Lymphopenic Hosts Research Articles

Stromal Notch ligands foster lymphopenia-driven functional plasticity and homeostatic proliferation of naive B cells.

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting the homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain incompletely understood. To evaluate homeostasis of the mature B cell pool during lymphopenia, we turned to an adoptive transfer model of purified follicular B cells into Rag2–/– mouse recipients. Highly purified follicular B cells transdifferentiated into marginal zone–like B cells when transferred into Rag2–/– lymphopenic hosts but not into wild-type hosts. In lymphopenic spleens, transferred B cells gradually lost their follicular phenotype and acquired characteristics of marginal zone B cells, as judged by cell surface phenotype, expression of integrins and chemokine receptors, positioning close to the marginal sinus, and an ability to rapidly generate functional plasma cells. Initiation of follicular to marginal zone B cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B cells and expression of Delta-like 1 Notch ligands by splenic Ccl19-Cre+ fibroblastic stromal cells. Gene expression analysis showed rapid induction of Notch-regulated transcripts followed by upregulated Myc expression and acquisition of broad transcriptional features of marginal zone B cells. Thus, naive mature B cells are endowed with plastic transdifferentiation potential in response to increased stromal Notch ligand availability during lymphopenia.

Stroma-derived Notch signals drive transdifferentiation of follicular B cells into marginal zone B cells during lymphopenia

Abstract In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain poorly understood. To evaluate the homeostasis of the mature B cell pool during lymphopenia, we developed an adoptive transfer model of follicular B (FoB) cells into Rag2−/− mouse recipients. We report that highly purified FoB cells transdifferentiate into marginal zone (MZ)-like B cells when transferred into Rag2−/− lymphopenic hosts, but not into wild type hosts. In lymphopenic spleens, transferred B cells gradually lost their FoB phenotype and acquired multiple characteristics of MZ B cells, as judged by their cell surface phenotype (sIgMhi sIgDlow CD21hi CD1dhi CD23low), expression of integrins and chemokine receptors, positioning close to the marginal sinus, and functional ability to rapidly generate plasma cells. Initiation of FoB to MZ B cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B cells and expression of Delta-like 1 Notch ligands by Ccl19-Cre+ fibroblastic stromal cells, a pathway also required for MZ B cell development in steady-state conditions. We postulate that stromal Notch ligands are critical to regulate the size and composition of the splenic B cell pool via interactions with Notch2 receptors in B cells. Thus, FoB cells are not locked in their FoB cell fate, but are are endowed with plastic transdifferentiation potential in response to increased stromal Dll1 availability in lymphopenic environments.

T cell-intrinsic role for Nod2 in protection against Th17-mediated uveitis

Mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) cause Blau syndrome, an inflammatory disorder characterized by uveitis. The antimicrobial functions of Nod2 are well-established, yet the cellular mechanisms by which dysregulated Nod2 causes uveitis remain unknown. Here, we report a non-conventional, T cell-intrinsic function for Nod2 in suppression of Th17 immunity and experimental uveitis. Reconstitution of lymphopenic hosts with Nod2−/− CD4+ T cells or retina-specific autoreactive CD4+ T cells lacking Nod2 reveals a T cell-autonomous, Rip2-independent mechanism for Nod2 in uveitis. In naive animals, Nod2 operates downstream of TCR ligation to suppress activation of memory CD4+ T cells that associate with an autoreactive-like profile involving IL-17 and Ccr7. Interestingly, CD4+ T cells from two Blau syndrome patients show elevated IL-17 and increased CCR7. Our data define Nod2 as a T cell-intrinsic rheostat of Th17 immunity, and open new avenues for T cell-based therapies for Nod2-associated disorders such as Blau syndrome.

In vitro treatment of CD8+ T cells with interleukin-7 enhances their in vivo anti-tumor capabilities against an animal model of Ehrlich ascites carcinoma

Background: Developing optimal strategies for generating T cells capable of effectively controlling tumors is one of the most important prerequisites for the clinical application of adoptive cell therapies in cancer patients. However, the generation of sufficient numbers of tumor-reactive T cells capable of efficient tumor regression and long-term persistence remains a significant impediment to widespread clinical implementation. Aim: The main aim of the present study was to evaluate the beneficial anti-tumor effects of a simplified combinatorial approach that involves a short activation of naive CD8+ T cells with the T cell mitogen concanavalin A (CON-A; 4 ug/mL) and the survival cytokine IL-7 (10 ng/mL), after a single intraperitoneal injection of cyclophosphamide (CTX; 4 mg/mouse) after their adoptive transfer into Ehrlich ascites carcinoma (EAC) tumor-bearing CD1 mice. Results: We found that adoptive transfer of in vitro IL-7-conditioned T cells into EAC-bearing (3-day) mice previously treated with a single dose of CTX induced a delay in the progression of EAC, and the establishment of long-term immunological memory, which has the efficiency to provide full protection for mice against cancer. Our results indicated that in the presence of IL-7, the short-term T-cell receptor signaling mediates promiscuous qualities in naive cytotoxic CD8+T cells. Conclusion: The data indicate that upon the adoptive transfer of IL-7 conditioned T cells into lymphopenic hosts, they were able to eradicate tumors and also to generate long-term tumor-specific memory.

PTPN22 Acts in a Cell Intrinsic Manner to Restrict the Proliferation and Differentiation of T Cells Following Antibody Lymphodepletion.

Lymphopenic insult has been shown to precipitate the initiation of autoimmune disease in murine models such as the Non-obese diabetic mouse. Similarly, in man lymphopenia induced by mAb therapy, for instance Alemtuzumab as treatment for Multiple Sclerosis, can precipitate development of secondary autoimmune disease in up to 30 % of patients. We asked whether an identified autoimmune susceptibility locus might increase the risk of developing autoimmunity in the context of mAb-induced lymphopenia in a mouse model. A single nucleotide polymorphism (SNP) in the gene encoding the tyrosine phosphatase PTPN22 (R620W) is associated with multiple human autoimmune diseases, and PTPN22 has been shown to modulate T cell responses, particularly to weak antigens. In keeping with this, PTPN22-deficient or PTPN22 R619W mutant murine T cells adoptively transferred into immunodeficient lymphopenic hosts showed a higher lymphopenia-induced proliferation rate than WT cells. We induced lymphopenia by treating wild-type or PTPN22 knock-out mice with T cell depleting antibodies and monitored reconstitution of the T cell pool. We found that PTPN22 deficient T cells acquired a more activated effector phenotype, with significantly more IFNγ producing cells. This resulted from expansion driven by self-peptide MHC, as it was evident when the contribution of IL-7 to lymphopenic expansion was blocked with IL-7R Ab. Interestingly, Foxp3+ Tregs were also considerably expanded in PTPN22-deficient and PTPN22 R619W mice, as was the frequency of both CD25+ and CD25− CD4 T cells that produce IL-10. Using bone marrow chimeric mice, we showed that PTPN22 influenced development of both regulatory and effector T cell functions in a cell-intrinsic manner. Overall the expansion of Tregs is likely to keep the expanded T effector populations in check and sparing Treg during therapeutic mAb depletion may be a useful strategy to prevent occurrence of secondary autoimmunity.

Lineage Tracking the Generation of T Regulatory Cells From Microbial Activated T Effector Cells in Naïve Mice.

Regulatory T cells (Tregs) are essential for the maintenance of gut homeostasis by suppressing conventional CD4+ helper T cells (Tconvs) that are activated by microbial antigens. Although thymus is the major source of the peripheral Tregs, peripheral conversion from Tconvs to Tregs have also been shown to occur under various experimental conditions. It remains less clear about the frequency of lineage conversion from Tconvs to Tregs in naïve animals. Here we used a newly established reporter system to track a group of post expansion Tregs (eTregs), which exhibited a stronger suppressive ability than the non-lineage marked Tregs. Notably, microbial antigens are the primary driver for the formation of eTregs. TCR repertoire analysis of Peyer's patch T cells revealed that eTregs are clonally related to Tconvs, but not to the non-lineage tracked Tregs. Adoptive transfer of Tconvs into lymphopenic hosts demonstrated a conversion from Tconvs to eTregs. Thus, our lineage tracking method was able to capture the lineage conversion from microbial activated effector T cells to Tregs in naïve animals. This study suggests that a fraction of clonally activated T cells from the natural T cell repertoire exhibits lineage conversion to Tregs in response to commensal microbes under homeostatic conditions.

Interleukin-27 promotes CD8+ T cell reconstitution following antibody-mediated lymphoablation.

Antibody-mediated lymphoablation is used in solid organ and stem cell transplantation and autoimmunity. Using murine anti-thymocyte globulin (mATG) in a mouse model of heart transplantation, we previously reported that the homeostatic recovery of CD8+ T cells requires help from depletion-resistant memory CD4+ T cells delivered through CD40-expressing B cells. This study investigated the mechanisms by which B cells mediate CD8+ T cell proliferation in lymphopenic hosts. While CD8+ T cell recovery required MHC class I expression in the host, the reconstitution occurred independently of MHC class I, MHC class II, or CD80/CD86 expression on B cells. mATG lymphoablation upregulated the B cell expression of several cytokine genes, including IL-15 and IL-27, in a CD4-dependent manner. Neither treatment with anti-CD122 mAb nor the use of IL-15Rα-/- recipients altered CD8+ T cell recovery after mATG treatment, indicating that IL-15 may be dispensable for T cell proliferation in our model. Instead, IL-27 neutralization or the use of IL-27Rα-/- CD8+ T cells inhibited CD8+ T cell proliferation and altered the phenotype and cytokine profile of reconstituted CD8+ T cells. Our findings uncover what we believe is a novel role of IL-27 in lymphopenia-induced CD8+ T cell proliferation and suggest that targeting B cell-derived cytokines may increase the efficacy of lymphoablation and improve transplant outcomes.

Differential Cytokine Utilization and Tissue Tropism Results in Distinct Repopulation Kinetics of Naïve vs. Memory T Cells in Mice.

Naïve and memory T cells co-exist in the peripheral T cell pool, but the cellular mechanisms that maintain the balance and homeostasis of these two populations remain mostly unclear. To address this question, here, we assessed homeostatic proliferation and repopulation kinetics of adoptively transferred naïve and memory T cells in lymphopenic host mice. We identified distinct kinetics of proliferation and tissue-distribution between naïve and memory donor T cells, which resulted in the occupancy of the peripheral T cell pool by mostly naïve-origin T cells in short term (<1 week), but, in a dramatic reversal, by mostly memory-origin T cells in long term (>4 weeks). To explain this finding, we assessed utilization of the homeostatic cytokines IL-7 and IL-15 by naïve and memory T cells. We found different efficiencies of IL-7 signaling between naïve and memory T cells, where memory T cells expressed larger amounts of IL-7Rα but were significantly less potent in activation of STAT5 that is downstream of IL-7 signaling. Nonetheless, memory T cells were superior in long-term repopulation of the peripheral T cell pool, presumably, because they preferentially migrated into non-lymphoid tissues upon adoptive transfer and additionally utilized tissue IL-15 for rapid expansion. Consequently, co-utilization of IL-7 and IL-15 provides memory T cells a long-term survival advantage. We consider this mechanism important, as it permits the memory T cell population to be maintained in face of constant influx of naïve T cells to the peripheral T cell pool and under competing conditions for survival cytokines.

Spontaneous Proliferation of CD4+ T Cells in RAG-Deficient Hosts Promotes Antigen-Independent but IL-2-Dependent Strong Proliferative Response of Naïve CD8+ T Cells.

The fast and intense proliferative responses have been well documented for naïve T cells adoptively transferred into chronic lymphopenic hosts. This response known as spontaneous proliferation (SP), unlike antigen-independent lymphopenia-induced proliferation (LIP), is driven in a manner dependent on antigens derived from commensal microbiota. However, the precise nature of the SP response and its impact on homeostasis and function for T cells rapidly responding under this lymphopenic condition are still unclear. Here we demonstrate that, when naïve T cells were adoptively transferred into specific pathogen-free (SPF) but not germ-free (GF) RAG−/− hosts, the SP response of these cells substantially affects the intensity and tempo of the responding T cells undergoing LIP. Therefore, the resulting response of these cells in SPF RAG−/− hosts was faster and stronger than the typical LIP response observed in irradiated B6 hosts. Although the intensity and tempo of such augmented LIP in SPF RAG−/− hosts were analogous to those of antigen-dependent SP, the former was independent of antigenic stimulation but most importantly, dependent on IL-2. Similar observations were also apparent in other acute lymphopenic settings where antigen-dependent T cell activation can strongly occur and induce sufficient levels of IL-2 production. Consequently, the resulting T cells undergoing IL-2-driven strong proliferative responses showed the ability to differentiate into functional effector and memory cells that can control infectious pathogens. These findings therefore reveal previously unappreciated role of IL-2 in driving the intense form of T cell proliferative responses in chronic lymphopenic hosts.

Spontaneous T Cell Proliferation: A Physiologic Process to Create and Maintain Homeostatic Balance and Diversity of the Immune System.

Naive T lymphocytes undergo heterogeneous proliferative responses when introduced into lymphopenic hosts, referred to as “homeostatic proliferation” and “spontaneous proliferation.” Spontaneous proliferation is a unique process through which the immune system generates memory phenotype cells with increasing T cell receptors repertoire complexity. Here, the mechanisms that initiate and control spontaneous proliferation are discussed.

Density dependent re-tuning of autoreactive T cells alleviates their pathogenicity in a lymphopenic environment

Peripheral T cell tolerance is challenging to induce in partially lymphopenic hosts and this is relevant for clinical situations involving transplant tolerance. While the shortage of regulatory cells is thought to be one reason for this, T cell-intrinsic tolerance processes such as anergy are also poorly triggered in such hosts. In order to understand the latter, we used a T cell deficient mouse model system where adoptively transferred autoreactive T cells are significantly tolerized in a cell intrinsic fashion, without differentiation to regulatory T cells. Intriguingly these T cells often retain sufficient effector functions to trigger autoimmune pathology. Here we find that the high population density of the autoreactive T cells that accumulated in such a host limits the progression of the cell-intrinsic tolerance process in T cells. Accordingly, reducing the cell density during a second transfer allowed T cells to further tune down their responsiveness to antigenic stimulation. The retuning of T cells was reflected by a loss of the T cell’s abilities to proliferate, produces cytokines or help B cells. We further suggest, based on altering the levels of chronic antigen using miniosmotic pumps, that the effects of cell-density on T cell re-tuning may reflect the effective changes in the antigen dose perceived by individual T cells. This could proportionally elicit more negative feedback downstream of the TCR. Consistent with this, the retuned T cells showed signaling defects both proximal and distal to the TCR. Therefore, similar to the immunogenic activation of T cells, cell-intrinsic T cell tolerance may also involve a quantitative and progressive process of tuning down its antigen-responsiveness. The progress of such tuning seems to be stabilized at multiple intermediate stages by factors such as cell density, rather than just absolute antigen levels.

PD-1 Controls Tonic Signaling and Lymphopenia-Induced Proliferation of T Lymphocytes.

Recovery of the T lymphocyte compartment within a lymphopenic host by lymphopenia-induced proliferation (LIP) is regulated by inter- and intraclonal competition for limited resources, including homeostatic cytokines and peptide:MHC (pMHC) complexes with which the TCR can interact at least weakly to yield a tonic signal. Importantly, the process of LIP can synergize with other factors that promote T cell activation to drive inflammatory disease. While reconstitution of the lymphoid compartment of immune deficient Rag−/− mice by transfer of wild-type hematopoietic stem cells (HSC) does not generally result in an overt disease phenotype, transfer of HSC deficient in expression of the co-inhibitory molecule PD-1 results in severe systemic autoimmunity driven by newly generated T cells that emerge from the thymus into the periphery and undergo LIP. Importantly, autoimmunity does not appear to depend on a response to exogenous (i.e., gut flora-derived) antigens. PD-1 is well known to be upregulated during T cell activation in response to cognate antigens, but it is unclear whether PD-1 has a role in controlling LIP of T cells in the absence of cognate antigen, i.e., in response to tonic pMHC. We examined whether PD-1 controls LIP of newly generated T cells by controlling the response to tonic pMHC or the homeostatic cytokine IL-7. We found that PD-1-deficient T cells have a proliferative advantage over WT T cells during LIP and this effect is MHC-II dependent and independent of IL-7Rα signaling. Furthermore, our data suggest that signals through IL-7Rα can be dispensable for LIP and may instead be of increased importance for T cell survival in conditions of high competition for limited pMHC (e.g., post-LIP, in a lymphoreplete host). We hypothesize that autoimmunity post-PD-1−/− HSC transplant is the result of an overzealous T cell response to normally tonic self-pMHC precipitated by the synergy of LIP and PD-1 deficiency. Furthermore, potentiation of TCR signals in response to normally tonic self-pMHC may contribute to the success of PD-1 blockade in cancer immunotherapy.

Abstract B107: Immunotransplant: Merging checkpoint blockade and T-cell transfer into lymphodepleted recipients

Abstract Lymphomas, with an estimated 80,000 new cases, are the 5th most common cancer in the United States and the majority of lymphoma subtypes are incurable and in need of novel, mechanistically distinct therapies. Two approaches to potently increase the anti-tumor potential of T cells are: checkpoint blockade (e.g. anti-CTLA-4 and anti-PD-1 antibodies) and adoptive T-cell transfer into lympho-depleted recipients. We have developed a novel therapy combining these approaches into ‘checkpoint-blockade-primed immunotransplant’ comprised of: -treatment of tumor-bearing host with anti-CTLA-4 and/or anti-PD-1 antibodies -splenocyte harvest and transfer to lympho-depleted recipient Whereas checkpoint blockade alone has minimal anti-tumor effect in the pre-clinical model, our results show that the combined therapy results in superior anti-tumor immunity as seen by increased production of tumor-reactive IFN γ positive T-cells. Treatment of both tumor-bearing donor and recipient with checkpoint antibodies induces cure of the majority of recipients, in a CD8, NK, and IFNγ-dependent manner. Furthermore, we have demonstrated that CD8 T cells exposed to checkpoint blockade and transfer into the lymphopenic host demonstrate greater: -in vivo serum levels of IL-15 and IL-7 -surface expression levels of IL-15R and IL-7R -in vitro STAT5 phosphorylation in response to common γ-chain cytokines -in vivo proliferation in response to exposure to cognate tumor antigen Ongoing studies will seek to assess the dependence of the above observations (cytokine production, proliferation, anti-tumor effect) on specific common γ-chain cytokines, the role of lymphopenia in inducing T cell trafficking to tumor versus healthy tissue, and guide development of the immunotransplant model to optimize the amplification of anti-tumor immunity for near-term clinical translation. Citation Format: Netonia Marshall, Thomas Marron, Judith Agudo, Brian Brown, Joshua Brody. Immunotransplant: Merging checkpoint blockade and T-cell transfer into lymphodepleted recipients [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B107.

Distinct IL-7 signaling in recent thymic emigrants versus mature naïve Tcells controls T-cell homeostasis.

IL-7 is essential for T-cell survival but its availability is limited in vivo. Consequently, all peripheral Tcells, including recent thymic emigrants (RTEs) are constantly competing for IL-7 to survive. RTEs are required to replenish TCR diversity and rejuvenate the peripheral T-cell pool. However, it remains unknown how RTEs successfully compete with resident mature Tcells for IL-7. Moreover, RTEs express low levels of IL-7 receptors, presumably rendering them even less competitive. Here, we show that, surprisingly, RTEs are more responsive to IL-7 than mature naïve Tcells as demonstrated by markedly increased STAT5 phosphorylation upon IL-7 stimulation. Nonetheless, adoptive transfer of RTE cells into lymphopenic host mice resulted in slower IL-7-induced homeostatic proliferation and diminished expansion compared to naïve donor Tcells. Mechanistically, we found that IL-7 signaling in RTEs preferentially upregulated expression of Bcl-2, which is anti-apoptotic but also anti-proliferative. In contrast, naïve Tcells showed diminished Bcl-2 induction but greater proliferative response to IL-7. Collectively, these data indicate that IL-7 responsiveness in RTE is designed to maximize survival at the expense of reduced proliferation, consistent with RTE serving as a subpopulation of Tcells rich in diversity but not in frequency.

Atg5 Is Essential for the Development and Survival of Innate Lymphocytes.

Autophagy is an essential cellular survival mechanism that is required for adaptive lymphocyte development; however, its role in innate lymphoid cell (ILC) development remains unknown. Furthermore, the conditions that promote lymphocyte autophagy during homeostasis are poorly understood. Here, we demonstrate that Atg5, an essential component of the autophagy machinery, is required for the development of mature natural killer (NK) cells and group 1, 2, and 3 innate ILCs. Although inducible ablation of Atg5 was dispensable for the homeostasis of lymphocyte precursors and mature lymphocytes in lymphoreplete mice, we found that autophagy is induced in both adaptive and innate lymphocytes during homeostatic proliferation in lymphopenic hosts to promote their survival by limiting cell-intrinsic apoptosis. Induction of autophagy through metformin treatment following homeostatic proliferation increased lymphocyte numbers through an Atg5-dependent mechanism. These findings highlight the essential role for autophagy in ILC development and lymphocyte survival during lymphopenia.

The histone lysine methyltransferase G9a maintains the transcriptional profile of mature CD8 T cells

Abstract Lineage identities of developing lymphocytes are established by both activation of lineage specific genes and repression of alternative lineage genes. The latter process depends on active gene repression by continued expression of transcriptional repressors or epigenetic silencing mediated by methylation of cytosine on DNA and lysine residues in histone tails, including Lys9 (K9) of histone H3. Here we show that the histone H3K9 methyltransferase G9a is essential for silencing of helper lineage-related genes in mature CD8+ T cells. Despite the development of normal CD4− CD8+ T cells, a proportion of G9a-deficient CD8+ T cells, when forced to divide with anti-CD3e and anti-CD28 stimulation, depressed surface CD4 in vitro. Furthermore, a substantial fraction of G9a-deficient CD8+ T cells carrying an OT-I T cell receptor derepressed surface CD4 during homeostatic proliferation following an adoptive transfer to lymphopenic host mice. These results suggest that G9a establish stable silencing of alternative lineage genes in dividing mature CD8+ T cells. We are currently conducting gene expression analysis to assess the requirement for G9a in establishment of the lineage identity of CD8+ T cells at a genome-wide level.

Baicalein exhibits anti-inflammatory effects via inhibition of NF-κB transactivation

NF-κB is a crucial mediator of inflammatory and immune responses and a number of phytochemicals that can suppress this immune-regulatory transcription factor are known to have promising anti-inflammatory potential. However, we report that inducer of pro-inflammatory transcription factor NF-κB functions as an anti-inflammatory agent. Our findings reveal that a plant derived flavonoid baicalein could suppress mitogen induced T cell activation, proliferation and cytokine secretion. Treatment of CD4+ T cells with baicalein prior to transfer in to lymphopenic allogenic host significantly suppressed graft versus host disease. Interestingly, addition of baicalein to murine splenic lymphocytes induced DNA binding of NF-κB but did not suppress Concanavalin A induced NF-κB. Since baicalein did not inhibit NF-κB binding to DNA, we hypothesized that baicalein may be suppressing NF-κB trans-activation. Thioredoxin system is implicated in the regulation of NF-κB trans-activation potential and therefore inhibition of thioredoxin system may be responsible for suppression of NF-κB dependent genes. Baicalein not only inhibited TrxR activity in cell free system but also suppressed mitogen induced thioredoxin activity in the nuclear compartment of lymphocytes. Similar to baicalein, pharmacological inhibitors of thioredoxin system also could suppress mitogen induced T cell proliferation without inhibiting DNA binding of NF-κB. Further, activation of cellular thioredoxin system by the use of pharmacological activator or over-expression of thioredoxin could abrogate the anti-inflammatory action of baicalein. We propose a novel strategy using baicalein to limit NF-κB dependent inflammatory responses via inhibition of thioredoxin system.

Proteome and Protein Network Analyses of Memory T Cells Find Altered Translation and Cell Stress Signaling in Treated Human Immunodeficiency Virus Patients Exhibiting Poor CD4 Recovery.

Background. Human immunodeficiency virus (HIV) patients who experience poor CD4 T-cell recovery despite viral suppression during antiretroviral therapy (ART) are known as immunological nonresponders. The molecular mechanism(s) underlying incomplete immune restoration during ART is not fully understood.Methods. Label-free quantitative proteomics on single-cell type central memory T cells were used to reveal relative protein abundance changes between nonresponder, responder (good CD4 recovery during ART), and healthy individuals. Proteome changes were analyzed by protein pathway and network analyses and verified by selected reaction monitoring mass spectrometry.Results. Proteomic analysis across groups detected 155 significant proteins from 1500 nonredundant proteins. Pathway and network analyses revealed dysregulation in mammalian target of rapamycin and protein translation-related proteins and decreases in stress response-related proteins for nonresponder subjects compared with responders and controls. Actin cytoskeleton signaling was increased for HIV responders and nonresponders alike.Conclusions. Memory T cells from immunologic nonresponders have increases in proteins related to motility and protein translation and decreases in proteins capable of responding to cellular stresses compared with responders and controls. The potential for T cells to manage stress and modulate metabolism may contribute to their capacity to reconstitute a lymphopenic host.

Requirement of interleukin 7 signaling for anti-tumor immune response under lymphopenic conditions in a murine lung carcinoma model.

Induction of lymphopenia before adoptive transfer of T cells was followed by lymphopenia-induced proliferation (LIP) and generated a potent anti-tumor immune response in rodents and in a clinical setting. Previously, we reported that CD28 signaling is essential for the differentiation of functional effector cytotoxic T lymphocytes (CTLs) under lymphopenic conditions and sequential LIP of T cells. In this study, to clarify the correlation between LIP and the anti-tumor effect, LIP was inhibited with interleukin 7 (IL7) receptor blockade at various stages, and the anti-tumor effect then assessed. We confirmed that IL7 signaling at the start of LIP is crucial for the anti-tumor immune response. In contrast, continuous IL7 signaling was not required for tumor regression, although LIP of naïve CD8+ T cells is usually regulated by IL7. The expansion and migration of CTLs in lymphopenic hosts depend on IL7 signaling during the induction phase. Here, we propose that IL7 signaling and subsequent LIP of T cells have distinct roles in the induction of T cell immunity during lymphopenia.

Unleashing the power of anti-tumor CD4+ T cells: novel insights into the curative mechanisms of chemoimmunotherapy for cancer

Meeting abstracts CD4+ T cells are critical mediators of anti-tumor immunity and orchestrate a broad range of immune responses against cancer. Previous studies from our lab and others have demonstrated that, adoptive transfer of tumor specific CD4+ T cells to lymphopenic hosts led to eradication of