Murine Splenic Natural Killer Cells Do Not Develop Immunological Memory after Re-Encounter with Mycobacterium bovis BCG

Several lines of evidence have recently suggested that natural killer (NK) cells develop immunological memory against viral infections. However, there is no apparent evidence that NK cells acquire specific memory against Mycobacterium bovis bacillus Calmette—Guérin (BCG), the only currently licensed vaccine for preventing tuberculosis. In the present study, we investigated whether murine splenic NK cells can be activated by BCG in a dendritic cell (DC)-independent or -dependent manner, and furthermore examined whether these NK cells acquire specific memory following BCG vaccination. NK cells isolated from spleens of BCG-immunized mice produced interferon (IFN)γ through direct BCG stimulation in the absence of antigen-presenting cells; however, NK cells from control animals similarly directly responded to BCG, and the response level was not statistically significant between the immunized and the naïve NK cells. When purified NK cells that had been exposed to BCG were cocultured with RAW murine macrophages infected with BCG, the antibacterial activity of the macrophages was strongly enhanced; however, its level was similar to that by naïve NK cells, which had not been exposed to BCG. When splenocytes harvested from BCG-immunized mice were stimulated with purified protein derivative (PPD) derived from Mycobacterium tuberculosis, a specific IFNγ response was clearly observed, mainly attributed to NK cells and memory CD4⁺ T cells. To investigate whether these NK cells as well as the T cells are activated by cell−cell interaction with DCs presenting mycobacterial antigens, NK cells isolated from BCG-immunized mice were cocultured with splenocytes harvested from naïve mice in the presence of PPD stimulation. However, no IFNγ response was found in the NK cells. These results suggest that murine splenic NK cells do not develop BCG-specific immunological memory in either a DC-independent or -dependent manner.     

The Polyfunctionality of Human Memory CD8+ T Cells Elicited by Acute and Chronic Virus Infections Is Not Influenced by Age

As humans age, they experience a progressive loss of thymic function and a corresponding shift in the makeup of the circulating CD8+ T cell population from naïve to memory phenotype. These alterations are believed to result in impaired CD8+ T cell responses in older individuals; however, evidence that these global changes impact virus-specific CD8+ T cell immunity in the elderly is lacking. To gain further insight into the functionality of virus-specific CD8+ T cells in older individuals, we interrogated a cohort of individuals who were acutely infected with West Nile virus (WNV) and chronically infected with Epstein Barr virus (EBV) and Cytomegalovirus (CMV). The cohort was stratified into young (<40 yrs), middle-aged (41–59 yrs) and aged (>60 yrs) groups. In the aged cohort, the CD8+ T cell compartment displayed a marked reduction in the frequency of naïve CD8+ T cells and increased frequencies of CD8+ T cells that expressed CD57 and lacked CD28, as previously described. However, we did not observe an influence of age on either the frequency of virus-specific CD8+ T cells within the circulating pool nor their functionality (based on the production of IFNγ, TNFα, IL2, Granzyme B, Perforin and mobilization of CD107a). We did note that CD8+ T cells specific for WNV, CMV or EBV displayed distinct functional profiles, but these differences were unrelated to age. Collectively, these data fail to support the hypothesis that immunosenescence leads to defective CD8+ T cell immunity and suggest that it should be possible to develop CD8+ T cell vaccines to protect aged individuals from infections with novel emerging viruses.     

IFN-β Inhibits the Increased Expression of IL-9 during Experimental Autoimmune Uveoretinitis

Purpose

It has been shown that IL-9 plays a proinflammatory role in the pathogenesis of certain autoimmune diseases. This study was designed to investigate the possible role of IL-9 in the development of experimental autoimmune uveoretinitis (EAU) and the effect of IFN-β on its expression.

Methods

EAU was induced in B10RIII mice by immunization with interphotoreceptor retinoid-binding protein peptide 161–180 (IRBP_161–180). IFN-β was administered subcutaneously to IRBP_161–180 immunized mice every other day from day one before immunization to the end of the study. Splenocytes and draining lymph node (DLN) cells from EAU mice or control mice or EAU mice treated with IFN-β or PBS were stimulated with anti-CD3/CD28 or IRBP_161–180 for 3 days. Naïve T cells cultured under Th1 or Th17 polarizing conditions were incubated in the presence or absence of IFN-β for 4 days. Effector/memory T cells were activated by anti-CD3/CD28 in the presence or absence of IFN-β for 3 days. IFN-β-treated monocytes were cocultured with naïve T cells or effector/memory T cells for 3 days. Culture supernatants were collected and IL-9 was detected by ELISA.

Results

IL-9 expression in splenocytes and DLN cells was increased in EAU mice during the inflammatory phase and returned back to lower levels during the recovery phase. IFN-β in vivo treatment significantly inhibited EAU activity in association with a down-regulated expression of IL-9. In vitro polarized Th1 and Th17 cells both secreted IL-9 and the addition of IFN-β suppressed production of IL-9 by both Th subsets. Beside its effect on polarized Th cells, IFN-β also suppressed the secretion of IL-9 by effector/memory T cells. However, IFN-β-treated monocytes had no effect on the production of IL-9 when cocultured with naïve or effector/memory T cells.

Conclusion

IL-9 expression is increased during EAU which could be suppressed by IFN-β.     

CD40 Activation Rescues Antiviral CD8+ T Cells from PD-1-Mediated Exhaustion

The intrahepatic immune environment is normally biased towards tolerance. Nonetheless, effective antiviral immune responses can be induced against hepatotropic pathogens. To examine the immunological basis of this paradox we studied the ability of hepatocellularly expressed hepatitis B virus (HBV) to activate immunologically naïve HBV-specific CD8⁺ T cell receptor (TCR) transgenic T cells after adoptive transfer to HBV transgenic mice. Intrahepatic priming triggered vigorous in situ T cell proliferation but failed to induce interferon gamma production or cytolytic effector function. In contrast, the same T cells differentiated into cytolytic effector T cells in HBV transgenic mice if Programmed Death 1 (PD-1) expression was genetically ablated, suggesting that intrahepatic antigen presentation per se triggers negative regulatory signals that prevent the functional differentiation of naïve CD8⁺ T cells. Surprisingly, coadministration of an agonistic anti-CD40 antibody (αCD40) inhibited PD-1 induction and restored T cell effector function, thereby inhibiting viral gene expression and causing a necroinflammatory liver disease. Importantly, the depletion of myeloid dendritic cells (mDCs) strongly diminished the αCD40 mediated functional differentiation of HBV-specific CD8⁺ T cells, suggesting that activation of mDCs was responsible for the functional differentiation of HBV-specific CD8⁺ T cells in αCD40 treated animals. These results demonstrate that antigen-specific, PD-1-mediated CD8⁺ T cell exhaustion can be rescued by CD40-mediated mDC-activation.     

Superior Cervical Ganglia Neurons Induce Foxp3+ Regulatory T Cells via Calcitonin Gene-Related Peptide

The nervous and immune systems communicate bidirectionally, utilizing diverse molecular signals including cytokines and neurotransmitters to provide an integrated response to changes in the body’s internal and external environment. Although, neuro-immune interactions are becoming better understood under inflammatory circumstances and it has been evidenced that interaction between neurons and T cells results in the conversion of encephalitogenic T cells to T regulatory cells, relatively little is known about the communication between neurons and naïve T cells. Here, we demonstrate that following co-culture of naïve CD4⁺ T cells with superior cervical ganglion neurons, the percentage of Foxp3 expressing CD4⁺CD25⁺ cells significantly increased. This was mediated in part by immune-regulatory cytokines TGF-β and IL-10, as well as the neuropeptide calcitonin gene-related peptide while vasoactive intestinal peptide was shown to play no role in generation of T regulatory cells. Additionally, T cells co-cultured with neurons showed a decrease in the levels of pro-inflammatory cytokine IFN-γ released upon in vitro stimulation. These findings suggest that the generation of Tregs may be promoted by naïve CD4⁺ T cell: neuron interaction through the release of neuropeptide CGRP.     

CD8+ T-Cells Expressing Interferon Gamma or Perforin Play Antagonistic Roles in Heart Injury in Experimental Trypanosoma Cruzi-Elicited Cardiomyopathy

In Chagas disease, CD8⁺ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8⁺ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8⁺ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8⁺ T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8⁺ T-cells segregated into IFNγ⁺ perforin (Pfn)^neg or IFNγ^negPfn⁺ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8⁺Pfn⁺ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8 ^−/− recipients showed that the CD8⁺ cells from infected ifnγ^−/− pfn ^+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8⁺ cells from ifnγ ^+/+ pfn ^−/− donors. Moreover, the reconstitution of naïve cd8 ^−/− mice with CD8⁺ cells from naïve ifnγ ^+/+ pfn ^−/− donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ⁺ cells accumulation, whereas reconstitution with CD8⁺ cells from naïve ifnγ ^−/− pfn ^+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn⁺ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8⁺Pfn⁺ and CD8⁺IFNγ⁺ cells during CCC. CD8⁺IFNγ⁺ cells may exert a beneficial role, whereas CD8⁺Pfn⁺ may play a detrimental role in T. cruzi-elicited heart injury.     

Contrasted TCRβ Diversity of CD8+ and CD8? T Cells in Rainbow Trout

Teleost fish express highly diverse naive TCRβ (TRB) repertoires and mount strong public and private clonal responses upon infection with pathogens. Fish T cells express typical markers such as CD8, CD4-1 and CD4-2, CD3, CD28 and CTLA4. Fish CD8⁺ T cells have been shown to be responsible for antigen-specific cell-mediated cytotoxicity in in vitro systems using histo-compatible effector and target cells. We compare here the complexity of TRB repertoires between FACS sorted CD8⁺ and CD8⁻ T cells from spleen and pronephros of rainbow trout. In contrast to human, while the TRB repertoire is highly diverse and polyclonal in CD8⁺ T cells of naïve fish, it appeared very different in CD8⁻ lymphocytes with irregular CDR3 length distributions suggesting a dominance of activated clones already in naïve fish or the presence of non conventional T cells. After infection with a systemic virus, CD8⁺ T cells mount a typical response with significant skewing of CDR3 length profiles. The infection also induces significant modifications of the TRB repertoire expressed by the CD8⁻ fraction, but for a different set of V/J combinations. In this fraction, the antiviral response results in an increase of the peak diversity of spectratypes. This unusual observation reflects the presence of a number of T cell expansions that rise the relative importance of minor peaks of the highly skewed distributions observed in unchallenged animals. These results suggest that the diversity of TRB expressed by CD8⁺ and CD8⁻ αβ T cells may be subjected to different regulatory patterns in fish and in mammals.     

Expression of CD11c Is Associated with Unconventional Activated T Cell Subsets with High Migratory Potential

CD11c is an α integrin classically employed to define myeloid dendritic cells. Although there is little information about CD11c expression on human T cells, mouse models have shown an association of CD11c expression with functionally relevant T cell subsets. In the context of genital tract infection, we have previously observed increased expression of CD11c in circulating T cells from mice and women. Microarray analyses of activated effector T cells expressing CD11c derived from naïve mice demonstrated enrichment for natural killer (NK) associated genes. Here we find that murine CD11c⁺ T cells analyzed by flow cytometry display markers associated with non-conventional T cell subsets, including γδ T cells and invariant natural killer T (iNKT) cells. However, in women, only γδ T cells and CD8⁺ T cells were enriched within the CD11c fraction of blood and cervical tissue. These CD11c⁺ cells were highly activated and had greater interferon (IFN)-γ secretory capacity than CD11c^- T cells. Furthermore, circulating CD11c⁺ T cells were associated with the expression of multiple adhesion molecules in women, suggesting that these cells have high tissue homing potential. These data suggest that CD11c expression distinguishes a population of circulating T cells during bacterial infection with innate capacity and mucosal homing potential.     

High Frequencies of Virus-Specific CD8+ T-Cell Precursors

A productive CD8⁺ T-cell response to a viral infection requires rapid division and proliferation of virus-specific CD8⁺ T cells. Tetramer-based enrichment assays have recently given estimates of the numbers of peptide-major histocompatibility complex-specific CD8⁺ T cells in naïve mice, but precursor frequencies for entire viruses have been examined only by using in vitro limiting-dilution assays (LDAs). To examine CD8⁺ T-cell precursor frequencies for whole viruses, we developed an in vivo LDA and found frequencies of naïve CD8⁺ T-cell precursors of 1 in 1,444 for vaccinia virus (VV) (∼13,850 VV-specific CD8⁺ T cells per mouse) and 1 in 2,958 for lymphocytic choriomeningitis virus (LCMV) (∼6,761 LCMV-specific CD8⁺ T cells per mouse) in C57BL/6J mice. In mice immune to VV, the number of VV-specific precursors, not surprisingly, dramatically increased to 1 in 13 (∼1,538,462 VV-specific CD8⁺ T cells per mouse), consistent with estimates of VV-specific memory T cells. In contrast, precursor numbers for LCMV did not increase in VV-immune mice (1 in 4,562, with ∼4,384 LCMV-specific CD8⁺ T cells per VV-immune mouse). Using H-2D^b-restricted LCMV GP33-specific P14-transgenic T cells, we found that, after donor T-cell take was accounted for, approximately every T cell transferred underwent a full proliferative expansion in response to LCMV infection. This high efficiency was also seen with memory populations, suggesting that most antigen-specific T cells will proliferate extensively at a limiting dilution in response to infections. These results show that frequencies of naïve and memory CD8⁺ T cell precursors for whole viruses can be remarkably high.The immune response to a viral infection often involves the rapid proliferation of CD8⁺ effector T cells that recognize virus-infected targets expressing 8- to 11-amino-acid-long peptides on class I major histocompatibility complex (MHC) molecules. This recognition is mediated by membrane-bound T-cell receptors (TCRs) that are generated through largely random DNA recombination events of the many TCRα and -β genes, encoding polypeptide chains that heterodimerize to form the recognition structure of T cells. The recombination of the segments also involves addition or deletion of nucleotides during the joining process, causing even greater diversity, and these processes allow for a very broad range of T-cell specificities, with a calculated theoretical diversity of ∼10¹⁵ TCRs in the mouse (7). By use of PCR, CDR3 spectratyping, and sequencing techniques, it was estimated that there are approximately 2 × 10⁶ distinct TCR specificities in a mouse spleen (1, 5). This is far below the theoretical level of T-cell diversity, but considering estimates of T-cell degeneracy that propose that a single TCR can recognize up to 10⁶ peptide-MHC (pMHC) complexes (17, 36), it is likely that the functional diversity is much greater than the number of individual TCRs.It has been of interest to calculate the number of T cells that would either recognize or respond to a pathogen or to a specific pMHC complex. Early estimates of numbers of CD8⁺ T cells that are specific to a single virus, i.e., precursor frequencies, took advantage of an in vitro limiting-dilution assay (LDA) and calculated CD8⁺ T-cell virus-specific precursor frequencies to be on the order of 1 in 100,000 in naïve mice and predicted that these cells needed to undergo about 15 divisions to reach the higher precursor frequencies found at day 8 postinfection (29, 30). The efficiency of such assays, however, is relatively poor. Later studies estimated the number of pMHC-specific CD8⁺ T cells in a naïve mouse by CDR3 sequencing. H-2K^d-restricted T cells specific to HLA residues 170 to 179 (HLA 170-179) were sorted by tetramer from human tumor-immunized mice, and their Vβ CDR3 regions were sequenced. After a plateau suggesting that the majority of the different TCRs had been sequenced was reached, exhaustive sequencing was then used to identify the frequencies of these sequences in naïve mice. These studies found that there were about 600 CD8⁺ T cells specific for that pMHC complex in naïve mice (4). A second strategy used an in vivo competition assay with H-2D^b-restricted lymphocytic choriomeningitis virus (LCMV) GP33-specific P14-transgenic T cells to estimate the number of GP33-specific CD8 T cells in naïve mice and calculated the number to be between 100 to 200 cells per mouse (2).Others estimated numbers of pMHC-specific T cells by sequencing the CDR3β regions of antigen-specific T cells that had expanded during an acute infection. By calculating a measure of CDR3 diversity and then assuming a logarithmic distribution of diversity, they extrapolated the number of T-cell clones that responded to an acute infection. With this technique, 300 to 500 H-2D^b-restricted mouse hepatitis virus (MHV)-encoded S510 clonotypes were calculated to be in the central nervous systems of acutely infected mice, with ∼100 to 900 clonotypes calculated to be in chronically infected mice (24). Later studies used a gamma interferon (IFN-γ) capture assay instead of tetramer sorting and estimated 1,100 to 1,500 H-2D^b-restricted S510-specific clonotypes and 600 to 900 clonotypes of the subdominant H-2K^b-restricted MHV S598 peptide-specific T cells in the spleens of acutely infected mice (25). Those studies also estimated that there were 1,000 to 1,200 different H-2D^b-restricted GP33-specific clonotypes that could respond to an LCMV infection.More-recent studies have taken advantage of magnetic tetramer binding enrichment and double tetramer staining of cells from the spleen and lymph nodes of naïve mice to determine pMHC precursor frequencies, with the assumption that most CD8⁺ T cells in a naïve mouse reside in lymphoid organs and will react with tetramers. This technique was first described by Moon et al. for CD4⁺ T cells, and it detected ∼190 I-A^b 2W1S 52-68-specific T cells, ∼20 I-A^b Salmonella enterica serovar Typhimurium FLiC 427-441-specific T cells, and ∼16 I-A^b chicken ovalbumin (OVA) 323-339-specific T cells per mouse (19). This same technique was then used to determine numbers of pMHC-specific CD8⁺ T cells for epitopes derived from a variety of viruses and found 15 to 1,070 pMHC-specific CD8⁺ T cells per mouse, depending on the specificity of the pMHC tetramer (10, 15, 23). Determinations of CD8⁺ T-cell precursor frequencies in humans are currently not experimentally attainable, but exhaustive sequencing of an HLA-A2.1-restricted influenza A virus (IAV) M1 58-66-specific T-cell response has suggested that there are at least 141 different clonotypes that can grow out in response to an in vitro stimulation with peptide, providing a minimum number of T cells that can respond to this pMHC complex in humans (22).Most of the assays estimate the number of T cells specific to single peptides in individual mice. These assays, therefore, do not determine the numbers of CD8⁺ T cells that can proliferate in response to an entire virus, especially if the virus is known to have many epitopes or if epitopes for the virus have not been described. By examining the average number of pMHC-specific CD8⁺ T cells in a naïve mouse and comparing this to the number of pMHC-specific CD8⁺ T cells that are in a mouse at the peak of the T-cell response, it can be calculated that CD8⁺ T cells divide approximately 12 to 14 times after virus infection (23). Considering that the progeny of one precursor after only 12 divisions can result in just over 4,000 cells, and since recent experiments using H-2K^b-restricted chicken OVA 257-264-specific OT-1-transgenic T cells have confirmed that the progeny from a single cell can be detected in a mouse after infection (31), an in vivo LDA was set up to take advantage of the extensive division and proliferation of virus-specific CD8⁺ T cells in order to determine virus-specific CD8⁺ T-cell precursor frequencies.Here, we show that by transferring limiting amounts of carboxyfluorescein succinimidyl ester (CFSE)-labeled Thy1.1⁺ Ly5.2⁺ heterogeneous CD8⁺ T cells into Thy1.2⁺ Ly5.1⁺ hosts, we are able to calculate CD8⁺ T-cell precursor frequencies for whole viruses. Our calculations are based on finding the number of donor CD8⁺ T cells that results in low-level-CFSE (CFSElo) (i.e., proliferated) donor CD8 T cells in 50% of the hosts. Using probit or Reed and Muench 50% endpoint calculations (3, 26), we are able to calculate CD8⁺ T-cell precursor frequencies. We show here that frequencies of naïve CD8⁺ T-cell precursors for whole viruses are quite high and that our in vivo LDA calculates whole-virus precursor frequencies in line with determinations using other methods with naïve and immune mice.     

Composition and Function of T Cell Subpopulations Are Slow to Change Despite Effective Antiretroviral Treatment of HIV Disease

The ability to reconstitute a normal immune system with antiretroviral therapy in the setting of HIV infection remains uncertain. This study aimed to characterize quantitative and qualitative aspects of various T cell subpopulations that do not improve despite effective ART. CD4∶CD8 ratio was evaluated in HIV-infected subjects with viral loads >10,000 copies/µl (“non-controllers”, n = 42), those with undetectable viral loads on ART (“ART-suppressed”, n = 53), and HIV-uninfected subjects (n = 22). In addition, T cell phenotype and function were examined in 25 non-controllers, 18 ART-suppressed, and 7 HIV-uninfected subjects. CD4∶CD8 ratio in non-controllers, ART-suppressed, and HIV-uninfected subjects was 0.25, 0.48, and 1.95 respectively (P<0.0001 for all comparisons). The increased ratio in ART-suppressed compared to non-controllers was driven by an increase of CD4+ T cells, with no change in the expanded CD8+ T cell population. Expansion of differentiated (CD28−CD27−CD45RA+/−CCR7−) T cell subpopulations persisted despite ART and minimal changes were noted in naïve T cell frequencies over time. Increased number of CD8+CD28− T cells and increased CD8+ CMV-specific T cell responses were associated with a decreased CD4∶CD8 ratio. Measures of T cell function demonstrated persistence of high frequencies of CD8+ T cells producing IFN–γ. Lastly, though all CD8+ subpopulations demonstrated significantly lower Ki67 expression in ART-suppressed subjects, CD4+ T cell subpopulations did not consistently show this decrease, thus demonstrating different proliferative responses in the setting of T cell depletion. In summary, this study demonstrated that CD4∶CD8 ratios remained significantly decreased and naïve T cell numbers were slow to increase despite long-term viral suppression on ART. In addition, there is a evidence of differential regulation of the CD4+ and CD8+ T cell subpopulations, suggesting independent homeostatic regulation of the two compartments.     

The Metalloprotease ADAM12 Regulates the Effector Function of Human Th17 Cells

A key modulator of immune homeostasis, TGFβ has an important role in the differentiation of regulatory T cells (Tregs) and IL-17-secreting T cells (Th17). How TGFβ regulates these functionally opposing T cell subsets is not well understood. We determined that an ADAM family metalloprotease called ADAM12 is specifically and highly expressed in both Tregs and CCR6+ Th17 cells. ADAM12 is induced in vitro upon differentiation of naïve T cells to Th17 cells or IL-17-secreting Tregs. Remarkably, silencing ADAM12 expression in CCR6+ memory T cells enhances the production of Th17 cytokines, similar to suppressing TGFβ signaling. Further, ADAM12 knockdown in naïve human T cells polarized towards Th17/Treg cells, or ectopically expressing RORC, greatly enhances IL-17-secreting cell differentiation, more potently then inhibiting TGFβ signals. Together, our findings reveal a novel regulatory role for ADAM12 in Th17 cell differentiation or function and may have implications in regulating their aberrant responses during immune pathologies.     

Abacavir-Reactive Memory T Cells Are Present in Drug Na?ve Individuals

BackgroundFifty-five percent of individuals with HLA-B*57:01 exposed to the antiretroviral drug abacavir develop a hypersensitivity reaction (HSR) that has been attributed to naïve T-cell responses to neo-antigen generated by the drug. Immunologically confirmed abacavir HSR can manifest clinically in less than 48 hours following first exposure suggesting that, at least in some cases, abacavir HSR is due to re-stimulation of a pre-existing memory T-cell population rather than priming of a high frequency naïve T-cell population.MethodsTo determine whether a pre-existing abacavir reactive memory T-cell population contributes to early abacavir HSR symptoms, we studied the abacavir specific naïve or memory T-cell response using HLA-B*57:01 positive HSR patients or healthy controls using ELISpot assay, intra-cellular cytokine staining and tetramer labelling.ResultsAbacavir reactive CD8+ T-cell responses were detected in vitro in one hundred percent of abacavir unexposed HLA-B*57:01 positive healthy donors. Abacavir-specific CD8+ T cells from such donors can be expanded from sorted memory, and sorted naïve, CD8+ T cells without need for autologous CD4+ T cells.ConclusionsWe propose that these pre-existing abacavir-reactive memory CD8+ T-cell responses must have been primed by earlier exposure to another foreign antigen and that these T cells cross-react with an abacavir-HLA-B*57:01-endogenous peptide ligand complex, in keeping with the model of heterologous immunity proposed in transplant rejection.     

Th1-Th17 Cells Contribute to the Development of Uropathogenic Escherichia coli-Induced Chronic Pelvic Pain

The etiology of chronic prostatitis/chronic pelvic pain syndrome in men is unknown but may involve microbes and autoimmune mechanisms. We developed an infection model of chronic pelvic pain in NOD/ShiLtJ (NOD) mice with a clinical Escherichia coli isolate (CP-1) from a patient with chronic pelvic pain. We investigated pain mechanisms in NOD mice and compared it to C57BL/6 (B6) mice, a strain resistant to CP-1-induced pain. Adoptive transfer of CD4+ T cells, but not serum, from CP-1-infected NOD mice was sufficient to induce chronic pelvic pain. CD4+ T cells in CP-1-infected NOD mice expressed IFN-γ and IL-17A but not IL-4, consistent with a Th1/Th17 immune signature. Adoptive transfer of ex-vivo expanded IFN-γ or IL-17A-expressing cells was sufficient to induce pelvic pain in naïve NOD recipients. Pelvic pain was not abolished in NOD-IFN-γ-KO mice but was associated with an enhanced IL-17A immune response to CP1 infection. These findings demonstrate a novel role for Th1 and Th17-mediated adaptive immune mechanisms in chronic pelvic pain.     

GB Virus C Infection Is Associated with Altered Lymphocyte Subset Distribution and Reduced T Cell Activation and Proliferation in HIV-Infected Individuals

GBV-C infection is associated with prolonged survival and with reduced T cell activation in HIV-infected subjects not receiving combination antiretroviral therapy (cART). The relationship between GBV-C and T cell activation in HIV-infected subjects was examined. HIV-infected subjects on cART with non-detectable HIV viral load (VL) or cART naïve subjects were studied. GBV-C VL and HIV VL were determined. Cell surface markers of activation (CD38⁺/HLA-DR⁺), proliferation (Ki-67+), and HIV entry co-receptor expression (CCR5+ and CXCR4+) on total CD4+ and CD8+ T cells, and on naïve, central memory (CM), effector memory (EM), and effector CD4+ and CD8+ subpopulations were measured by flow cytometry. In subjects with suppressed HIV VL, GBV-C was consistently associated with reduced activation in naïve, CM, EM, and effector CD4+ cells. GBV-C was associated with reduced CD4+ and CD8+ T cell surface expression of activation and proliferation markers, independent of HIV VL classification. GBV-C was also associated with higher proportions of naïve CD4+ and CD8+ T cells, and with lower proportions of EM CD4+ and CD8+ T cells. In conclusion, GBV-C infection was associated with reduced activation of CD4+ and CD8+ T cells in both HIV viremic and HIV RNA suppressed patients. Those with GBV-C infection demonstrated an increased proportion of naive T cells and a reduction in T cell activation and proliferation independent of HIV VL classification, including those with suppressed HIV VL on cART. Since HIV pathogenesis is thought to be accelerated by T cell activation, these results may contribute to prolonged survival among HIV infected individuals co-infected with GBV-C. Furthermore, since cART therapy does not reduce T cell activation to levels seen in HIV-uninfected people, GBV-C infection may be beneficial for HIV-related diseases in those effectively treated with anti-HIV therapy.     

B-lymphocytes as Key Players in Chemical-Induced Asthma

T-lymphocytes and B-lymphocytes are key players in allergic asthma, with B-lymphocytes producing antigen-specific immunoglobulins E (IgE). We used a mouse model of chemical-induced asthma and transferred B-lymphocytes from sensitized animals into naïve wild type mice, B-lymphocyte knock-out (B-KO) mice or severe combined immunodeficiency (SCID) mice. On days 1 and 8, BALB/c mice were dermally sensitized with 0.3% toluene diisocyanate (TDI) (20µl/ear). On day 15, mice were euthanized and the auricular lymph nodes isolated. B-lymphocytes (CD19⁺) were separated from the whole cell suspension and 175,000 cells were injected in the tail vein of naïve wild type, B-KO or SCID mice. Three days later, the mice received a single oropharyngeal challenge with 0.01% TDI (20µl) or vehicle (acetone/olive oil (AOO)) (controls). Airway reactivity to methacholine and total and differential cell counts in the bronchoalveolar lavage (BAL) fluid were measured 24 hours after challenge. B-lymphocytes of AOO or TDI-sensitized mice were characterized for the expression of surface markers and production of cytokines. We found that transfer of B-cells obtained from mice dermally sensitized to toluene diisocyanate (TDI) into naïve wild type mice, B-KO mice or SCID mice led, within three days, to an acute asthma-like phenotype after an airway challenge with TDI. This response was specific and independent of IgE. These B-lymphocytes showed antigen presenting capacities (CD80/CD86 and CD40) and consisted of B effector (Be)2- (IL-4) and Be1-lymphocytes (IFN-γ). The transferred B-lymphocytes were visualized near large airways, 24 hours after TDI challenge. Thus, B-lymphocytes can provoke an asthmatic response without the action of T-lymphocytes and without major involvement of IgE.     

Mitochondrial reactive oxygen is critical for IL-12/IL-18-induced IFN-γ production by CD4+ T cells and is regulated by Fas/FasL signaling

Mitochondrial activation and the production of mitochondrial reactive oxygen species (mROS) are crucial for CD4⁺ T cell responses and have a role in naïve cell signaling after TCR activation. However, little is known about mROS role in TCR-independent signaling and in recall responses. Here, we found that mROS are required for IL-12 plus IL-18-driven production of IFN-γ, an essential cytokine for inflammatory and autoimmune disease development. Compared to TCR stimulation, which induced similar levels of mROS in naïve and memory-like cells, IL-12/IL-18 showed faster and augmented mROS production in memory-like cells. mROS inhibition significantly downregulated IFN-γ and CD44 expression, suggesting a direct mROS effect on memory-like T cell function. The mechanism that promotes IFN-γ production after IL-12/IL-18 challenge depended on the effect of mROS on optimal activation of downstream signaling pathways, leading to STAT4 and NF-κB activation. To relate our findings to IFN-γ-driven lupus-like disease, we used Fas-deficient memory-like CD4⁺ T cells from lpr mice. Importantly, we found significantly increased IFN-γ and mROS production in lpr compared with parental cells. Treatment of WT cells with FasL significantly reduced mROS production and the activation of signaling events leading to IFN-γ. Moreover, Fas deficiency was associated with increased mitochondrial levels of cytochrome C and caspase-3 compared with WT memory-like cells. mROS inhibition significantly reduced the population of disease-associated lpr CD44^hiCD62L^loCD4⁺ T cells and their IFN-γ production. Overall, these findings uncovered a previously unidentified role of Fas/FasL interaction in regulating mROS production by memory-like T cells. This apoptosis-independent Fas activity might contribute to the accumulation of CD44^hiCD62L^loCD4⁺ T cells that produce increased IFN-γ levels in lpr mice. Overall, our findings pinpoint mROS as central regulators of TCR-independent signaling, and support mROS pharmacological targeting to control aberrant immune responses in autoimmune-like disease.Subject terms: Autoimmunity, Cytokines     

The Cortical Actin Determines Different Susceptibility of Na?ve and Memory CD4+ T Cells to HIV-1 Cell-to-Cell Transmission and Infection

Memory CD4+ T cells are preferentially infected by HIV-1 compared to naïve cells. HIV-1 fusion and entry is a dynamic process in which the cytoskeleton plays an important role by allowing virion internalization and uncoating. Here, we evaluate the role of the cortical actin in cell-to-cell transfer of virus antigens and infection of target CD4+ T cells. Using different actin remodeling compounds we demonstrate that efficiency of HIV-internalization was proportional to the actin polymerization of the target cell. Naïve (CD45RA+) and memory (CD45RA−) CD4+ T cells could be phenotypically differentiated by the degree of cortical actin density and their capacity to capture virus. Thus, the higher cortical actin density of memory CD4+ T cells was associated to increased efficiency of HIV-antigen internalization and the establishment of a productive infection. Conversely, the lower cortical actin density in naïve CD4+ T cells restricted viral antigen transfer and consequently HIV-1 infection. In conclusion, the cortical actin density differentially affects the susceptibility to HIV-1 infection in naïve and memory CD4+ T cells by modulating the efficiency of HIV antigen internalization.     

A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8⁺ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8⁺ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8⁺ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8⁺ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8⁺ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8⁺ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.