Decreased T-cell Responses Research Articles

Decreased T-cell response against latentcytomegalovirus infection does not correlate with anti-IFN autoantibodies in patients with APECED.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is an inborn error of immunity affecting both multiple endocrine organs and susceptibility to candidiasis, each with an autoimmune basis. Recently, high titer neutralizing anti-type I interferon (IFN) autoantibodies have been linked with increased severity of SARS-CoV-2 and varicella zoster virus infections in APECED patients. Examining immunity against cytomegalovirus (CMV), we found a higher prevalence of anti-CMV IgG antibodies in patients with APECED (N = 19) than in 44 healthy controls (90% vs 64%, p = 0.04); the similar difference in their IgG levels did not achieve significance (95 ± 74 vs 64 ± 35 IU/mL, ns.). In contrast, the frequency of CMV-specific Tcells was lower (804 ± 718/million vs 1591 ± 972/million PBMC p = 0.03). We saw no correlations between levels of anti-CMV IgG and anti-IFN antibodies in APECED patients or in a separate cohort of patients with thymoma (n = 70), over 60% of whom also had anti-IFN antibodies. Our results suggest a dysregulated response to CMV in APECED patients and highlight immunodeficiency to viral infections as part of the disease spectrum.

Compromised DNA Repair Promotes the Accumulation of Regulatory T Cells With an Aging-Related Phenotype and Responsiveness.

Decline of immune function during aging has in part been ascribed to the accumulation of regulatory T cells (Tregs) and decreased T-cell responses with age. Aside from changes to T cells that occur over a lifetime, the impact of intracellular aging processes such as compromised DNA repair on T cells remains incompletely defined. Here we aimed to define the impact of compromised DNA repair on T-cell phenotype and responsiveness by studying T cells from mice with a deficiency in their DNA excision-repair gene Ercc1. These Ercc1 mutant (Ercc1 −/Δ7 ) mice show accumulation of nuclear DNA damage resulting in accelerated aging. Similarly to wild-type aged mice, Ercc1 −/Δ7 mice accumulated Tregs with reduced CD25 and increased PD-1 expression among their naive T cells. Ercc1-deficiency limited the capacity of Tregs, helper T cells, and cytotoxic T cells to proliferate and upregulate CD25 in response to T-cell receptor- and IL-2-mediated stimulation. The recent demonstration that the mammalian target of rapamycin (mTOR) may impair DNA repair lead us to hypothesize that changes induced in the T-cell population by compromised DNA repair may be slowed down or reversed by blocking mTOR with rapamycin. In vivo dietary treatment of Ercc1 −/Δ7 mice with rapamycin did not reduce Treg levels, but highly increased the proportion of CD25+ and PD-1+ memory Tregs instead. Our study elucidates that compromised DNA repair promotes the accumulation of Tregs with an aging-related phenotype and causes reduced T-cell responsiveness, which may be independent of mTOR activation.

2572. Decreased T-Cell Response, but Appropriate Antibody Production to Tetanus Vaccine Among HIV-Exposed Uninfected Infants in Botswana

BackgroundIn Botswana, more than 10% of HIV-exposed, uninfected infants (HEU) are hospitalized or die in the first 6 months of life, largely due to infectious causes. Vaccine responses can act as a marker of the immune response to infectious antigens. Previous studies of vaccine responses in HEU have had conflicting results. We compared T-cell responses to tetanus and purified protein derivative (PPD), and antibody titers to tetanus, between HEU and HIV-unexposed infants (HUU).MethodsA total of 443 HIV-infected and 451 HIV-uninfected mothers and their 453 HEU/457 HUU live-born infants were followed from pregnancy/delivery through 24 months postpartum in a prospective observational study in Botswana (“Tshipidi”). T-cell expression of interferon (IFN)-γ and interleukin (IL)-2 were measured at 6 months using FluoroSpot after stimulation with tetanus toxoid or PPD. Quantitative tetanus toxoid IgG was measured in plasma at 18 months using ELISA. Mean spot-forming units (sfu) per 105 cells and geometric mean antibody titers were compared between HEU and HUU infants who had received 3 doses of tetanus and 1 dose of Bacille Calmette–Guérin (BCG) vaccines by 6 months, and 3 or 4 doses of tetanus vaccine by 18 months. Results. Peripheral blood mononuclear cells were available at 6 months for 63 HEU and 18 HUU. Plasma was available at 18 months for 39 HEU and 42 HUU. HEU infants had significantly lower IL-2 expression after tetanus stimulation than HUU infants (4.4 vs. 16.1 sfu/105 cells, P = 0.004; figure), but no difference in IFNγ expression. There were no differences in T-cell responses to PPD between HEU and HUU. There were no differences in tetanus geometric mean antibody titers between HEU and HUU. Conclusion. In this cohort of infants from Botswana, we found decreased T-cell responses, but not antibody responses, to tetanus toxoid and no differences in T-cell responses to PPD. Cell-mediated immune defects may play a greater role than humoral immune defects in the increased susceptibility to infection among HEU. BCG vaccine produces robust T-helper 1 responses, which may overcome cell-mediated immune defects in HEU. Disclosures All authors: No reported disclosures.

Black seed oil ameliorates allergic airway inflammation by inhibiting T-cell proliferation in rats

The black seeds, from the Ranunculaceae family, have been traditionally used by various cultures as a natural remedy for several ailments. In this study, we examined the effect of black seed oil as an immunomodulator in a rat model of allergic airway inflammation. Rats sensitized to ovalbumin and challenged intranasally with ovalbumin to induce an allergic inflammatory response were compared to ovalbumin-sensitized, intranasally ovalbumin-exposed rats pretreated with intraperitoneally administered black seed oil and to control rats. The levels of IgE, IgG1 and ova-specific T-cell proliferation in spleen were measured by ELISA. The pro-inflammatory cytokine IL-4, IL-5, IL-6 and TGF-β1 mRNA expression levels were measured by reverse transcription polymerase chain reaction. The intraperitoneal administration of black seed oil inhibited the Th2 type immune response in rats by preventing inflammatory cell infiltration and pathological lesions in the lungs. It significantly decreased the nitric oxide production in BALF, total serum IgE, IgG1 and OVA-specific IgG1 along with IL-4, IL-5, IL-6 and TGF-β1 mRNA expression. Black seed oil treatment resulted in decreased T-cell response evident by lesser delayed type hypersensitivity and lower T-cell proliferation in spleen. In conclusion, black seed oil exhibited a significant reduction in all the markers of allergic inflammation mainly by inhibiting the delayed type hypersensitivity and T-cell proliferation. The data suggests that inhibition of T-cell response may be responsible for immunomodulatory effect of black seed oil in the rat model of allergic airway inflammation.

Expression of the E3L Gene of Vaccinia Virus in Transgenic Mice Decreases Host Resistance to Vaccinia Virus and Leishmania major Infections

The E3L gene of vaccinia virus (VACV) encodes the E3 protein that in cultured cells inhibits the activation of interferon (IFN)-induced proteins, double-stranded RNA-dependent protein kinase (PKR), 2'-5'-oligoadenylate synthetase/RNase L (2-5A system) and adenosine deaminase (ADAR-1), thus helping the virus to evade host responses. Here, we have characterized the in vivo E3 functions in a murine inducible cell culture system (E3L-TetOFF) and in transgenic mice (TgE3L). Inducible E3 expression in cultured cells conferred on cells resistance to the antiviral action of IFN against different viruses, while expression of the E3L gene in TgE3L mice triggered enhanced sensitivity of the animals to pathogens. Virus infection monitored in TgE3L mice by different inoculation routes (intraperitoneal and tail scarification) showed that transgenic mice became more susceptible to VACV infection than control mice. TgE3L mice were also more susceptible to Leishmania major infection, leading to an increase in parasitemia compared to control mice. The enhanced sensitivity of TgE3L mice to VACV and L. major infections occurred together with alterations in the host immune system, as revealed by decreased T-cell responses to viral antigens in the spleen and lymph nodes and by differences in the levels of specific innate cell populations. These results demonstrate that expression of the E3L gene in transgenic mice partly reverses the resistance of the host to viral and parasitic infections and that these effects are associated with immune alterations.

Increased TGF-β, Cbl-b and CTLA-4 levels and immunosuppression in association with chronic immune activation

In this study we investigated the mechanisms mediating T-cell hyporesponsiveness in chronically immune-activated individuals. We analyzed in healthy and persistently helminth-infected individuals the relationship between immune activation and general T-cell hyporesponsiveness, Th3/regulatory T-cell expression, transforming growth factor-beta (TGF-beta) secretion, CTL-associated antigen 4 (CTLA-4) levels, Casitas B-cell lymphoma-b (Cbl-b) (a negative regulator of T-cell activation) levels and phosphorylation of mitogen-activated protein kinases/extracellular signal-regulated kinase (ERK)-1 and -2. We found a very significant increase in plasma levels of TGF-beta and intracellular pools of CTLA-4 and Cbl-b in association with immune activation, which correlates with decreased T-cell responses to anti-CD3 stimulation. We demonstrate that the impaired activity of ERK of peripheral T cells in highly immune-activated individuals is associated with increased levels of CTLA-4 and Cbl-b. Interestingly, in some, but not in all, of these immune-activated individuals, induction of Cbl-b intracellular pools occurs by TGF-beta or CTLA-4 stimulation. We suggest that the higher levels of CTLA-4 and TGF-beta, both involved in the induction of Cbl-b, point at potential mechanisms underlying general and antigen-specific immune hyporesponsiveness in chronically infected individuals.

Cytotoxic T-lymphocyte antigen 4 gene and recovery from hepatitis B virus infection.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is an inhibitory T-cell receptor expressed by activated and regulatory T cells. We hypothesized that single-nucleotide polymorphisms (SNPs) in the gene encoding CTLA-4 may affect the vigor of the T-cell response to hepatitis B virus (HBV) infection, thus influencing viral persistence. To test this hypothesis, we genotyped six CTLA4 SNPs, from which all frequent haplotypes can be determined, using a large, matched panel of subjects with known HBV outcomes. Haplotypes with these SNPs were constructed for each subject using PHASE software. The haplotype distribution differed between those with viral persistence and those with clearance. Two haplotypes were associated with clearance of HBV infection, which was most likely due to associations with the SNPs -1722C (odds ratio [OR] = 0.60, P = 0.06) and +49G (OR = 0.73, P = 0.02). The wild-type haplotype, which contains an SNP leading to a decreased T-cell response (+6230A), was associated with viral persistence (OR = 1.32, P = 0.04). These data suggest that CTLA4 influences recovery from HBV infection, which is consistent with the emerging role of T regulatory cells in the pathogenesis of disease.

Regulation of T-cell differentiation by IL-4Rα-chain single nucleotide polymorphisms

Chronic inflammation in rheumatoid arthritis (RA) is mediated by repeatedly activated proinflammatory Th1 cells. In contrast, Th2 cells that might downmodulate the chronic autoimmune response are rarely found in RA. It has been previously documented that RA T cells are severely impaired in their ability to differentiate into Th2 effectors while exerting enhanced Th1 differentiation. The mechanisms underlying this functional abnormality, however, have not been delineated. As IL-4 is a most critical determinant in regulating immune responses by promoting Th2 cell development and inhibiting Th1 cell differentiation, we analyzed the role of single nucleotide polymorphisms (SNP) in the IL-4 receptor α-chain, which is critical for binding of IL-4 and for IL-4 signal transduction, in the differentiation of human T cells. Naive and memory CD4 T cells were isolated from the peripheral blood of 348 healthy individuals and genotyped by allele-specific PCR for the two IL-4R α-chain SNPs that are located in functionally important regions of the IL-4R α-chain – the I50 V SNP50 and the Q551R SNP551 in the IL-4-binding and STAT6-binding domains, respectively. To analyze the functional role of IL-4R α-chain SNPs for T-cell differentiation, CD4-positive T cells were purified from the peripheral blood from the individuals who were homozygous for either allele at SNP50 and SNP551, and primed for 5 days with mAbs to CD28 and/or CD3 in the presence or absence of exogenous IL-4. The phenotype of the resulting differentiated effector cells was then analyzed by flow cytometric analysis of cytoplasmic cytokines. The SNP551 alleles did not significantly affect T-cell differentiation. In marked contrast, the inhibitory effect of IL-4 on Th1 cell differentiation was significantly diminished in CD4 T cells that were homozygous for the mutated allele at SNP50 (50 V) as compared with those with the wild-type allele (I50). Likewise, the augmenting effect of IL-4 on Th2 cell differentiation was markedly enhanced on T cells that were homozygous for the wild-type allele as compared with T cells expressing the mutant allele. The data indicate that the mutated allele of the IL-4R α-chain SNP50 is associated with a decreased T-cell response to IL-4. Thus, SNP50 of the IL-4R α-chain might regulate T-cell differentiation by altering T-cell responses to IL-4 and contribute to the development of unbalanced Th subset activation, as characteristic for autoimmune diseases, such as RA.

Detection of viral DNA and immune responses to the human herpesvirus 6 101-kilodalton virion protein in patients with multiple sclerosis and in controls.

Human herpesvirus 6 (HHV-6), a latent lymphotropic and neurotropic virus, has been suspected as an etiologic agent in multiple sclerosis (MS). The study was undertaken to correlate virologic evidence for HHV-6 activity with the state of host immunity to HHV-6 in MS patients and control subjects. The study revealed that cell-free DNA of HHV-6 was detected more frequently in both serum and cerebrospinal fluid of MS patients than in those of control subjects. T cells recognizing the recombinant 101-kDa protein (101K) corresponding to the major immunoreactive region unique to HHV-6 occurred at significantly lower precursor frequency in MS patients than in control subjects. The resulting HHV-6-specific T-cell lines obtained from MS patients exhibited skewed cytokine profiles characterized by the inability to produce interleukin-4 (IL-4) and IL-10. The decreased T-cell responses to HHV-6 and the altered cytokine profile were consistent with significantly declined serum immunoglobulin G (IgG) titers for HHV-6 of MS patients compared to those of control subjects. In contrast, elevated serum IgM titers for HHV-6 were detected in the majority of MS patients, which may reflect frequent exposure of B cells to HHV-6. The findings suggest that the decreased immune responses to HHV-6 may be responsible for ineffective clearance of HHV-6 in MS patients.

Low T-cell responses to CD3 plus CD28 monoclonal antibodies are predictive of development of AIDS.

Decreased T-cell reactivity in vitro is strongly associated with progression to AIDS and low CD4+ T-cell numbers. Low T-cell responses in vitro induced by CD3 monoclonal antibody (mAb) are predictive for progression to AIDS independent of low CD4+ T-cell counts and high HIV-1 RNA levels. We developed a whole-blood lymphocyte culture system in which T cells were stimulated by a combination of CD3 and CD28 mAb. Combined stimulation of CD28, a costimulatory molecule, and CD3 considerably enhances T-cell responses in vitro and reduces variation coefficients, which may increase the prognostic power of T-cell responses. A prospective study of HIV-1-infected homosexual men followed for 35 months. The predictive value of low T-cell responses to CD3 plus CD28 mAb relative to low CD4+ T-cell counts, high HIV-1 RNA levels and the presence of syncytium-inducing (SI) HIV-1 variants was evaluated longitudinally in 202 HIV-1-infected homosexual men followed for 35 months. In multivariate analysis, decreased T-cell responses at baseline were predictive of development of AIDS, independent of low CD4+ T-cell numbers and high HIV-1 RNA levels. In a time-dependent model, HIV-1 RNA levels lost their predictive value, whereas low T-cell responses, low CD4+ T-cell numbers and the presence of SI HIV-1 variants independently predicted AIDS. These data demonstrate that combined use of virological and immunological markers may be useful in monitoring disease progression and response to antiretroviral therapy.

Expression and signal transduction of T-cell antigen receptor (TCR)/CD3 complexes on fresh or in vitro expanded T lymphocytes from patients with Hodgkin's and non-Hodgkin's lymphomas.

T-cell responses against soluble antigens, alloantigens and mitogens are frequently diminished in patients with certain types of cancer. In the present study, the authors investigated possible mechanisms for the partial T-cell immunodeficiency in patients with Hodgkin's or non-Hodgkin's lymphomas. It was found that T-cells from lymphoma patients had significantly reduced proliferative responses to EBV-transformed B-cell lines and to anti-TCR/CD3 MoAb; a 30-50% reduction of cells expressing membrane T-cell receptor (TCR) complexes; and a significantly reduced signal transduction function. Long-term in vitro culture conditions were developed to expand T cells in TCR/CD3-dependent or TCR/CD3-independent manners. With such methods, it was found that the decreased T-cell responses in patients with Hodgkin's and non-Hodgkin's lymphomas appeared to be an intrinsic T-cell defect (not at the antigen presenting cell level), and the T-cell responses could be recovered after only a few days in culture. Thus, it is suggested that the T-cell response-defect in Hodgkin or non-Hodgkin lymphoma patients is a reversible phenomenon, dependent on the patient's tumour-bearing environment.

Characterization of regulatory T cells in patients with B-cell chronic lymphocytic leukemia

The status of the immune system of patients with B-cell chronic lymphocytic leukemia (B-CLL) is not yet sufficiently characterized. Clinically, B-CLL patients present immunodeficiency increasing along with disease progression and signs of autoimmunity. In the current study, we evaluated the expression of FOXP3 in CD4+CD25hi T regulatory lymphocytes (Treg) and their influence on immune response against tumor and viral antigens in the complex system of peripheral blood mononuclear cells. In 80 B-CLL patients, the frequency of Treg (CD4+CD25hi FOXP3+) cells was significantly higher in B-CLL patients when compared to healthy volunteers (HV) and increased with the progression of the disease (median: 8.24% in stage A, 11.24% in stage B and 12.57% in stage C according to the Binet classification). The frequency of Treg showed no correlation with prognostic markers such as ZAP-70, CD38 and HLA-G. Notably, Treg frequency correlated with serum levels of TNF (r(2)=0.45, p=0.001). T-cell immune responses against epitopes derived from the tumor-associated antigens survivin, fibromodulin and RHAMM as well as from the influenza matrix protein were evaluated. Functionally, higher frequencies of Treg correlated with decreased T-cell responses against viral and tumor antigens. In conclusion, we detected higher frequencies of Treg in B-CLL patients than in HV. Furthermore, Treg constitute the crucial mechanism of immunosuppression in B-CLL patients.

Aberrant Cytokine Production By Sezary Syndrome Patients: Cytokine Secretion Pattern Resembles Murine Th2 Cells

Sezary syndrome (SzS), the leukemic stage of cutaneous T-cell lymphoma (CTCL), is typically a CD4+ T-cell lympho-proliferative disease characterized by numerous immunologic abnormalities, including decreased T-cell responses to antigens and mitogens, decreased natural killer and lymphocyte-activated killer cell activities, eosinophilia, and increased levels of immunoglobulins, particularly IgE and IgA. Because this constellation of abnormalities is reminiscent of the pleiotropic in vitro activities of IL-4 and IL-5, we examined the possibility that malignant T cells in SzS may be producing increased amounts of IL-4 with a concomitant decrease in IL-2 and IFN-gamma production. Such a cytokine secretion pattern would be similar to that produced by murine Th2 cells. Serum IL-4 enzyme-linked immunosorbent assay measurements revealed that 33% of SzS patients (n = 21) had levels of IL-4 significantly higher (mean, 7.2 pg/ml; range, 0-48, p less than 0.05) than normal controls (mean, 1.59; range, 0-3.1). Although the majority of tested patients had elevated serum IgE, no direct correlation between serum IL-4 levels and serum IgE levels was observed. Peripheral blood mononuclear cells (PBMC) isolated from SzS patients and stimulated with phytohemagglutinin (PHA) produced significantly higher levels of interleukin (IL)-4 and significantly lower levels of IL-2 and interferon (IFN)-gamma than did PBMC from normal controls (p less than 0.02, p less than 0.05, and p less than 0.02, respectively). PBMC from SzS patients in remission produced IL-4 and IL-2 levels similar to that of the normal controls. To determine if IFN-gamma could inhibit the increased IL-4 production by PHA-stimulated PBMC from SzS patients, IFN-gamma was added to culture at 0, 24, or 48 h prior to the addition of PHA. Significant decreases in IL-4 production were seen only in cultures incubated with IFN-gamma for 24 and 48 h prior to the addition of PHA. IL-2 levels were not affected by IFN-gamma incubation. The increased IL-4 and decreased IL-2 and IFN-gamma production by PBMC from SzS patients suggests that Sezary cells have a cytokine profile similar to murine Th2 cells. Moreover, this cytokine secretion pattern may play an integral role in the immunopathogenesis of advanced CTCL. The results also suggest that IFN-gamma be a useful component in the spectrum of therapeutic approaches to SzS.

Dose and time related effects of T-2 toxin on mitogenic response of murine splenic cells in vitro

The effects of T-2 toxin in vitro on murine splenic cells were investigated. Cells from syngeneic male NFS/N mice were cultured with three concentrations of either lipopolysaccharide (LPS), pokeweed mitogen (PWM), phytohaemagglutinin (PHA), or concanavalin A (Con A), for 48 h. T-2 toxin (10 −12−10 −8M) was added at the beginning or 24 h after initiation of the cultures. Exposure to T-2 toxin (10 −11−10 −10M) after 24 h increased 3H-thymidine uptake by splenic cells. Stimulation by PWM increased dramatically while the response to LPS was increased to a lesser extent in this system. Conversely, exposure to T-2 toxin decreased T-cell responses to both PHA and Con A. Exposing cells to T-2 toxin at the initiation of culture increased Con A stimulation; LPS and PWM responses decreased following 48 h exposure. When splenic cells were pre-treated for 60 or 90 min at 0°C with 10 −9M T-2 toxin the responses to both PWM and LPS increased. Increased 3H-thymidine uptake was also observed following 90 min exposure to T-2 toxin alone at either 0 or 37°C. The increased PWM response and the mitogenic phenomena observed when cells were exposed only to T-2 toxin were consistent with previously observed effects of in vivo T-2 toxin exposure.

Impaired T-cell responses in chronic lymphocytic leukemia: Lack of suppressor cell effect

T-Lymphocyte responses in B-cell chronic lymphocytic leukemia (CLL) are abnormal by several criteria. We investigated the possibility that impaired T-cell responses might be due to increased suppressor cell activity by: (a) measuring the responses to phytohemagglutinin (PHA) stimulation of normal mononuclear cells cocultured with CLL lymphocytes compared to normal cells alone, (b) preincubation of lymphocytes in tissue culture medium prior to the addition of PHA, (c) measuring the responses of lymphocytes to PHA in the presence of indomethacin. Cells from 8 patients with CLL cocultured with normal cells did not suppress responses to PHA. Both preincubation in tissue culture medium and addition of indomethacin to the cell cultures have been shown to block different suppressor cell activities. As expected, both of these procedures significantly increased the PHA responses of lymphocytes from 7 normal individuals in contrast to essentially no change in the responses of lymphocytes from 9 and 11 patients with CLL. Since T cells are diluted by the predominant B lymphocytes in CLL, the mononuclear cells were fractionated with enrichment of T lymphocytes to 75–85%. No increases in PHA responses of the enriched T cells were observed after preincubation in two cases or with the addition of indomethacin in four cases. We were therefore unable to demonstrate global suppressor cell activity in CLL affecting PHA responses, and there was evidence of less than normal activity in two specific suppressor cell systems. We suggest that decreased T-cell responses to PHA in CLL represent an innate cellular abnormality.