scholarly journals Assessment of T-cell immunity to SARS-CoV-2 in COVID-19 convalescents and vaccinated subjects, using TigraTest® SARS-CoV-2 ELISPOT kit

2021 ◽  
Vol 21 (3) ◽  
pp. 178-192
Author(s):  
D. A. Poteryaev ◽  
S. G. Abbasova ◽  
P. E. Ignatyeva ◽  
O. M. Strizhakova ◽  
S. V. Kolesnik ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Cell Response ◽  
Human Peripheral Blood ◽  
T Cell Response ◽  
T Cell Immunity ◽  
Cell Immunity

With the onset of the COVID-19 pandemic, a number of molecular-based tests have been developed to diagnose SARS-CoV-2 infection. However, numerous available serological tests lack sufficient sensitivity or specificity. They do not detect specific antibodies in a significant proportion of patients with PCR-confirmed COVID-19. There is evidence that some convalescents have a relatively short-lived humoral immunity. In contrast, a number of publications have shown that T-cell response to human coronaviruses, including SARS-CoV-1, MERS, and SARS-CoV-2, can be strong and long-term. Assessment of T-cell immunity to SARS-CoV-2 is important not only for stratification of risks and identification of potentially protected populations with immunity acquired as a result of previous infection, but also for determining immunogenicity and potential efficacy of vaccines under development. The existing methods of quantitative or semi-quantitative assessment of specific T-cell response are mainly used in scientific research and are not standardised. The aim of the study was to develop and verify experimentally a test kit to be used in a standardised procedure for in vitro determination of T-cells specific to SARS-CoV-2 antigens, in human peripheral blood. Materials and methods: the TigraTest® SARS-CoV-2 kit developed by GENERIUM, which determines the number of T-cells secreting interferon gamma in vitro, was tested in the study. Samples of venous blood of volunteers from three different groups were analysed in the study: presumably healthy volunteers; COVID-19 convalescents; individuals vaccinated against SARS-CoV-2. Results: the authors developed the TigraTest® SARS-CoV-2 kit for in vitro determination of T-cells specific to SARS-CoV-2 antigens in human peripheral blood, demonstrated its specificity and performed preliminary assessment of its sensitivity. The study analysed the range and magnitude of the T-cell response in convalescent and vaccinated individuals. A pronounced T-cell response was also shown in some individuals with no symptoms or with unconfirmed diagnosis. It was discovered that the mean T-cell response to peptides of the spike protein (S-protein) was higher in the vaccinated individuals than in the convalescent patients. A correlation was determined between the severity of the disease and the level of T-cell response. Specific contributions of various groups of antigens to the T-cell response after COVID-19 infection were also determined. Conclusions: the TigraTest® SARS-CoV-2 kit is a specific and sensitive tool for the assessment of T-cell immunity to the SARS-CoV-2 virus, which can also be used for vaccinated individuals. The kit may be used in clinical practice for comprehensive assessment of immunity to SARS-CoV-2.

scholarly journals Lack of CD4+ T Cells Does Not Affect Induction of CD8+ T-Cell Immunity against Encephalitozoon cuniculi Infection

2000 ◽  
Vol 68 (11) ◽  
pp. 6223-6232 ◽  
Author(s):  
Magali Moretto ◽  
Lori Casciotti ◽  
Brigit Durell ◽  
Imtiaz A. Khan
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Response ◽  
T Cell Response ◽  
T Cell Immunity ◽  
Cell Mediated Immunity ◽  
Wild Type ◽  
Encephalitozoon Cuniculi ◽  
Cell Immunity ◽  
Mouse Tissues

ABSTRACT Cell-mediated immunity has been reported to play an important role in defense against Encephalitozoon cuniculi infection. Previous studies from our laboratory have underlined the importance of cytotoxic CD8+ T lymphocytes (CTL) in survival of mice infected with E. cuniculi. In the present study, immune response against E. cuniculi infection in CD4+T-cell-deficient mice was evaluated. Similar to resistant wild-type animals, CD4−/− mice were able to resolve E. cuniculi infection even at a very high challenge dose (5 × 107 spores/mouse). Tissues from infected CD4−/− mice did not exhibit higher parasite loads in comparison to the parental wild-type mice. Conversely, at day 21 postinfection, susceptible CD8−/− mice had 1014 times more parasites in the liver compared to control wild-type mice. Induction of the CD8+ T-cell response in CD4−/− mice against E. cuniculi infection was studied. Interestingly, a normal antigen-specific CD8+T-cell response to E. cuniculi infection was observed in CD4−/− mice (precursor proliferation frequency, 1/2.5 × 104 versus 1/104 in wild-type controls). Lack of CD4+ T cells did not alter the magnitude of the antigen-specific CTL response (precursor CTL frequency; 1/1.4 × 104 in CD4−/− mice versus 1/3 × 104 in control mice). Adoptive transfer of immune CD8+ T cells from both CD4−/− and wild-type animals prevented the mortality in CD8−/− mice.E. cuniculi infection thus offers an example of an intracellular parasitic infection where CD8+ T-cell immunity can be induced in the absence of CD4+ T cells.

scholarly journals Children and adults with mild COVID-19 symptoms develop memory T cell immunity to SARS-CoV-2

2021 ◽  
Author(s):  
Patricia Kaaijk ◽  
Veronica Olivo Pimentel ◽  
Maarten E. Emmelot ◽  
Martien Poelen ◽  
Alper Cevirgel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Response ◽  
T Cell Response ◽  
T Cell Immunity ◽  
Immune Memory ◽  
Effector Memory ◽  
Longitudinal Assessment ◽  
Memory T Cell ◽  
Cell Immunity

Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to considerable morbidity/mortality worldwide, but most infections, especially among children, have a mild course. However, it remains largely unknown whether infected children develop cellular immune memory. Methods: To determine whether a memory T cell response is being developed as an indicator for long-term immune protection, we performed a longitudinal assessment of the SARS-CoV-2-specific T cell response by IFN-γ ELISPOT and activation marker expression analyses of peripheral blood samples from children and adults with mild-to-moderate COVID-19. Results: Upon stimulation of PBMCs with heat-inactivated SARS-CoV-2 or overlapping peptides of spike (S-SARS-CoV-2) and nucleocapsid proteins, we found S-SARS-CoV-2-specific IFN-ɣ T cell responses in most infected children (83%) and all adults (100%) that were absent in unexposed controls. Frequencies of SARS-CoV-2-specific T cells were higher in infected adults, especially in those with moderate symptoms, compared to infected children. The S-SARS-CoV-2 IFN-ɣ T cell response correlated with S1-SARS-CoV-2-specific serum IgM, IgG, and IgA antibody concentrations. Predominantly, effector memory CD4+ T cells of a Th1 phenotype were activated upon exposure to SARS-CoV-2 antigens, which persisted for 4-8 weeks after symptom onset. We detected very low frequencies of SARS-CoV-2-reactive CD8+ T cells in these individuals. Conclusions: Our data indicate that an antigen-specific memory CD4+ T cell response is induced in children and adults with mild SARS-CoV-2 infection. T cell immunity induced after mild COVID-19 could contribute to protection against re-infection.

scholarly journals Lack of Interleukin-12 in p40-Deficient Mice Leads to Poor CD8+ T-Cell Immunity against Encephalitozoon cuniculi Infection

2010 ◽  
Vol 78 (6) ◽  
pp. 2505-2511 ◽  
Author(s):  
Magali M. Moretto ◽  
Elizabeth M. Lawlor ◽  
Imtiaz A. Khan
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Response ◽  
T Cell Response ◽  
Scid Mice ◽  
T Cell Immunity ◽  
Interleukin 12 ◽  
Cell Immune Response ◽  
Encephalitozoon Cuniculi ◽  
Cell Immunity

ABSTRACT A CD8+ T-cell response is critical for protection against Encephalitozoon cuniculi infection. However, the factors responsible for the generation of CD8+ T-cell immunity during E. cuniculi infection and the cytokines involved in this process have not been identified. In the present study, we demonstrated that p40-deficient animals, which are unable to produce interleukin-12 (IL-12), have a serious defect in expansion of the CD8+ T-cell response which compromises the survival of an infected host. Adoptive transfer of CD8+ T cells from immunocompetent donors protected SCID mice infected with E. cuniculi, whereas administration of CD8+ T cells from p40−/− mice failed to protect infected SCID mice. In vitro dendritic cell (DC) cultures from knockout mice pulsed with E. cuniculi spores were unable to develop a robust CD8+ T-cell immune response. Addition of exogenous IL-12 or transfer of CD8+ T cells that were initially primed with DC from p40−/− animals to DC cultures from immunocompetent mice (directly or via transwells) led to optimal expansion of these cells. This IL-12-mediated reinstatement of CD8+ T-effector immunity was independent of gamma interferon (IFN-γ) as addition of antibody to the cultures failed to have an effect. These studies demonstrated that IL-12 plays a predominant role in the expansion of effector CD8+ T-cell immunity against E. cuniculi, which is critical for host survival. These findings are very important for understanding the protective immune mechanisms needed to protect an immunocompromised host against an opportunistic infection and can be extended to other microsporidial pathogens.

Histone Methyltransferase Ezh2 Controls T-Cell Immunity by Regulating Bioenergetic Metabolism

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 953-953
Author(s):  
Shan He ◽  
Fang Xie ◽  
Qing Tong ◽  
Kazuhiro Mochizuki ◽  
Yongnian Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cellular Metabolism ◽  
T Cell Response ◽  
T Cell Immunity ◽  
Adoptive T Cell Therapy ◽  
Activated T Cells ◽  
Cell Immunity ◽  
Bioenergetic Metabolism

Abstract Abstract 953 Adoptive T cell therapy has the potential to enhance antitumor immunity and improve vaccine efficacy, of which a key challenge is to generate sufficient numbers of T cells that can persist in vivo after transfer. Cellular metabolism plays important roles in regulating T cell proliferation and survival. T cells responding to antigen activation dramatically upregulate both glycolysis and oxidative phosphorylation (OXPHOS), leading to increased production of adenosine triphosphate (ATP) and metabolic intermediates that are required for cell growth and proliferation. Without sufficient support for their demands, activated T cells may be deleted or become quiescent. Thus, better understanding of the mechanism that regulates cellular metabolism in T cell response will lead to new strategies to improve the efficacy of adoptive T cell therapy. Here we explore the functional impact of an epigenetic pathway in cellular metabolism in antigen-driven T cells and tumor immunity. Using genetic approaches and experimental mouse models, we demonstrate that Ezh2, which is a histone methyltransferase that represses the transcription of cohorts of developmental regulators, promotes the survival and expansion of antigen-driven T cells through regulating bioenergetic metabolism. Conditional deletion of Ezh2 caused selective apoptosis in T cells upon activation with alloantigens in vivo and in vitro or with T cell receptor (TCR)-ligation in vitro. Ezh2 deficiency resulted in markedly increased expression of proapoptotic gene Bim, but had no significant impact on the expression of other Bcl-2 family members (e.g., anti-apoptotic genes Bcl-2 and Bcl-xL,). Genetic inactivation of Bim only slightly improved the survival of alloantigen-activated Ezh2-deficient T cells, suggesting that Ezh2 may control T-cell immunity largely through a Bim-independent mechanism. This differs from our recent observations showing that Bim is required for increased apoptosis in activated T cells treated with a pharmacologic inhibitor of Ezh2 and histone methylation 3-Deazaneplanocin A (Blood, 2012). Our prior studies and others suggest that impaired cellular metabolism may lead to increased apoptosis of antigen-activated T cells. We observed that upon TCR-ligation Ezh2 null T cells were incapable to upregulate OXPHOS as compared to wild-type (WT) T cells, which was accompanied with reduced ATP levels and increased reactive oxygen species (ROS). Neutralization of ROS by N-acetylcysteine significantly improved the survival of TCR-activated Ezh2 null T cells. Interestingly, overexpression of WT Ezh2 in TCR-activated Ezh2 null T cells, but not enzymatically inactive H689A Ezh2 mutant or nuclear localization-inactive Ezh2 mutant, restored the ability of Ezh2-deficient T cells to upregulate OXPHOS, reduced ROS levels, and rescued their survival capability in vitro. These results suggest that Ezh2 is important for regulating bioenergetic metabolism in activated T cells. Furthermore, the nuclear but not cytoplasmic Ezh2 is required to regulate bioenergetic metabolism in activated T cells during clonal expansion phase, although Ezh2 in the cell cytoplasm could be involved in regulating actin polymerization. In mouse models of graft-versus-host disease (GVHD) and leukemia, transfer of donor T cells lacking Ezh2 failed to mediate GVHD and anti-leukemia activity in mice receiving allogeneic bone marrow transplantation. In addition, Ezh2 deficiency also ablated the ability of adoptively transferred antigen-specific CD8 T cells to control tumor growth in mice with established melanoma. Importantly, the absence of Ezh2 did not impair the development of effector T cells producing IFN-γ, granzyme B, Fas ligand and Trail, ruling out the possibility that impaired T-cell immunity of Ezh2 null T cells results from defective effector differentiation. Our findings identify the critical role of Ezh2 in regulating bioenergetic metabolism in antigen-driven T cells, therefore for the first time linking the epigenetic pathway to cellular metabolism in T cell response. Thus, Ezh2 and its-regulated bioenergetic metabolism may represent novel targets to improve the efficacy of adoptive T-cell immunotherapy. Modulation of Ezh2 and its activity may have broad implications in the treatment of many other inflammatory disorders, such as graft rejection after organ transplantation, GVHD and autoimmune diseases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD8+ T Cell Immunity Is Compromised by Anti-CD20 Treatment and Rescued by Interleukin-17A

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Facundo Fiocca Vernengo ◽  
Cristian G. Beccaria ◽  
Cintia L. Araujo Furlan ◽  
Jimena Tosello Boari ◽  
Laura Almada ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Response ◽  
T Cell Response ◽  
T Cell Immunity ◽  
Content Type ◽  
Cell Immunity ◽  
Anti Cd20

ABSTRACT Treatment with anti-CD20, used in many diseases in which B cells play a pathogenic role, has been associated with susceptibility to intracellular infections. Here, we studied the effect of anti-CD20 injection on CD8+ T cell immunity using an experimental model of Trypanosoma cruzi infection, in which CD8+ T cells play a pivotal role. C57BL/6 mice were treated with anti-CD20 for B cell depletion prior to T. cruzi infection. Infected anti-CD20-treated mice exhibited a CD8+ T cell response with a conserved expansion phase followed by an early contraction, resulting in a strong reduction in total and parasite-specific CD8+ T cell numbers at 20 days postinfection. Anti-CD20 injection increased the frequency of apoptotic CD8+ T cells, decreased the number of effector and memory CD8+ T cells, and reduced the frequency of proliferating and cytokine-producing CD8+ T cells. Accordingly, infected anti-CD20-treated mice presented lower cytotoxicity of T. cruzi peptide-pulsed target cells in vivo. All of these alterations in CD8+ T cell immunity were associated with increased tissue parasitism. Anti-CD20 injection also dampened the CD8+ T cell response, when this had already been generated, indicating that B cells were involved in the maintenance rather than the induction of CD8+ T cell immunity. Anti-CD20 injection also resulted in a marked reduction in the frequency of interleukin-6 (IL-6)- and IL-17A-producing cells, and recombinant IL-17A (rIL-17A) injection partially restored the CD8+ T cell response in infected anti-CD20-treated mice. Thus, anti-CD20 reduced CD8+ T cell immunity, and IL-17A is a candidate for rescuing deficient responses either directly or indirectly. IMPORTANCE Monoclonal antibody targeting the CD20 antigen on B cells is used to treat the majority of non-Hodgkin lymphoma patients and some autoimmune disorders. This therapy generates adverse effects, notably opportunistic infections and activation of viruses from latency. Here, using the infection murine model with the intracellular parasite Trypanosoma cruzi, we report that anti-CD20 treatment affects not only B cell responses but also CD8+ T cell responses, representing the most important immune effectors involved in control of intracellular pathogens. Anti-CD20 treatment, directly or indirectly, affects cytotoxic T cell number and function, and this deficient response was rescued by the cytokine IL-17A. The identification of IL-17A as the cytokine capable of reversing the poor response of CD8+ T cells provides information about a potential therapeutic treatment aimed at enhancing defective immunity induced by B cell depletion.

scholarly journals CD8+ T cell immunity is compromised by anti-CD20 treatment and rescued by IL-17A

2019 ◽  
Author(s):  
Facundo Fiocca Vernengo ◽  
Cristian G. Beccaria ◽  
Cintia L. Araujo Furlan ◽  
Jimena Tosello Boari ◽  
Laura Almada ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Response ◽  
T Cell Response ◽  
T Cell Immunity ◽  
B Cell Depletion ◽  
Cell Immunity ◽  
Anti Cd20

AbstractTreatment with anti-CD20, used in many diseases in which B cells play a pathogenic role, has been associated with susceptibility to intracellular infections. Here, we studied the effect of anti-CD20 injection on CD8+ T cell immunity using an experimental model of Trypanosoma cruzi infection, in which CD8+ T cells play a pivotal role. C57BL/6 mice were treated with anti-CD20 for B cell depletion prior to T. cruzi infection. Infected anti-CD20-treated mice exhibited a CD8+ T cell response with a conserved expansion phase followed by an early contraction, resulting in a strong reduction in total and parasite-specific CD8+ T cells at 20 days postinfection. Anti-CD20 injection decreased the number of effector and memory CD8+ T cells and reduced the frequency of proliferating and cytokine producing CD8+ T cells. Accordingly, infected anti-CD20-treated mice presented a lower cytotoxicity of T. cruzi peptide-pulsed target cells in vivo. All of these alterations in CD8+ T cell immunity were associated with increased tissue parasitism. Anti-CD20 injection also dampened an established CD8+ T cell response, indicating that B cells were involved in the maintenance rather than the induction of CD8+ T cell immunity. Anti-CD20 injection also resulted in a marked reduction in the frequency of IL-6- and IL-17A-producing cells, and only rIL-17A injection partially restored the CD8+ T cell response in infected anti-CD20-treated mice. Thus, anti-CD20 reduced CD8+ T cell immunity, and IL-17A is a candidate for rescuing deficient responses either directly or indirectly.ImportanceMonoclonal antibody targeting the CD20 antigen on B cells is used to treat the majority of Non-Hodgkin lymphoma patients and some autoimmune disorders. This therapy generates adverse effects, notably opportunistic infections and activation of viruses from latency. Here, using the infection murine model with the intracellular parasite Trypanosoma cruzi, we report that anti-CD20 treatment not only affects B cell response but also CD8+ T cells, the most important immune effectors involved in control of intracellular pathogens. Anti-CD20 treatment, directly or indirectly, affects cytotoxic T cell number and function and this deficient response was rescued by the cytokine IL-17A. The identification of IL-17A as the cytokine capable of reversing the poor response of CD8+ T cells provide information about a potential therapeutic treatment aimed at enhancing defective immunity induced by B cell depletion.

scholarly journals CD8+-T-Cell Immunity against Toxoplasma gondii Can Be Induced but Not Maintained in Mice Lacking Conventional CD4+ T Cells

2002 ◽  
Vol 70 (2) ◽  
pp. 434-443 ◽  
Author(s):  
Lori Casciotti ◽  
Kenneth H. Ely ◽  
Martha E. Williams ◽  
Imtiaz A. Khan
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Toxoplasma Gondii ◽  
Cell Response ◽  
T Cell Response ◽  
T Cell Immunity ◽  
Cell Immune Response ◽  
Cell Immunity ◽  
Ifn Γ

ABSTRACT T-cell immunity is critical for survival of hosts infected with Toxoplasma gondii. Among the cells in the T-cell population, CD8+ T cells are considered the major effector cells against this parasite. It is believed that CD4+ T cells may be crucial for induction of the CD8+-T-cell response against T. gondii. In the present study, CD4−/− mice were used to evaluate the role of conventional CD4+ T cells in the immune response against T. gondii infection. CD4−/− mice infected with T. gondii exhibited lower gamma interferon (IFN-γ) messages in the majority of their tissues. As a result, mortality due to a hyperinflammatory response was prevented in these animals. Interestingly, T. gondii infection induced a normal antigen-specific CD8+-T-cell immune response in CD4−/− mice. No difference in generation of precursor cytotoxic T lymphocytes (pCTL) or in IFN-γ production by the CD8+-T-cell populations from the knockout and wild-type animals was observed. However, the mutant mice were not able to sustain CD8+-T-cell immunity. At 180 days after infection, the CD8+-T-cell response in the knockout mice was depressed, as determined by pCTL and IFN-γ assays. Loss of CD8+-T-cell immunity at this time was confirmed by adoptive transfer experiments. Purified CD8+ T cells from CD4−/− donors that had been immunized 180 days earlier failed to protect the recipient mice against a lethal infection. Our study demonstrated that although CD8+-T-cell immunity can be induced in the absence of conventional CD4+ T cells, it cannot be maintained without such cells.

scholarly journals Mechanisms that allow vaccination against an oncolytic vesicular stomatitis virus-encoded transgene to enhance safety without abrogating oncolysis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Amanda W. K. AuYeung ◽  
Robert C. Mould ◽  
Ashley A. Stegelmeier ◽  
Jacob P. van Vloten ◽  
Khalil Karimi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Vesicular Stomatitis Virus ◽  
Cell Response ◽  
Viral Infections ◽  
T Cell Response ◽  
Oncolytic Viruses ◽  
Oncolytic Virotherapy ◽  
Cell Immunity ◽  
Vesicular Stomatitis

AbstractVaccination can prevent viral infections via virus-specific T cells, among other mechanisms. A goal of oncolytic virotherapy is replication of oncolytic viruses (OVs) in tumors, so pre-existing T cell immunity against an OV-encoded transgene would seem counterproductive. We developed a treatment for melanomas by pre-vaccinating against an oncolytic vesicular stomatitis virus (VSV)-encoded tumor antigen. Surprisingly, when the VSV-vectored booster vaccine was administered at the peak of the primary effector T cell response, oncolysis was not abrogated. We sought to determine how oncolysis was retained during a robust T cell response against the VSV-encoded transgene product. A murine melanoma model was used to identify two mechanisms that enable this phenomenon. First, tumor-infiltrating T cells had reduced cytopathic potential due to immunosuppression. Second, virus-induced lymphopenia acutely removed virus-specific T cells from tumors. These mechanisms provide a window of opportunity for replication of oncolytic VSV and rationale for a paradigm change in oncolytic virotherapy, whereby immune responses could be intentionally induced against a VSV-encoded melanoma-associated antigen to improve safety without abrogating oncolysis.

Cultured Human B Cell APCs Expanded Using Il-4 and CD40 Ligand Preferentially Promote a Type 2 T Cell Response to Alloimmune Stimulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2668-2668
Author(s):  
Abdul Tawab ◽  
Yoshiyuki Takahashi ◽  
Childs Richard ◽  
Kurlander J. Roger
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Response ◽  
Cd40 Ligand ◽  
T Cell Response ◽  
Ifn Γ

Abstract In vitro stimulation of human peripheral blood B cells with recombinant IL-4 and CD40 ligand (CD40L) markedly increases their expression of MHC and costimulatory molecules, thus enhancing antigenic peptide presentation to T cells. Because these cells proliferate extensively in vitro (unlike monocytes or dendritic cells), they represent a promising and convenient reagent for the generation and maintenance of antigen-specific T cells for use in a variety of experimental or therapeutic settings. However, the impact of this type of B cell APC on cytokine production by responder T cells has hitherto not been examined. To address this issue, we stimulated normal human T cells with either allogeneic B cells (generated in vitro) or with MNCs obtained from the same donor. After 7 days, T cells were washed and re-challenged with the same APCs. The resulting alloreactive cytokine response was measured using quantitative ELISPOT methods and expressed as the frequencies of IFN-γ, IL-4, and IL-5 producing cells per thousand responder cells added. B cell- and MNC-primed cell lines both produced vigorous lymphokine responses, but B cell-stimulated T cells consistently produced more IL-5 spots (mean of 265 vs. 98/1000 responders, p<0.002) and fewer IFN-γ spots (163 vs 386/1000 cells, p<0.005) than MNC-stimulated cells. Further, the ratio of IFN-γ to IL-5 spots was almost ten-fold lower in B cell-stimulated cultures compared to MNC-induced cultures (0.67 vs. 5.2, p<0.001). ELISPOT studies assessing the ratio of IFN-γ to IL-4 spots and ELISA assays comparing IFN-γ and IL-5 levels from culture supernatants demonstrated the same pattern of marked type 2 skewing by B cells. This pattern was unaffected by the presence of anti-IL-4 antibody suggesting type 2 skewing was not mediated by IL-4. Cytokine skewing produced by B cells or MNC could be partially reversed by swapping MNC and B cells during re-stimulation on day 7, but this plasticity was markedly reduced after 3 (weekly) cycles of B cell or MNC re-stimulation in vitro. Type 2 skewing by B cells was enhanced when monocytes were removed from responder T cell populations by either depleting CD14+ positive cells or by positive selection of T cells prior to stimulation. In contrast, type 2 polarization could be prevented using recombinant IL-12. Not all cells of B-cell origin share the same propensity to type 2 skewing observed with IL-4/CD40L-stimulated B cells; under identical conditions, EBV-transformed B cells stimulated alloimmune T cells to produce a strong type 1 cytokine response comparable to that produced by MNCs. In summary, IL-4/CD40L-stimulated B cells strongly promote a type 2 T cell response during primary alloimmune challenge; this skewing can become fixed after repeated B cell stimulation. Investigators using these cells as APC should be aware of this potential phenomenon, particularly during primary T cell responses. It is also important to consider the factors described above that may exacerbate or ameliorate this effect.

T Cell Recognition of HCMV: Pan-Genome Analysis of Immunogenic Open-Reading Frames.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 606-606 ◽  
Author(s):  
Louis J. Picker ◽  
Andrew W. Sylwester ◽  
Bridget L. Mitchell ◽  
Cara Taormina ◽  
Christian Pelte ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cell Response ◽  
Cd8 T Cell ◽  
Cross Reactivity ◽  
T Cell Response ◽  
Cd4 T Cell ◽  
Cell Responses

Abstract Human Cytomegalovirus (HCMV) is among the largest and most complex of known viruses with 150–200nm virions enclosing a double stranded 230kb DNA genome capable of coding for >200 proteins. HCMV infection is life-long, and for the vast majority of immune competent individuals clinically benign. Disease occurs almost exclusively in the setting of immune deficiency, suggesting that the stable host-parasite relationship that characterizes these infections is the result of an evolutionarily “negotiated” balance between viral mechanisms of pathogenesis and the host immune response. In keeping with, and perhaps because of this balance, the human CD4+ T cell response to whole HCMV viral lysates is enormous, with median peripheral blood frequencies of HCMV-specific cells ~5–10 fold higher than for analogous preparations of other common viruses. Although certain HCMV ORFs have been identified as targets of either the CD4+ or CD8+ T cell response, the specificities comprising the CD4+ T cell response, and both the total frequencies and component parts of the CD8+ T cell response are unknown. Here, we used cytokine flow cytometry and ~14,000 overlapping 15mer peptides comprising all 213 HCMV ORFs encoding proteins >100 amino acids in length to precisely define the total CD4+ and CD8+ HCMV-specific T cell responses and the HCMV ORFs responsible for these responses in 33 HCMV-seropositive, HLA-disparate donors. An additional 9 HCMV seronegative donors were similarly examined to define the extent to which non-HCMV responses cross-react with HCMV-encoded epitopes. We found that when totaled, the median frequencies of HCMV-specific CD4+ and CD8+ T cells in the peripheral blood of the seropositive subjects were 4.0% and 4.5% for the total CD4+ or CD8+ T cell populations, respectively (which corresponds to 9.1% and 10.5% of the memory populations, respectively). The HCMV-specific CD4+ and CD8+ T cell responses included a median 12 and 7 different ORFs, respectively, and all told, 73 HCMV ORFs were identified as targets for both CD4+ and CD8+ T cells, 26 ORFs as targets for CD8+ T cells alone, and 43 ORFS as targets for CD4+ T cells alone. UL55, UL83, UL86, UL99, and UL122 were the HCMV ORFs most commonly recognized by CD4+ T cells; UL123, UL83, UL48, UL122 and UL28 were the HCMV ORFs most commonly recognized by CD8+ T cells. The relationship between immunogenicity and 1) HLA haplotype and 2) ORF expression and function will be discussed. HCMV-seronegative individuals were non-reactive with the vast majority of HCMV peptides. Only 7 potentially cross-reactive responses were identified (all by CD8+ T cells) to 3 ORFs (US32, US29 and UL116) out of a total of almost 4,000 potential responses, suggesting fortuitous cross-reactivity with HCMV epitopes is uncommon. These data provide the first glimpse of the total human T cell response to a complex infectious agent, and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans.

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