had a trend toward increased levels of IL-27. Regardless of aging and CMV-infectious status, plasma levels of IL-27 correlated highly with plasma levels of IFN-γ. These findings suggest the maintenance of CMV pp65-specific CD8⁺ T cell proliferation and cytokine production with aging as well as the sustaining of circulatory IL-27 levels and its biological link to IFN-γ in young and elderly people irrespective of CMV infection.