Biodistribution of VEGF-4-1BB conjugate. Survival assay in MCA induced fibrosarcoma model. Cytokine levels in treated animals. Tumor volume studies with combinatorial therapy in Balb/c model. Histology and morphology for toxicity profiles.

BACKGROUND & AIMSImmunotherapies are ineffective against pancreatic cancer. We investigated whether the activity of nuclear factor (NF)κB in pancreatic stromal cells contributes to an environment that suppresses antitumor immune response.METHODSPancreata of C57BL/6 or Rag1-/- mice were given pancreatic injections of a combination of KrasG12D/+; Trp53 R172H/+; Pdx-1cre (KPC) pancreatic cancer cells and pancreatic stellate cells (PSCs) extracted from C57BL/6 (control) or mice with disruption of the gene encoding the NFκB p50 subunit (Nfkb1 or p50-/- mice). Tumor growth was measured as an endpoint. Other mice were given injections of Lewis lung carcinoma (LLC) lung cancer cells or B16-F10 melanoma cells with control or p50-/- fibroblasts. Cytotoxic T cells were depleted from C57BL/6 mice by administration of antibodies against CD8 (anti-CD8), and growth of tumors from KPC cells, with or without control or p50-/- PSCs, was measured. Some mice were given an inhibitor of CXCL12 (AMD3100) and tumor growth was measured. T-cell migration toward cancer cells was measured using the Boyden chamber assay.RESULTSC57BL/6 mice coinjected with KPC cells (or LLC or B16-F10 cells) and p50-/- PSCs developed smaller tumors than mice given injections of the cancer cells along with control PSCs. Tumors that formed when KPC cells were injected along with p50-/- PSCs had increased infiltration by activated cytotoxic T cells along with decreased levels of CXCL12, compared with tumors grown from KPC cells injected along with control PSCs. KPC cells, when coinjected with control or p50-/- PSCs, developed the same-size tumors when CD8+ T cells were depleted from C57BL/6 mice or in Rag1-/- mice. The CXCL12 inhibitor slowed tumor growth and increased tumor infiltration by cytotoxic T cells. In vitro expression of p50 by PSCs reduced T-cell migration toward and killing of cancer cells. When cultured with cancer cells, control PSCs expressed 10-fold higher levels of CXCL12 than p50-/- PSCs. The CXCL12 inhibitor increased migration of T cells toward KPC cells in culture.CONCLUSIONSIn studies of mice and cell lines, we found that NFκB activity in PSCs promotes tumor growth by increasing expression of CXCL12, which prevents cytotoxic T cells from infiltrating the tumor and killing cancer cells. Strategies to block CXCL12 in pancreatic tumor cells might increase antitumor immunity.

Uptake of tumor antigens by tumor-infiltrating dendritic cells is limiting step in the induction of tumor immunity, which can be mediated through Fc receptor (FcR) triggering by antibody-coated tumor cells. Here we describe an approach to potentiate tumor immunity whereby hapten-specific polyclonal antibodies are recruited to tumors by coating tumor cells with the hapten. Vaccination of mice against dinitrophenol (DNP) followed by systemic administration of DNP targeted to tumors by conjugation to a VEGF or osteopontin aptamer elicits potent FcR dependent, T cell mediated, antitumor immunity. Recruitment of αGal-specific antibodies, the most abundant naturally occurring antibodies in human serum, inhibits tumor growth in mice treated with a VEGF aptamer-αGal hapten conjugate, and recruits antibodies from human serum to human tumor biopsies of distinct origin. Thus, treatment with αGal hapten conjugated to broad-spectrum tumor targeting ligands could enhance the susceptibility of a broad range of tumors to immune elimination.

T cell immunoglobulin-3 (TIM-3) is a negative regulator of interferon-gamma (IFN-gamma) secreting CD4(+) T cells and CD8(+) T cyto-toxic cells. Recent studies have highlighted the role of TIM-3 as an important mediator of CD8(+) T cell exhaustion in the setting of chronic viral infections and cancer. In murine tumor models, antibody blockade of TIM-3 with anti-TIM-3 antibodies as monotherapy has no or minimal antitumor activity, suggesting that TIM-3 signaling exerts an accessory or amplifying effect in keeping immune responses in check. Using a combined bead and cell-based systemic evolution of ligands by exponential enrichment (SELEX) protocol, we have isolated nuclease-resistant oligonucleotide aptamer ligands that bind to cell-associated TIM-3 with high affinity and specificity. A trimeric form of the TIM-3 aptamer blocked the interaction of TIM-3 with Galectin-9, reduced cell death, and enhanced survival, proliferation, and cytokine secretion in vitro. In tumor-bearing mice, the aptamer delayed tumor growth as monotherapy and synergized with PD-1 antibody in prolonging the survival of the tumor-bearing mice. Both in vitro and in vivo, the trimeric aptamer displayed superior activity compared to the currently used RMT3-23 monoclonal antibody. This study suggests that multi-valent aptamers could represent an alternative platform to generate potent ligands to manipulate the function of TIM-3 and other immune modulatory receptors.

The ability to control lentivirus replication may be determined, in part, by the extent to which individual viral proteins are targeted by the immune system. Consequently, defining the antigens that elicit the most protective immune responses may facilitate the design of effective HIV-1 vaccines. Here we vaccinated four groups of rhesus macaques with a heterologous vector prime/boost/boost/boost (PBBB) regimen expressing the following simian immunodeficiency virus (SIV) genes: env, gag, vif, rev, tat, and nef (Group 1); env, vif, rev, tat, and nef (Group 2); gag, vif, rev, tat, and nef (Group 3); or vif, rev, tat, and nef (Group 4). Following repeated intrarectal challenges with a marginal dose of the neutralization-resistant SIVmac239 clone, vaccinees in Groups 1-3 became infected at similar rates compared to control animals. Unexpectedly, vaccinees in Group 4 became infected at a slower pace than the other animals, although this difference was not statistically significant. Group 1 exhibited the best post-acquisition virologic control of SIV infection, with significant reductions in both peak and chronic phase viremia. Indeed, 5/8 Group 1 vaccinees had viral loads of less than 2,000 vRNA copies/mL of plasma in the chronic phase. Vaccine regimens that did not contain gag (Group 2), env (Group 3), or both of these inserts (Group 4) were largely ineffective at decreasing viremia. Thus, vaccine-induced immune responses against both Gag and Env appeared to maximize control of immunodeficiency virus replication. Collectively, these findings are relevant for HIV-1 vaccine design as they provide additional insights into which of the lentiviral proteins might serve as the best vaccine immunogens.

Radiotherapy (RT)-potentiated immunomodulation is an emerging field in solid tumor malignancies. RT induces peripheral antitumor immunity that can lead to the reduction of distant, non-irradiated tumor lesions (abscopal response). This has provided rationale for current early phase trials combining existing checkpoint blockade and co-stimulatory mAbs with stereotactic RT. Objective responses remain suboptimal with checkpoint blockade monotherapy, and dose limiting toxicity is widely observed with these modalities.  We hypothesize that targeting immunotherapy to the tumor using oligonucleotide aptamers that bind to RT induced tumor stress products will significantly enhance the therapeutic index.  We used the aptamer platform to generate a bi-specific construct containing a costimulatory aptamer ligand specific to murine 4-1BB which was conjugated to an aptamer specific to products secreted into the tumor stroma (VEGF or osteopontin). Tumor targeting, local control, abscopal responses and toxicity were compared to unconjugated 4-1BB-VEGF constructs, or mAB based therapies, when used in combination with RT in murine subcutaneous and autochthonous MCA tumor models. RT induced VEGF upregulation was demonstrated in null/low VEGF secreting tumors that led to preferential accumulation of VEGF-41BB aptamers in the irradiated tumor.12Gy x1 elicited optimal tumor targeting of bi-specific VEGF-4-1BB conjugate. Significant anti-tumor immune responses were observed in s.c. murine tumor models. This conjugate showed dramatic reduction in systemic toxicity compared to the equivalent gold standard 4-1BB mAb. RT-VEGF-4-1BB treatment significantly improved local control, overall survival and induced potent abscopal responses compared to unconjugated and non-irradiated controls in the Balb/c 4T1 and MCA murine tumor models. Furthermore, RT-VEGF-4-1BB treatment showed similar anti-tumor effects as CTLA-4 mAB with significantly less systemic toxicity. This approach increased intra-tumoral CD4+ and CD8+ T cells, the CD8+ to Treg ratio, and induced significantly more tumor cell death. In conclusion, this strategy has been shown to induce potent anti-tumor immune responses in relevant murine tumor models and significantly improves the therapeutic index over non-targeted mABs. Furthermore, radiotherapy can expand the scope of tumor targeted immune modulation to virtually all solid malignancies and induce potent abscopal responses. These results provide the rationale for developing clinical grade bispecific aptamers for future testing. Citation Format: Brett Schrand, Bhavna Verma, Agata Levay, Shradha Patel, Iris Castro, Ana Paula Benaduce, Randall Brenneman, Oliver Umland, Hideo Yagita, Eli Gilboa, Adrian Ishkanian. Radiation-induced vegf-targeted 4-1bb costimulation enhances immune control of tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1700. doi:10.1158/1538-7445.AM2017-1700

Abstract Radiotherapy can elicit systemic immune control of local tumors and distant nonirradiated tumor lesions, known as the abscopal effect. Although this effect is enhanced using checkpoint blockade or costimulatory antibodies, objective responses remain suboptimal. As radiotherapy can induce secretion of VEGF and other stress products in the tumor microenvironment, we hypothesized that targeting immunomodulatory drugs to such products will not only reduce toxicity but also broaden the scope of tumor-targeted immunotherapy. Using an oligonucleotide aptamer platform, we show that radiation-induced VEGF-targeted 4-1BB costimulation potentiated both local tumor control and abscopal responses with equal or greater efficiency than 4-1BB, CTLA-4, or PD1 antibodies alone. Although 4-1BB and CTLA-4 antibodies elicited organ-wide inflammatory responses and tissue damage, VEGF-targeted 4-1BB costimulation produced no observable toxicity. These findings suggest that radiation-induced tumor-targeted immunotherapy can improve the therapeutic index and extend the reach of immunomodulatory agents. Cancer Res; 77(6); 1310–21. ©2017 AACR.

Baboons naturally infected with simian T lymphotropic virus (STLV) are a potentially useful model system for the study of vaccination against human T lymphotropic virus (HTLV). Here we expanded the number of available full-length baboon STLV-1 sequences from one to three and related the T cell responses that recognize the immunodominant Tax protein to the tax sequences present in two individual baboons. Continuously growing T cell lines were established from two baboons, animals 12141 and 12752. Next-generation sequencing (NGS) of complete STLV genome sequences from these T cell lines revealed them to be closely related but distinct from each other and from the baboon STLV-1 sequence in the NCBI sequence database. Overlapping peptides corresponding to each unique Tax sequence and to the reference baboon Tax sequence were used to analyze recognition by T cells from each baboon using intracellular cytokine staining (ICS). Individual baboons expressed more gamma interferon and tumor necrosis factor alpha in response to Tax peptides corresponding to their own STLV-1 sequence than in response to Tax peptides corresponding to the reference baboon STLV-1 sequence. Thus, our analyses revealed distinct but closely related STLV-1 genome sequences in two baboons, extremely low heterogeneity of STLV sequences within each baboon, no evidence for superinfection within each baboon, and a ready ability of T cells in each baboon to recognize circulating Tax sequences. While amino acid substitutions that result in escape from CD8+ T cell recognition were not observed, premature stop codons were observed in 7% and 56% of tax sequences from peripheral blood mononuclear cells from animals 12141 and 12752, respectively.IMPORTANCE It has been estimated that approximately 100,000 people suffer serious morbidity and 10,000 people die each year from the consequences associated with human T lymphotropic virus (HTLV) infection. There are no antiviral drugs and no preventive vaccine. A preventive vaccine would significantly impact the global burden associated with HTLV infections. Here we provide fundamental information on the simian T lymphotropic virus (STLV) naturally transmitted in a colony of captive baboons. The limited viral sequence heterogeneity in individual baboons, the identity of the viral gene product that is the major target of cellular immune responses, the persistence of viral amino acid sequences that are the major targets of cellular immune responses, and the emergence in vivo of truncated variants in the major target of cellular immune responses all parallel what are seen with HTLV infection of humans. These results justify the use of STLV-infected baboons as a model system for vaccine development efforts.

ABSTRACT There are currently 5 million to 10 million human T-lymphotropic virus type 1 (HTLV-1)-infected people, and many of them will develop severe complications resulting from this infection. A vaccine is urgently needed in areas where HTLV-1 is endemic. Many vaccines are best tested in nonhuman primate animal models. As a first step in designing an effective HTLV-1 vaccine, we defined the CD8+ and CD4+ T cell response against simian T-lymphotropic virus type 1 (STLV-1), a virus closely related to HTLV-1, in olive baboons (Papio anubis). Consistent with persistent antigenic exposure, we observed that STLV-1-specific CD8+ T cells displayed an effector memory phenotype and usually expressed CD107a, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α). To assess the viral targets of the cellular immune response in STLV-1-infected animals, we used intracellular cytokine staining to detect responses against overlapping peptides covering the entire STLV-1 proteome. Our results show that, similarly to humans, the baboon CD8+ T cell response narrowly targeted the Tax protein. Our findings suggest that the STLV-1-infected baboon model may recapitulate some of the important aspects of the human response against HTLV-1 and could be an important tool for the development of immune-based therapy and prophylaxis. IMPORTANCE HTLV-1 infection can lead to many different and often fatal conditions. A vaccine deployed in areas of high prevalence might reduce the incidence of HTLV-1-induced disease. Unfortunately, there are very few animal models of HTLV-1 infection useful for testing vaccine approaches. Here we describe cellular immune responses in baboons against a closely related virus, STLV-1. We show for the first time that the immune response against STLV-1 in naturally infected baboons is largely directed against the Tax protein. Similar findings in humans and the sequence similarity between the human and baboon viruses suggest that the STLV-1-infected baboon model might be useful for developing a vaccine against HTLV-1.

Splenic transitional B-cells (T1 and T2) are selected to avoid self-reactivity and to safeguard against autoimmunity, then differentiate into mature follicular (FO-I and FO-II) and marginal zone (MZ) subsets. Transcriptomic analysis by RNA-seq of the five B-cell subsets revealed T1 cell signature genes included RAG suggesting a potential for receptor revision. T1 to T2 B-cell differentiation was marked by a switch from Myb to Myc, increased expression of the PI3K adapter DAP10 and MHC class II. FO-II may be an intermediate in FO-I differentiation and may also become MZ B-cells as suggested by principle component analysis. MZ B-cells possessed the most distinct transcriptome including down-regulation of CD45 phosphatase-associated protein (CD45-AP/PTPRC-AP), as well as upregulation of IL-9R and innate molecules TLR3, TLR7, and bactericidal Perforin-2 (MPEG1). Among the endosomal TLRs, stimulation via TLR3 further enhanced Perforin-2 expression exclusively in MZ B-cells. Using gene-deleted and overexpressing transgenic mice we show that IL-9/IL-9R interaction resulted in rapid activation of STAT1, 3, and 5, primarily in MZ B-cells. Importantly, CD45-AP mutant mice had reduced transitional and increased mature MZ and FO B-cells, suggesting that it prevents premature entry of transitional B-cells to the mature B-cell pool or their survival and proliferation. Together, these findings suggest, developmental plasticity among splenic B-cell subsets, potential for receptor revision in peripheral tolerance whereas enhanced metabolism coincides with T2 to mature B-cell differentiation. Further, unique core transcriptional signatures in MZ B-cells may control their innate features.

Recent studies have underscored the importance of memory T cells in mediating protective immunity against pathogens and cancer. Pharmacological inhibition of regulators that mediate T cell differentiation promotes the differentiation of activated CD8(+) T cells into memory cells. Nonetheless, pharmacological agents have broad targets and can induce undesirable immunosuppressive effects. Here, we tested the hypothesis that aptamer-targeted siRNA inhibition of mTOR complex 1 (mTORC1) function in CD8(+) T cells can enhance their differentiation into memory T cells and potentiate antitumor immunity more effectively than the pharmacologic inhibitor rapa,mycin. To specifically target activated cells, we conjugated an siRNA targeting the mTORC1 component raptor to an aptamer that binds 4-1BB, a costimulatory molecule that is expressed on CD8(+) T cells following TCR stimulation. We found that systemic administration of the 4-1BB aptamer-raptor siRNA to mice downregulated mTORC1 activity in the majority of CD8(+) T cells, leading to the generation of a potent memory response that exhibited cytotoxic effector functions and enhanced vaccine-induced protective immunity in tumor-bearing mice. In contrast, while treatment with the general mTORC1 inhibitor rapamycin also enhanced antigen-activated CD8(+) T cell persistence, the cytotoxic effector functions of the reactivated memory cells were reduced and the alloreactivity of DCs was diminished. Consistent with the immunological findings, mice treated with rapamycin, but not with 4-1BB aptamer-raptor siRNA, failed to reject a subsequent tumor challenge.

B-lymphocytes are integral to host defense against microbial pathogens and are associated with many autoimmune diseases. The B-cell receptor implements B-cell self-tolerance based on the antigen specificity, and B-cell-activating factor receptor (BAFF-R) imposes homeostatic control. While shaping the repertoire, the immune tolerance process also culls mature B cells into distinct populations. The activation response of B cells is tailored to the type of pathogen attack and is facilitated by T-cell help via CD40/CD40L interaction and/or innate cell help via toll-like receptors in conjunction with BAFF receptors and ligands. Activated effector B cells not only produce antibodies, but also produce a variety of cytokines to enhance and suppress the immune response. Not surprisingly, B cells play multiple roles in both humoral and cellular immune responses during infection and autoimmune pathogenesis. Here, we discuss how gene expression and signaling networks regulate peripheral B-cell tolerance, B-cell effector functions and emerging therapies targeting B-cell signaling in autoimmune diseases.

Abstract Recent studies in mice, nonhuman primates, and clinical trials in human patients have emphasized the importance of the persistence of the vaccine-induced immune response, immunological memory, in mediating protective immunity against infectious diseases and cancer. Multiple extrinsic pathways control the differentiation of CD8+ cytotoxic T lymphocytes (CTL) into effector or memory cells by regulating the balanced expression of intracellular mediators in the antigen-activated T cells. Notably, inhibition of mediators of effector differentiation like mTOR, GSK3β or T-bet using genetic means or whenever available pharmacological agents, not only prevented the accumulation of the short-lived effectors but also redirected the activated T cells to differentiate along the memory pathway. For example, pharmacological inhibition of mTOR or GSK3β in mice with rapamycin or TSW119, respectively, led to the differentiation of antigen activated CD8+ T cells into long lasting memory cells that exhibited enhanced antiviral and antitumor immunity. Nevertheless, pharmacological agents often exhibit undesirable effects reflecting the broad distribution of their targets. For example, rapamycin inhibition of mTOR promotes the development of immune suppressive regulatory CD4+ T cells and GSK3β; inhibition polarizes dendritic cells (DC) to a tolerogenic state, arguably counterproductive in the setting of vaccination. In addition, development of pharmacological agents to modulate the function of intracellular targets that are not accessible to antibodies (“undruggable targets”) is highly challenging, and their availability especially for clinical use is limited. Here we describe a versatile, broadly applicable, and clinically feasible approach to promote the generation of memory T cell responses that addresses the main limitations of pharmacological agents. RNAi was used to downregulate intracellular mediators of effector differentiation that were targeted to CD8+ T cells by conjugation to aptamer ligands. We have shown that a CD8+ T cell targeted 4-1BB aptamer-raptor siRNA conjugate administered to mice by tail vein injection downregulated mTORC1, while preserving mTORC2, activity in at least 60% of adoptively transferred OVA-specific transgenic OT-I cells while sparing host cells. Both rapamycin and aptamer-siRNA conjugate led to the development of an enhanced memory response in mice as judged by the ability of the memory cells to proliferate in response to re-exposure to antigen at a latter time point. However, the rapamycin generated CTL were defective in their cytotoxic effector functions and failed to elicit protective immunity against a tumor challenge. In contrast, the aptamer-raptor siRNA generated CTL exhibited normal cytotoxic effector functions and enhanced vaccine-induce protective antitumor immunity in both prophylactic and therapeutic tumor models. Reflecting the lack of cell specificity of mTOR inhibition by rapamycin, and providing a potential mechanisms underlying the effector defect of rapamycin generated CTL, dendritic cells from mice treated with rapamycin, but not with aptamer-raptor siRNA conjugate, exhibited reduced MLR activity, consistent with the known suppressive effects of rapamycin induced mTOR inhibition on DC. These studies demonstrate the feasibility and efficiency of aptamer targeted delivery of siRNAs to immune cells, and underscore the potential advantages of aptamer-targeted siRNA delivery over nontargeted administration of pharmacological agents. Citation Format: Alexey Berezhnoy, Iris Castro, Agata Levay, Thomas Malek, Eli Gilboa. Aptamer-targeted siRNA-mediated inhibition of mTORC1 in antigen-primed CD8+ cytotoxic T lymphocytes enhances immunological memory and antitumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B68.

Abstract Recent studies have highlighted the importance of immunological memory in mediating protective immunity in chronic infectious diseases and cancer. Inhibition of intracellular mediators of T cell differentiation with pharmacological agents was shown to promote memory differentiation in mice. However, given their often pleiotropic effects, non-targeted administration of pharmacological agents could compromise their immune promoting properties. We have shown that a CD8+ T cell targeted 4-1BB aptamer -raptor siRNA conjugate downregulated mTORC1, while preserving mTORC2, activity in 60% of adoptively transferred OVA-primed OT-I cells while sparing host cells. Both rapamycin and aptamer-siRNA conjugate led to the development of an enhanced proliferative recall response. However, the rapamycin-generated memory cells were defective in their in vivo cytotoxic effector functions which correlated with reduced perforin expression. Likewise, rapamycin or aptamer-siRNA conjugate treatment enhanced the in vivo differentiation of gp100-primed Pmel-1 cells into memory cells. Consistent with a cytotoxic defect in the rapamycin-generated memory cells, the rapamycin-treated mice were not able to inhibit a challenge with gp100-expressing B16 melanoma tumor cells. These observations support the premise that in vivo cell-targeted inhibition of mTOR, and conceivably other mediators of T cell differentiation, will circumvent undesirable effects of non-targeted inhibition by pharmacological agents.

Much is known concerning the cellular and molecular basis for CD8+ T memory immune responses. Nevertheless, conditions that selectively support memory generation have remained elusive. In this study, we show that an immunization regimen that delivers TCR signals through a defined antigenic peptide, inflammatory signals through LPS, and growth and differentiation signals through the IL-2R initially favors Ag-specific CD8+ T cells to develop rapidly and substantially into T effector-memory cells by TCR transgenic OVA-specific OT-I CD8+ T cells. Amplified CD8+ T memory development depends upon a critical frequency of Ag-specific T cells and direct responsiveness to IL-2. A homologous prime-boost immunization protocol with transiently enhanced IL-2R signaling in normal mice led to persistent polyclonal Ag-specific CD8+ T cells that supported protective immunity to Listeria monocytogenes. These results identify a general approach for amplified T memory development that may be useful to optimize vaccines aimed at generating robust cell-mediated immunity.

Recent work suggests that IL-2 and IL-15 induce distinctive levels of signaling through common receptor subunits and that such varied signaling directs the fate of Ag-activated CD8(+) T cells. In this study, we directly examined proximal signaling by IL-2 and IL-15 and CD8(+) T cell primary and memory responses as a consequence of varied CD122-dependent signaling. Initially, IL-2 and IL-15 induced similar p-STAT5 and p-S6 activation, but these activities were only sustained by IL-2. Transient IL-15-dependent signaling is due to limited expression of IL-15Rα. To investigate the outcome of varied CD122 signaling for CD8(+) T cell responses in vivo, OT-I T cells were used from mouse models where CD122 signals were attenuated by mutations within the cytoplasmic tail of CD122 or intrinsic survival function was provided in the absence of CD122 expression by transgenic Bcl-2. In the absence of CD122 signaling, generally normal primary response occurred, but the primed CD8(+) T cells were not maintained. In marked contrast, weak CD122 signaling supported development and survival of T central-memory (T(CM)) but not T effector-memory (T(EM)) cells. Transgenic expression of Bcl-2 in CD122(-/-) CD8(+) T cells also supported the survival and persistence of T(CM) cells but did not rescue T(EM) development. These data indicate that weak CD122 signals readily support T(CM) development largely through providing survival signals. However, stronger signals, independent of Bcl-2, are required for T(EM) development. Our findings are consistent with a model whereby low, intermediate, and high CD122 signaling support T(CM) memory survival, T(EM) programming, and terminal T effector cell differentiation, respectively.

Abstract Splenic immature B cells traverse through the early transitional (T1) and late transitional (T2) stages to produce the mature B cell repertoire. BCR specificity-based selection ensures elimination of pathogenic B cells by apoptosis, receptor editing or anergy. These processes are tightly regulated by anti- and pro-apoptotic members of the Bcl-2 family. Conventional gene deletion of the pro-apoptotic member Bim lead to prolonged life-span of autoreactive B cells, defective anergy and production of autoantibodies. Whether these pathophysiological conditions arise as a result of B cell autonomous function of Bim remains unclear. We have found that T1 cells express higher levels of Bim and display increased susceptibility to BCR-induced apoptosis than more mature B cells. These properties are shared by BCR signaling defective Bruton’s tyrosine kinase (Btk) deficient B cells. We investigated the B cell-intrinsic role of Bim in the regulation of B cell maturation and survival in mice carrying floxed Bim and CD19-Cre in Btk-sufficient or deficient background. B lineage-specific deletion of Bim increased the B cell compartment through selective expansion of mature B cells and significantly restored mature B cells in Btk-mutant mice. These studies demonstrate a B cell autonomous role for Bim in BCR-dependent regulation of B cell homeostasis. Analysis of these mice in the context of BCR transgene will reveal the B cell autonomous function of Bim in tolerance and autoimmunity.

Interleukin-2 receptor (IL-2R) signaling regulates tolerance and immunity. Here, we review recent work concerning the structure, signaling, and function of the IL-2R, emphasizing the contribution of IL-2 for T cell-dependent activity in vivo. IL-2R signaling influences two discrete aspects of immune responses by CD8(+) T cells, terminal differentiation of effector cells in primary responses, and aspects of memory recall responses. IL-2 also delivers essential signals for thymic development of regulatory T (Treg) cells and later to promote their homeostasis and function. Each of these outcomes on T effector and Treg cells requires distinct amounts of IL-2R signaling, with low IL-2R signaling sufficient for many key aspects of Treg cells. Thus, tolerance is readily maintained and favored with limited IL-2.