Precursor Frequency Research Articles

The contribution of the HSCs niche to bone marrow failure in the IL-2 deficient model

Abstract Bone marrow failure (BMF) is a devastating disease where regeneration of peripheral blood lineages is compromised, resulting in anemia and mortality. In IL-2 KO BALB/c mice, the balance between early myeloid and lymphoid progenitor cell populations is skewed resulting in insufficient myelopoiesis with accumulation of CD8 T cells in the BM. It is widely accepted that crosstalk between cells of the bone microenvironment and hematopoietic cells affects each other’s behavior. However, it remains unknown how the BM niche in this mouse model contribute to the BMF. Multipotent stromal cells (MSCs), osteoblasts (OBs) and endothelial cells (ECs) are the three types of BM stromal cells (BMSCs) that may serve as the distinct functional niche to maintain HSCs. Repots have shown the interplay between CD8 T cells and MSCs cause a change in myelopoiesis and that MSCs promote the formation of Tregs. Our data also indicates that IL-2 KO CD8 T cells are important for the expansion of CD4 T cells in the bone. Since IL-2 KO bones are extremely brittle and CD4 T cells produce RANKL, the ligand needed for osteoclast (OC) differentiation, we evaluated the frequency of BMSCs and OCs. Our preliminary data shows clear differences in BMSCs profiles of IL-2 KO mice, namely elevated stromal cell numbers and a shift in Sca-1 expression in ECs and CD51 expression in OBs. Additionally, OC precursor frequency is elevated in the BM, indicating an increase in bone resorption. The ability of the different subsets of the BMSCs to support the differentiation of HSCs in vitro and the role of CD8 T cells in promoting these changes in vivo is being currently evaluated. Overall, our preliminary data suggests that the change in the bone microenvironment may contribute to the BMF observed in these mice.

Defining limits on efficacy of CD8 T cell-based HIV vaccines

Abstract It is generally accepted that an effective HIV vaccine should be able to induce neutralizing antibodies and memory CD8 T cells. Yet, basic features of protective CD8 T cell responses remain poorly defined. Using recently published data from a cohort of HIV-infected patients that were followed from the onset of symptoms into the chronic phase, we determined the basic quantitative in vivo features of HIV-specific CD8 T cell responses. First, we found that the number of responses recognizing different viral epitopes (immune response breadth) varied greatly between patients and on average, 8 epitopes were recognized by CD8 T cells. Surprisingly, we found that in most patients the breadth did not change significantly over the course of first year of infection suggesting that most HIV-specific T cell responses were primed in acute infection. There was a strong negative correlation between the predicted precursor frequency and the rate of expansion of epitope-specific T cells suggesting intraclonal competition. Finally, we found that the majority of CD8 T cell populations expanded and contracted in unison; however, about 20% of all CD8 T cell responses had a discordant dynamics suggesting interclonal competition. Taken together, our results suggest that effective control of HIV infection can be achieved with broad CD8 T cell response if vaccine-induced breadth is larger than that induced by a natural infection. However, broader T cell response will likely lead to interclonal competition between responses, and boosting the magnitude of epitope-specific T cell responses may reduce the expansion rate of these responses due to intraclonal competition. Our results thus suggest potential limits on the efficacy of CD8 T cell-based HIV vaccines.

Increasing strength of antigenic stimulation negatively impacts memory CD8 T cell formation and function during persistent viral infection

Abstract Memory CD8 T cells are critical for keeping persistent viral infections in check. Here, we used a novel approach to directly study the impact of T cell stimulation strength on memory T cell differentiation in the setting of a persistent viral infection. Using site-directed mutagenesis, we generated a series of MPyV variants with substitutions in a subdominant Db-restricted epitope that modulated level of activation of TCR transgenic CD8 T cells specific for a corresponding epitope in SV40 Large T Antigen. By altering a subdominant epitope constituting approximately 1% of the total anti-MPyV CD8 T cell response, we avoided perturbing MPyV-associated inflammation, virus infection levels, antiviral CD4 T cell responses, and the dominant anti-MPyV CD8 T cell response. Moreover, by using physiologic numbers of naïve donor monoclonal CD8 T cells for adoptive transfer into immunocompetent C57BL/6 recipients, we also controlled for variability in naïve T cell precursor frequency and TCR affinity inherent in T cell recruitment to infection. Our data demonstrate an inverse relationship between antigenic stimulation strength and expression of memory precursor effector phenotype (CD127highKLRG1low) by virus-specific CD8 T cells during acute infection. Although stronger antigenic stimulation augmented expansion of functional CD8 T cells in acute infection, lower stimulation favored functional competence of memory antiviral CD8 T cells during persistent infection. Together, these data provide clear in vivo evidence that the strength of antigenic stimulation during recruitment of MPyV-specific CD8 T cells dictates the functional integrity of memory T cells during persistent infection.

Adoptive cell transfer enhances antitumor response generated by Cytomegalovirus-based vaccine

Abstract Methods for generating long-lived T cell responses to tumor antigen have been highly sought for potential therapy against many cancers. Our group has recently developed a novel vaccine against melanoma using recombinant Mouse Cytomegalovirus (MCMV) expressing a modified tumor antigen which generated life-long CD8+ T cell responses to the melanoma antigen, gp100. While this vaccine delays tumor growth in a mouse model of B16 metastatic melanoma, mice eventually succumb to the disease. Adoptive cell therapy has shown clinical efficacy in some patients with melanoma; however, it requires in vitro activation of large numbers of T cells to be effective. We hypothesized that combining these therapies would result in more effective tumor rejection by 1) increasing precursor frequency of tumor specific T cells, thus inducing a more robust T cell response following vaccination and 2) promoting life-long persistence of transferred cells following transfer. To test this, C57BL/6 mice received 105 CD8+ T cells expressing a transgenic T cell receptor recognizing gp100 (PMEL) followed by vaccination. Five days after transfer, PMEL cells constituted up to 20% of all CD8+ T cells in peripheral blood following MCMV vaccination in contrast to 7.5% following vaccination with another viral vaccine, Vesicular Stomatitis Virus. Our results also show that adoptive transfer and MCMV vaccination delays B16 tumor growth and improves survival in a solid tumor model. In summary, this study tests a novel method for stimulating adoptively transferred antitumor T cells, while avoiding the need for long-term in vitroculture of cells. Future work will also examine if immune checkpoint inhibitors or costimulatory agonists further improve this therapy.

HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen.

Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

Type I Interferons Regulate the Magnitude and Functionality of Mouse Polyomavirus-Specific CD8 T Cells in a Virus Strain-Dependent Manner.

Mouse polyomavirus (MPyV) is a ubiquitous persistent natural mouse pathogen. A glutamic acid (E)-to-glycine (G) difference at position 91 of the VP1 capsid protein shifts the profile of tumors induced by MPyV from an epithelial to a mesenchymal cell origin. Here we asked if this tropism difference affects the MPyV-specific CD8 T cell response, which controls MPyV infection and tumorigenesis. Infection by the laboratory MPyV strain RA (VP1-91G) or a strain A2 mutant with an E-to-G substitution at VP1 residue 91 [A2(91G)] generated a markedly smaller virus-specific CD8 T cell response than that induced by A2(VP1-91E) infection. Mutant A2(91G)-infected mice showed a higher frequency of memory precursor (CD127(hi) KLRG1(lo)) CD8 T cells and a higher recall response than those of A2-infected mice. Using T cell receptor (TCR)-transgenic CD8 T cells and immunization with peptide-pulsed dendritic cells, we found that early bystander inflammation associated with A2 infection contributed to recruitment of the larger MPyV-specific CD8 T cell response. Beta interferon (IFN-β) transcripts were induced early during A2 or A2(91G) infections. IFN-β inhibited replication of A2 and A2(91G) in vitro Using mice lacking IFN-αβ receptors (IFNAR(-/-)), we showed that type I IFNs played a role in controlling MPyV replication in vivo but differentially affected the magnitude and functionality of virus-specific CD8 T cells recruited by A2 and A2(91G) viral infections. These data indicate that type I IFNs are involved in protection against MPyV infection and that their effect on the antiviral CD8 T cell response depends on capsid-mediated tropism properties of the MPyV strain. Isolates of the human polyomavirus JC virus from patients with the frequently fatal demyelinating brain disease progressive multifocal leukoencephalopathy (PML) carry single amino acid substitutions in the domain of the VP1 capsid protein that binds the sialic acid moiety of glycoprotein/glycolipid receptors on host cells. These VP1 mutations may alter neural cell tropism or enable escape from neutralizing antibodies. Changes in host cell tropism can affect recruitment of virus-specific CD8 T cells. Using mouse polyomavirus, we demonstrate that a single amino acid difference in VP1 known to shift viral tropism profoundly affects the quantity and quality of the anti-polyomavirus CD8 T cell response and its differentiation into memory cells. These findings raise the possibility that CD8 T cell responses to infections by human polyomaviruses may be influenced by VP1 mutations involving domains that engage host cell receptors.

Low-Affinity Memory CD8+ T Cells Mediate Robust Heterologous Immunity.

Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide-MHC, the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming, low-affinity priming elicited fully differentiated memory T cells with a CD45RB(hi) status. High CD45RB status enabled robust secondary responses in vivo, as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity-primed mice, but not in high affinity-primed mice. Mechanistically, low affinity-primed memory CD8(+) T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RB(hi) status was also a stable marker of priming affinity within polyclonal CD8(+) T cell populations. Following high-affinity rechallenge, low affinity-primed CD45RB(hi) cells became CD45RB(lo), demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8(+) T cells. Thus, these data establish a novel mechanism by which CD45 isoforms tune low affinity-primed memory CD8(+) T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency, low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge.

Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes

Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5–10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues.

CCR7 Controls Thymus Recirculation, but Not Production and Emigration, of Foxp3(+) T Cells.

SummaryCurrent models of Foxp3+ regulatory T cell (Treg) development involve CCR7-mediated migration of thymocytes into the thymus medulla to enable essential interactions with medullary epithelium. However, increased Foxp3+ thymic Treg numbers in Ccr7−/− mice challenge this view, and the role of CCR7 in Treg development, emigration, and/or recirculation is unknown. Here, we have examined CCR7 and Rag2pGFP levels during Treg development and generated Rag2pGFPCcr7−/− mice to study its impact on the intrathymic Treg pool. We reveal surprising developmental heterogeneity in thymocytes described as Treg precursors, showing that they contain recirculating CCR6+CCR7−Rag2pGFP− T cells. Although CCR7 defines bona fide Rag2GFP+ Treg precursors, it is not required for Treg production and emigration. Rather, we show that lack of CCR7 renders the thymus more receptive to Treg thymus homing. Our study reveals a role for CCR7 in limiting Treg recirculation back to the thymus and enables separation of the mechanisms controlling Treg production and thymic recirculation.

Virus-Specific CD8(+) T Cells Cross-Reactive to Donor-Alloantigen Are Transiently Present in the Circulation of Kidney Transplant Recipients Infected With CMV and/or EBV.

T cells play a dual role in transplantation: They mediate transplant rejection and are crucial for virus control. Memory T cells generated in response to pathogens can cross-react to alloantigen, a phenomenon called heterologous immunity. Virus-specific CD8(+) T cells cross-reacting to donor-alloantigen might affect alloimmune responses and hamper tolerance induction following transplantation. Here, we longitudinally studied these cross-reactive cells in peripheral blood of 25 kidney transplant recipients with a cytomegalovirus and/or Epstein-Barr virus infection. Cross-reactive T cells were identified by flow cytometry as virus-specific T cells that proliferate in response to donor cells in a mixed-lymphocyte reaction. In 13 of 25 patients, we found cross-reactivity to donor cells for at least 1 viral epitope before (n = 7) and/or after transplantation (n = 8). Cross-reactive T cells were transiently present in the circulation, and their precursor frequency did not increase following transplantation or viral infection. Cross-reactive T cells expressed interferon-γ and CD107a in response to both alloantigen and viral peptide and resembled virus-specific T cells in phenotype and function. Their presence was not associated with impaired renal function, proteinuria, or rejection. In conclusion, virus-specific T cells that cross-react to donor-alloantigen are transiently detectable in the circulation of kidney transplant recipients.

Heightened self-reactivity associated with selective survival, but not expansion, of naïve virus-specific CD8+ T cells in aged mice

In advanced age, decreased CD8(+) cytotoxic T-lymphocyte (CTL) responses to novel pathogens and cancer is paralleled by a decline in the number and function of naïve CTL precursors (CTLp). Although the age-related fall in CD8(+) T-cell numbers is well established, neither the underlying mechanisms nor the extent of variation for different epitope specificities have been defined. Furthermore, naïve CD8(+) T cells expressing high levels of CD44 accumulate with age, but it is unknown whether this accumulation reflects their preferential survival or an age-dependent driver of CD8(+) T-cell proliferation. Here, we track the number and phenotype of four influenza A virus (IAV)-specific CTLp populations in naïve C57BL/6 (B6) mice during aging, and compare T-cell receptor (TCR) clonal diversity for the CD44hi and CD44lo subsets of one such population. We show differential onset of decline for several IAV-specific CD8(+) T-cell populations with advanced age that parallel age-associated changes in the B6 immunodominance hierarchy, suggestive of distinct impacts of aging on different epitope-specific populations. Despite finding no evidence of clonal expansions in an aged, epitope-specific TCR repertoire, nonrandom alterations in TCR usage were observed, along with elevated CD5 and CD8 coreceptor expression. Collectively, these data demonstrate that naïve CD8(+) T cells expressing markers of heightened self-recognition are selectively retained, but not clonally expanded, during aging.

Sirt6 regulates dendritic cell differentiation, maturation, and function.

Dendritic cells (DCs) are antigen-presenting cells that critically influence decisions about immune activation or tolerance. Impaired DC function is at the core of common chronic disorders and contributes to reduce immunocompetence during aging. Knowledge on the mechanisms regulating DC generation and function is necessary to understand the immune system and to prevent disease and immunosenescence. Here we show that the sirtuin Sirt6, which was previously linked to healthspan promotion, stimulates the development of myeloid, conventional DCs (cDCs). Sirt6-knockout (Sirt6KO) mice exhibit low frequencies of bone marrow cDC precursors and low yields of bone marrow-derived cDCs compared to wild-type (WT) animals. Sirt6KO cDCs express lower levels of class II MHC, of costimulatory molecules, and of the chemokine receptor CCR7, and are less immunostimulatory compared to WT cDCs. Similar effects in terms of differentiation and immunostimulatory capacity were observed in human monocyte-derived DCs in response to SIRT6 inhibition. Finally, while Sirt6KO cDCs show an overall reduction in their ability to produce IL-12, TNF-α and IL-6 secretion varies dependent on the stimulus, being reduced in response to CpG, but increased in response to other Toll-like receptor ligands. In conclusion, Sirt6 plays a crucial role in cDC differentiation and function and reduced Sirt6 activity may contribute to immunosenescence.

Clonal Abundance of Tumor-Specific CD4+ T Cells Potentiates Efficacy and Alters Susceptibility to Exhaustion

Current approaches to cancer immunotherapy aim to engage the natural T cell response against tumors. One limitation is the elimination of self-antigen-specific T cells from the immune repertoire. Using a system in which precursor frequency can be manipulated in a murine melanoma model, we demonstrated that the clonal abundance of CD4(+) T cells specific for self-tumor antigen positively correlated with antitumor efficacy. At elevated precursor frequencies, intraclonal competition impaired initial activation and overall expansion of the tumor-specific CD4(+) T cell population. However, through clonally derived help, this population acquired a polyfunctional effector phenotype and antitumor immunity was enhanced. Conversely, development of effector function was attenuated at low precursor frequencies due to irreversible T cell exhaustion. Our findings assert that the differential effects of T cell clonal abundance on phenotypic outcome should be considered during the design of adoptive T cell therapies, including use of engineered T cells.

Role of the Immune Response in Disease Progression and Therapy in Multiple Myeloma.

Multiple myeloma (MM) is a hematologic cancer derived from malignant plasma cells within the bone marrow. Unlike most solid tumors, which originate from epithelial cells, the myeloma tumor is a plasma cell derived from the lymphoid cell lineage originating from a post-germinal B-cell. As such, the MM plasma cell represents an integral component of the immune system in terms of both antibody production and antigen presentation, albeit not efficiently. This fundamental difference has significant implications when one considers the implications of immunotherapy. In the case of lymphoid malignancies such as myeloma, immune-based strategies must take into consideration this important difference, potentially necessitating immunotherapy targeted toward MM to be altered from that targeted at solid tumors. Typically, the immune system "surveys" cells within our body and is able to recognize and attack cancerous cells that may arise. However, some cancer cells are able to evade immune surveillance and continue to flourish, causing disease. The major mechanism leading to an effective tumor-specific response is one that enables effective antigen processing and presentation with subsequent T-cell activation, expansion, and effective trafficking to the tumor site. Plasma cells employ several mechanisms to escape immune surveillance which include altered interactions with T-cells, DCs, bone marrow stromal cells (BMSC's), and natural killer cells (NK Cells) that can be mediated by immunosuppressive cells such as and myeloid-derived suppressor cells (MDSC's) and cytokines such as IL-10, TGFβ, and IL-6 as well as down-regulation of the antigen processing machinery. Many therapies have been developed to reestablish a functional immune system in MM patients. These include adoptive T-cell therapies to deliver more tumor-specific T-cells, vaccines to increase the tumor-specific precursor frequency of the endogenous T-cell population, immunomodulatory agents (IMiDs) such as thalidomide and lenalidomide to enhance global endogenous immunity, immunostimulatory cytokines, and antibodies to specifically target tumor-specific cell-surface proteins or cytokines. This review will dissect these various approaches currently being explored in MM as well as highlight some future directions for myeloma-specific immune-based strategies.

Measuring Lymphocyte Proliferation in Response to Specific Antigen and Mitogen Stimuli Using Flow Cytometry.

We present a multivariate test system using flow cytometry after in-vitro lymphocyte stimulation using 5 mitogens and 7 antigens to describe in-vitro immunofunction. The present work is a crucial step towards establishing a simple, CFSE-based, multivariate test system that can describe the dynamics of stimulus-induced lymphocyte proliferation with considerably more precision than is possible with the radionucleotide method using 3H-thymidine. Using multicolour flow cytometry, our method allows additional phenotyping of the proliferating cells and quantifies the proliferation behaviour by precisely resolving daughter generations and determining the precursor frequency. </i> Taking the calculated apoptosis parameters into account not only provides additional information about the stimulus-specific response behaviour but also improves the validity of the commonly used proliferation indices. Not only can we confirm previous findings that healthy people have marked differences in a multivariate test system in terms of the individual in-vitro reactivity to various stimuli but also substantiate that the response pattern of an individual is remarkably constant. In follow-up studies we can show for the first time that the results of immunofunctional testing do not change over a period of at least 6 months and appear to be an inherent characteristic of the individual and thus possibly have a genetic basis.

A Temporal Switch in the Germinal Center Determines Differential Output of Memory B and Plasma Cells

There is little insight into or agreement about the signals that control differentiation of memory B cells (MBCs) and long-lived plasma cells (LLPCs). By performing BrdU pulse-labeling studies, we found that MBC formation preceded the formation of LLPCs in an adoptive transfer immunization system, which allowed for a synchronized Ag-specific response with homogeneous Ag-receptor, yet at natural precursor frequencies. We confirmed these observations in wild-type (WT) mice and extended them with germinal center (GC) disruption experiments and variable region gene sequencing. We thus show that the GC response undergoes a temporal switch in its output as it matures, revealing that the reaction engenders both MBC subsets with different immune effector function and, ultimately, LLPCs at largely separate points in time. These data demonstrate the kinetics of the formation of the cells that provide stable humoral immunity and therefore have implications for autoimmunity, for vaccine development, and for understanding long-term pathogen resistance.

Role of the MHC restriction during maturation of antigen-specific human T cells in the thymus.

In the thymus, a T-cell repertoire able to confer protection against infectious and noninfectious agents in a peptide-dependent, self-MHC-restricted manner is selected. Direct detection of Ag-specific thymocytes, and analysis of the impact of the expression of the MHC-restricting allele on their frequency or function has never been studied in humans because of the extremely low precursor frequency. Here, we used a tetramer-based enrichment protocol to analyze the ex vivo frequency and activation-phenotype of human thymocytes specific for self, viral and tumor-antigens presented by HLA-A*0201 (A2) in individuals expressing or not this allele. Ag-specific thymocytes were quantified within both CD4CD8 double or single-positive compartments in every donor. Our data indicate that the maturation efficiency of Ag-specific thymocytes is poorly affected by HLA-A2 expression, in terms of frequencies. Nevertheless, A2-restricted T-cell lines from A2(+) donors reacted to A2(+) cell lines in a highly peptide-specific fashion, whereas their alloreactive counterparts showed off-target activity. This first ex vivo analysis of human antigen-specific thymocytes at different stages of human T-cell development should open new perspectives in the understanding of the human thymic selection process.

Lateral Gene Transfer Contributing to Leukemogenesis

The uncontrolled proliferation of genetically mutated cells is the commonly understood mechanism for cancer growth and invasion, with accumulation of new mutations in daughter cells leading to clonal diversity of cancer derived from a single founding event. The genetic alterations are passed to new generations by cell division and vertical gene transfer. Viral transmission of oncogenes represents a known mechanism of lateral gene transfer in cancer initiation. Some experimental systems have also suggested that circulating DNA or micro-vesicles may contribute to lateral oncogene transfer in tumorigenesis. We hypothesized that interactions between leukemic cells and adjacent normal hematopoietic stem or progenitor cells may provide an alternative mechanism for the accumulation of mutated genes and the multiplicity of distinct clones in leukemia.To test this hypothesis, we performed experiments to determine whether tumorigenic properties could be transferred from a tumor cell line to normal mouse bone marrow cells using both in vivo and in vitro and systems. B6-GFP+ mice were injected i.v. with 200,000 C1498-Luc cells (a B6-derived NKT-cell-like mouse tumor cell line expressing luciferase and DSRed). Bioluminescent imaging was used to monitor the progression of tumor cell growth in recipients. At 1 month after tumor-cell inoculation, marrow from these mice was harvested and FACS-sorted for GFP+ cells (to eliminate C1498 cells), and then cultured on irradiated stromal cell layers in 96-well plates in a limiting dilution analysis for Poisson analysis of GFP+ clonogenic precursor frequency on day 9. On day 10, cells were harvested from culture and GFP+ cells resorted onto fresh stromal layers for second and third determinations of GFP+ clonogenic precursor frequency on days 15 and 18. As shown in Figure 1, the frequency of clonogenic precursors increased with each successive determination for marrow from C1498-injected mice, while control cultures from non-injected mice showed no increase in precursor frequency, suggesting that exposure to C1498 cells conferred a growth advantage to the marrow cells in the tumor-cell injected mice. Similar results were obtained using an in vitro system of co-culture using C1498 cells and GFP+ bone marrow cells, followed by serial rounds of GFP+ sorting and Poisson analysis, showing increases in clonogenic frequency over 5 successive sorts and re-cultures over a 2-month period, while control cultures showed decreased clonogenic frequencies over the course of the experiment.To confirm these observations in vivo, B6-GFP mice were injected with C1498-Luc and marrow was harvested after a month and sorted for GFP+ cells. The sorted marrow was transplanted into 11Gy-irradiated (FVB x B6albino)F1 recipients (5 x 106 cells per recipient, n=5). Control recipients were irradiated and transplanted with GFP+ marrow from non-injected donors. All recipients developed full hematopoietic engraftment with GFP+ cells. At 6 months post-transplant, a tumor was observed near the left shoulder of one of the recipients of C1498-exposed GFP+ marrow. Figure 2 shows IVIS GFP imaging of this mouse with the GFP+ tumor along with control animals. The tumor was not positive for luciferase expression. The mouse was sacrificed and the tumor excised and a portion was dissociated for flow cytometric analysis and culturing (with other segments reserved for subsequent histological and genetic analysis). Both GFP+ and non-GFP cells were found in the dissociated tumor cell suspension. The GFP+ cells were hematopoietic in origin (CD45+) and exhibited a mixed phenotype containing markers expressed on C1498 (DX5+) and myeloid lineage cells (CD11b+) as well as Sca-1, a stem cell marker. Cultures of the GFP+ tumor yielded a population of GFP+ mononuclear cells.These data are consistent with a model in which growth-promoting or transforming genes from cancer cells become incorporated within a healthy hematopoietic stem or progenitor cell, which contributes to the genetic diversity of the cancer through the initiation a new transformed clone. Genetic analysis with deep sequencing will compare the DNA sequences between the parental C1498 cell line, sorted populations of clonogenic GFP+ cells obtained from the in vitro and in vivo experiments, and the GFP+ tumor cells to confirm the transformation of healthy bone marrow hematopoietic stem cells with genetic sequences derived from the C1498 cells. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bystander hyperactivation of preimmune CD8+ T cells in chronic HCV patients.

Chronic infection perturbs immune homeostasis. While prior studies have reported dysregulation of effector and memory cells, little is known about the effects on naïve T cell populations. We performed a cross-sectional study of chronic hepatitis C (cHCV) patients using tetramer-associated magnetic enrichment to study antigen-specific inexperienced CD8(+) T cells (i.e., tumor or unrelated virus-specific populations in tumor-free and sero-negative individuals). cHCV showed normal precursor frequencies, but increased proportions of memory-phenotype inexperienced cells, as compared to healthy donors or cured HCV patients. These observations could be explained by low surface expression of CD5, a negative regulator of TCR signaling. Accordingly, we demonstrated TCR hyperactivation and generation of potent CD8(+) T cell responses from the altered T cell repertoire of cHCV patients. In sum, we provide the first evidence that naïve CD8(+) T cells are dysregulated during cHCV infection, and establish a new mechanism of immune perturbation secondary to chronic infection.

Exogenous antigens bind MHC class II first, and are processed by cathepsins later.

The field of antigen processing and presentation is likely one of the most well defined areas in immunology based on decades of intense molecular and structural studies. Many molecules contributing to antigen processing and presentation have been discovered and their mechanisms of action been largely defined, yet a major question, which lies at the very core of the field has remained hard to pin down. The question is what determines immunodominance? Immunodominance is defined as a few specific epitopes being selected to represent an antigen to the immune system and provide targets for T cells. Many studies have aimed at understanding how epitopes are selected. A range of hypotheses related to the structural features of antigens, sensitivity to proteases, epitope affinity for MHC II, T cell precursor frequency, and T cell receptor affinity for peptide/MHC II have been considered. However, because of the variety of proteins and factors involved in antigen processing and enormous complexity, finding an answer has been challenging. Here we make an effort to tease out the sequence of events in antigen processing that promote selection of immunodominant epitopes for exogenous antigens.