Antigen-driven Proliferation Research Articles

A minority of proliferating human CD4+ T cells in antigen-driven proliferation assays are antigen specific

Antigen-driven T-cell proliferation is often measured using fluorescent dye dilution assays, such as the CFSE-based proliferation assay. Dye dilution assays have been powerful tools to detect human CD4+ T-cell responses, particularly against autoantigens. However, it is not known how many cells within the proliferating population are specific for the stimulating antigen. Here we determined the frequency of CD4+ T cells specific for the stimulating antigen within the antigen-responsive population of CFSE-based proliferation assays. We compared CD4+ T-cell responses to a type 1 diabetes autoantigen (proinsulin C-peptide) and to a vaccine antigen (tetanus toxoid). The TCRs expressed by antigen-responsive CD4+ T cells were sequenced, and their antigen specificity was tested functionally by expressing them in a reporter T-cell line. Responses to C-peptide were weak, but detectable, in PBMC from individuals with T1D, whereas responses to tetanus toxoid were much stronger. The frequency of antigen-specific CD4+ T cells correlated with the strength of the response to antigen in the proliferation assay. However, antigen-specific CD4+ T cells were rare among antigen-responsive CD4+ T cells. For C-peptide, an average frequency of 7.5% (1%–11%, n = 4) of antigen-responsive CD4+ T cells were confirmed to be antigen specific. In the tetanus-toxoid-stimulated cultures, on average, 45% (16%–78%, n = 5) of the antigen-responsive CD4+ T cells were tetanus toxoid specific. These data show that antigen-specific CD4+ T cells are a minority of the cells that proliferate in response to antigen and have important implications for in vitro CD4+ T-cell proliferation assays.

CD4+ T cells with latent HIV-1 have reduced proliferative responses to T cell receptor stimulation.

The latent reservoir for HIV-1 in resting CD4+ T cells persists despite antiretroviral therapy as a barrier to cure. The antigen-driven proliferation of infected cells is a major mechanism of reservoir persistence. However, activation through the T cell antigen receptor (TCR) can induce latent proviruses, leading to viral cytopathic effects and immune clearance. In single-cell studies, we show that, relative to uninfected cells or cells with a defective provirus, CD4+ T cells with an intact provirus have a profound proliferative defect in response to TCR stimulation. Virion production was observed in only 16.5% of cultures with an intact provirus, but proliferation was reduced even when no virion production was detected. Proliferation was inversely correlated with in vivo clone size. These results may reflect the effects of previous in vivo proliferation and do not support attempts to reduce the reservoir with antiproliferative agents, which may have greater effects on normal T cell responses.

Normalizing pathogen exposure via pet shop mouse cohousing leads to preferential maintenance of long-lived effector memory CD8 T cells

Abstract Studies of the CD8 T cell memory compartment in mice have primarily been done in young mice maintained in specific pathogen free (SPF) environment. The murine compartment is skewed towards central memory CD8 T cells, whereas adult human memory T cells are dominated by effector memory T cells. The murine memory compartment is akin to that of neonate humans, suggesting there are factors missing in SPF mice that are required to mature the T cell compartment. We wanted to ask how pathogen exposure changes the memory compartment of mice, so we utilized a model of pet shop mouse co-housing (COH), which exposes mice to a wide variety of normal mouse pathogens. COH mice display a substantial increase in long-lived effector CD8 T cells (LLEC). LLEC are a CD8 T cell memory population described by the Hamilton Lab that is phenotypically more effector-like and are highly effective at clearing systemic infections when compared to other memory T cells. However, LLEC display reduced homeostatic and antigen-driven proliferation in SPF mice leading to a lower representation in the memory pool. The factors in COH that support LLEC expansion and persistence are currently unknown, and it is also unclear if COH impacts LLEC function. Thus, the goal of this study is to understand what intrinsic and environmental factors contribute to LLEC formation and maintenance, as well as determine how LLEC function contributes to memory responses in COH mice. I have found that COH LLEC are more polyfunctional compared to SPF LLEC, and my preliminary work has found that LLEC turnover is increased in COH mice. Future studies will determine what specific inflammatory cues lead to LLEC expansion, and if this process can be altered to improve T cell responses to infection and vaccination. Supported by grants from the NIH (R01 AI155468)

Diverse mutational landscapes in human lymphocytes

The lymphocyte genome is prone to many threats, including programmed mutation during differentiation1, antigen-driven proliferation and residency in diverse microenvironments. Here, after developing protocols for expansion of single-cell lymphocyte cultures, we sequenced whole genomes from 717 normal naive and memory B and T cells and haematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells, with higher burdens in memory cells than in naive cells, and with T cells accumulating mutations at a higher rate throughout life. Off-target effects of immunological diversification accounted for approximately half of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every on-target IGHV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than in stem cells, with around 15% of deletions being attributable to off-target recombinase-activating gene activity. DNA damage from ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory cells. The mutation burden and signatures of normal B cells were broadly similar to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes that are active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.

Pharmacologic control of homeostatic and antigen-driven proliferation to target HIV-1 persistence

The presence of latent human immunodeficiency virus 1 (HIV-1) in quiescent memory CD4 + T cells represents a major barrier to viral eradication. Proliferation of memory CD4 + T cells is the primary mechanism that leads to persistence of the latent reservoir, despite effective antiretroviral therapy (ART). Memory CD4 + T cells are long-lived and can proliferate through two mechanisms: homeostatic proliferation via γc-cytokine stimulation or antigen-driven proliferation. Therefore, therapeutic modalities that perturb homeostatic and antigen-driven proliferation, combined with ART, represent promising strategies to reduce the latent reservoir. In this study, we investigated a library of FDA-approved oncology drugs to determine their ability to inhibit homeostatic and/or antigen-driven proliferation. We confirmed potential hits by evaluating their effects on proliferation in memory CD4 + T cells from people living with HIV-1 on ART (PLWH) and interrogated downstream signaling of γc-cytokine stimulation. We found that dasatinib and ponatinib, tyrosine kinase inhibitors, and trametinib, a MEK inhibitor, reduced both homeostatic and antigen-driven proliferationby >65%, with a reduction in viability <45%, ex vivo. In memory CD4 + T cells from PLWH, only dasatinib restricted both homeostatic and antigen-driven proliferation and prevented spontaneous rebound, consistent with promoting a smaller reservoir size. We show that dasatinib restricts IL-7 induced proliferation through STAT5 phosphorylation inhibition. Our results establish that the anti-cancer agent dasatinib is an exciting candidate to be used as an anti-proliferative drug in a clinical trial, since it efficiently blocks proliferation and iswell tolerated in patients with chronic myeloid leukemia (CML).

Reliance on Cox10 and oxidative metabolism for antigen-specific NK cell expansion.

Natural killer (NK) cell effector functions are dependent on metabolic regulation of cellular function; however, less is known about invivo metabolic pathways required for NK cell antiviral function. Mice with an inducible NK-specific deletion of Cox10, which encodes a component of electron transport chain complex IV, were generated to investigate the role of oxidative phosphorylation in NK cells during murine cytomegalovirus (MCMV) infection. Ncr1-Cox10Δ/Δ mice had normal numbers of NK cells but impaired expansion of antigen-specific Ly49H+ NK cells and impaired NK cell memory formation. Proliferation invitro and homeostatic expansion were intact, indicating a specific metabolic requirement for antigen-driven proliferation. Cox10-deficient NK cells upregulated glycolysis, associated with increased AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) activation, although this was insufficient to protect the host. These data demonstrate that oxidative metabolism is required for NK cell antiviral responses invivo.

Exposure to pesticides in utero impacts the fetal immune system and response to vaccination in infancy

The use of pesticides to reduce mosquito vector populations is a cornerstone of global malaria control efforts, but the biological impact of most pesticides on human populations, including pregnant women and infants, is not known. Some pesticides, including carbamates, have been shown to perturb the human immune system. We measure the systemic absorption and immunologic effects of bendiocarb, a commonly used carbamate pesticide, following household spraying in a cohort of pregnant Ugandan women and their infants. We find that bendiocarb is present at high levels in maternal, umbilical cord, and infant plasma of individuals exposed during pregnancy, indicating that it is systemically absorbed and trans-placentally transferred to the fetus. Moreover, bendiocarb exposure is associated with numerous changes in fetal immune cell homeostasis and function, including a dose-dependent decrease in regulatory CD4 T cells, increased cytokine production, and inhibition of antigen-driven proliferation. Additionally, prenatal bendiocarb exposure is associated with higher post-vaccination measles titers at one year of age, suggesting that its impact on functional immunity may persist for many months after birth. These data indicate that in utero bendiocarb exposure has multiple previously unrecognized biological effects on the fetal immune system.

AMBRA1 controls antigen-driven activation and proliferation of naive T cells.

AMBRA1 (activating molecule in Beclin1-regulated autophagy) is a member of the BECN1 (BECLIN1) protein complex, and it plays a role in autophagy, cell death, tumorigenesis and proliferation. We recently reported that on T-cell receptor (TCR) stimulation, AMBRA1 controlled both autophagy and the cell cycle with metabolic regulation. Accumulating evidence has shown that autophagy and metabolic control are pivotal for T-cell activation, clonal expansion and effector/memory cell fate decision. However, it is unknown whether AMBRA1 is involved in T-cell function under physiological conditions. We found that T cells in Ambra1-conditional knockout (cKO) mice induced an exacerbated graft versus host response when they were transplanted into allogeneic BALB/c mice. Furthermore, Ambra1-deficient T cells showed increased proliferation and cytotoxic capability toward specific antigens in response to in vivo stimulation using allogeneic spleen cells. This enhanced immune response mainly contributed to naive T-cell hyperactivity. The T-cell hyperactivity observed in this study was similar to those in some metabolic factor-deficient mice, but not those in other pro-autophagic factor-deficient mice. Under the static condition, however, naive T cells were reduced in Ambra1-cKO mice, the same as in pro-autophagic factor-deficient mice. Collectively, these results suggested that AMBRA1 was involved in regulating T cell-mediated immune responses through autophagy-dependent and -independent mechanisms.

Role of MMP-13 in Graft-Versus-Host Disease

Matrix metalloproteinases (MMPs) have been initially recognized for their role in degradation of extracellular matrix (ECM) and collagen remodeling. However, MMPs have been shown to play a crucial role in inflammation, tumor cell invasion, adaptive and innate immunity. Acute and chronic Graft versus Host Disease (GVHD) are characterized by distinctive histopathological features involving tissue infiltration with donor cells, tissue damage and remodeling. We therefore hypothesized that GVHD-associated organ damage may involve MMPs. We have now identified a novel immunomodulatory function for MMP-13 (alternatively called collagenase-3)and have uncovered a previously unknown role of MMP-13 in regulating GVHD.To address the function of MMP-13 in GVHD we first assesed the effect of MMP-13 on alloresponses in vitro. Using fully Major Histocompatibility Complex (MHC)-mismatched standard mixed lymphocyte reaction we demonstated that antigen presentig cells (APC) from B6.MMP-13-/-(H2b) mice led to signifcantly enhanced antigen-driven activation and proliferation of Carboxyfluorescein succinimidyl ester (CFSE)-labeled Balb/c responder splenocytes. Thus, MMP-13 deficiency in either splenocytes or bone marrow-derived dendritic cells used as stimulators resulted in enhanced proliferation, activation and IFN-gproduction in the allo-reactive lymphocyte responders. Similarly, exogenous MMP13 reduced proliferation of responder T cells as determined tested by CFSEdilution (CFSEloof CD4+T cells from 62.3% decreased to 40.6%, CFSEloof CD8+T cells from 74.1% down to 47.9%). We next assessed the impact of MMP-13 in vivousing fully MHC-mismatched rodent acute GVHD models. To study the role of host-derived MMP-13 we induced GVHD in B6.MMP-13-/-or B6.WT recipient mice following lethal TBI (1075 rad) using splenic T cells from Balb/cdonors. We observed signifcantly accelerated GVHD-related mortality (Median Survival Time 7 vs. 47 days post-transplant, p&lt;0.05) in MMP-13-deficient recipients. Most importantly, donor T cells expanded more vigorously in the secondary lymphoid organs (Spleen and mesenteric lymphnoodes) of MMP13-/-compared to wildtype recipient mice (e.g. spleen: absolute donor CD4+Tcells 1.5x104± 7.3 x 103 (WT) vs. 5.83 x104±1.65 x104[MMP-13-/-] and CD8+5.5 x104± 3.8 x104(WT) vs 3.4 x105±1.4 x105[MMP-13-/-], p&lt;0.01). Enhanced donor lymphocyte expansion was further confirmed by bioluminescence imaging. To further delineate the underlying mechanisms, we analyzed the effects of MMP-13deficiency and exogenous MMP-13 on maturation of mouse bone marrow derived-dendritic cells (BMDC) and macrophages in vitro. We noted decreased expression of inhibitory molecules PD-L1 and PD-1H on GM-CSF/LPS cultured BMDC. Similarly, bone marrow-derived MMP-13-/-macrophages also showed reduced PD-L1 and PD-1H expression upon LPS stimulation when compared to their WT counterparts. In summary we posit that recipient myeloid cell-derived MMP-13 mitigates GVHD and limits donor T cell expansion. Further studies are warranted to determine how MMP-13 suppresses expansion of donor T cells and impacts Graft-versus-Leukemia responses. Disclosures Lentzsch: Caelum Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Abbvie: Consultancy; Clinical Care Options: Speakers Bureau; Sanofi: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Honoraria; International Myeloma Foundation: Honoraria; Karyopharm: Research Funding; Columbia University: Patents &amp; Royalties: 11-1F4mAb as anti-amyloid strategy; Proclara: Consultancy; BMS: Consultancy.

A late B lymphocyte action in dysfunctional tissue repair following kidney injury and transplantation

The mechanisms initiating late immune responses to an allograft are poorly understood. Here we show, via transcriptome analysis of serial protocol biopsies from kidney transplants, that the initial responses to kidney injury correlate with a late B lymphocyte signature relating to renal dysfunction and fibrosis. With a potential link between dysfunctional repair and immunoreactivity, we investigate the immunological consequences of dysfunctional repair examining chronic disease in mouse kidneys 18 months after a bilateral ischemia/reperfusion injury event. In the absence of foreign antigens, a sustained immune response involving both innate and adaptive immune systems accompanies a transition to chronic kidney damage. At late stages, B lymphocytes exhibite an antigen-driven proliferation, selection and maturation into broadly-reacting antibody-secreting cells. These findings reveal a previously unappreciated role for dysfunctional tissue repair in local immunomodulation that may have particular relevance to transplant-associated immunobiology.

CXCR3high CD8+ T cells with naïve phenotype and high capacity for IFN-γ production are generated during homeostatic T-cell proliferation.

Naïve phenotype (NP) Tcells spontaneously initiate homeostatic proliferation (HP) as T-cell output is reduced because of physiologic thymic involution with age. However, the effects of sustained HP on overall immune function are poorly understood. We demonstrated that the NP CD8+ Tcell population in adult thymectomized mice showing accelerated HP has an increased capacity for TCR-mediated interferon-γ and tumor necrosis factor α production, which is attributed to an increase in CXCR3+ cells in the NP CD8+ Tcell population. The CXCR3+ NP CD8+ Tcells developed during persistent HP with a slow cell division rate, but rarely during robust antigen-driven proliferation with a fast cell division rate. In ontogeny, the proportions of CXCR3+ cells in the NP CD8+ Tcell population showed a biphasic profile, which was high at the newborn and aged stages. Upon transfer, CXCR3+ NP CD8+ Tcells, but not CXCR3- NP CD8+ Tcells, potently enhanced Th17-mediated inflammatory tissue reactions in vivo. Furthermore, CXCR3high NP CD8+ Tcells with similar features were also detected at variable levels in healthy human blood. These results suggest that CXCR3+ NP CD8+ Tcells generated during physiological HP significantly impact overall immunity at the immunologically vulnerable neonatal and aged stages.

Cross sectional association of arsenic and seroprevalence of hepatitis B infection in the United States (NHANES 2003–2014)

BackgroundArsenic alters immunological parameters including antibody formation and antigen-driven T-cell proliferation. ObjectiveWe evaluated the cross-sectional relationship between urinary arsenic and the seroprevalence of hepatitis B (HBV) infection in the United States using data from six pooled cycles of the National Health and Nutrition Examination Survey (2003–2014, N = 12,447). MethodsUsing serological data, participants were classified as susceptible, immune due to vaccination, or immune due to past natural infection. We used multinomial logistic regression to evaluate the association between urinary DMA and HBV classification. A sensitivity analysis using total urinary arsenic (TUA) was also conducted. Both DMA and TUA were adjusted for arsenobetaine using a residual regression method ResultsA 1-unit increase in the natural logarithm (ln) of DMA was associated with 40% greater adjusted odds of having immunity due to natural infection compared to being susceptible (Odds Ratio [aOR]: 1.40, 95% Confidence Intervals [CI] 1.15, 1.69), 65% greater odds of having immunity due to a natural infection (aOR: 1.65, 95% CI: 1.34, 2.04) and 18% greater odds of being susceptible (aOR: 1.18, 95% CI: 1.05, 1.33) compared to being immune due to vaccination after adjusting for creatinine, age, sex, race, income, country of birth, BMI, survey cycle, serum cotinine, recent seafood intake, and self-reported HBV immunization status. ConclusionIn the U.S. general public, higher urinary arsenic levels were associated with a greater odds of having a serological classification consistent with a past natural hepatitis B infection after adjusting for other risk factors. Additionally, higher urinary arsenic levels were linked to a greater odds of not receiving hepatitis B vaccinations. Given the cross-sectional nature of this analysis, more research is needed to test the hypothesis that environmentally relevant exposure to arsenic modulates host susceptibility to hepatitis B virus.

C-Reactive Protein Impairs Dendritic Cell Development, Maturation, and Function: Implications for Peripheral Tolerance.

C-reactive protein (CRP) is the prototypical acute phase reactant, increasing in blood concentration rapidly and several-fold in response to inflammation. Recent evidence indicates that CRP has an important physiological role even at low, baseline levels, or in the absence of overt inflammation. For example, we have shown that human CRP inhibits the progression of experimental autoimmune encephalomyelitis (EAE) in CRP transgenic mice by shifting CD4+ T cells away from the TH1 and toward the TH2 subset. Notably, this action required the inhibitory Fcγ receptor IIB (FcγRIIB), but did not require high levels of human CRP. Herein, we sought to determine if CRP’s influence in EAE might be explained by CRP acting on dendritic cells (DC; antigen presenting cells known to express FcγRIIB). We found that CRP (50 µg/ml) reduced the yield of CD11c+ bone marrow-derived DCs (BMDCs) and CRP (≥5 μg/ml) prevented their full expression of major histocompatibility complex class II and the co-stimulatory molecules CD86 and CD40. CRP also decreased the ability of BMDCs to stimulate antigen-driven proliferation of T cells in vitro. Importantly, if the BMDCs were genetically deficient in mouse FcγRIIB then (i) the ability of CRP to alter BMDC surface phenotype and impair T cell proliferation was ablated and (ii) CD11c-driven expression of a human FCGR2B transgene rescued the CRP effect. Lastly, the protective influence of CRP in EAE was fully restored in mice with CD11c-driven human FcγRIIB expression. These findings add to the growing evidence that CRP has important biological effects even in the absence of an acute phase response, i.e., CRP acts as a tonic suppressor of the adaptive immune system. The ability of CRP to suppress development, maturation, and function of DCs implicates CRP in the maintenance of peripheral T cell tolerance.

Expanded cellular clones carrying replication-competent HIV-1 persist, wax, and wane

The latent reservoir for HIV-1 in resting CD4+ T cells is a major barrier to cure. Several lines of evidence suggest that the latent reservoir is maintained through cellular proliferation. Analysis of this proliferative process is complicated by the fact that most infected cells carry defective proviruses. Additional complications are that stimuli that drive T cell proliferation can also induce virus production from latently infected cells and productively infected cells have a short in vivo half-life. In this ex vivo study, we show that latently infected cells containing replication-competent HIV-1 can proliferate in response to T cell receptor agonists or cytokines that are known to induce homeostatic proliferation and that this can occur without virus production. Some cells that have proliferated in response to these stimuli can survive for 7 d while retaining the ability to produce virus. This finding supports the hypothesis that both antigen-driven and cytokine-induced proliferation may contribute to the stability of the latent reservoir. Sequencing of replication-competent proviruses isolated from patients at different time points confirmed the presence of expanded clones and demonstrated that while some clones harboring replication-competent virus persist longitudinally on a scale of years, others wax and wane. A similar pattern is observed in longitudinal sampling of residual viremia in patients. The observed patterns are not consistent with a continuous, cell-autonomous, proliferative process related to the HIV-1 integration site. The fact that the latent reservoir can be maintained, in part, by cellular proliferation without viral reactivation poses challenges to cure.

HIV dynamics linked to memory CD4+ T cell homeostasis.

The dynamics of latent HIV is linked to infection and clearance of resting memory CD4+ T cells. Infection also resides within activated, non-dividing memory cells and can be impacted by antigen-driven and homeostatic proliferation despite suppressive antiretroviral therapy (ART). We investigated whether plasma viral level (pVL) and HIV DNA dynamics could be explained by HIV’s impact on memory CD4+ T cell homeostasis. Median total, 2-LTR and integrated HIV DNA levels per μL of peripheral blood, for 8 primary (PHI) and 8 chronic HIV infected (CHI) individuals enrolled on a raltegravir (RAL) based regimen, exhibited greatest changes over the 1st year of ART. Dynamics slowed over the following 2 years so that total HIV DNA levels were equivalent to reported values for individuals after 10 years of ART. The mathematical model reproduced the multiphasic dynamics of pVL, and levels of total, 2-LTR and integrated HIV DNA in both PHI and CHI over 3 years of ART. Under these simulations, residual viremia originated from reactivated latently infected cells where most of these cells arose from clonal expansion within the resting phenotype. Since virion production from clonally expanded cells will not be affected by antiretroviral drugs, simulations of ART intensification had little impact on pVL. HIV DNA decay over the first year of ART followed the loss of activated memory cells (120 day half-life) while the 5.9 year half-life of total HIV DNA after this point mirrored the slower decay of resting memory cells. Simulations had difficulty reproducing the fast early HIV DNA dynamics, including 2-LTR levels peaking at week 12, and the later slow loss of total and 2-LTR HIV DNA, suggesting some ongoing infection. In summary, our modelling indicates that much of the dynamical behavior of HIV can be explained by its impact on memory CD4+ T cell homeostasis.

H1N1 influenza vaccination in HIV-infected women on effective antiretroviral treatment did not induce measurable antigen-driven proliferation of the HIV-1 proviral reservoir

ObjectivesAntigen-induced activation and proliferation of HIV-1-infected cells is hypothesized to be a mechanism of HIV persistence during antiretroviral therapy. The objective of this study was to determine if proliferation of H1N1-specific HIV-infected cells could be detected following H1N1 vaccination.MethodsThis study utilized cryopreserved PBMC from a previously conducted trial of H1N1 vaccination in HIV-infected pregnant women. HIV-1 DNA concentrations and 437 HIV-1 C2V5 env DNA sequences were analyzed from ten pregnant women on effective antiretroviral therapy, before and 21 days after H1N1 influenza vaccination.ResultsHIV-1 DNA concentration did not change after vaccination (median pre- vs. post-vaccination: 95.77 vs. 41.28 copies/million PBMC, p = .37). Analyses of sequences did not detect evidence of HIV replication or proliferation of infected cells.ConclusionsAntigenic stimulation during effective ART did not have a detectable effect on the genetic makeup of the HIV-1 DNA reservoir. Longitudinal comparison of the amount and integration sites of HIV-1 in antigen-specific cells to chronic infections (such as herpesviruses) may be needed to definitively evaluate whether antigenic stimulation induces proliferation of HIV-1 infected cells.

Antigen Exposure History Defines CD8 T Cell Dynamics and Protection during Localized Pulmonary Infections.

Unlike systemic infections, little is known about the role of repeated localized infections on (re)shaping pathogen-specific memory CD8 T cell responses. Here, we used primary (1°) and secondary (2°) intranasal influenza virus infections of mice as a model to study intrinsic memory CD8 T cell properties. We show that secondary antigen exposure, relative to a single infection, generates memory CD8 T cell responses of superior magnitude in multiple tissue compartments including blood, spleen, draining lymph nodes, and lung. Unexpectedly, regardless of the significantly higher number of 2° memory CD8 T cells, similar degree of protection against pulmonary challenge was observed in both groups of mice containing 1° or 2° memory CD8 T cells. Mechanistically, using pertussis toxin-induced migration block, we showed that superior antigen-driven proliferation and ability to relocate to the site of infection allowed 1° memory CD8 T cells to accumulate in the infected lung during the first few days after challenge, compensating for the initially lower cell numbers. Taken together, the history of antigen exposures to localized pulmonary infections, through altering basic cell biology, dictates dynamic properties of protective memory CD8 T cell responses. This knowledge has important implications for a design of novel and an improvement of existing vaccines and immunization strategies.

Loss of Normal T-Cell Subset Distribution in Myelofibrosis Is Recovered By the Immunomodulatory Effects of JAK1/2 Inhibition

▪Introduction: Myeloproliferative neoplasms (MPN) are clonal disorders of the hematopoietic system characterized by an excessive proliferation of myeloid cells and progressive bone marrow fibrosis. MPNs are result of an abnormal constitutive activation of the JAK/STAT signaling pathway. Ruxolitinib is the first JAK1/2 inhibitor approved for the treatment of myelofibrosis (MF), the most aggressive of MPNs. However, the beneficial effects of ruxolitinib cannot be attributed to its anticlonal activity but rather to its reduction in inflammatory cytokine production, spleen size and symptom burden. Helper (CD4+) and cytotoxic (CD8+) T-cells are key mediator elements in the adaptive immune system. Disrupted homeostasis of functionally diverse T-cell subpopulations (naïve, memory and effector) can result in abnormal cytokine production. The aim of this study was to determine the baseline T-cell subset composition in patients with MF and to monitor the immunomodulatory effects of JAK1/2 inhibition.Methods: CD4+ and CD8+T-cell subpopulations were measured in PB samples from healthy controls (n=16) and PB and BM samples from patients (n=47) with MF treated on a phase I/II clinical trial of ruxolitinib, using multiparametric flow cytometry. Subsets were immunophenotypically defined based on the cell-surface expression of CD45RO and CD62L as follows: naïve T-cells, CD45RO-CD62L+; central memory (CM), CD45RO+CD62L+; effector memory (EM), CD45RO+CD62L-; and terminally differentiated effector memory T-cells (TEM) CD45RO-CD62L-.Results: Our results showed no significant difference in the distribution of helper vs. cytotoxic T-cells between untreated MF patients and healthy subjects. Nevertheless, profound alterations in both the CD4+ and CD8+ compartments were found in MF patients. Patients with MF had significantly fewer antigen inexperienced naïve (38.7±3.1% vs. 12.9±2.0%, p<0.0001; 26.5±2.6% vs. 7.0±1.2%, p<0.0001) and central memory (23.9±1.7% vs. 6.9±0.9%, p<0.0001; 10.7±1.1% vs. 2.7±0.4%, p<0.0001) T-cells than control subjects. At the same time, terminally differentiated effector memory T-cells were significantly increased in MF patients (13.0±1.0% vs. 44.0±2.5%, p<0.0001, 26.6±2.4% vs. 58.3±2.2%, p<0.0001).To determine the effects of JAK1/2 inhibition on the T-cell subset distribution, we compared baseline (n=47) T-cell subsets with on-treatment (n=49) patient samples. Median follow-up time was 2.8 years (range: 0.2-8.0 years). We found that ruxolitinib administration increased the naïve and CM T-cells in both the CD4+ (12.9±2.0% vs. 20.1±1.4%, p=0.011; 6.9±0.9% vs. 18.3±1.2%, p<0.0001) and CD8+ populations (7.0±1.2% vs. 11.4±1.1%, p=0.02; 2.7±0.4% vs. 5.4±0.5%, p=0.0001), whereas it decreased TEM (44.0±2.5% vs. 25.5±1.7%, p<0.0001; 58.3±2.2% vs. 48.8±2.4%, p=0.0072).Remarkably, only patients who achieved a ≥50% spleen size reduction (SR) had a significant increase in naïve CD4+ (11.0±2.5% vs. 24.2±1.8%, p=0.0002, compared with 17.0±5.2% vs. 16.0±2.4%, p=0.98 for SR <50%) and CD8+ T-cells (5.4±1.3% vs. 12.3±1.5%, p=0.0036, compared with 10.4±3.2% vs. 9.3±2.3%, p=0.96 for SR <50%) during ruxolitinib treatment.Conclusions: In patientswith MF, T-cell subsets are skewed towards the effector phenotype. It has been shown that terminally differentiated effector memory T-cells are generated as a result of cytokine-driven rather than antigen-driven proliferation and differentiation stimuli. These highly efficient effector cells produce vast amounts of pro-inflammatory cytokines that account for and/or contribute to the chronic inflammatory milieu commonly found in MF. The JAK1/2 inhibitor reverses the equilibrium towards naïve T-cell phenotype to some extent, possibly contributing to the diminished cytokine production seen after JAK1/2 inhibition. This effect is more pronounced in patients with a better response, as measured by the degree of spleen size reduction.In conclusion, even though MF is a disease of the myeloid lineage, we show evidence of severe immune derangements in T-cell subpopulations. Ruxolitinib might exert its benefit for MF patients due to its modulating effect on T-cells known to produce high cytokine levels. DisclosuresNo relevant conflicts of interest to declare.

Characterization of Human CD8 T Cell Responses in Dengue Virus-Infected Patients from India.

ABSTRACTEpidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DR− CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-γ) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-γ unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-γ by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-γ unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue.IMPORTANCE Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-γ in vitro. Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects.

Regulatory T Cell Numbers in Inflamed Skin Are Controlled by Local Inflammatory Cues That Upregulate CD25 and Facilitate Antigen-Driven Local Proliferation.

CD4(+)Foxp3(+) regulatory T cells (Tregs) are key immune suppressors that regulate immunity in diverse tissues. The tissue and/or inflammatory signals that influence the magnitude of the Treg response remain unclear. To define signals that promote Treg accumulation, we developed a simple system of skin inflammation using defined Ags and adjuvants that induce distinct cytokine milieus: OVA protein in CFA, aluminum salts (Alum), and Schistosoma mansoni eggs (Sm Egg). Polyclonal and Ag-specific Treg accumulation in the skin differed significantly between adjuvants. CFA and Alum led to robust Treg accumulation, with >50% of all skin CD4(+) T cells being Foxp3(+) In contrast, Tregs accumulated poorly in the Sm Egg-inflamed skin. Surprisingly, we found no evidence of inflammation-specific changes to the Treg gene program between adjuvant-inflamed skin types, suggesting a lack of selective recruitment or adaptation to the inflammatory milieu. Instead, Treg accumulation patterns were linked to differences in CD80/CD86 expression by APC and the regulation of CD25 expression, specifically in the inflamed skin. Inflammatory cues alone, without cognate Ag, differentially supported CD25 upregulation (CFA and Alum > Sm Egg). Only in inflammatory milieus that upregulated CD25 did the provision of Ag enhance local Treg proliferation. Reduced IL-33 in the Sm Egg-inflamed environment was shown to contribute to the failure to upregulate CD25. Thus, the magnitude of the Treg response in inflamed tissues is controlled at two interdependent levels: inflammatory signals that support the upregulation of the important Treg survival factor CD25 and Ag signals that drive local expansion.